Lipocalins constitute the vast majority of mammalian dander allergens. Cytoplasmic fatty acid-binding proteins are distantly related to extracellular lipocalins and β-lactoglobulins. They were identified as minor allergens in mites (group 13).

Triabins are extracellular proteins distantly related to lipocalins that were identified in insects. In the saliva of hematophagous species, they function as serine protease inhibitors that interfere with blood clotting in the host (Figure 24).³⁴⁶

* Examples for mammalian allergenic lipocalins are Equ c 1 from horse, Bos d 2 from cattle, Can f 1 and Can f 2 from dog, Fel d 4 from cat and Mus m 1 from mouse (see chapter C07).

* The prototypic allergenic member of the β-lactoglobulin subfamily is Bos d 5 from cow's milk (see chapter B10).

* Allergenic cytoplasmic fatty acid-binding proteins are Der p 13, Der f 13, and Blo t 13 from house dust mites (see chapter B04).

* Allergens from the triabin family include the minor allergens Per a 4 and Bla g 4 from American and German cockroach as well as Tria p 1 (procalin) from the California kissing bug (see chapter B05).

## 2.8. DPBB (double-psi beta barrel) superfamily

Members of the DPBB superfamily fold into six-stranded β-barrels defined by their distinct strand connections³⁴⁷ [40]. They are composed of two psi-loop motifs that consist of three β-strands that assume the form of the Greek letter psi (Ψ) (Figure 25). The grass pollen group 1 allergens belong to the β-expansins and contain two domains. The amino-terminal domain assumes the six-stranded double-psi β-barrel topology, and the carboxy-terminal domain consists of two stacked β-sheets with an immunoglobulin-like fold.³⁴⁸ Expansins possess cell wall loosening activities in growing cells including penetration of pollen tubes through the stigma and style.³⁴⁹ Group 2 and 3 grass pollen allergens are related to the C-terminal domain of β-expansins but lack the N-terminal DPBB domain.³⁴⁸

The kiwellin family derives its name from the kiwifruit, in which the first family members were characterized. Kiwellin contains a small cysteine-rich N-terminal domain linked to a C-terminal DPBB domain and is cleaved into its domains by the main kiwifruit protease actinidin.³⁵⁰ Kiwellins are part of the plant defense system as shown for an anti-fungal kiwellin from maize.³⁵¹

Another family of plant-defense related proteins is the barwin (barley wound-induced) family, also named pathogenesis-related proteins PR-4. Barwin-like proteins contain a DPBB domain that in some members (PR-4 class II), such as the major rubber latex allergen Hev b 6 (prohevein), is linked to an N-terminal cysteine-rich hevein-like domain and cleaved upon activation into its constituent domains.³⁵²

* Examples for group 1 grass pollen allergens are Lol p 1 from ryegrass, Phl p 1 from Timothy grass, and Poa p 1 from Kentucky bluegrass. Group 2/3 grass pollen allergens include Dac g 2 and Dac g 3 from orchard grass, Lol p 2 and Lol p 3 from ryegrass, and Phl p 2 and Phl p 3 from Timothy grass. Members of both groups are major allergens (see chapter B02).

* Allergenic members of the kiwellin family are the minor allergens Act c 5 and Act d 5 from gold and green kiwifruit, respectively.

* The most important allergenic member of the barwin family is the major natural rubber latex allergen Hev b 6 (see chapter B22).

## 2.9. The CAP superfamily

The CAP (CRISP, Antigen 5, PR-1) superfamily of proteins comprises the cysteine-rich secretory proteins (CRISPs) in the mammalian male reproductive tract and in the venom secretory ducts in many snakes, lizards, and other vertebrates, the insect venom antigen 5 family, and the plant pathogenesis-related PR-1 proteins.³⁵³ Members of this superfamily are widely spread across the bacterial, fungal, plant and animal kingdoms.³⁵⁴ The CAP domains, which are α-β-α sandwiches where two layers of α-helices flank the central three-stranded anti-parallel β-sheet, are involved in a large variety of biological processes such as reproduction, tumor suppression, and immune regulation.

Most allergens from this superfamily are major insect venom allergens including group 5 wasp allergens and group 3 ant allergens. Examples are Ves v 5 from yellow jacket, Dol m 5 from the white face hornet, and Sol i 3 from the red imported fire ant (Figure 26); (see chapter B21). Allergens from the plant PR-1 family are minor allergens. They include the Bermuda grass pollen allergen Cyn d 24, the mugwort pollen allergen Art v 2 and the muskmelon allergen Cuc m 3 (Figure 26).

### Secreted lipocalins

<figure><img src="image_74.png" /><figcaption>Equ c 1</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>Dog</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>Cat</figcaption></figure>

<figure><img src="image_77.png" /><figcaption>Horse</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>Rodents</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>Milk</figcaption></figure>

### Cytosolic fatty acid binding proteins

### Triabins

<figure><img src="image_80.png" /><figcaption>Der f 13</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>Mites</figcaption></figure>

<figure><img src="image_82.png" /><figcaption>Per a 4</figcaption></figure>

<figure><img src="image_83.png" /><figcaption>Cockroaches</figcaption></figure>

**FIGURE 24** Distribution of allergens from the calycin superfamily. The representative structures shown are Equ c 1, an extracellular lipocalin from horse dander (PDB: 1ew3), Der f 13, a cytosolic fatty acid-binding protein from house dust mite (PDB: 2a0a), and Per a 4, a triabin like allergen from American cockroach (PDB: 3ebw).
Lipocalins constitute the vast majority of mammalian dander allergens. Cytoplasmic fatty acid-binding proteins are distantly related to extracellular lipocalins and β-lactoglobulins. They were identified as minor allergens in mites (group 13).

Triabins are extracellular proteins distantly related to lipocalins that were identified in insects. In the saliva of hematophagous species, they function as serine protease inhibitors that interfere with blood clotting in the host (Figure 24).³⁴⁶

* Examples for mammalian allergenic lipocalins are Equ c 1 from horse, Bos d 2 from cattle, Can f 1 and Can f 2 from dog, Fel d 4 from cat and Mus m 1 from mouse (see chapter C07).

* The prototypic allergenic member of the β-lactoglobulin subfamily is Bos d 5 from cow's milk (see chapter B10).

* Allergenic cytoplasmic fatty acid-binding proteins are Der p 13, Der f 13, and Blo t 13 from house dust mites (see chapter B04).

* Allergens from the triabin family include the minor allergens Per a 4 and Bla g 4 from American and German cockroach as well as Tria p 1 (procalin) from the California kissing bug (see chapter B05).

## 2.8. DPBB (double-psi beta barrel) superfamily

Members of the DPBB superfamily fold into six-stranded β-barrels defined by their distinct strand connections³⁴⁷ [40]. They are composed of two psi-loop motifs that consist of three β-strands that assume the form of the Greek letter psi (Ψ) (Figure 25). The grass pollen group 1 allergens belong to the β-expansins and contain two domains. The amino-terminal domain assumes the six-stranded double-psi β-barrel topology, and the carboxy-terminal domain consists of two stacked β-sheets with an immunoglobulin-like fold.³⁴⁸ Expansins possess cell wall loosening activities in growing cells including penetration of pollen tubes through the stigma and style.³⁴⁹ Group 2 and 3 grass pollen allergens are related to the C-terminal domain of β-expansins but lack the N-terminal DPBB domain.³⁴⁸

The kiwellin family derives its name from the kiwifruit, in which the first family members were characterized. Kiwellin contains a small cysteine-rich N-terminal domain linked to a C-terminal DPBB domain and is cleaved into its domains by the main kiwifruit protease actinidin.³⁵⁰ Kiwellins are part of the plant defense system as shown for an anti-fungal kiwellin from maize.³⁵¹

Another family of plant-defense related proteins is the barwin (barley wound-induced) family, also named pathogenesis-related proteins PR-4. Barwin-like proteins contain a DPBB domain that in some members (PR-4 class II), such as the major rubber latex allergen Hev b 6 (prohevein), is linked to an N-terminal cysteine-rich hevein-like domain and cleaved upon activation into its constituent domains.³⁵²

* Examples for group 1 grass pollen allergens are Lol p 1 from ryegrass, Phl p 1 from Timothy grass, and Poa p 1 from Kentucky bluegrass. Group 2/3 grass pollen allergens include Dac g 2 and Dac g 3 from orchard grass, Lol p 2 and Lol p 3 from ryegrass, and Phl p 2 and Phl p 3 from Timothy grass. Members of both groups are major allergens (see chapter B02).

* Allergenic members of the kiwellin family are the minor allergens Act c 5 and Act d 5 from gold and green kiwifruit, respectively.

* The most important allergenic member of the barwin family is the major natural rubber latex allergen Hev b 6 (see chapter B22).

## 2.9. The CAP superfamily

The CAP (CRISP, Antigen 5, PR-1) superfamily of proteins comprises the cysteine-rich secretory proteins (CRISPs) in the mammalian male reproductive tract and in the venom secretory ducts in many snakes, lizards, and other vertebrates, the insect venom antigen 5 family, and the plant pathogenesis-related PR-1 proteins.³⁵³ Members of this superfamily are widely spread across the bacterial, fungal, plant and animal kingdoms.³⁵⁴ The CAP domains, which are α-β-α sandwiches where two layers of α-helices flank the central three-stranded anti-parallel β-sheet, are involved in a large variety of biological processes such as reproduction, tumor suppression, and immune regulation.

Most allergens from this superfamily are major insect venom allergens including group 5 wasp allergens and group 3 ant allergens. Examples are Ves v 5 from yellow jacket, Dol m 5 from the white face hornet, and Sol i 3 from the red imported fire ant (Figure 26); (see chapter B21). Allergens from the plant PR-1 family are minor allergens. They include the Bermuda grass pollen allergen Cyn d 24, the mugwort pollen allergen Art v 2 and the muskmelon allergen Cuc m 3 (Figure 26).

### Secreted lipocalins

<figure><img src="image_74.png" /><figcaption>Equ c 1</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>Dog</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>Cat</figcaption></figure>

<figure><img src="image_77.png" /><figcaption>Horse</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>Rodents</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>Milk</figcaption></figure>

### Cytosolic fatty acid binding proteins

### Triabins

<figure><img src="image_80.png" /><figcaption>Der f 13</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>Mites</figcaption></figure>

<figure><img src="image_82.png" /><figcaption>Per a 4</figcaption></figure>

<figure><img src="image_83.png" /><figcaption>Cockroaches</figcaption></figure>

**FIGURE 24** Distribution of allergens from the calycin superfamily. The representative structures shown are Equ c 1, an extracellular lipocalin from horse dander (PDB: 1ew3), Der f 13, a cytosolic fatty acid-binding protein from house dust mite (PDB: 2a0a), and Per a 4, a triabin like allergen from American cockroach (PDB: 3ebw).
### Expansin and expansin-like

<figure><img src="image_84.png" /></figure>

Phl p 1

<figure><img src="image_85.png" /></figure>

Phl p 2

<figure><img src="image_86.png" /></figure>

Grass pollen

Kiwellin

Barwin

<figure><img src="image_87.png" /></figure>

Act d 5

<figure><img src="image_88.png" /></figure>

Kiwifruit

<figure><img src="image_89.png" /></figure>

Cari p barwin

<figure><img src="image_90.png" /></figure>

Latex

**FIGURE 25** Distribution of allergens from the DPBB superfamily. The representative structures shown are Phl p 1 (PDB: 1n10) and Phl p 2 (PDB: 1who), a β-expansin and expansin like protein from Timothy grass pollen, Act d 5, a kiwellin from kiwifruit (PDB: 4x9u), and Cari p barwin, a nonallergenic barwin-like protein from papaya (PDB: 4jp7). No structures of barwin domains from allergens have been determined.

### 2.10. Pectate lyase-like superfamily

This large and functionally diverse superfamily is defined by the common core structure of its members, a parallel right-handed β-helix.³⁵⁵ Its members are found in eukaryotes, bacteria and viruses. Many pectate lyase-like enzymes are involved in carbohydrate metabolism. Allergenic members of this superfamily were identified mainly in pollen (Figure 27).

Pectate lyases are responsible for the eliminative cleavage of pectate and involved in pollen tube growth and fruit ripening but also expressed in plant pathogenic microorganisms. Allergenic pectate lyases are present in pollen of conifers from the Cupressaceae family (cypress and cedar) and weeds from the Asteraceae family (ragweed, sunflower and mugwort). They are highly abundant and represent dominant allergens in their pollen allergen sources (except Art v 6 from mugwort). Analogous to sequence identity, IgE cross-reactivity between botanically related members is observed, while limited between allergens from conifers and weeds.³⁵⁶,³⁵⁷

Allergens were found in two other pectate-lyase-like families that contain enzymes taking part in plants in cell wall modification and breakdown, while their homologues in plant pathogenic fungi

Venom antigen 5

<figure><img src="image_91.png" /></figure>

Ves v 5

<figure><img src="image_92.png" /></figure>

Wasps

<figure><img src="image_93.png" /></figure>

Ants

Plant PR-1

<figure><img src="image_94.png" /></figure>

Sola I PR-1

<figure><img src="image_95.png" /></figure>

Mugwort

<figure><img src="image_96.png" /></figure>

Bermuda grass

<figure><img src="image_97.png" /></figure>

Musk melon

**FIGURE 26** Distribution of allergens from the CAP superfamily. Representative structures shown are Ves v 5 from yellow jacket (PDB: 1qnx) and Sola I PR-1, a nonallergenic PR-1 from tomato (PDB: 1cfe). No structures of allergenic PR-1 proteins have been determined.

### Pectate lyase

<figure><img src="image_98.png" /></figure>

Jun a 1

<figure><img src="image_99.png" /></figure>

Cypress

<figure><img src="image_100.png" /></figure>

Juniper

<figure><img src="image_101.png" /></figure>

Ragweed

### Pectinesterase

<figure><img src="image_102.png" /></figure>

Dau c PE

<figure><img src="image_103.png" /></figure>

Saltwort

<figure><img src="image_104.png" /></figure>

Olive pollen

<figure><img src="image_105.png" /></figure>

Kiwifruit

### Polygalacturonase

<figure><img src="image_106.png" /></figure>

Asp n PG

<figure><img src="image_107.png" /></figure>

Grasses

<figure><img src="image_108.png" /></figure>

Cypress

<figure><img src="image_109.png" /></figure>

Juniper

<figure><img src="image_110.png" /></figure>

Plane tree

**FIGURE 27** Distribution of allergens from the pectate-lyase-like superfamily. The representative structures shown are Jun a 1, a pectate lyase from mountain cedar (PDB: 1pxz), Asp n PG, a nonallergenic polygalacturonase from *Aspergillus niger* (PDB: 1nhc) and Dau c PE, a nonallergenic pectinesterase from carrot (PDB: 1gq8). No structures of allergenic polygalacturonases and pectinesterases have been determined.
### Expansin and expansin-like

<figure><img src="image_84.png" /></figure>

Phl p 1

<figure><img src="image_85.png" /></figure>

Phl p 2

<figure><img src="image_86.png" /></figure>

Grass pollen

Kiwellin

Barwin

<figure><img src="image_87.png" /></figure>

Act d 5

<figure><img src="image_88.png" /></figure>

Kiwifruit

<figure><img src="image_89.png" /></figure>

Cari p barwin

<figure><img src="image_90.png" /></figure>

Latex

**FIGURE 25** Distribution of allergens from the DPBB superfamily. The representative structures shown are Phl p 1 (PDB: 1n10) and Phl p 2 (PDB: 1who), a β-expansin and expansin like protein from Timothy grass pollen, Act d 5, a kiwellin from kiwifruit (PDB: 4x9u), and Cari p barwin, a nonallergenic barwin-like protein from papaya (PDB: 4jp7). No structures of barwin domains from allergens have been determined.

### 2.10. Pectate lyase-like superfamily

This large and functionally diverse superfamily is defined by the common core structure of its members, a parallel right-handed β-helix.³⁵⁵ Its members are found in eukaryotes, bacteria and viruses. Many pectate lyase-like enzymes are involved in carbohydrate metabolism. Allergenic members of this superfamily were identified mainly in pollen (Figure 27).

Pectate lyases are responsible for the eliminative cleavage of pectate and involved in pollen tube growth and fruit ripening but also expressed in plant pathogenic microorganisms. Allergenic pectate lyases are present in pollen of conifers from the Cupressaceae family (cypress and cedar) and weeds from the Asteraceae family (ragweed, sunflower and mugwort). They are highly abundant and represent dominant allergens in their pollen allergen sources (except Art v 6 from mugwort). Analogous to sequence identity, IgE cross-reactivity between botanically related members is observed, while limited between allergens from conifers and weeds.³⁵⁶,³⁵⁷

Allergens were found in two other pectate-lyase-like families that contain enzymes taking part in plants in cell wall modification and breakdown, while their homologues in plant pathogenic fungi

Venom antigen 5

<figure><img src="image_91.png" /></figure>

Ves v 5

<figure><img src="image_92.png" /></figure>

Wasps

<figure><img src="image_93.png" /></figure>

Ants

Plant PR-1

<figure><img src="image_94.png" /></figure>

Sola I PR-1

<figure><img src="image_95.png" /></figure>

Mugwort

<figure><img src="image_96.png" /></figure>

Bermuda grass

<figure><img src="image_97.png" /></figure>

Musk melon

**FIGURE 26** Distribution of allergens from the CAP superfamily. Representative structures shown are Ves v 5 from yellow jacket (PDB: 1qnx) and Sola I PR-1, a nonallergenic PR-1 from tomato (PDB: 1cfe). No structures of allergenic PR-1 proteins have been determined.

### Pectate lyase

<figure><img src="image_98.png" /></figure>

Jun a 1

<figure><img src="image_99.png" /></figure>

Cypress

<figure><img src="image_100.png" /></figure>

Juniper

<figure><img src="image_101.png" /></figure>

Ragweed

### Pectinesterase

<figure><img src="image_102.png" /></figure>

Dau c PE

<figure><img src="image_103.png" /></figure>

Saltwort

<figure><img src="image_104.png" /></figure>

Olive pollen

<figure><img src="image_105.png" /></figure>

Kiwifruit

### Polygalacturonase

<figure><img src="image_106.png" /></figure>

Asp n PG

<figure><img src="image_107.png" /></figure>

Grasses

<figure><img src="image_108.png" /></figure>

Cypress

<figure><img src="image_109.png" /></figure>

Juniper

<figure><img src="image_110.png" /></figure>

Plane tree

**FIGURE 27** Distribution of allergens from the pectate-lyase-like superfamily. The representative structures shown are Jun a 1, a pectate lyase from mountain cedar (PDB: 1pxz), Asp n PG, a nonallergenic polygalacturonase from *Aspergillus niger* (PDB: 1nhc) and Dau c PE, a nonallergenic pectinesterase from carrot (PDB: 1gq8). No structures of allergenic polygalacturonases and pectinesterases have been determined.
and bacteria are responsible for maceration and soft-rotting of plant tissue. Polygalacturonases, also known as pectinases or glycoside hydrolases family 28, catalyse the hydrolysis of pectate. Allergens from this family are glycosylated minor allergens found in pollen from various plant families such as Cupressaceae (group 2) and grasses (group 13). Pectinesterases catalyse the de-esterification of pectin into pectate and methanol. Allergenic pectinesterases were identified in pollen and plant foods.

* Examples of allergenic pectate lyases are Jun a 1 from mountain cedar, Cry j 1 from Japanese cedar and Amb a 1 from ragweed (see chapter B01 and chapter B03).

* Allergenic polygalacturonases include Jun a 2 from mountain cedar, Cry j 2 from Japanese cedar, Pla a 2 from plane tree and Phl p 13 from Timothy grass.

* Allergenic pectinesterases are Sal k 1 and Ole e 11, major allergens from saltwort and olive pollen, respectively, as well as Act d 7, a minor allergen from kiwifruit.

## 2.11. Transthyretin superfamily

The transthyretin superfamily (Pfam clan CL0287) is a functionally diverse superfamily whose members are found in all domains of life and defined by their common 7-stranded β-sandwich fold. The only family that contains allergens is the Ole e 1-like family. Proteins containing Ole e 1-like domains, also known as PAC (Proline-Rich, Arabinogalactan Proteins, Conserved Cysteines) domains are found in all land plants and proposed to have a role in pollen tube development and glycan-related modifications in the cell wall<sup>1650</sup>. They comprise a structurally conserved disulfide-stabilized β-barrel and loop regions of varying length and sequences.<sup>358</sup> Ole e 1 from olive pollen was the first identified allergenic member, and Ole e 1-like proteins represent major and minor allergens in pollen of ash, privet, plantain, grasses, chenopod, and Russian thistle (Figure 28). Sequence identity and IgE cross-reactivity is high between olive and ash, while for other Ole e 1-like allergens limited or no cross-reactivity is observed.<sup>358</sup> Recently, TLR-independent activation of innate immune cells was reported for Lig v 1 from privet.<sup>359</sup>

Ole e 1-related allergens include Ole e 1 from olive, Fra e 1 from ash, Pla I 1 from plantain and Phl p 11 from Timothy grass (see chapters B01, B02, and B03).

<figure><img src="image_111.png" /><figcaption>**FIGURE 28** Distribution of allergens from the Ole e 1 family. The depicted structure is Pla I 1 from narrowleaf plantain pollen (PDB: 4z8w).</figcaption></figure>

## 2.12. Papain-like cysteine superfamily

Cysteine proteases contain a cysteine residue at their active catalytic site and catalyze the hydrolysis of peptides and proteins. Despite structural similarities involving the residues that surround the catalytic site, cysteine proteases possess only low levels of overall sequence similarities.<sup>360</sup> There are eight superfamilies of cysteine proteases (https://www.ebi.ac.uk/merops), named CA to CP.<sup>361</sup> Papain belongs to the C1 family of the CA superfamily. Members of the papain-like cysteine protease (PLCP) family are wide-spread and have been found in baculovirus, bacteria, yeast, plants and animals. PLCPs, such as papain, ficin and bromelain, are the most abundant family of cysteine proteases in plants and play essential roles in biotic/abiotic stress responses, growth and senescence.<sup>362</sup>

Allergenic papain-like cysteine proteases include plant food allergens such as actinidin (Act d 1), the major allergen from kiwifruit, bromelain (Ana c 2) from pineapple, ficin from fig and chymopapain (Cari p 2) from papaya, as well as group 1 mite allergens such as the major dust mite allergen Der p 1 (Figure 29) (see chapters B15 and B04).

## 2 - Databases

During the latest two decades, various databases covering allergens and allergen-related data have been established by academic institutions and the industry. These databases contain overlapping data, but address different user groups, use varying criteria for including allergens, and some provide additional tools such as sequence comparisons. The most widely-used, freely accessible databases are summarized below. A more extensive discussion of allergen databases was published recently.<sup>363</sup>

### 2.1 WHO/IUIS allergen nomenclature database (http://www.allergen.org/)

The allergen nomenclature database is a repository of allergens that underwent a submission and evaluation process and were accepted by the WHO/IUIS Allergen Nomenclature Sub-Committee, a panel of experts in allergen characterization, structure, and function. This is the only body officially authorized to assign allergen designations. The database contains links for each allergen to the nucleotide and protein databases of the NCBI (National Center

<figure><img src="image_112.png" /><figcaption>**FIGURE 29** Distribution of allergenic members of the papain-like cysteine protease family. The depicted structure is Act d 1, actinidin from kiwifruit (PDB: 2act).</figcaption></figure>
and bacteria are responsible for maceration and soft-rotting of plant tissue. Polygalacturonases, also known as pectinases or glycoside hydrolases family 28, catalyse the hydrolysis of pectate. Allergens from this family are glycosylated minor allergens found in pollen from various plant families such as Cupressaceae (group 2) and grasses (group 13). Pectinesterases catalyse the de-esterification of pectin into pectate and methanol. Allergenic pectinesterases were identified in pollen and plant foods.

* Examples of allergenic pectate lyases are Jun a 1 from mountain cedar, Cry j 1 from Japanese cedar and Amb a 1 from ragweed (see chapter B01 and chapter B03).

* Allergenic polygalacturonases include Jun a 2 from mountain cedar, Cry j 2 from Japanese cedar, Pla a 2 from plane tree and Phl p 13 from Timothy grass.

* Allergenic pectinesterases are Sal k 1 and Ole e 11, major allergens from saltwort and olive pollen, respectively, as well as Act d 7, a minor allergen from kiwifruit.

## 2.11. Transthyretin superfamily

The transthyretin superfamily (Pfam clan CL0287) is a functionally diverse superfamily whose members are found in all domains of life and defined by their common 7-stranded β-sandwich fold. The only family that contains allergens is the Ole e 1-like family. Proteins containing Ole e 1-like domains, also known as PAC (Proline-Rich, Arabinogalactan Proteins, Conserved Cysteines) domains are found in all land plants and proposed to have a role in pollen tube development and glycan-related modifications in the cell wall<sup>1650</sup>. They comprise a structurally conserved disulfide-stabilized β-barrel and loop regions of varying length and sequences.<sup>358</sup> Ole e 1 from olive pollen was the first identified allergenic member, and Ole e 1-like proteins represent major and minor allergens in pollen of ash, privet, plantain, grasses, chenopod, and Russian thistle (Figure 28). Sequence identity and IgE cross-reactivity is high between olive and ash, while for other Ole e 1-like allergens limited or no cross-reactivity is observed.<sup>358</sup> Recently, TLR-independent activation of innate immune cells was reported for Lig v 1 from privet.<sup>359</sup>

Ole e 1-related allergens include Ole e 1 from olive, Fra e 1 from ash, Pla I 1 from plantain and Phl p 11 from Timothy grass (see chapters B01, B02, and B03).

<figure><img src="image_111.png" /><figcaption>**FIGURE 28** Distribution of allergens from the Ole e 1 family. The depicted structure is Pla I 1 from narrowleaf plantain pollen (PDB: 4z8w).</figcaption></figure>

## 2.12. Papain-like cysteine superfamily

Cysteine proteases contain a cysteine residue at their active catalytic site and catalyze the hydrolysis of peptides and proteins. Despite structural similarities involving the residues that surround the catalytic site, cysteine proteases possess only low levels of overall sequence similarities.<sup>360</sup> There are eight superfamilies of cysteine proteases (https://www.ebi.ac.uk/merops), named CA to CP.<sup>361</sup> Papain belongs to the C1 family of the CA superfamily. Members of the papain-like cysteine protease (PLCP) family are wide-spread and have been found in baculovirus, bacteria, yeast, plants and animals. PLCPs, such as papain, ficin and bromelain, are the most abundant family of cysteine proteases in plants and play essential roles in biotic/abiotic stress responses, growth and senescence.<sup>362</sup>

Allergenic papain-like cysteine proteases include plant food allergens such as actinidin (Act d 1), the major allergen from kiwifruit, bromelain (Ana c 2) from pineapple, ficin from fig and chymopapain (Cari p 2) from papaya, as well as group 1 mite allergens such as the major dust mite allergen Der p 1 (Figure 29) (see chapters B15 and B04).

## 2 - Databases

During the latest two decades, various databases covering allergens and allergen-related data have been established by academic institutions and the industry. These databases contain overlapping data, but address different user groups, use varying criteria for including allergens, and some provide additional tools such as sequence comparisons. The most widely-used, freely accessible databases are summarized below. A more extensive discussion of allergen databases was published recently.<sup>363</sup>

### 2.1 WHO/IUIS allergen nomenclature database (http://www.allergen.org/)

The allergen nomenclature database is a repository of allergens that underwent a submission and evaluation process and were accepted by the WHO/IUIS Allergen Nomenclature Sub-Committee, a panel of experts in allergen characterization, structure, and function. This is the only body officially authorized to assign allergen designations. The database contains links for each allergen to the nucleotide and protein databases of the NCBI (National Center

<figure><img src="image_112.png" /><figcaption>**FIGURE 29** Distribution of allergenic members of the papain-like cysteine protease family. The depicted structure is Act d 1, actinidin from kiwifruit (PDB: 2act).</figcaption></figure>
for Biotechnology Information; https://www.ncbi.nlm.nih.gov/), the UniProt database (https://www.uniprot.org/), and the Protein Databank (PDB) of macromolecular structures (https://www.rcsb.org/).

## 2.2 Allergen Online (https://www.allergenonline.org/)
AllergenOnline provides access to a peer-reviewed allergen list and sequence searchable database intended for the identification of proteins that may present a potential risk of allergenic cross-reactivity. The website was designed to help in assessing the safety of proteins that may be introduced into foods through genetic engineering or through food processing methods.

## 2.3 Allergome (https://www.allergome.org/)
The Allergome database has the most extensive collection of information on allergens and allergen sources, including data on sequences, structures, cross-reactivity, epidemiology, and an annotated list of references. It is based on the literature published since the early sixties but also contains many genomic and putative cDNA sequences listed as allergens that have been identified by bioinformatics searches from sources related to species containing allergens. Each allergen record contains an allergenicity score that helps the user in judging the relevance of the respective allergen.

## 2.4 COMprehensive Protein Allergen REsource (COMPARE; https://comparedatabase.org/)

Similar to AllergenOnline, the COMPARE database aims at providing a peer-reviewed list of allergen sequences associated with bioinformatics tools for sequence search.

AllerBase (http://bioinfo.unipune.ac.in/AllerBase/) The AllerBase knowledgebase is an extensively cross-linked collection of data on allergens, their sequences, structures, physico-chemical and immunological characterization, cross-reactivity and IgE epitopes, as well as IgE antibodies. Data are compiled from the literature and other databases and manually curated.

## 2.5 AllFam (allergen protein families, http://www.meduniwien.ac.at/allfam/)

The AllFam database is a resource for classifying allergens into protein families. It is curated by the host scientists. AllFam groups allergens from the WHO/IUIS database and AllergenOnline according to the protein family classification from the Pfam database (https://www.ebi.ac.uk/interpro/). Allfam provides a good overview on allergen families and their member proteins.

## 2.6 Immune Epitope Database (IEDB; http://www.iedb.org/)

The IEDB is a comprehensive collection of data on experimentally determined B cell and T cell epitopes in the context of infectious diseases, allergy, autoimmunity and transplantation. Data are extracted from the literature and curated by a board of reviewers following detailed published criteria. IEDB's sophisticated user interface allows for targeted searches for specific information on epitopes of allergens.
for Biotechnology Information; https://www.ncbi.nlm.nih.gov/), the UniProt database (https://www.uniprot.org/), and the Protein Databank (PDB) of macromolecular structures (https://www.rcsb.org/).

## 2.2 Allergen Online (https://www.allergenonline.org/)
AllergenOnline provides access to a peer-reviewed allergen list and sequence searchable database intended for the identification of proteins that may present a potential risk of allergenic cross-reactivity. The website was designed to help in assessing the safety of proteins that may be introduced into foods through genetic engineering or through food processing methods.

## 2.3 Allergome (https://www.allergome.org/)
The Allergome database has the most extensive collection of information on allergens and allergen sources, including data on sequences, structures, cross-reactivity, epidemiology, and an annotated list of references. It is based on the literature published since the early sixties but also contains many genomic and putative cDNA sequences listed as allergens that have been identified by bioinformatics searches from sources related to species containing allergens. Each allergen record contains an allergenicity score that helps the user in judging the relevance of the respective allergen.

## 2.4 COMprehensive Protein Allergen REsource (COMPARE; https://comparedatabase.org/)

Similar to AllergenOnline, the COMPARE database aims at providing a peer-reviewed list of allergen sequences associated with bioinformatics tools for sequence search.

AllerBase (http://bioinfo.unipune.ac.in/AllerBase/) The AllerBase knowledgebase is an extensively cross-linked collection of data on allergens, their sequences, structures, physico-chemical and immunological characterization, cross-reactivity and IgE epitopes, as well as IgE antibodies. Data are compiled from the literature and other databases and manually curated.

## 2.5 AllFam (allergen protein families, http://www.meduniwien.ac.at/allfam/)

The AllFam database is a resource for classifying allergens into protein families. It is curated by the host scientists. AllFam groups allergens from the WHO/IUIS database and AllergenOnline according to the protein family classification from the Pfam database (https://www.ebi.ac.uk/interpro/). Allfam provides a good overview on allergen families and their member proteins.

## 2.6 Immune Epitope Database (IEDB; http://www.iedb.org/)

The IEDB is a comprehensive collection of data on experimentally determined B cell and T cell epitopes in the context of infectious diseases, allergy, autoimmunity and transplantation. Data are extracted from the literature and curated by a board of reviewers following detailed published criteria. IEDB's sophisticated user interface allows for targeted searches for specific information on epitopes of allergens.
# A09 - Immunotherapy and molecular allergy approaches

Lars K. Poulsen, Sarita Patil, Stefan Vieths, Ronald van Ree

## Highlights

* Allergen-specific immunotherapy started in 1911 and since then, numerous studies contributed to improve efficacy and safety of this treatment.

* In parallel, characterization and quality assurance of allergen extracts have been improved and regulatory requirements have been developed for market authorization.

* So far only extract based formulations have been approved for immunotherapy and *in vivo* diagnosis.

## 1 - Introduction

Allergen-specific immunotherapy (AIT)—in the following referred to as immunotherapy—grew out of the successes of vaccination against infectious diseases in the late nineteenth century. The first successful attempt to ameliorate allergic symptoms was published by Noon in 1911, where grass-pollen allergic patients were vaccinated with watery extracts of grass pollen and eventually developed some form of tolerance.³⁶⁴,³⁶⁵ While the rationale behind the experiments—to immunize against a putative contagion—may not have been correct, the benefit for the patients made the treatment principle grow and was already in the first part of the twentieth century greatly expanded to the most common allergenic sources, such as tree and weed pollens, mold spores, cat, dog and horse dander and “house dust”—only later to be related to house dust mites.

The impact of molecular allergology on the field of immunotherapy has been enormous, but in spite of more than 20 years with recombinant allergens, still no product based on recombinant technology has yet been authorized for clinical use. There are, however, a wealth of clinical studies from which many important lessons can be learned.

In the present chapter we will focus on clinical studies, where molecular allergology has been applied to development of immunotherapy. As demonstrated in (Figure 30) a crude characterization can be made of the development lines: Starting from the crude allergen extract the search for the active components lead to an understanding that extracts contained many proteins some of which were allergens. The immunological science provided tools in the form of animal antibodies, and after the discovery of IgE in the late 1960s, the ability to measure IgE further boosted research. Combined with the development in biophysical separation and characterization techniques, this allowed detailed studies of allergens that eventually helped the manufacturers of allergen extracts to standardize the qualitative and quantitative contents of their products, and thereby ascertain an improved efficacy and safety. With the advent

of the first recombinant molecules in the 1980s, a revolution in the possibilities of identifying, characterizing and producing allergens became available, but in spite of many attempts, the barriers for implementation in immunotherapy seem to be much more substantial than for *in vitro* diagnosis.

The interplay between the development of biotechnology to produce new molecules with an increasing understanding of the immunological mechanisms of allergy and immunotherapy sets the scene for new products of immunotherapy, but it is important also to mention the evolution in smaller increments where clinical studies have optimized dose schedules and regimens (See newest EAACI guidelines on www.eaaci.org) and combination of immunotherapy with pharmacological³⁶⁶ or immunopharmacological³⁶⁷,³⁶⁸ therapy. Finally, also studies of the administration routes: subcutaneous, sublingual, oral, intranasal or intralymphatic have given rise to a large body of literature, but this generally falls outside the realm of molecular allergology and will not be discussed further in the present chapter.

## 2 - The standardized immunotherapy extract: Ideals and reality

One of the most tangible contributions molecular allergology has so far made to the improvement of immunotherapy is to product characterization and standardization. By the time most of the major allergens of the most important allergen sources had been identified, it became clear that their (consistent) presence in immunotherapy products as important active ingredients is essential and should be monitored. Traditionally, standardization of immunotherapy products has been focused on total IgE-binding potency of extracts, using in-house reference preparations (extracts) and associated company-specific units. Competitive IgE-binding assays using pooled sera of allergic patients are at the basis of this approach. This approach stems from a time when major allergens had not yet been identified and was very much safety driven: IgE-binding potencies should not vary too much to prevent adverse events caused by too potent extracts. Overall, IgE-binding potencies do not provide insight into the content of individual major allergens but of all allergens together. In particular the development of major allergen-specific monoclonal antibodies (mAbs), provided the tools to monitor major allergen content of immunotherapy products. Slowly, companies started implementing mAb-based sandwich ELISAs to measure major allergens in their products. Gaining insight into the content of these active ingredients was a major step forward. It was, however, realized that quantification of major allergens is not as straightforward due to a number of factors. Firstly, major allergens are often present in multiple isoforms and it turned out that not all are picked up with the same sensitivity by major allergen ELISAs. Secondly, many immunotherapy products are composed of mixtures of different species, e.g., a mix of pollen from 5 or even 10 different grass pollen species or a mix of *Dermatophagoides pteronyssinus* and *farinae* house dust mites. As for isoforms, homologous
# A09 - Immunotherapy and molecular allergy approaches

Lars K. Poulsen, Sarita Patil, Stefan Vieths, Ronald van Ree

## Highlights

* Allergen-specific immunotherapy started in 1911 and since then, numerous studies contributed to improve efficacy and safety of this treatment.

* In parallel, characterization and quality assurance of allergen extracts have been improved and regulatory requirements have been developed for market authorization.

* So far only extract based formulations have been approved for immunotherapy and *in vivo* diagnosis.

## 1 - Introduction

Allergen-specific immunotherapy (AIT)—in the following referred to as immunotherapy—grew out of the successes of vaccination against infectious diseases in the late nineteenth century. The first successful attempt to ameliorate allergic symptoms was published by Noon in 1911, where grass-pollen allergic patients were vaccinated with watery extracts of grass pollen and eventually developed some form of tolerance.³⁶⁴,³⁶⁵ While the rationale behind the experiments—to immunize against a putative contagion—may not have been correct, the benefit for the patients made the treatment principle grow and was already in the first part of the twentieth century greatly expanded to the most common allergenic sources, such as tree and weed pollens, mold spores, cat, dog and horse dander and “house dust”—only later to be related to house dust mites.

The impact of molecular allergology on the field of immunotherapy has been enormous, but in spite of more than 20 years with recombinant allergens, still no product based on recombinant technology has yet been authorized for clinical use. There are, however, a wealth of clinical studies from which many important lessons can be learned.

In the present chapter we will focus on clinical studies, where molecular allergology has been applied to development of immunotherapy. As demonstrated in (Figure 30) a crude characterization can be made of the development lines: Starting from the crude allergen extract the search for the active components lead to an understanding that extracts contained many proteins some of which were allergens. The immunological science provided tools in the form of animal antibodies, and after the discovery of IgE in the late 1960s, the ability to measure IgE further boosted research. Combined with the development in biophysical separation and characterization techniques, this allowed detailed studies of allergens that eventually helped the manufacturers of allergen extracts to standardize the qualitative and quantitative contents of their products, and thereby ascertain an improved efficacy and safety. With the advent

of the first recombinant molecules in the 1980s, a revolution in the possibilities of identifying, characterizing and producing allergens became available, but in spite of many attempts, the barriers for implementation in immunotherapy seem to be much more substantial than for *in vitro* diagnosis.

The interplay between the development of biotechnology to produce new molecules with an increasing understanding of the immunological mechanisms of allergy and immunotherapy sets the scene for new products of immunotherapy, but it is important also to mention the evolution in smaller increments where clinical studies have optimized dose schedules and regimens (See newest EAACI guidelines on www.eaaci.org) and combination of immunotherapy with pharmacological³⁶⁶ or immunopharmacological³⁶⁷,³⁶⁸ therapy. Finally, also studies of the administration routes: subcutaneous, sublingual, oral, intranasal or intralymphatic have given rise to a large body of literature, but this generally falls outside the realm of molecular allergology and will not be discussed further in the present chapter.

## 2 - The standardized immunotherapy extract: Ideals and reality

One of the most tangible contributions molecular allergology has so far made to the improvement of immunotherapy is to product characterization and standardization. By the time most of the major allergens of the most important allergen sources had been identified, it became clear that their (consistent) presence in immunotherapy products as important active ingredients is essential and should be monitored. Traditionally, standardization of immunotherapy products has been focused on total IgE-binding potency of extracts, using in-house reference preparations (extracts) and associated company-specific units. Competitive IgE-binding assays using pooled sera of allergic patients are at the basis of this approach. This approach stems from a time when major allergens had not yet been identified and was very much safety driven: IgE-binding potencies should not vary too much to prevent adverse events caused by too potent extracts. Overall, IgE-binding potencies do not provide insight into the content of individual major allergens but of all allergens together. In particular the development of major allergen-specific monoclonal antibodies (mAbs), provided the tools to monitor major allergen content of immunotherapy products. Slowly, companies started implementing mAb-based sandwich ELISAs to measure major allergens in their products. Gaining insight into the content of these active ingredients was a major step forward. It was, however, realized that quantification of major allergens is not as straightforward due to a number of factors. Firstly, major allergens are often present in multiple isoforms and it turned out that not all are picked up with the same sensitivity by major allergen ELISAs. Secondly, many immunotherapy products are composed of mixtures of different species, e.g., a mix of pollen from 5 or even 10 different grass pollen species or a mix of *Dermatophagoides pteronyssinus* and *farinae* house dust mites. As for isoforms, homologous
**FIGURE 30** Quasi-historical developmental history of therapeutics for allergen immunotherapy (AIT). The development has taken place as an interplay between the clinical research and documentation (left column) and the biochemical description and manufacturing of the allergens (right column). Left: The monitoring of patients treated with AIT has developed from increasingly more quantitative clinical observations and skin prick tests, over serological assays to extensive immunological and inflammatory studies of biomarkers. Right: The control of the administered allergen molecules has developed from simply descriptive of the qualitative and quantitative allergen content of the natural source extract, over standardized extracts and the potential for production of wild-type allergen molecules, to synthetic designer molecules being developed based on scientific hypotheses on the vaccinology and the immune system.

<figure><img src="image_113.png" /></figure>

major allergens from different species are usually not measured with the same sensitivity. Thirdly, each individual ELISA with its own mAbs differs with respect to performance characteristics. Finally, references used in sandwich ELISAs can be natural extracts, natural purified major allergens, or specific recombinant isoforms. It does not require too much imagination to realize that this has great impact on the outcome of these assays. The consequence of this was that 10 µg of a major allergen in product A is not necessarily equivalent to 10 µg in product B, measured with different assays. The EU-funded project, CREATE, aimed at providing an answer to these challenges.³⁶⁹ For four allergen sources (birch, grass and olive pollen and house dust mite) the project set out to characterize and compare natural and recombinant versions of eight major allergens (Bet v 1, Phl p 1 and 5, Ole e 1, Der p and f 1 and Der p and f 2) as candidates to become certified reference materials.

The second objective was to compare different available sandwich ELISAs for each of the allergens, using both natural and recombinant references, to ultimately allow selection of assays that were best equipped to measure different natural isoforms in extracts from different companies. The project was followed up by the BSP 090 program under the guidance of EDQM(European Directorate for the Quality of Medicines and HealthCare),³⁷⁰ to establish the two first changed to first two recombinant references with linked sandwich ELISAs, i.e., for Bet v 1³⁷¹,³⁷² and for Phl p 5.³⁷³ These references and associated ELISAs can now be used by companies to calibrate their in-house references and, if used, evaluate their in-house ELISA assays. These developments are a major first step towards application of molecular allergology into standardization and quality control of immunotherapy products, allowing more reliable comparison between competitor products.
**FIGURE 30** Quasi-historical developmental history of therapeutics for allergen immunotherapy (AIT). The development has taken place as an interplay between the clinical research and documentation (left column) and the biochemical description and manufacturing of the allergens (right column). Left: The monitoring of patients treated with AIT has developed from increasingly more quantitative clinical observations and skin prick tests, over serological assays to extensive immunological and inflammatory studies of biomarkers. Right: The control of the administered allergen molecules has developed from simply descriptive of the qualitative and quantitative allergen content of the natural source extract, over standardized extracts and the potential for production of wild-type allergen molecules, to synthetic designer molecules being developed based on scientific hypotheses on the vaccinology and the immune system.

<figure><img src="image_113.png" /></figure>

major allergens from different species are usually not measured with the same sensitivity. Thirdly, each individual ELISA with its own mAbs differs with respect to performance characteristics. Finally, references used in sandwich ELISAs can be natural extracts, natural purified major allergens, or specific recombinant isoforms. It does not require too much imagination to realize that this has great impact on the outcome of these assays. The consequence of this was that 10 µg of a major allergen in product A is not necessarily equivalent to 10 µg in product B, measured with different assays. The EU-funded project, CREATE, aimed at providing an answer to these challenges.³⁶⁹ For four allergen sources (birch, grass and olive pollen and house dust mite) the project set out to characterize and compare natural and recombinant versions of eight major allergens (Bet v 1, Phl p 1 and 5, Ole e 1, Der p and f 1 and Der p and f 2) as candidates to become certified reference materials.

The second objective was to compare different available sandwich ELISAs for each of the allergens, using both natural and recombinant references, to ultimately allow selection of assays that were best equipped to measure different natural isoforms in extracts from different companies. The project was followed up by the BSP 090 program under the guidance of EDQM(European Directorate for the Quality of Medicines and HealthCare),³⁷⁰ to establish the two first changed to first two recombinant references with linked sandwich ELISAs, i.e., for Bet v 1³⁷¹,³⁷² and for Phl p 5.³⁷³ These references and associated ELISAs can now be used by companies to calibrate their in-house references and, if used, evaluate their in-house ELISA assays. These developments are a major first step towards application of molecular allergology into standardization and quality control of immunotherapy products, allowing more reliable comparison between competitor products.
## 3 – Current practices and documentation requirements

When an allergen extract is put on the market to be used for AIT or in vivo diagnostics such as skin prick testing, intradermal testing or target organ challenges, it is considered a biological medicinal product, and as such it needs a market authorization. In the European Union this is taken care of by the European Medicines Agency (EMA) in collaboration with the national drug agencies, and in the US by the Food Drug Administration (FDA). Several aspects of the required documentation and evaluation have been harmonized between EU and USA, but some differences remain (reviewed in³⁷⁴).

Since no recombinant allergen molecules have yet reached the market (see below), this section will focus on the regulatory issues pertaining to extract-based therapy and diagnosis. Some of the key elements in the documentation of these are:

1. Thorough description of the source material, including species documentation and quality assurance levels for pollutants such as other species (i.e., % nonrelevant pollens, or nonrelevant house dust mites).

2. Description of the production processes involved. Since even minor differences in e.g., extraction procedures may have pro- found effects on the yield of different individual allergens, the process is considered an inherent part of the product.

3. Establishment of an in-house reference preparation (IHRP), to which different production batches may be compared and stand- ardized. As described in the previous section this may be done in terms of total allergenic potency of an extract or by measuring the individual allergens. Typically, a variation from 50 to 150% has been accepted for the former, and 50-200% for the latter, assum- ing 100% for the IRHP. It should be noticed that these variances must be adhered to for the whole shelf-life of the product.

4. Some leniency is introduced by allowing similar allergenic sources (such as birch pollen-related tree pollen, botanically related grasses, or different house dust mite species) to be grouped thus allowing for a somewhat reduced burden of documentation.

5. Clinical efficacy and side effects. For the clinical documentation the whole area has suffered from a large variability in the level of documentation. Many products have been on the market for many years and at the time they were introduced, the require- ments for documentation were much smaller. Moreover, some in vitro diagnostic products, such as skin prick test extracts for rare allergen sources, may never reach a market value that eco- nomically justifies large clinical studies. Recently, the EU Heads of Medicines Agency (HMA) Co-ordination Group for Mutual Recognition and Decentralised Procedures Human (CMDh) has issued Recommendations on common regulatory approaches for allergen products (reviewed in¹⁷⁰³) in which it is suggested to require full clinical documentation for extracts from important allergen sources (pollen from the grasses, birch-related trees, ol- ive- and cypress-related tree pollen, ragweed and Parietaria weed pollen, bee and wasp venom, cat allergens, and the food allergens

peanut and peach). On the other hand, well-established products on the market, and other extracts may require somewhat less clinical documentation (reviewed in³⁷⁵).

## 4 – Immunotherapy for food allergy

More recently, immunotherapy has been employed in the treat- ment of IgE-mediated food allergies. This approach models the im- munotherapy approach for aeroallergens, using a build-up phase followed by an extended maintenance phase with daily allergen administration to achieve desensitization. Previous efforts using subcutaneous forms of food immunotherapy were largely aban- doned due to safety concerns.³⁷⁶,¹⁶⁵² The best studied form, oral immunotherapy, was first developed using whole foods for admin- istration. Other immunotherapy approaches studied for the treat- ment of food allergy include both, sublingual and epicutaneous administration.

Most oral immunotherapy protocols have involved the use of allergen in a flour form, which is then mixed into a food as a ve- hicle for ingestion. This formulation has been commonly used with high protein containing foods, including egg,³⁷⁷,³⁷⁸,³⁹³ pea- nut,³⁷⁹,³⁸⁰,¹⁶⁵³,¹⁷⁰¹ and tree nuts.³⁸² On the other hand, other aller- gens have been administered as whole food allergens, such as in milk oral immunotherapy.³⁸³,³⁸⁴,¹⁷⁰²

However, the high rates of adverse reactions in whole food immunotherapy have promoted the study of using modified food allergens for oral immunotherapy, based on observations on the allergenicity of naturally modified foods. In about 70% of children with milk or egg allergy, extensively heated cow's milk and baked eggs, such as in cakes, muffins, and cookies, are tolerated. The pro- cess of heating, in the case of milk, or forming a gluten-containing food matrix, in the case of egg, modify their allergenicity by altering the protein structure and abrogating IgE binding to conformational epitopes and thereby decreasing their ability to activate allergen effector responses³⁸⁵⁻³⁹⁰. The oral introduction of modified milk and egg allergens in allergic patients have been shown to acceler- ate the development of tolerance³⁹¹,³⁹², though not as well as oral immunotherapy.³⁹³

Modified natural allergens, with reduced allergenicity, have also been studied in oral immunotherapy. For instance, boiled peanuts have been found to have decreased IgE-binding capacity due to a loss of key allergenic components, such as Ara h 1, Ara h 2, and Ara h 6, into the cooking water.³⁹⁴,³⁹⁵ Immunotherapy with boiled pea- nuts have been shown to be safe in children with peanut allergy,³⁹⁶ and currently, a phase 2/3 clinical trial using boiled peanut in im- munotherapy is ongoing (NCT02149719). Similarly, a low allergen hydrolyzed hen's egg preparation using a combination of heat and enzymatic digestion,³⁹⁷ has been used in a clinical trial for treatment of egg allergy.³⁹⁸

Engineering of recombinant food allergens, by introduction of chemical modifications or site-directed mutagenesis for abrogation of IgE-binding sites while preserving the T cell epitopes, has achieved
## 3 - Current practices and documentation requirements

When an allergen extract is put on the market to be used for AIT or in vivo diagnostics such as skin prick testing, intradermal testing or target organ challenges, it is considered a biological medicinal product, and as such it needs a market authorization. In the European Union this is taken care of by the European Medicines Agency (EMA) in collaboration with the national drug agencies, and in the US by the Food Drug Administration (FDA). Several aspects of the required documentation and evaluation have been harmonized between EU and USA, but some differences remain (reviewed in³⁷⁴).

Since no recombinant allergen molecules have yet reached the market (see below), this section will focus on the regulatory issues pertaining to extract-based therapy and diagnosis. Some of the key elements in the documentation of these are:

1. Thorough description of the source material, including species documentation and quality assurance levels for pollutants such as other species (i.e., % nonrelevant pollens, or nonrelevant house dust mites).

2. Description of the production processes involved. Since even minor differences in e.g., extraction procedures may have profound effects on the yield of different individual allergens, the process is considered an inherent part of the product.

3. Establishment of an in-house reference preparation (IHRP), to which different production batches may be compared and standardized. As described in the previous section this may be done in terms of total allergenic potency of an extract or by measuring the individual allergens. Typically, a variation from 50 to 150% has been accepted for the former, and 50-200% for the latter, assuming 100% for the IRHP. It should be noticed that these variances must be adhered to for the whole shelf-life of the product.

4. Some leniency is introduced by allowing similar allergenic sources (such as birch pollen-related tree pollen, botanically related grasses, or different house dust mite species) to be grouped thus allowing for a somewhat reduced burden of documentation.

5. Clinical efficacy and side effects. For the clinical documentation the whole area has suffered from a large variability in the level of documentation. Many products have been on the market for many years and at the time they were introduced, the requirements for documentation were much smaller. Moreover, some in vitro diagnostic products, such as skin prick test extracts for rare allergen sources, may never reach a market value that economically justifies large clinical studies. Recently, the EU Heads of Medicines Agency (HMA) Co-ordination Group for Mutual Recognition and Decentralised Procedures Human (CMDh) has issued Recommendations on common regulatory approaches for allergen products (reviewed in¹⁷⁰³) in which it is suggested to require full clinical documentation for extracts from important allergen sources (pollen from the grasses, birch-related trees, olive- and cypress-related tree pollen, ragweed and Parietaria weed pollen, bee and wasp venom, cat allergens, and the food allergens peanut and peach). On the other hand, well-established products on the market, and other extracts may require somewhat less clinical documentation (reviewed in³⁷⁵).

## 4 - Immunotherapy for food allergy

More recently, immunotherapy has been employed in the treatment of IgE-mediated food allergies. This approach models the immunotherapy approach for aeroallergens, using a build-up phase followed by an extended maintenance phase with daily allergen administration to achieve desensitization. Previous efforts using subcutaneous forms of food immunotherapy were largely abandoned due to safety concerns.³⁷⁶,¹⁶⁵² The best studied form, oral immunotherapy, was first developed using whole foods for administration. Other immunotherapy approaches studied for the treatment of food allergy include both, sublingual and epicutaneous administration.

Most oral immunotherapy protocols have involved the use of allergen in a flour form, which is then mixed into a food as a vehicle for ingestion. This formulation has been commonly used with high protein containing foods, including egg,³⁷⁷,³⁷⁸,³⁹³ peanut,³⁷⁹,³⁸⁰,¹⁶⁵³,¹⁷⁰¹ and tree nuts.³⁸² On the other hand, other allergens have been administered as whole food allergens, such as in milk oral immunotherapy.³⁸³,³⁸⁴,¹⁷⁰²

However, the high rates of adverse reactions in whole food immunotherapy have promoted the study of using modified food allergens for oral immunotherapy, based on observations on the allergenicity of naturally modified foods. In about 70% of children with milk or egg allergy, extensively heated cow's milk and baked eggs, such as in cakes, muffins, and cookies, are tolerated. The process of heating, in the case of milk, or forming a gluten-containing food matrix, in the case of egg, modify their allergenicity by altering the protein structure and abrogating IgE binding to conformational epitopes and thereby decreasing their ability to activate allergen effector responses³⁸⁵⁻³⁹⁰. The oral introduction of modified milk and egg allergens in allergic patients have been shown to accelerate the development of tolerance³⁹¹,³⁹², though not as well as oral immunotherapy.³⁹³

Modified natural allergens, with reduced allergenicity, have also been studied in oral immunotherapy. For instance, boiled peanuts have been found to have decreased IgE-binding capacity due to a loss of key allergenic components, such as Ara h 1, Ara h 2, and Ara h 6, into the cooking water.³⁹⁴,³⁹⁵ Immunotherapy with boiled peanuts have been shown to be safe in children with peanut allergy,³⁹⁶ and currently, a phase 2/3 clinical trial using boiled peanut in immunotherapy is ongoing (NCT02149719). Similarly, a low allergen hydrolyzed hen's egg preparation using a combination of heat and enzymatic digestion,³⁹⁷ has been used in a clinical trial for treatment of egg allergy.³⁹⁸

Engineering of recombinant food allergens, by introduction of chemical modifications or site-directed mutagenesis for abrogation of IgE-binding sites while preserving the T cell epitopes, has achieved
reduced allergenicity. This modification has been proposed as a way to increase the safety of oral immunotherapy while promoting adaptive immune responses. Several efforts to produce engineered recombinant allergens are underway with peanut, fish, apple, and peach. For instance, a phase I trial of rectally administered modified major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, encapsulated in heat/phenol killed *E. coli* (EMP-123) for treatment of peanut allergy resulted in frequent allergic reactions, including anaphylaxis.³⁹⁹

Finally, sublingual immunotherapy with aqueous allergen extracts has been employed for treatment of food allergy. Previous studies have used aqueous food allergen extracts for immunotherapy to treat hazelnut,⁴⁰⁰ peach,⁴⁰¹ and peanut⁴⁰²⁻⁴⁰⁴,¹⁶⁵¹ allergy. Alternatively, protein powder has been used sublingually for milk.⁴⁰⁵,⁴⁰⁶

## 5 – Immunotherapy products based on wild type recombinant allergens

The first placebo-controlled clinical trial with recombinant allergen molecules was employing a multi-allergen grass pollen mixture of recombinant allergens of *Phleum pratense*, using approximately equimolar concentrations of the recombinant allergens Phl p 1, Phl p 2, Phl p 5a, Phl p 5b, and Phl p 6, all expressed in *E. coli*.⁴⁰⁷ The mixture was subcutaneously administered in a classical dose increase with 10 weekly intervals up to a maintenance dose of around 40 mg total recombinant protein (10+5+10+10+5 mg of the five allergens). The allergens were adsorbed to aluminium hydroxide and the treatment controlled by placebo (aluminium hydroxide plus histamine) was given for about 30 months with a 50% dose reduction during grass pollen seasons. Using combined symptom-medication scores as outcome parameter, the experimental product, i.e., the mixture of recombinants showed significantly better scores compared with placebo when tested on hay fever patients allergic to grass pollen. Also immunologically, a strong induction of both IgG1 and IgG4 was demonstrated (60-fold for IgG1 and 4000-fold for IgG4). In spite of these interesting findings, subsequent studies with the experimental product failed to demonstrate superiority over placebo, and the development program was discontinued.

For birch pollen a similar product—although simpler since it only contained recombinant Bet v 1, which is the dominating allergen in birch pollen extract—was developed.⁴⁰⁸ With this product, a four-arm randomized clinical trial was developed, in which the recombinant Bet v 1, was compared with the purified natural Bet v 1 and a standardized birch pollen extract as well as placebo. The three active arms were given up to what corresponds to 15 ug Bet v 1 as maintenance dose, where the allergens/extracts were adsorbed to aluminium hydroxide. The study was run for two birch pollen seasons as a multi-center study of birch pollen hay fever patients in Northern and Central Europe. In both pollen seasons, the symptoms and medication use were significantly reduced in all three study arms receiving active allergen compared with placebo. No differences were found between full birch pollen extract, recombinant or purified natural

Bet v 1. As for the grass pollen study, strong inductions of Bet v 1-specific IgG1 and IgG4 responses were seen accompanying the treatments.⁴⁰⁸ In this case no follow-up studies were made, and an immunotherapy treatment based on the wildtype recombinant Bet v 1 has never reached the market.

The two examples presented above would suggest that it is indeed technically and clinically feasible to produce well-performing immunotherapy products based on recombinant or wild-type allergenic molecules. It is likely that overcoming economical and perhaps regulatory barriers in the future will determine whether this potential will be fulfilled to same degree in immunotherapy as we have seen *in vitro* diagnosis as exemplified by these guidelines. A summary of recombinant allergen approaches in AIT has been published by Nandy et al.⁴⁰⁹

## 6 – Can we learn from immunotherapy biomarkers in the monitoring of immunotherapy and for the design of new molecules?

The search for biomarkers of immunotherapy has focused on clinical need: to identify markers of clinical efficacy characterized by longer, or sustained responses after immunotherapy and to identify risk factors for increased adverse effects. On a biological level, biomarkers also promise to provide insights into clinically relevant mechanisms of disease.

Allergen-specific antibody induction after immunotherapy has been long-recognized as one of the first immunological changes to occur in immunotherapy.⁴¹⁰ Since then, increases in whole allergen-specific IgG, particularly IgG4, have been reproducibly observed in many forms of immunotherapy, including subcutaneous,⁴¹¹ oral,³⁷⁷ sublingual,⁴⁰² and epicutaneous⁴¹² forms, for treatment of several IgE-mediated diseases, including environmental, venom, and food allergies. Next, discovery of immunodominant protein allergens led to our ability to demonstrate increases in component-specific antibodies for immunotherapy to both aeroallergens and foods. However, the changes in induced antibody levels occur almost uniformly during immunotherapy and did not correlate with clinical efficacy.¹⁶⁵³ One well-studied example has been that peanut-specific IgG4 and Ara h 2-specific IgG4 increases do correlate with clinical efficacy.¹⁸⁶

Drilling down to the specific epitope-based recognition of allergens, the importance of linear epitope recognition by allergen-specific antibodies has been increasingly recognized, particularly in food allergy. Increased diversity of linear epitope recognition by IgE has correlated to clinical severity of oral food challenges in peanut allergy.¹³⁸,⁴¹³ Moreover, increased linear epitope recognition has correlated with more persistent milk allergy as well. However, linear epitope recognition has not always correlated with clinical efficacy, in either IgE or IgG epitopes after peanut oral immunotherapy.⁴¹⁴

In individuals with sensitization to allergens, immunotherapy preparations containing the particular allergen more effectively drive clinical efficacy. For instance, honey bee venom patients with Api m 10 IgE sensitization were more likely to have treatment failure,
reduced allergenicity. This modification has been proposed as a way to increase the safety of oral immunotherapy while promoting adaptive immune responses. Several efforts to produce engineered recombinant allergens are underway with peanut, fish, apple, and peach. For instance, a phase I trial of rectally administered modified major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, encapsulated in heat/phenol killed *E. coli* (EMP-123) for treatment of peanut allergy resulted in frequent allergic reactions, including anaphylaxis.³⁹⁹

Finally, sublingual immunotherapy with aqueous allergen extracts has been employed for treatment of food allergy. Previous studies have used aqueous food allergen extracts for immunotherapy to treat hazelnut,⁴⁰⁰ peach,⁴⁰¹ and peanut⁴⁰²⁻⁴⁰⁴,¹⁶⁵¹ allergy. Alternatively, protein powder has been used sublingually for milk.⁴⁰⁵,⁴⁰⁶

## 5 – Immunotherapy products based on wild type recombinant allergens

The first placebo-controlled clinical trial with recombinant allergen molecules was employing a multi-allergen grass pollen mixture of recombinant allergens of *Phleum pratense*, using approximately equimolar concentrations of the recombinant allergens Phl p 1, Phl p 2, Phl p 5a, Phl p 5b, and Phl p 6, all expressed in *E. coli*.⁴⁰⁷ The mixture was subcutaneously administered in a classical dose increase with 10 weekly intervals up to a maintenance dose of around 40 mg total recombinant protein (10+5+10+10+5 mg of the five allergens). The allergens were adsorbed to aluminium hydroxide and the treatment controlled by placebo (aluminium hydroxide plus histamine) was given for about 30 months with a 50% dose reduction during grass pollen seasons. Using combined symptom-medication scores as outcome parameter, the experimental product, i.e., the mixture of recombinants showed significantly better scores compared with placebo when tested on hay fever patients allergic to grass pollen. Also immunologically, a strong induction of both IgG1 and IgG4 was demonstrated (60-fold for IgG1 and 4000-fold for IgG4). In spite of these interesting findings, subsequent studies with the experimental product failed to demonstrate superiority over placebo, and the development program was discontinued.

For birch pollen a similar product—although simpler since it only contained recombinant Bet v 1, which is the dominating allergen in birch pollen extract—was developed.⁴⁰⁸ With this product, a four-arm randomized clinical trial was developed, in which the recombinant Bet v 1, was compared with the purified natural Bet v 1 and a standardized birch pollen extract as well as placebo. The three active arms were given up to what corresponds to 15 ug Bet v 1 as maintenance dose, where the allergens/extracts were adsorbed to aluminium hydroxide. The study was run for two birch pollen seasons as a multi-center study of birch pollen hay fever patients in Northern and Central Europe. In both pollen seasons, the symptoms and medication use were significantly reduced in all three study arms receiving active allergen compared with placebo. No differences were found between full birch pollen extract, recombinant or purified natural

Bet v 1. As for the grass pollen study, strong inductions of Bet v 1-specific IgG1 and IgG4 responses were seen accompanying the treatments.⁴⁰⁸ In this case no follow-up studies were made, and an immunotherapy treatment based on the wildtype recombinant Bet v 1 has never reached the market.

The two examples presented above would suggest that it is indeed technically and clinically feasible to produce well-performing immunotherapy products based on recombinant or wild-type allergenic molecules. It is likely that overcoming economical and perhaps regulatory barriers in the future will determine whether this potential will be fulfilled to same degree in immunotherapy as we have seen *in vitro* diagnosis as exemplified by these guidelines. A summary of recombinant allergen approaches in AIT has been published by Nandy et al.⁴⁰⁹

## 6 – Can we learn from immunotherapy biomarkers in the monitoring of immunotherapy and for the design of new molecules?

The search for biomarkers of immunotherapy has focused on clinical need: to identify markers of clinical efficacy characterized by longer, or sustained responses after immunotherapy and to identify risk factors for increased adverse effects. On a biological level, biomarkers also promise to provide insights into clinically relevant mechanisms of disease.

Allergen-specific antibody induction after immunotherapy has been long-recognized as one of the first immunological changes to occur in immunotherapy.⁴¹⁰ Since then, increases in whole allergen-specific IgG, particularly IgG4, have been reproducibly observed in many forms of immunotherapy, including subcutaneous,⁴¹¹ oral,³⁷⁷ sublingual,⁴⁰² and epicutaneous⁴¹² forms, for treatment of several IgE-mediated diseases, including environmental, venom, and food allergies. Next, discovery of immunodominant protein allergens led to our ability to demonstrate increases in component-specific antibodies for immunotherapy to both aeroallergens and foods. However, the changes in induced antibody levels occur almost uniformly during immunotherapy and did not correlate with clinical efficacy.¹⁶⁵³ One well-studied example has been that peanut-specific IgG4 and Ara h 2-specific IgG4 increases do correlate with clinical efficacy.¹⁸⁶

Drilling down to the specific epitope-based recognition of allergens, the importance of linear epitope recognition by allergen-specific antibodies has been increasingly recognized, particularly in food allergy. Increased diversity of linear epitope recognition by IgE has correlated to clinical severity of oral food challenges in peanut allergy.¹³⁸,⁴¹³ Moreover, increased linear epitope recognition has correlated with more persistent milk allergy as well. However, linear epitope recognition has not always correlated with clinical efficacy, in either IgE or IgG epitopes after peanut oral immunotherapy.⁴¹⁴

In individuals with sensitization to allergens, immunotherapy preparations containing the particular allergen more effectively drive clinical efficacy. For instance, honey bee venom patients with Api m 10 IgE sensitization were more likely to have treatment failure,
so treatment with Api m 10 containing extracts has been recom-
mended for those patients.¹⁰¹

Functional cellular assays have provided an *in vitro*, integrated
tool for assessment of clinical reactivity. By evaluating how allergen-
specific IgE binding is outcompeted by allergen-specific antibod-
ies, these assays have been shown to be better biomarkers of
clinical efficacy than the measurement of whole allergen-specific
antibody serum levels. Two well-studied biomarker assays have cor-
related with clinical efficacy of immunotherapy. The first is a flow
cytometry-based assay, IgE-FAB, which was developed as an *in vitro*
assay of IgE-facilitated antigen presentation and activation of T cells
during aeroallergen immunotherapy,⁴¹⁵ and which correlates with
clinical efficacy after grass pollen immunotherapy.⁴¹⁶

The second assay, basophil activation testing (BAT) uses baso-
phils, an allergen effector cell in the peripheral blood coated with
surface IgE that can be cross-linked by allergen for activation, mea-
sured as CD63 upregulation by flow cytometry. Blocking antibodies
can prevent IgE-cross-linking, thereby suppressing reactivity (see
chapter A05). Suppression of basophil reactivity due to blocking an-
tibodies, as well as basophil-intrinsic modulation of reactivity, occurs
reproducibly during immunotherapy.¹⁸⁴,³⁷⁷ However, basophil sen-
sitivity, measured as a shift in the dose response curve of basophil
activation to allergen stimulation, is an early biomarker of clinical
efficacy in peanut oral immunotherapy.¹⁸⁶ Moreover, basophil sen-
sitivity to whole peanut was not as useful as the change in basophil
sensitivity to the immunodominant allergen Ara h 2.¹⁸⁶ Other simi-
lar assays, which use allergen-specific cell lines, such as the LAD2
cell line, have been used to create the inhibition of mast cell activa-
tion test (iMAT), which is also used to assess blocking antibodies in
immunotherapy.⁴¹⁷

Other cellular biomarkers of immunotherapy have been primar-
ily aimed at understanding the mechanisms underlying the develop-
ment of allergic tolerance. Reduction in circulating allergen-specific
type 2 helper (Th2) cells have been identified in both aeroallergen
and food immunotherapy.¹⁸⁵,⁴¹⁸,⁴¹⁹,⁴²⁰ Assays to profile allergen-
specific T cells have included the use of T cell tetramers⁴²⁰ and the
use of CD154 assays, where upregulation of CD154 after allergen
stimulation identified allergen-specific T cells.⁴¹⁸

In summary, the application of biomarkers of immunotherapy,
based on molecular identification of allergenic protein components
and allergen recognition, are emerging as correlates of clinical effi-
cacy. Advances in our understanding of the adaptive mechanisms of
allergic tolerance and detailed molecular typing of allergic responses
have the potential to lead to significant improvement in biomarker
discovery in immunotherapy.

7 – New allergen-derived molecules for
immunotherapy

A. Bits of allergens

One of the earliest initiatives to introduce molecular approaches
into immunotherapy was centered on the use of cocktails of short

synthetic peptides representing dominant T-cell epitopes of major
allergens. This was mainly evaluated for the major cat allergen Fel d
1 but to a lesser extent also for the major ragweed allergen Amb a
1. Already before the turn of the century, first clinical studies were
performed with cat Fel d 1 peptides,⁴²¹,⁴²² and this was later fur-
ther pursued, albeit at lower peptide concentrations. The idea be-
hind T-cell-targeted peptide immunotherapy was to down-regulate
allergen-specific Th2 cell activity, and reduce the risk of allergic side-
effects due to the inability of short peptides to induce cross-linking
of IgE on effector cells. At the initial higher dosages, however, signif-
icant late-phase adverse events were observed, and the field moved
to lower dosages. These proved to be quite effective in Phase II clin-
ical trials⁴²³ but finally did not reach its primary outcome in Phase III.
This discrepancy was possibly explained by choices made for patient
selection in Phase III rather than that the concept failed.

Two other peptide-based approaches were evaluated up to
Phase II clinical trials. The first one consisted of short peptides rep-
resenting minor B-cell epitopes of major grass pollen allergen, con-
jugated to a hepatitis antigen for T-cell help.⁴²⁴ The other approach
made use of larger peptide fragments of major allergens, containing
both B- and T-cell epitopes.⁴²⁵,⁴²⁶ In both approaches allergenicity
was significantly reduced, and some degree of efficacy could be
demonstrated. In Phase II, the first approach did not reach its pri-
mary endpoint and further development was in the end stopped.
Also, the second approach has been abandoned due to disappointing
results. Overall, the development of peptide-based immunotherapy
has thus far not lived up to its initial promises.

B. Hypoallergens

The idea of creating low-allergenic molecules has a long his-
tory in allergy (historical studies reviewed in⁴²⁷) being inspired by
the vaccinology field that has a century-long tradition of creation
of toxoids, i.e., modified bacterial toxins, which could be used for
vaccination to raise a protective immune response but without the
serious pathogenic effects of the native toxins. The allergenic coun-
terpart, sometimes referred to as allergoids or more commonly as
hypoallergens, is an allergen-related molecule that has the capabil-
ity to raise an immune response, preferably both in the B- (IgG) and
T-cell compartment of the immune system but without the elicitat-
tion of the well-known allergic effects when administered to the
allergic patient. Before the advent of the DNA-based recombinant
technologies combined with a detailed structural knowledge of
the allergens, more crude techniques employing heat, radiation or
chemical treatments such as reduction/alkylation, formaldehyde or
glutaraldehyde.

In a large EU-funded project of collaborating academical, clinical
and commercial groups it was attempted to develop hypoallergenic
versions of the molecules Pru p 3 from peach⁴²⁸ and Cyp c 1 from
carp.⁴²⁹

Several strategies were applied for the peach LTP molecule⁴²⁸:
reduction/alkylation, heat treatment, glutaraldehyde-treatment
(creating an allergoid, see above and chapterA02), replacement by
a natural less-allergenic homologue (Fra a 3, an LTP-molecule from
so treatment with Api m 10 containing extracts has been recom-
mended for those patients.¹⁰¹

Functional cellular assays have provided an *in vitro*, integrated
tool for assessment of clinical reactivity. By evaluating how allergen-
specific IgE binding is outcompeted by allergen-specific antibod-
ies, these assays have been shown to be better biomarkers of
clinical efficacy than the measurement of whole allergen-specific
antibody serum levels. Two well-studied biomarker assays have cor-
related with clinical efficacy of immunotherapy. The first is a flow
cytometry-based assay, IgE-FAB, which was developed as an *in vitro*
assay of IgE-facilitated antigen presentation and activation of T cells
during aeroallergen immunotherapy,⁴¹⁵ and which correlates with
clinical efficacy after grass pollen immunotherapy.⁴¹⁶

The second assay, basophil activation testing (BAT) uses baso-
phils, an allergen effector cell in the peripheral blood coated with
surface IgE that can be cross-linked by allergen for activation, mea-
sured as CD63 upregulation by flow cytometry. Blocking antibodies
can prevent IgE-cross-linking, thereby suppressing reactivity (see
chapter A05). Suppression of basophil reactivity due to blocking an-
tibodies, as well as basophil-intrinsic modulation of reactivity, occurs
reproducibly during immunotherapy.¹⁸⁴,³⁷⁷ However, basophil sen-
sitivity, measured as a shift in the dose response curve of basophil
activation to allergen stimulation, is an early biomarker of clinical
efficacy in peanut oral immunotherapy.¹⁸⁶ Moreover, basophil sen-
sitivity to whole peanut was not as useful as the change in basophil
sensitivity to the immunodominant allergen Ara h 2.¹⁸⁶ Other simi-
lar assays, which use allergen-specific cell lines, such as the LAD2
cell line, have been used to create the inhibition of mast cell activa-
tion test (iMAT), which is also used to assess blocking antibodies in
immunotherapy.⁴¹⁷

Other cellular biomarkers of immunotherapy have been primar-
ily aimed at understanding the mechanisms underlying the develop-
ment of allergic tolerance. Reduction in circulating allergen-specific
type 2 helper (Th2) cells have been identified in both aeroallergen
and food immunotherapy.¹⁸⁵,⁴¹⁸,⁴¹⁹,⁴²⁰ Assays to profile allergen-
specific T cells have included the use of T cell tetramers⁴²⁰ and the
use of CD154 assays, where upregulation of CD154 after allergen
stimulation identified allergen-specific T cells.⁴¹⁸

In summary, the application of biomarkers of immunotherapy,
based on molecular identification of allergenic protein components
and allergen recognition, are emerging as correlates of clinical effi-
cacy. Advances in our understanding of the adaptive mechanisms of
allergic tolerance and detailed molecular typing of allergic responses
have the potential to lead to significant improvement in biomarker
discovery in immunotherapy.

7 – New allergen-derived molecules for
immunotherapy

A. Bits of allergens

One of the earliest initiatives to introduce molecular approaches
into immunotherapy was centered on the use of cocktails of short

synthetic peptides representing dominant T-cell epitopes of major
allergens. This was mainly evaluated for the major cat allergen Fel d
1 but to a lesser extent also for the major ragweed allergen Amb a
1. Already before the turn of the century, first clinical studies were
performed with cat Fel d 1 peptides,⁴²¹,⁴²² and this was later fur-
ther pursued, albeit at lower peptide concentrations. The idea be-
hind T-cell-targeted peptide immunotherapy was to down-regulate
allergen-specific Th2 cell activity, and reduce the risk of allergic side-
effects due to the inability of short peptides to induce cross-linking
of IgE on effector cells. At the initial higher dosages, however, signif-
icant late-phase adverse events were observed, and the field moved
to lower dosages. These proved to be quite effective in Phase II clin-
ical trials⁴²³ but finally did not reach its primary outcome in Phase III.
This discrepancy was possibly explained by choices made for patient
selection in Phase III rather than that the concept failed.

Two other peptide-based approaches were evaluated up to
Phase II clinical trials. The first one consisted of short peptides rep-
resenting minor B-cell epitopes of major grass pollen allergen, con-
jugated to a hepatitis antigen for T-cell help.⁴²⁴ The other approach
made use of larger peptide fragments of major allergens, containing
both B- and T-cell epitopes.⁴²⁵,⁴²⁶ In both approaches allergenicity
was significantly reduced, and some degree of efficacy could be
demonstrated. In Phase II, the first approach did not reach its pri-
mary endpoint and further development was in the end stopped.
Also, the second approach has been abandoned due to disappointing
results. Overall, the development of peptide-based immunotherapy
has thus far not lived up to its initial promises.

B. Hypoallergens

The idea of creating low-allergenic molecules has a long his-
tory in allergy (historical studies reviewed in⁴²⁷) being inspired by
the vaccinology field that has a century-long tradition of creation
of toxoids, i.e., modified bacterial toxins, which could be used for
vaccination to raise a protective immune response but without the
serious pathogenic effects of the native toxins. The allergenic coun-
terpart, sometimes referred to as allergoids or more commonly as
hypoallergens, is an allergen-related molecule that has the capabil-
ity to raise an immune response, preferably both in the B- (IgG) and
T-cell compartment of the immune system but without the elicitat-
tion of the well-known allergic effects when administered to the
allergic patient. Before the advent of the DNA-based recombinant
technologies combined with a detailed structural knowledge of
the allergens, more crude techniques employing heat, radiation or
chemical treatments such as reduction/alkylation, formaldehyde or
glutaraldehyde.

In a large EU-funded project of collaborating academical, clinical
and commercial groups it was attempted to develop hypoallergenic
versions of the molecules Pru p 3 from peach⁴²⁸ and Cyp c 1 from
carp.⁴²⁹

Several strategies were applied for the peach LTP molecule⁴²⁸:
reduction/alkylation, heat treatment, glutaraldehyde-treatment
(creating an allergoid, see above and chapterA02), replacement by
a natural less-allergenic homologue (Fra a 3, an LTP-molecule from
strawberry), trimerization, and mutations directed against either
cysteine or surface molecules. The manufacture candidates were
compared with native Pru p 3 and a wild-type recombinant molecule
in a preclinical phase using IgE-binding assays and biological assays
for effector cell (basophil histamine release) to screen for allergenic-
ity in vitro. The immunogenicity was tested by rabbit and murine im-
munizations, and the molecules underwent a thorough biochemical
characterization. Sera were obtained from relevant patient groups
from different centers in Europe. While it was indeed possible to
strongly reduce the allergenicity, this often went hand in hand with
a similar reduced immunogenicity if not a downright destruction of
the molecule making it unfit for pharmaceutical use.

For the parvalbumin molecule similar attempts were slightly
more successful, and a single candidate (called m-(modified) Cyp
c 1)⁴³⁰ was selected for toxicity studies and further clinical devel-
opment. In an initial first-in-man safety study the low to absent al-
lerogenicity of the mCyp c 1 was confirmed initially by skin testing
and later by actual dosing up to ug-dosages in fish-allergic patients.
Moreover, promising responses of IgG were demonstrated, includ-
ing demonstration that the raised IgG not only bound mCyp c 1 but
also the native parvalbumin molecules from fish (see chapters B12,
C11) to which the patients reacted. A subsequent study on efficacy
proved less conclusive, however, and the preliminary conclusion
would suggest that further dose-finding studies may be necessary.

To summarize the quest for hypoallergens there are some inher-
ent problems in that some allergenic molecules exemplified by LTP
are difficult to target with strategies for reducing allergenicity due
to their structure. In many cases it is not possible to modify/reduce
allergenicity without at the same time to eliminate the immunoge-
nicity of the molecule. Even in the case of success with reduction/
elimination of allergenicity while retaining the immunogenicity, the
variability within the allergic population may represent a challenge:
People may react highly individually to not only different allergenic
molecules making the selection of candidates difficult but also dif-
ferently to different epitopes on a single allergen. This may result
in highly varying allergenic potencies of the same hypoallergen in
different patients. It is therefore likely that at least the same precau-
tions of careful titration will be necessary for hypoallergenic prod-
ucts as is the case for subcutaneous allergen immunotherapy with
conventional allergen extracts.

C. Ligation to adjuvants and other new constructs

Modern vaccinology has produced a plethora of adjuvants and
adjuvating principles that have also inspired the allergy field. Among
the examples are chimeric molecules where B-cell epitopes of grass
allergens are mixed with immunogenic viral proteins from hepa-
titis⁴²⁴ earlier alluded to. Initial clinical trials demonstrated strong
immunogenicity but failed to reach statistical significance of primary
efficacy outcomes in grass pollen-allergic hay fever patients in a ran-
domized placebo-controlled trial. Another interesting example is in-
tralymphatic immunotherapy with a modular allergen translocation
vaccine in cat allergy⁴³¹,⁴³² revealing promising results in an initial
clinical trial but not taken further to pivotal studies.

8 – Conclusions

There is a striking difference between the progress and success of
molecular allergology in the diagnostic and the therapeutic field.
This chapter has hinted at some of the challenges and barriers to
develop an allergen immunotherapy based on molecular methods
rather than the well-known and long used allergen extracts.

An important difference is the regulatory demands: while it is
relatively easy with modern biotechnology to produce a recombi-
nant molecule and to use it in an *in vitro* diagnostic test, the de-
mands for preclinical testing, toxicity studies, and finally phase 1-3
in clinical randomized studies may easily last a decade and cost up
to hundreds of millions of €. The strength of molecular allergy in the
diagnosis, where the hyped ideal of personal medicine almost be-
comes true, with the possibility of obtaining a totally individualized
molecular profile of IgE reactivity to individual allergens in the pa-
tient, becomes the Achilles' heel for the therapy: If allergy patients
are so different, how can we ever dream of obtaining sufficient pur-
chasing power to support the costly product development?

With the advent of the *in vitro* directive for the European Union,
the increased demands for evidence-based medicine and the in-
creasing scrutiny on each € or $ spent in the health care sector, the
diagnostic smorgasbord that is described in the remainder of these
guidelines may also become reduced, but this does not help us in the
therapeutic field.

With some relaxation of the present regulation, which has been
seen, e.g., in the oncology field, one way forward could be a personal-
ized approach with recombinant allergens: individual recombinant al-
lergens produced under GMP and mixed of the shelf or used to fortify
existing extracts according to the sensitization profile of the patient.

Another route of development may be an improved understand-
ing of the immunological mechanisms of the beneficial effects of
immunotherapy, which may lead to a more focused product de-
velopment. The history of immunotherapy began with the famous
study by Noon in 1911,³⁶⁴ and while it started a long journey to-
wards increasingly more efficacious and safe immunotherapy prod-
ucts, we have to admit that rationale behind Noon's study was the
incorrect notion that allergens are toxins. We have come some way
in our immunological understanding, but as demonstrated in this
chapter, even a clear decision on whether to go for B-cell epitopes
and antibody responses or T-cell epitopes and reprogramming of
the T-cell profiles (or both?) are lacking in the allergy community.
In this respect it is interesting that for both of these pathways,
new developments of specific allergy treatment may lie ahead:
Specific IgG-antibodies to allergens can now be generated *in vitro*
and administered in high dosages with seemingly high efficacy,⁴³³
Likewise from the T-cell field the concept of CAR T-cell therapy in
oncology, may be transferred to allergy with infusion of *in vitro* gen-
erated and tailored T-cells based on a sample of the patients own
cells. To accomplish such developments would need an even more
thorough knowledge of the clinically relevant allergenic molecules
and a molecular-based description of the intimate reactions with the
allergen-specific receptors of the immune system.
strawberry), trimerization, and mutations directed against either
cysteine or surface molecules. The manufacture candidates were
compared with native Pru p 3 and a wild-type recombinant molecule
in a preclinical phase using IgE-binding assays and biological assays
for effector cell (basophil histamine release) to screen for allergenic-
ity in vitro. The immunogenicity was tested by rabbit and murine im-
munizations, and the molecules underwent a thorough biochemical
characterization. Sera were obtained from relevant patient groups
from different centers in Europe. While it was indeed possible to
strongly reduce the allergenicity, this often went hand in hand with
a similar reduced immunogenicity if not a downright destruction of
the molecule making it unfit for pharmaceutical use.

For the parvalbumin molecule similar attempts were slightly
more successful, and a single candidate (called m-(modified) Cyp
c 1)⁴³⁰ was selected for toxicity studies and further clinical devel-
opment. In an initial first-in-man safety study the low to absent al-
lerogenicity of the mCyp c 1 was confirmed initially by skin testing
and later by actual dosing up to ug-dosages in fish-allergic patients.
Moreover, promising responses of IgG were demonstrated, includ-
ing demonstration that the raised IgG not only bound mCyp c 1 but
also the native parvalbumin molecules from fish (see chapters B12,
C11) to which the patients reacted. A subsequent study on efficacy
proved less conclusive, however, and the preliminary conclusion
would suggest that further dose-finding studies may be necessary.

To summarize the quest for hypoallergens there are some inher-
ent problems in that some allergenic molecules exemplified by LTP
are difficult to target with strategies for reducing allergenicity due
to their structure. In many cases it is not possible to modify/reduce
allergenicity without at the same time to eliminate the immunoge-
nicity of the molecule. Even in the case of success with reduction/
elimination of allergenicity while retaining the immunogenicity, the
variability within the allergic population may represent a challenge:
People may react highly individually to not only different allergenic
molecules making the selection of candidates difficult but also dif-
ferently to different epitopes on a single allergen. This may result
in highly varying allergenic potencies of the same hypoallergen in
different patients. It is therefore likely that at least the same precau-
tions of careful titration will be necessary for hypoallergenic prod-
ucts as is the case for subcutaneous allergen immunotherapy with
conventional allergen extracts.

C. Ligation to adjuvants and other new constructs

Modern vaccinology has produced a plethora of adjuvants and
adjuvating principles that have also inspired the allergy field. Among
the examples are chimeric molecules where B-cell epitopes of grass
allergens are mixed with immunogenic viral proteins from hepa-
titis⁴²⁴ earlier alluded to. Initial clinical trials demonstrated strong
immunogenicity but failed to reach statistical significance of primary
efficacy outcomes in grass pollen-allergic hay fever patients in a ran-
domized placebo-controlled trial. Another interesting example is in-
tralymphatic immunotherapy with a modular allergen translocation
vaccine in cat allergy⁴³¹,⁴³² revealing promising results in an initial
clinical trial but not taken further to pivotal studies.

8 – Conclusions

There is a striking difference between the progress and success of
molecular allergology in the diagnostic and the therapeutic field.
This chapter has hinted at some of the challenges and barriers to
develop an allergen immunotherapy based on molecular methods
rather than the well-known and long used allergen extracts.

An important difference is the regulatory demands: while it is
relatively easy with modern biotechnology to produce a recombi-
nant molecule and to use it in an *in vitro* diagnostic test, the de-
mands for preclinical testing, toxicity studies, and finally phase 1-3
in clinical randomized studies may easily last a decade and cost up
to hundreds of millions of €. The strength of molecular allergy in the
diagnosis, where the hyped ideal of personal medicine almost be-
comes true, with the possibility of obtaining a totally individualized
molecular profile of IgE reactivity to individual allergens in the pa-
tient, becomes the Achilles' heel for the therapy: If allergy patients
are so different, how can we ever dream of obtaining sufficient pur-
chasing power to support the costly product development?

With the advent of the *in vitro* directive for the European Union,
the increased demands for evidence-based medicine and the in-
creasing scrutiny on each € or $ spent in the health care sector, the
diagnostic smorgasbord that is described in the remainder of these
guidelines may also become reduced, but this does not help us in the
therapeutic field.

With some relaxation of the present regulation, which has been
seen, e.g., in the oncology field, one way forward could be a personal-
ized approach with recombinant allergens: individual recombinant al-
lergens produced under GMP and mixed of the shelf or used to fortify
existing extracts according to the sensitization profile of the patient.

Another route of development may be an improved understand-
ing of the immunological mechanisms of the beneficial effects of
immunotherapy, which may lead to a more focused product de-
velopment. The history of immunotherapy began with the famous
study by Noon in 1911,³⁶⁴ and while it started a long journey to-
wards increasingly more efficacious and safe immunotherapy prod-
ucts, we have to admit that rationale behind Noon's study was the
incorrect notion that allergens are toxins. We have come some way
in our immunological understanding, but as demonstrated in this
chapter, even a clear decision on whether to go for B-cell epitopes
and antibody responses or T-cell epitopes and reprogramming of
the T-cell profiles (or both?) are lacking in the allergy community.
In this respect it is interesting that for both of these pathways,
new developments of specific allergy treatment may lie ahead:
Specific IgG-antibodies to allergens can now be generated *in vitro*
and administered in high dosages with seemingly high efficacy,⁴³³
Likewise from the T-cell field the concept of CAR T-cell therapy in
oncology, may be transferred to allergy with infusion of *in vitro* gen-
erated and tailored T-cells based on a sample of the patients own
cells. To accomplish such developments would need an even more
thorough knowledge of the clinically relevant allergenic molecules
and a molecular-based description of the intimate reactions with the
allergen-specific receptors of the immune system.
# A10 – Cross-reactive carbohydrate determinants

Ronald van Ree, Friedrich Altmann, Uta Jappe, Ekaterina Potapova, Paolo M Matricardi

## Highlights

* Cross-reactive carbohydrate determinants (CCDs).

* IgE antibodies to CCDs result in a broad cross-reactivity with poor clinical relevance.

* Some diagnostic tests add a CCD-like inhibitor to rule out detection of CCD-specific IgE.

## 1 – Introduction

The following lines explain how the current view of the structures relevant for antibody binding by plant/insect CCDs evolved, how it became common ground that IgE binding to these CCDs does not entail allergic symptoms and have no or little clinical relevance. At the very beginning of this story, there was considerable excitement about the discovery of a highly widespread immunogenic determinant. As for IgE, the wide cross-reactivity of carbohydrate determinants was already described in 1981.¹⁶ It was a decade later that the plant typical glycoprotein structural features xylose and core α1,3-fucose were recognized as relevant epitopes and that sera reacting with these traits also bound to certain tissues in insects, notably neurons of drosophila larvae.⁴³⁴ Soon after, the structures of the N-glycans of the major allergen of honeybee venom were exposed and found to contain the same core α1,3-fucose as plant glycoproteins (Figure 31 and 32). Inhibition experiments then showed that this fucose residue was indeed essential for IgE binding and that the GlcNAc residue to which it is bound has to be in an intact ring conformation, in other words it has to be bound to asparagine.⁴³⁵ This complicates experimental work as it precludes the use of reduced or derivatized glycans, which are the subjects of efficient separation and handling procedures. Thus, some fine details about the role for IgE binding

of other structural details such as α1,6-fucose or terminal GlcNAc residues are still unanswered. However, a biosynthetic approach revealed that—unlike deliberately immunized rabbits—humans do not usually produce IgE against xylose.⁴³⁶ In how far this finding reflects the route of sensitization—insect stings necessarily would only elicit anti-fucose antibodies—is a matter of speculation. The argument could be that subcutaneous contact with an allergen is more likely to induce sensitization than oral contact. Sensitization to galactose-α1,3-galactose (αGal) by tick bites rather than by milk or meat consumption is a highly plausible example for this mechanism.⁴³⁷,⁴³⁸ Meanwhile, the excitement about a newly discovered super epitope had vanished as evidence accumulated that anti-CCD IgE was of little or no clinical relevance.⁵¹,⁴³⁹ Then, however, histamine-release assays demonstrated functionality of anti-CCD IgE.⁴⁴⁰⁻⁴⁴² This discrepancy probably results from the unphysiological conditions of histamine-release tests in which other components such as competing IgG are removed from the stage. Fact is that two decades of vigilance towards finding cases of substantial adverse reaction to the glycan moieties of glyco-allergens did not unearth unambiguous evidence in this direction. This is fantastic news for all allergic patients with anti-CCD IgE. They will not suffer from each plant food, plant pollen, insect remnant and so on. However, exactly because of that, this patient group experiences troubles when it comes to serum-based allergy diagnosis⁴⁴³ and even the more sophisticated cellular test systems are prone to errors caused by CCDs.⁴⁴⁴,⁴⁴⁵

But let us first contemplate the reasons why anti-CCD does not or nearly not elicit clinical symptoms. Is it the very carbohydrate nature of the epitope? In analogy to the binding of most lectins to their carbohydrate ligands, the reason indeed could lie in low-binding affinities. To answer this question, patients' IgE and IgG to CCD were affinity purified, and the affinities of these pools were tested with glycoprotein ligands demonstrating the affinity of anti-CCD IgE.⁴⁴⁶ The eye-catching difference to peptide epitopes rather was the clearly higher affinity of anti-CCD IgG as compared to anti-protein IgG.

Of note, after αGal and its significance has been identified, it was suggested that the CCD types known before α-GAL should be called classical CCD,⁴⁴⁷ a term that has been accepted in a Position Paper on CCD¹⁷.

<figure><img src="image_114.png" /><figcaption>FIGURE 31 Prototypical N-glycan structures: A: example of a nonimmunogenic mammalian N-glycan; B: example of an N-glycan with an α1,3-galactose epitope from a nonprimate vertebrate; C: the typical plant CCD structure called MMXF or short MMXF; D: CCD-structure from insect venom with both types of core-fucose. Regions deemed pivotal for antibody binding are indicated by concentric half-circles.</figcaption></figure>
# A10 – Cross-reactive carbohydrate determinants

Ronald van Ree, Friedrich Altmann, Uta Jappe, Ekaterina Potapova, Paolo M Matricardi

## Highlights

* Cross-reactive carbohydrate determinants (CCDs).

* IgE antibodies to CCDs result in a broad cross-reactivity with poor clinical relevance.

* Some diagnostic tests add a CCD-like inhibitor to rule out detection of CCD-specific IgE.

## 1 – Introduction

The following lines explain how the current view of the structures relevant for antibody binding by plant/insect CCDs evolved, how it became common ground that IgE binding to these CCDs does not entail allergic symptoms and have no or little clinical relevance. At the very beginning of this story, there was considerable excitement about the discovery of a highly widespread immunogenic determinant. As for IgE, the wide cross-reactivity of carbohydrate determinants was already described in 1981.¹⁶ It was a decade later that the plant typical glycoprotein structural features xylose and core α1,3-fucose were recognized as relevant epitopes and that sera reacting with these traits also bound to certain tissues in insects, notably neurons of drosophila larvae.⁴³⁴ Soon after, the structures of the N-glycans of the major allergen of honeybee venom were exposed and found to contain the same core α1,3-fucose as plant glycoproteins (Figure 31 and 32). Inhibition experiments then showed that this fucose residue was indeed essential for IgE binding and that the GlcNAc residue to which it is bound has to be in an intact ring conformation, in other words it has to be bound to asparagine.⁴³⁵ This complicates experimental work as it precludes the use of reduced or derivatized glycans, which are the subjects of efficient separation and handling procedures. Thus, some fine details about the role for IgE binding

of other structural details such as α1,6-fucose or terminal GlcNAc residues are still unanswered. However, a biosynthetic approach revealed that—unlike deliberately immunized rabbits—humans do not usually produce IgE against xylose.⁴³⁶ In how far this finding reflects the route of sensitization—insect stings necessarily would only elicit anti-fucose antibodies—is a matter of speculation. The argument could be that subcutaneous contact with an allergen is more likely to induce sensitization than oral contact. Sensitization to galactose-α1,3-galactose (αGal) by tick bites rather than by milk or meat consumption is a highly plausible example for this mechanism.⁴³⁷,⁴³⁸ Meanwhile, the excitement about a newly discovered super epitope had vanished as evidence accumulated that anti-CCD IgE was of little or no clinical relevance.⁵¹,⁴³⁹ Then, however, histamine-release assays demonstrated functionality of anti-CCD IgE.⁴⁴⁰⁻⁴⁴² This discrepancy probably results from the unphysiological conditions of histamine-release tests in which other components such as competing IgG are removed from the stage. Fact is that two decades of vigilance towards finding cases of substantial adverse reaction to the glycan moieties of glyco-allergens did not unearth unambiguous evidence in this direction. This is fantastic news for all allergic patients with anti-CCD IgE. They will not suffer from each plant food, plant pollen, insect remnant and so on. However, exactly because of that, this patient group experiences troubles when it comes to serum-based allergy diagnosis⁴⁴³ and even the more sophisticated cellular test systems are prone to errors caused by CCDs.⁴⁴⁴,⁴⁴⁵

But let us first contemplate the reasons why anti-CCD does not or nearly not elicit clinical symptoms. Is it the very carbohydrate nature of the epitope? In analogy to the binding of most lectins to their carbohydrate ligands, the reason indeed could lie in low-binding affinities. To answer this question, patients' IgE and IgG to CCD were affinity purified, and the affinities of these pools were tested with glycoprotein ligands demonstrating the affinity of anti-CCD IgE.⁴⁴⁶ The eye-catching difference to peptide epitopes rather was the clearly higher affinity of anti-CCD IgG as compared to anti-protein IgG.

Of note, after αGal and its significance has been identified, it was suggested that the CCD types known before α-GAL should be called classical CCD,⁴⁴⁷ a term that has been accepted in a Position Paper on CCD¹⁷.

<figure><img src="image_114.png" /><figcaption>FIGURE 31 Prototypical N-glycan structures: A: example of a nonimmunogenic mammalian N-glycan; B: example of an N-glycan with an α1,3-galactose epitope from a nonprimate vertebrate; C: the typical plant CCD structure called MMXF or short MMXF; D: CCD-structure from insect venom with both types of core-fucose. Regions deemed pivotal for antibody binding are indicated by concentric half-circles.</figcaption></figure>
<figure><img src="image_115.png" /><figcaption>**FIGURE 32** Biosynthesis of N-glycans in different groups of organisms. The top line shows the so called high-mannose N-glycans, whose conversion to complex-type N-glycans starts with the attachment of a GlcNAc residue (bold black arrow). From here on, the fate of glycans diverges between vertebrates, land plants and insects (and other protostomia). Structures with designations are of known relevance as CCDs.</figcaption></figure>

## 2 – Biochemistry

The term CCD was coined for asparagine-linked oligosaccharides, usually referred to as N-glycans because the sugar is attached to the peptide backbone via the nitrogen of an amide group. Although proteins can be glycosylated in a bewildering variety of ways and each of these structures could in theory elicit IgE, the most relevant and frequently encountered structures to date are N-glycans.

Here again, two types of structures must be clearly distinguished: (a) N-glycans with α1,3-fucose linked to the innermost N-acetylglucosamine (GlcNAc) residue (Figures 31 and 32); (b) N-glycans with terminal α1,3-galactose (Figure 32), termed “αGal,” play a rather different role as will be detailed in (Chapter B14).

If not clearly specified otherwise, the term CCDs will herein refer to N-glycans with core α1,3-fucose as they occur in all kinds and parts of land plants including mosses<sup>434,448,449,1704</sup> and in a wide variety of nonvertebrate animals from nematodes to mollusks and arthropods, the latter including stinging insects.<sup>450</sup> For the sake of clarity, these CCDs may be termed “classical CCDs”.<sup>17,447</sup>

The plural CCDs was chosen at a time when the structures involved were only vaguely defined.<sup>451</sup> Later, it became evident that the core α1,3-fucose constitutes the crucial element for the human immune system whereas the xylose residue—if at all—plays a much smaller role.<sup>436,446</sup> A substantiation of this notion came from a panel of peanut allergic patients. Peanuts contain predominantly N-glycans with xylose but without fucose.<sup>448</sup> None of the sera examined, however, substantially reacted with a biosynthetic xylose-only glycoprotein (Altmann F., Eiwegger T., unpublished observations). Even though, the use of the plural form appears all the more warranted

now that the structural basis of the reaction of glycoproteins with IgE has become clearer. IgE-reactive N-glycans with core α1,3-fucose occur in several different forms. Figure 31 just shows the most prototypical versions of plant and insect N-glycans. IgE-binding and nuclear magnetic resonance experiments with glycopeptides, free glycans and reduced glycans of various structures revealed the core α1,3-fucose as primarily relevant, and the α1,6-linked mannose residue as well as the intact ring structure of the innermost GlcNAc residue as likewise pivotal for antibody binding.<sup>434,435</sup> However, a look at the biosynthesis pathways of the flagship CCD structures (Figure 31) shows that a variety of structures exist that fulfill these criteria (Figure 32). Notable differences between CCDs from insects and plants are the presence of xylose or of α1,6-fucose (Figure 32). It should be added here, that xylose—though hardly an IgE epitope by itself—contributes to binding strength.<sup>446</sup> Further heterogeneity is introduced by removal of the α1,3-mannose from the conserved trimannosyl core-structure. Thereby the frequently found glycan MMXF is converted to MUXF (U indicating the unsubstituted 3-position), which is a structure of considerable practical importance (see chapter on competitive blocking of CCD-reactive IgE). The role of terminal substituents such as GlcNAc on either the 3- or 6-arm is totally unknown.

A survey of various allergen extracts revealed two things: all grass, weed, and tree pollens (Poa pratensis, Lolium perenne, rye, ragweed, birch, horse chestnut, pine, olive), as well as vegetable foods contained the MUXF and MMXF glycans with the exception of pea (high-mannose only) and peanut and coconut (xylosylated only structures prevailing). Differences exist in the relative occurrence of glycans with terminal mannoses such as MMXF and biosynthetic
**FIGURE 32** Biosynthesis of N-glycans in different groups of organisms. The top line shows the so called high-mannose N-glycans, whose conversion to complex-type N-glycans starts with the attachment of a GlcNAc residue (bold black arrow). From here on, the fate of glycans diverges between vertebrates, land plants and insects (and other protostomia). Structures with designations are of known relevance as CCDs.

<figure><img src="image_115.png" /></figure>

## 2 – Biochemistry

The term CCD was coined for asparagine-linked oligosaccharides, usually referred to as N-glycans because the sugar is attached to the peptide backbone via the nitrogen of an amide group. Although proteins can be glycosylated in a bewildering variety of ways and each of these structures could in theory elicit IgE, the most relevant and frequently encountered structures to date are N-glycans.

Here again, two types of structures must be clearly distinguished: (a) N-glycans with α1,3-fucose linked to the innermost N-acetylglucosamine (GlcNAc) residue (Figures 31 and 32); (b) N-glycans with terminal α1,3-galactose (Figure 32), termed “αGal,” play a rather different role as will be detailed in (Chapter B14).

If not clearly specified otherwise, the term CCDs will herein refer to N-glycans with core α1,3-fucose as they occur in all kinds and parts of land plants including mosses<sup>434,448,449,1704</sup> and in a wide variety of nonvertebrate animals from nematodes to mollusks and arthropods, the latter including stinging insects.<sup>450</sup> For the sake of clarity, these CCDs may be termed “classical CCDs”.<sup>17,447</sup>

The plural CCDs was chosen at a time when the structures involved were only vaguely defined.<sup>451</sup> Later, it became evident that the core α1,3-fucose constitutes the crucial element for the human immune system whereas the xylose residue—if at all—plays a much smaller role.<sup>436,446</sup> A substantiation of this notion came from a panel of peanut allergic patients. Peanuts contain predominantly N-glycans with xylose but without fucose.<sup>448</sup> None of the sera examined, however, substantially reacted with a biosynthetic xylose-only glycoprotein (Altmann F., Eiwegger T., unpublished observations). Even though, the use of the plural form appears all the more warranted

now that the structural basis of the reaction of glycoproteins with IgE has become clearer. IgE-reactive N-glycans with core α1,3-fucose occur in several different forms. Figure 31 just shows the most prototypical versions of plant and insect N-glycans. IgE-binding and nuclear magnetic resonance experiments with glycopeptides, free glycans and reduced glycans of various structures revealed the core α1,3-fucose as primarily relevant, and the α1,6-linked mannose residue as well as the intact ring structure of the innermost GlcNAc residue as likewise pivotal for antibody binding.<sup>434,435</sup> However, a look at the biosynthesis pathways of the flagship CCD structures (Figure 31) shows that a variety of structures exist that fulfill these criteria (Figure 32). Notable differences between CCDs from insects and plants are the presence of xylose or of α1,6-fucose (Figure 32). It should be added here, that xylose—though hardly an IgE epitope by itself—contributes to binding strength.<sup>446</sup> Further heterogeneity is introduced by removal of the α1,3-mannose from the conserved trimannosyl core-structure. Thereby the frequently found glycan MMXF is converted to MUXF (U indicating the unsubstituted 3-position), which is a structure of considerable practical importance (see chapter on competitive blocking of CCD-reactive IgE). The role of terminal substituents such as GlcNAc on either the 3- or 6-arm is totally unknown.

A survey of various allergen extracts revealed two things: all grass, weed, and tree pollens (Poa pratensis, Lolium perenne, rye, ragweed, birch, horse chestnut, pine, olive), as well as vegetable foods contained the MUXF and MMXF glycans with the exception of pea (high-mannose only) and peanut and coconut (xylosylated only structures prevailing). Differences exist in the relative occurrence of glycans with terminal mannoses such as MMXF and biosynthetic
precursors with terminal GlcNAc residues, whose role as CCDs is
currently unknown. These results of structural analysis are in line
with the IgE-binding observed with multi-allergen tests, where all
pollen and all food allergens (see also chapters B01, B02, B03, B15,
B20 and B21)—and also extracts from the insect cockroach—give
more or less strong positive signals with CCD-reactive patients'
sera.⁴⁴³

3 – IgE to CCD in human pathology: allergic diseases and helminths

IgE to classical CCD in pollen allergic patients

In the largest study hitherto done on IgE to CCD, an overall prev-
alence of 23% positivity of IgE to cross-reacting carbohydrate deter-
minants was recorded.⁴³⁹ In this epidemiologic study, performed in
over 1800 patients, the prevalence of IgE antibodies to CCD varied
when different subsets of subjects were examined. Nonallergic in-
dividuals had the lowest prevalence (5%), followed by non-pollen-
allergic (10%), and pollen-allergic (31%), while subsets with multiple
pollen sensitization had a prevalence of 71%. Patients with an allergy
severe enough to require an allergen specific immunotherapy had
46%. Only minor differences in prevalence of IgE to CCD were found
when the patients were stratified by age and gender. This and many
other studies suggested that IgE to CCD in allergic patients is mainly
related to sensitization to pollen, although there is also quite some
evidence that insect venom allergy leads to CCD sensitization.⁴⁵² In
this study, the results between SPT and IgE detection to allergenic
extracts had significant differences, with almost all the negative skin
test outcomes turning into a positive IgE test outcome. A higher
correlation was observed for plant derived allergenic extracts, and
a lower one for mites and fungi. Interestingly, from the different
purified glycoproteins tested in vivo, only horseradish peroxidase
(HRP)-induced positive skin test results in 21% of the CCD-positive
subjects.⁴⁵³ There is some evidence that the consumption of alco-
hol has considerable boosting capacity for anti-CCD-IgE, the risk
of CCD sensitization being directly associated with the consumed
dose of alcohol.⁴⁵³ However, it seems that “alcohol-boostered” anti-
CCD-IgE only show negligible biologic activity in vivo (skin prick test
negativity, no clinically relevant allergy symptoms).⁴⁴⁷ In summary,
these observations bring us to the conclusion that IgE to CCDs are
common among the allergic population. On the other hand, the fact,
that patients with IgE to CCDs can provide positive results with in
vitro IgE test with an allergen extract but remain negative to the in
vivo SPT with the same extract, gives evidence of poor biological
activity of their IgE to CCDs.

Grass pollen as inducer of IgE to classical CCD

The study discovering that pollen sensitization can generate
CCD-specific IgE was published by Rob Aalberse in 1981, who also
proposed the definition and the abbreviation “CCD” still nowadays
globally used.¹⁶ Recently, the research question asking which pol-
len may be most frequently responsible for the induction of CCD-
specific IgE in pollen allergic patients has been further investigated

in detail with a molecular approach.⁴⁵⁴ In this study, experiments
with extended inhibition have been performed with the nonal-
lergenic, recombinant horse heart myoglobin-glycovariants ex-
pressed and purified from insect cells as monomeric and folded
proteins. IgE-reactivity and inhibition experiments established
a hierarchy of reactivity of patients' IgE antibodies to plant gly-
coallergens, as follows: nPhl p 4, nCyn d 1, nPla a 2, nJug r 2, nCup
a 1, and nCry j 1. A similar pattern of IgE recognition of plant gly-
coallergens has been observed earlier in allergic subjects from
Africa⁴⁵⁵ and from Asia.⁴⁵⁶ Those studies demonstrated not only
that the CCD recognized by the patients' IgE antibodies are het-
erogeneous, but also suggested that grass pollen might be the first
and most frequent inducer of this category of antibodies not only
in Europe but also worldwide.⁴⁵⁴ Both, group 1 and group 4 aller-
gen molecules in grass pollen are glycosylated, so the question
which of the two (or both) are contributing to the induction of IgE
antibodies to CCD remains open.⁴⁵⁷

IgE to classical CCD in insect venom allergic patients

Another category of allergic patients among which IgE to
CCD are frequently observed is that of patients allergic to insect
venom.¹⁶,⁴⁵² Indeed, the majority of cross-reactivities between
wasp and bee allergen extracts observed in venom allergic pa-
tients can be attributed to IgE antibodies to classical CCD.⁴⁵⁸ Most
Hymenoptera venom allergens are glycoproteins with one or more
of such carbohydrate structures, and this makes traditional diag-
nosis based on extracts quite confusing in many clinical cases. This
aspect is relevant as cross-reactivity often confounds the choice of
allergen specific immunotherapy for such patients. An interesting
study observed that IgE antibodies with specificity for the alpha-
1,3-fucose CCD epitope are responsible for about 75% of double
sensitizations to honeybee and yellow jacket.⁴⁵⁹ For diagnostics
purposes, it is very important to discriminate among (A) genuine
double sensitization to species-specific proteins of both honeybee
and yellow jacket, (B) cross-reactivity due to IgE sensitization to
protein epitopes expressed by homologous proteins in honeybee
and yellow jacket, and (C) cross-reactivity due to IgE sensitization
to carbohydrate epitopes only in both honeybee and yellow jacket.
In such cases, the use of CCD-free allergen molecules in the IgE
assays (component resolved diagnostics) is essential to define
which of the three conditions applies to the examined patient.⁴⁵²
Taken together, in most cases where the extract-based diagnos-
tics does not allow the identification of the culprit venom due to
cross-reactivity, the analysis on a molecular level applying species-
specific venom allergens, devoid of CCDs, enables the detailed
characterization of sensitization profiles and the identification
of the venom causing clinical symptoms.⁴⁵² Recent comparative
analysis on the natural (glycosylated) and recombinant bee venom
allergen Api m 1 revealed that glycosylation (of the natural vari-
ant) increased allergenicity by presenting more epitopes.⁴⁶⁰ Fur-
thermore, the glycosylated allergen induced a stronger basophil
activation.⁴⁶⁰ Whether this observation—together with others on
plant allergens (see below)—supports possible clinical relevance
of CCD-specific IgE has still to be determined.¹⁷ Clearly, venom
precursors with terminal GlcNAc residues, whose role as CCDs is
currently unknown. These results of structural analysis are in line
with the IgE-binding observed with multi-allergen tests, where all
pollen and all food allergens (see also chapters B01, B02, B03, B15,
B20 and B21)—and also extracts from the insect cockroach—give
more or less strong positive signals with CCD-reactive patients'
sera.⁴⁴³

3 – IgE to CCD in human pathology: allergic diseases and helminths

IgE to classical CCD in pollen allergic patients

In the largest study hitherto done on IgE to CCD, an overall prev-
ality of 23% positivity of IgE to cross-reacting carbohydrate deter-
minants was recorded.⁴³⁹ In this epidemiologic study, performed in
over 1800 patients, the prevalence of IgE antibodies to CCD varied
when different subsets of subjects were examined. Nonallergic in-
dividuals had the lowest prevalence (5%), followed by non-pollen-
allergic (10%), and pollen-allergic (31%), while subsets with multiple
pollen sensitization had a prevalence of 71%. Patients with an allergy
severe enough to require an allergen specific immunotherapy had
46%. Only minor differences in prevalence of IgE to CCD were found
when the patients were stratified by age and gender. This and many
other studies suggested that IgE to CCD in allergic patients is mainly
related to sensitization to pollen, although there is also quite some
evidence that insect venom allergy leads to CCD sensitization.⁴⁵² In
this study, the results between SPT and IgE detection to allergenic
extracts had significant differences, with almost all the negative skin
test outcomes turning into a positive IgE test outcome. A higher
correlation was observed for plant derived allergenic extracts, and
a lower one for mites and fungi. Interestingly, from the different
purified glycoproteins tested in vivo, only horseradish peroxidase
(HRP)-induced positive skin test results in 21% of the CCD-positive
subjects.⁴⁵³ There is some evidence that the consumption of alco-
hol has considerable boosting capacity for anti-CCD-IgE, the risk
of CCD sensitization being directly associated with the consumed
dose of alcohol.⁴⁵³ However, it seems that “alcohol-boostered” anti-
CCD-IgE only show negligible biologic activity in vivo (skin prick test
negativity, no clinically relevant allergy symptoms).⁴⁴⁷ In summary,
these observations bring us to the conclusion that IgE to CCDs are
common among the allergic population. On the other hand, the fact,
that patients with IgE to CCDs can provide positive results with in
vitro IgE test with an allergen extract but remain negative to the in
vivo SPT with the same extract, gives evidence of poor biological
activity of their IgE to CCDs.

Grass pollen as inducer of IgE to classical CCD

The study discovering that pollen sensitization can generate
CCD-specific IgE was published by Rob Aalberse in 1981, who also
proposed the definition and the abbreviation “CCD” still nowadays
globally used.¹⁶ Recently, the research question asking which pol-
len may be most frequently responsible for the induction of CCD-
specific IgE in pollen allergic patients has been further investigated

in detail with a molecular approach.⁴⁵⁴ In this study, experiments
with extended inhibition have been performed with the nonal-
lergenic, recombinant horse heart myoglobin-glycovariants ex-
pressed and purified from insect cells as monomeric and folded
proteins. IgE-reactivity and inhibition experiments established
a hierarchy of reactivity of patients' IgE antibodies to plant gly-
coallergens, as follows: nPhl p 4, nCyn d 1, nPla a 2, nJug r 2, nCup
a 1, and nCry j 1. A similar pattern of IgE recognition of plant gly-
coallergens has been observed earlier in allergic subjects from
Africa⁴⁵⁵ and from Asia.⁴⁵⁶ Those studies demonstrated not only
that the CCD recognized by the patients' IgE antibodies are het-
erogeneous, but also suggested that grass pollen might be the first
and most frequent inducer of this category of antibodies not only
in Europe but also worldwide.⁴⁵⁴ Both, group 1 and group 4 aller-
gen molecules in grass pollen are glycosylated, so the question
which of the two (or both) are contributing to the induction of IgE
antibodies to CCD remains open.⁴⁵⁷

IgE to classical CCD in insect venom allergic patients

Another category of allergic patients among which IgE to
CCD are frequently observed is that of patients allergic to insect
venom.¹⁶,⁴⁵² Indeed, the majority of cross-reactivities between
wasp and bee allergen extracts observed in venom allergic pa-
tients can be attributed to IgE antibodies to classical CCD.⁴⁵⁸ Most
Hymenoptera venom allergens are glycoproteins with one or more
of such carbohydrate structures, and this makes traditional diag-
nosis based on extracts quite confusing in many clinical cases. This
aspect is relevant as cross-reactivity often confounds the choice of
allergen specific immunotherapy for such patients. An interesting
study observed that IgE antibodies with specificity for the alpha-
1,3-fucose CCD epitope are responsible for about 75% of double
sensitizations to honeybee and yellow jacket.⁴⁵⁹ For diagnostics
purposes, it is very important to discriminate among (A) genuine
double sensitization to species-specific proteins of both honeybee
and yellow jacket, (B) cross-reactivity due to IgE sensitization to
protein epitopes expressed by homologous proteins in honeybee
and yellow jacket, and (C) cross-reactivity due to IgE sensitization
to carbohydrate epitopes only in both honeybee and yellow jacket.
In such cases, the use of CCD-free allergen molecules in the IgE
assays (component resolved diagnostics) is essential to define
which of the three conditions applies to the examined patient.⁴⁵²
Taken together, in most cases where the extract-based diagnos-
tics does not allow the identification of the culprit venom due to
cross-reactivity, the analysis on a molecular level applying species-
specific venom allergens, devoid of CCDs, enables the detailed
characterization of sensitization profiles and the identification
of the venom causing clinical symptoms.⁴⁵² Recent comparative
analysis on the natural (glycosylated) and recombinant bee venom
allergen Api m 1 revealed that glycosylation (of the natural vari-
ant) increased allergenicity by presenting more epitopes.⁴⁶⁰ Fur-
thermore, the glycosylated allergen induced a stronger basophil
activation.⁴⁶⁰ Whether this observation—together with others on
plant allergens (see below)—supports possible clinical relevance
of CCD-specific IgE has still to be determined.¹⁷ Clearly, venom
(or pollen) allergic patients with CCD-specific IgE cross-react to virtually all plant foods to a varying extent, but this does not lead to clinical food allergy to these foods. Basophil activation at higher glycoprotein concentrations cannot simply be considered as proof of possible clinical relevance. In experiments with human lactoferrin expressed in rice, hence (poly-) glycosylated with plant CCD, basophil activation could indeed be demonstrated at higher protein concentrations, but when the purified protein was orally administered to pollen allergic patients with high titers of CCD-specific IgE, the glycoprotein was tolerated at gram quantities.⁵³

### IgE to classical CCD in helminthiasis in rural Africa

Although IgE to CCD have been first described in allergic patients, it is becoming increasingly clear that humans frequently exposed to worms produce IgE responses against a broad variety of N-glycans.⁴⁶¹ This evidence emerged from a study showing that IgE sensitization to allergen extracts was highly prevalent (43%-73%) among the study population in Uganda but attributable not to established major allergenic components of the extracts but to CCD-bearing components instead. Experiments using glycan arrays scrutinized IgE responses to specific glycan moieties and uncovered a positive association between reactivity to classical CCD epitopes (core β-1,2-xylose; α-1,3-fucose) and sensitization to extracts, rural environment and infection to *Schistosoma mansoni*, while skin reactivity to extracts or sensitization to their major allergenic components presented no correlation. This study suggested therefore that, in this specific epidemiological setting, the worm infection, not allergens, was the inducer of the IgE response to CCD.⁴⁶¹

### Do IgEs to CCD play a protective role?

Considering that helminthiasis has been a normal condition during human evolution, the question arises as to whether this category of antibodies is just an epiphenomenon in allergy, while it plays a biological function in helminthiasis. There is IgE to CCD on schistosomes and schistosome eggs. Equally there is no good evidence that the symptoms that occur with nematodes entering the skin are related to IgE antibodies specific for oligosaccharides. The function of classical CCD, whether protective against helminths or against anaphylaxis, is still unknown.⁴⁶¹ Interestingly, an inverse association was found among the patients from Uganda between the presence of IgE to a subset of CCD (those with an alpha-1,3-fucose epitope) and asthma,⁴⁶¹ which may imply a protective role of IgE to CCD.

### Do IgE to CCD play an aggressive role?

Patients with schistosome infection sometimes suffer from urticaria, itching, cough, a general feeling of illness, symptoms like allergic reactions.⁴⁷⁴ It is not clear yet whether these symptoms are induced by anti-CCD-IgE.¹⁷ On the other hand, some studies have demonstrated that IgE to CCD can in a few cases induce basophil activation that correlates with clinical symptoms.⁴⁶² Similarly, a study reported five olive pollen allergic patients whose IgE antibodies to N-glycans of the major allergen of olive pollen (Ole e 1) induced basophil activation.⁴⁴⁰ In addition, a recent observation on Api m 1 showed similar results, see above.⁴⁶⁰ A clinically relevant exception is anyhow represented by the mammalian non-human disaccharide galactose-alpha1,3-galactose (αGal)⁴³⁸ (see Chapter B14).

## 4 – Methodological aspects: detection of IgE to CCDs and their confounding role in IVD

Cross-reactive IgE antibodies against plant and invertebrate carbohydrate structures were first reported by Aalberse et al. back in 1981, when the term “cross-reacting carbohydrate determinant” or CCD saw the light. Already in that seminal paper, it was reported that CCD-specific IgE resulted in broad cross-reactivity to plant foods that was not accompanied by clinical allergy to these foods. In their concluding remarks, the authors state: “For some reason, possibly continuous desensitization via oral exposure–this IgE antigen system will rarely, if ever, trigger mast cells or basophils. If further investigation should substantiate this hypothesis, it would be logical to disregard antibodies to this “allergen” for diagnostic purposes. In the RAST, this can be accomplished in principle by preabsorption of sera with buckwheat antigen as CCD source or a similar preparation, but complete absorption may be difficult to achieve. Alternatively, if the relevant allergen is periodate resistant, the sera might be tested with periodate-treated allergens”.¹⁶ Now, forty years later, some commercial diagnostic tests actually add a CCD-like inhibitor to their assays to prevent detection of CCD-specific IgE (Figures 33 and 34).⁴⁴³ One assay format offers the option to add a CCD inhibitor in an immunoblot format (RIDA qLine; r-Biopharm, Darmstadt, Germany), the other adds such an inhibitor by default (ALEX² by MacroArray Diagnostics, Vienna, Austria).

Since the first description of IgE antibodies against CCD, many studies have highlighted the poor clinical relevance of such cross-reactive antibodies. The strongest support for their poor clinical relevance was provided by Mari et al.⁵³ who performed double-blind oral challenges with human lactoferrin expressed in rice, carrying multiple CCD groups. Quantities of up to 1 gram of purified CCD-carrying human lactoferrin did not induce any symptoms in pollen-allergic patients with high IgE titers against CCD. Despite this convincing *in vivo* support, reports demonstrating biological activity of CCD-specific IgE antibodies in basophil or mast cell assays keep on fueling the discussion that they may have clinical relevance, simply because they can induce mediator release. It is important to realize that concentrations needed to achieve such activity are orders of magnitude higher than of “real” major allergens. Why CCD-specific IgE is of no clinical relevance is not yet really clarified, but it has been suggested that low antibody affinity is the most likely explanation. Independent from the question why, the consensus of poor clinical relevance has created a demand for diagnostic tests that identify CCD-specific IgE as the cause of polysensitization without clinical allergy. One way is to include CCD into screening allergy panels, as a sort of alarm that polysensitization may be caused by IgE against highly cross-reactive carbohydrate groups. A step further is to try to prevent binding to allergen extracts or purified glycoprotein allergens by addition of a CCD inhibitor, as was suggested by Aalberse et al., a long time ago. A potential disadvantage of the latter approach is that the sensitivity of the diagnostic test is decreased by the competitive format requiring serum dilution. A good alternative for microarray approaches is to avoid including purified natural glycoproteins,
(or pollen) allergic patients with CCD-specific IgE cross-react to virtually all plant foods to a varying extent, but this does not lead to clinical food allergy to these foods. Basophil activation at higher glycoprotein concentrations cannot simply be considered as proof of possible clinical relevance. In experiments with human lactoferrin expressed in rice, hence (poly-) glycosylated with plant CCD, basophil activation could indeed be demonstrated at higher protein concentrations, but when the purified protein was orally administered to pollen allergic patients with high titers of CCD-specific IgE, the glycoprotein was tolerated at gram quantities.⁵³

### IgE to classical CCD in helminthiasis in rural Africa

Although IgE to CCD have been first described in allergic patients, it is becoming increasingly clear that humans frequently exposed to worms produce IgE responses against a broad variety of N-glycans.⁴⁶¹ This evidence emerged from a study showing that IgE sensitization to allergen extracts was highly prevalent (43%-73%) among the study population in Uganda but attributable not to established major allergenic components of the extracts but to CCD-bearing components instead. Experiments using glycan arrays scrutinized IgE responses to specific glycan moieties and uncovered a positive association between reactivity to classical CCD epitopes (core β-1,2-xylose; α-1,3-fucose) and sensitization to extracts, rural environment and infection to *Schistosoma mansoni*, while skin reactivity to extracts or sensitization to their major allergenic components presented no correlation. This study suggested therefore that, in this specific epidemiological setting, the worm infection, not allergens, was the inducer of the IgE response to CCD.⁴⁶¹

### Do IgEs to CCD play a protective role?

Considering that helminthiasis has been a normal condition during human evolution, the question arises as to whether this category of antibodies is just an epiphenomenon in allergy, while it plays a biological function in helminthiasis. There is IgE to CCD on schistosomes and schistosome eggs. Equally there is no good evidence that the symptoms that occur with nematodes entering the skin are related to IgE antibodies specific for oligosaccharides. The function of classical CCD, whether protective against helminths or against anaphylaxis, is still unknown.⁴⁶¹ Interestingly, an inverse association was found among the patients from Uganda between the presence of IgE to a subset of CCD (those with an alpha-1,3-fucose epitope) and asthma,⁴⁶¹ which may imply a protective role of IgE to CCD.

### Do IgE to CCD play an aggressive role?

Patients with schistosome infection sometimes suffer from urticaria, itching, cough, a general feeling of illness, symptoms like allergic reactions.⁴⁷⁴ It is not clear yet whether these symptoms are induced by anti-CCD-IgE.¹⁷ On the other hand, some studies have demonstrated that IgE to CCD can in a few cases induce basophil activation that correlates with clinical symptoms.⁴⁶² Similarly, a study reported five olive pollen allergic patients whose IgE antibodies to N-glycans of the major allergen of olive pollen (Ole e 1) induced basophil activation.⁴⁴⁰ In addition, a recent observation on Api m 1 showed similar results, see above.⁴⁶⁰ A clinically relevant exception is anyhow represented by the mammalian non-human disaccharide galactose-alpha1,3-galactose (αGal)⁴³⁸ (see Chapter B14).

## 4 – Methodological aspects: detection of IgE to CCDs and their confounding role in IVD

Cross-reactive IgE antibodies against plant and invertebrate carbohydrate structures were first reported by Aalberse et al. back in 1981, when the term “cross-reacting carbohydrate determinant” or CCD saw the light. Already in that seminal paper, it was reported that CCD-specific IgE resulted in broad cross-reactivity to plant foods that was not accompanied by clinical allergy to these foods. In their concluding remarks, the authors state: “For some reason, possibly continuous desensitization via oral exposure–this IgE antigen system will rarely, if ever, trigger mast cells or basophils. If further investigation should substantiate this hypothesis, it would be logical to disregard antibodies to this “allergen” for diagnostic purposes. In the RAST, this can be accomplished in principle by preabsorption of sera with buckwheat antigen as CCD source or a similar preparation, but complete absorption may be difficult to achieve. Alternatively, if the relevant allergen is periodate resistant, the sera might be tested with periodate-treated allergens”.¹⁶ Now, forty years later, some commercial diagnostic tests actually add a CCD-like inhibitor to their assays to prevent detection of CCD-specific IgE (Figures 33 and 34).⁴⁴³ One assay format offers the option to add a CCD inhibitor in an immunoblot format (RIDA qLine; r-Biopharm, Darmstadt, Germany), the other adds such an inhibitor by default (ALEX² by MacroArray Diagnostics, Vienna, Austria).

Since the first description of IgE antibodies against CCD, many studies have highlighted the poor clinical relevance of such cross-reactive antibodies. The strongest support for their poor clinical relevance was provided by Mari et al.⁵³ who performed double-blind oral challenges with human lactoferrin expressed in rice, carrying multiple CCD groups. Quantities of up to 1 gram of purified CCD-carrying human lactoferrin did not induce any symptoms in pollen-allergic patients with high IgE titers against CCD. Despite this convincing *in vivo* support, reports demonstrating biological activity of CCD-specific IgE antibodies in basophil or mast cell assays keep on fueling the discussion that they may have clinical relevance, simply because they can induce mediator release. It is important to realize that concentrations needed to achieve such activity are orders of magnitude higher than of “real” major allergens. Why CCD-specific IgE is of no clinical relevance is not yet really clarified, but it has been suggested that low antibody affinity is the most likely explanation. Independent from the question why, the consensus of poor clinical relevance has created a demand for diagnostic tests that identify CCD-specific IgE as the cause of polysensitization without clinical allergy. One way is to include CCD into screening allergy panels, as a sort of alarm that polysensitization may be caused by IgE against highly cross-reactive carbohydrate groups. A step further is to try to prevent binding to allergen extracts or purified glycoprotein allergens by addition of a CCD inhibitor, as was suggested by Aalberse et al., a long time ago. A potential disadvantage of the latter approach is that the sensitivity of the diagnostic test is decreased by the competitive format requiring serum dilution. A good alternative for microarray approaches is to avoid including purified natural glycoproteins,
<figure><img src="image_116.png" /></figure>

FIGURE 33 Competitive suppression of CCD-based positive results: In the course of a solid-phase IgE assay serum containing antibodies with various specificities is incubated with an immobilized allergen (upper row). Green antibodies are protein-specific, red antibodies bind to clinically irrelevant CCD structure. Antibody binding leads to color development irrespective of the biological significance. A soluble glycoprotein functions as a CCD-blocker by adsorbing most of the CCD-directed antibodies, which are removed in the washing step prior to color development.

<figure><img src="image_117.png" /></figure>

FIGURE 34 Cross-reactive carbohydrate determinants (CCD) inhibition as observed on multi-allergen test strips. Custom-made test strips with CCD markers were incubated with serum in the absence (n) or presence (i) of inhibitor (20 µg/ml). The boxes mark allergens that may exhibit CCD-based IgE binding. Sera A, B and C were obtained from patients f16, m19, and f12 (a CCD-negative patient). The * denotes a mechanical scratch in panel C. The results of CAP tests performed with serum B show that CCD inhibition does not affect exclusively protein-based reactions with allergen components.⁴⁴⁴

and if possible, replace them by nonglycosylated recombinant alternatives. The newer release of the ImmunoCAP ISAC microarray (ThermoFisher Scientific, Uppsala, Sweden) have followed that approach and have removed natural pollen and food glycoproteins that were reported to give many false-positive test results (nJug r 2, nPla

a 2). It is important to realize that specific IgE tests serve as support for a diagnosis but cannot be regarded as establishing a diagnosis on their own. Sensitive-detection of specific IgE is the aim of a good serological test, and in this setting false positive would be a background issue with nonspecific IgE binding. This is of course not what
**FIGURE 33** Competitive suppression of CCD-based positive results: In the course of a solid-phase IgE assay serum containing antibodies with various specificities is incubated with an immobilized allergen (upper row). Green antibodies are protein-specific, red antibodies bind to clinically irrelevant CCD structure. Antibody binding leads to color development irrespective of the biological significance. A soluble glycoprotein functions as a CCD-blocker by adsorbing most of the CCD-directed antibodies, which are removed in the washing step prior to color development.

**FIGURE 34** Cross-reactive carbohydrate determinants (CCD) inhibition as observed on multi-allergen test strips. Custom-made test strips with CCD markers were incubated with serum in the absence (n) or presence (i) of inhibitor (20 µg/ml). The boxes mark allergens that may exhibit CCD-based IgE binding. Sera A, B and C were obtained from patients f16, m19, and f12 (a CCD-negative patient). The * denotes a mechanical scratch in panel C. The results of CAP tests performed with serum B show that CCD inhibition does not affect exclusively protein-based reactions with allergen components.444

<figure><img src="image_116.png" /></figure>

and if possible, replace them by nonglycosylated recombinant alternatives. The newer release of the ImmunoCAP ISAC microarray (ThermoFisher Scientific, Uppsala, Sweden) have followed that approach and have removed natural pollen and food glycoproteins that were reported to give many false-positive test results (nJug r 2, nPla

a 2). It is important to realize that specific IgE tests serve as support for a diagnosis but cannot be regarded as establishing a diagnosis on their own. Sensitive-detection of specific IgE is the aim of a good serological test, and in this setting false positive would be a background issue with nonspecific IgE binding. This is of course not what
is meant by false-positive in case of CCD-specific IgE: this is true specific IgE. In this case, false-positive is meant as clinically irrelevant. It can be argued that serological tests for specific IgE should stay away from avoiding detection of specific IgE considered to be of no clinical relevance. Should we detect specific IgE against profilins or try to avoid it because it often is of little clinical relevance? The point is: it is hard to generalize this. Perhaps therefore an approach in which specific IgE against CCD is separately detected, combined with CCD-containing extracts but as much as possible CCD-free recombinant major allergens.

## 5 - Clinical cases

### E1 - Perennial allergic rhino-conjunctivitis with seasonal exacerbations

Clinical history—A 19-year-old patient with hay fever symptoms throughout the year but with seasonal peaks in late spring only.

First series of IgE tests—The patient serum was tested with both a customized allergy strip (Mediwiss, Moers) and the ImmunoCAP Specific IgE test. The outcomes indicated that the patient had a very broad sensitization, with positive results for alder, birch, hazel, grass mix, rye, mugwort, ragweed, and plantain pollen, as well as *D. pteronyssinus*, *D. farinae*, cockroach, hazel, peanut, walnut, wheat flour, rye flour, soy, orange, apple, celery, carrot.

Diagnostic and therapeutic considerations—given the extremely broad sensitization profile, the patient could be defined as a highly atopic polysensitized subject, whose likelihood of successful response to allergen immunotherapy would have been quite low.

Further IgE tests—The patient serum was also tested for IgE antibodies to CCD and resulted highly positive. Hence, IgE tests were repeated after incubation of the serum with a CCD inhibitor, prepared from pineapple stem bromelain and human serum albumin. This time, the IgE reactions towards all tree pollens (alder, birch, hazel) and toward ragweed, cockroach and all foods (hazel, peanut, walnut, wheat flour, rye flour, soy, orange, apple, celery, and carrot) disappeared.

Testing IgE to allergen molecules—In agreement with the above listed outcomes of the IgE tests after incubation of the serum with CCD inhibitor, no IgE to rBet v 1, rBet v 2, rBet v 4 were detected. By contrast, over 40 kU/L of IgE to a mix of rPhl p 1 and rPhl p 5, as well as over 20 kU/L of IgE to nDer p 1 and over 40 kU/L of IgE to rDer p 2 were detected.

Diagnosis and therapy—The patient had perennial rhino-conjunctivitis due to allergy to house dust mites with seasonal exacerbations due to grass pollen allergy. Accordingly, prevention of exposure to HDM and allergen immunotherapy with HDM and grass pollen extracts could be taken into consideration.

### E2-Insect venom allergy: double-positivity to different hymenoptera species (Reproduced with permission from⁴⁶³)

**Clinical history**

A 35-year-old female patient was stung by an unidentified insect while walking on the edge of the forest. Within a few minutes,

there was a severe local swelling, and about eight hours later, a systemic reaction occurred. Pre-existing conditions: Tree pollen allergy with sensitization to birch, and a pollen-associated food allergy with oral allergy syndrome after consumption of peanuts and stone fruits.

First series of IgE tests—The patient's serum was tested with the ImmunoCAP Specific IgE test. Specific IgE antibodies were determined against whole bee and wasp venom extract: IgE to wasp venom extract: 6.25 kU/L (equivalent to CAP class 3); IgE to bee venom extract: 10.9 kU/L (CAP class 3). Total IgE: 1836 kU/l (reference range 0-100 kU/L). Serum tryptase: 6.03 µg/l (reference range 0-11.4 µg/l).

Diagnostic and therapeutic considerations—The IgE-detection assay revealed *in vitro* double positivity. The insect had not been identified, and the situation in which the patient was stung was not indicative either. There were weak positive reactions in skin tests to both venoms in different concentrations. A reliable statement concerning the culprit insect could not be made on the basis of these results.

Further IgE tests—The patient's serum was subsequently tested for IgE antibodies to CCD (MUXF3-component) and was found to be highly positive (12.80 kU/L).

Testing IgE to allergen molecules—Specific IgE antibodies against the major allergen of wasp venom, rVes v 5, were detected (2.16 kU/L) but not against the major allergen of the bee venom, rApi m 1. No sensitization was found against the second major allergen of wasp venom, rVes v 1. At that time, further bee venom allergens had not been available.

Cellular allergy diagnostic test—In addition, a basophil activation test was performed with both, bee and wasp venom. This showed a 14-fold increase in CD63 expression after stimulation with wasp venom. After incubation with bee venom, no significant stimulation of the basophils was induced.

Diagnosis and therapy—In the present case, neither the medical history nor the determination of specific IgE antibodies against bee and wasp venom extract nor the skin tests led to a clear identification of the insect venom responsible for the symptoms. Only by using the recombinant major allergens of bee and wasp venom, Api m 1, Ves v 5 and Ves v 1, could the wasp venom sensitization be diagnosed, which was confirmed by the basophil activation test. Specific immunotherapy with wasp venom was planned. By IgE determination against recombinant single allergens of bee (*Apis mellifera*) and wasp (*Vespula vulgaris*), as well as CCD, in our case, the CCD component MUXF of bromelain from pineapple, the culprit venom can be detected to a large extent. Api m 1, the major allergen of bee venom, induces sensitization in 69-80% of those allergic to bee venom. To Ves v 5, the major allergen of wasp venom, 88-90% of those allergic to wasp venom are sensitized. With a combination of Ves v 1 and Ves v 5, the sensitivity of IgE diagnostics can be increased from approx. 88% (anti-Ves v 5 IgE determination alone) to 96%. Due to the recombinant production in *E. coli*, the hymenoptera venom single allergens no longer exhibit CCD, the CCD-mediated cross-reactivity is thus⁴⁶³ eliminated.
is meant by false-positive in case of CCD-specific IgE: this is true specific IgE. In this case, false-positive is meant as clinically irrelevant. It can be argued that serological tests for specific IgE should stay away from avoiding detection of specific IgE considered to be of no clinical relevance. Should we detect specific IgE against profilins or try to avoid it because it often is of little clinical relevance? The point is: it is hard to generalize this. Perhaps therefore an approach in which specific IgE against CCD is separately detected, combined with CCD-containing extracts but as much as possible CCD-free recombinant major allergens.

## 5 - Clinical cases

### E1 - Perennial allergic rhino-conjunctivitis with seasonal exacerbations

Clinical history—A 19-year-old patient with hay fever symptoms throughout the year but with seasonal peaks in late spring only.

First series of IgE tests—The patient serum was tested with both a customized allergy strip (Mediwiss, Moers) and the ImmunoCAP Specific IgE test. The outcomes indicated that the patient had a very broad sensitization, with positive results for alder, birch, hazel, grass mix, rye, mugwort, ragweed, and plantain pollen, as well as *D. pteronyssinus*, *D. farinae*, cockroach, hazel, peanut, walnut, wheat flour, rye flour, soy, orange, apple, celery, carrot.

Diagnostic and therapeutic considerations—given the extremely broad sensitization profile, the patient could be defined as a highly atopic polysensitized subject, whose likelihood of successful response to allergen immunotherapy would have been quite low.

Further IgE tests—The patient serum was also tested for IgE antibodies to CCD and resulted highly positive. Hence, IgE tests were repeated after incubation of the serum with a CCD inhibitor, prepared from pineapple stem bromelain and human serum albumin. This time, the IgE reactions towards all tree pollens (alder, birch, hazel) and toward ragweed, cockroach and all foods (hazel, peanut, walnut, wheat flour, rye flour, soy, orange, apple, celery, and carrot) disappeared.

Testing IgE to allergen molecules—In agreement with the above listed outcomes of the IgE tests after incubation of the serum with CCD inhibitor, no IgE to rBet v 1, rBet v 2, rBet v 4 were detected. By contrast, over 40 kU/L of IgE to a mix of rPhl p 1 and rPhl p 5, as well as over 20 kU/L of IgE to nDer p 1 and over 40 kU/L of IgE to rDer p 2 were detected.

Diagnosis and therapy—The patient had perennial rhino-conjunctivitis due to allergy to house dust mites with seasonal exacerbations due to grass pollen allergy. Accordingly, prevention of exposure to HDM and allergen immunotherapy with HDM and grass pollen extracts could be taken into consideration.

### E2-Insect venom allergy: double-positivity to different hymenoptera species (Reproduced with permission from⁴⁶³)

**Clinical history**

A 35-year-old female patient was stung by an unidentified insect while walking on the edge of the forest. Within a few minutes,

there was a severe local swelling, and about eight hours later, a systemic reaction occurred. Pre-existing conditions: Tree pollen allergy with sensitization to birch, and a pollen-associated food allergy with oral allergy syndrome after consumption of peanuts and stone fruits.

First series of IgE tests—The patient's serum was tested with the ImmunoCAP Specific IgE test. Specific IgE antibodies were determined against whole bee and wasp venom extract: IgE to wasp venom extract: 6.25 kU/L (equivalent to CAP class 3); IgE to bee venom extract: 10.9 kU/L (CAP class 3). Total IgE: 1836 kU/l (reference range 0-100 kU/L). Serum tryptase: 6.03 µg/l (reference range 0-11.4 µg/l).

Diagnostic and therapeutic considerations—The IgE-detection assay revealed *in vitro* double positivity. The insect had not been identified, and the situation in which the patient was stung was not indicative either. There were weak positive reactions in skin tests to both venoms in different concentrations. A reliable statement concerning the culprit insect could not be made on the basis of these results.

Further IgE tests—The patient's serum was subsequently tested for IgE antibodies to CCD (MUXF3-component) and was found to be highly positive (12.80 kU/L).

Testing IgE to allergen molecules—Specific IgE antibodies against the major allergen of wasp venom, rVes v 5, were detected (2.16 kU/L) but not against the major allergen of the bee venom, rApi m 1. No sensitization was found against the second major allergen of wasp venom, rVes v 1. At that time, further bee venom allergens had not been available.

Cellular allergy diagnostic test—In addition, a basophil activation test was performed with both, bee and wasp venom. This showed a 14-fold increase in CD63 expression after stimulation with wasp venom. After incubation with bee venom, no significant stimulation of the basophils was induced.

Diagnosis and therapy—In the present case, neither the medical history nor the determination of specific IgE antibodies against bee and wasp venom extract nor the skin tests led to a clear identification of the insect venom responsible for the symptoms. Only by using the recombinant major allergens of bee and wasp venom, Api m 1, Ves v 5 and Ves v 1, could the wasp venom sensitization be diagnosed, which was confirmed by the basophil activation test. Specific immunotherapy with wasp venom was planned. By IgE determination against recombinant single allergens of bee (*Apis mellifera*) and wasp (*Vespula vulgaris*), as well as CCD, in our case, the CCD component MUXF of bromelain from pineapple, the culprit venom can be detected to a large extent. Api m 1, the major allergen of bee venom, induces sensitization in 69-80% of those allergic to bee venom. To Ves v 5, the major allergen of wasp venom, 88-90% of those allergic to wasp venom are sensitized. With a combination of Ves v 1 and Ves v 5, the sensitivity of IgE diagnostics can be increased from approx. 88% (anti-Ves v 5 IgE determination alone) to 96%. Due to the recombinant production in *E. coli*, the hymenoptera venom single allergens no longer exhibit CCD, the CCD-mediated cross-reactivity is thus⁴⁶³ eliminated.
# A11 – Small molecules as immunomodulators and allergen ligands

Pierre Rougé, Christiane Hilger, Karin Hoffmann-Sommergruber, Claudia Traidl-Hoffmann

## Highlights

Ligand allergen interaction can induce conformational changes and affect:

* Protein stability against gastric, thermal, and lysosomal degradation

* Accessibility of IgE antibodies

* The sensitization process

Lipids (free lipids or lipid ligands) in conjunction with allergens can act as promotors or enhancers of inflammatory (allergic) responses.

## 1 – Introduction and overview

The knowledge about allergens, their structures, biological functions and interactions with immune cells has tremendously increased in the last two decades. However, there is still a lack of understanding how “harmless,” nontoxic proteins can initiate an allergic sensitization in predisposed individuals. Among other factors such as impaired epithelial barriers, small molecules and allergen ligands can contribute to the onset of an allergic sensitization as it has been shown by recent findings.

In this context, small molecules include lipids, glycosylated flavonoids and derivatives thereof, steroids, fatty acids, and plant hormones. These molecules can be part of the allergen-surrounding matrix such as pollen matrix, food matrix and components from animal or plant derived dust. For some of those components direct interaction with allergens (protein – ligand) has been shown, while for others co-localization was described. For a number of small molecules their interaction with the immune system, including both, the innate and the adaptive arm, was shown. At present, a number of molecular structures from allergens have been characterized. This detailed structural analysis allows to investigate protein ligand interactions. Binding of a ligand into the cavity of an allergenic protein can induce local conformational changes. In case this affects surface exposed areas that are part of an IgE epitope, this may lead to better accessibility of IgE antibodies and increased IgE-binding activity.

Furthermore, it has been shown that ligand binding can increase protein stability against gastric, thermal, and lysosomal degradation, leading to prolonged availability of the protein to interact with the immune system. In addition, the ligand itself can interact with

immune cells, such as binding and activating surface exposed receptors of the innate immune system, e.g., Toll-like receptors (TLRs). Lipid ligands can activate certain T cell subsets via CD1 presentation and thus contribute to allergic sensitization.

This chapter summarizes recent findings about allergens and their ligands and their role in an immune response. Specifically, these new findings show that ligands can play a relevant role in mounting an allergic immune response. It collates current findings about structural data and how these interactions can influence IgE-binding activity, protein stability, and oligomerization, and thus contribute to both, the onset of an allergic sensitization process and the allergic effector phase, respectively. Furthermore, for some allergens it became evident that additional matrix components are required to induce a Th2 type immune response.

### Lipocalins

The majority of mammalian allergens belong to the lipocalin protein family.<sup>345</sup> Lipocalins are a highly diverse protein family with many functions, and members of the family are also found in arthropods, plants, and bacteria. Lipocalins are characterized by a common tertiary structure composed of a central β-barrel formed by eight anti-parallel β-strands. The internal binding pocket carries a broad range of small hydrophobic molecules such as retinol, steroids, lipids, pheromones, and odorants (Chapter C07). So far, only a few natural ligands have been characterized in detail. Although lipocalins are important mammalian allergens, the mechanism of their allergenicity is still elusive.<sup>26</sup> Lipocalin allergens were found to elicit weak adaptive cellular immune responses, e.g., T cell epitopes of Bos d 2 and Can f 1 were only recognized suboptimally by human T cells. It is thus likely that a major contribution to allergenicity may be based on innate immunity, receptor binding, or their role in ligand binding.<sup>464</sup> The milk allergen Bos d 5, β-lactoglobulin, was shown to bind many ligands, mainly long-chain fatty acids, resulting in changes in structure and resistance to denaturation.<sup>465</sup> However, Bos d 5 also binds quercetin-iron complexes and in this case the ligand load seems to provide an immune-regulatory effect and protection against allergic sensitization to birch pollen allergens in mice.<sup>466-468</sup>

Whereas Bos d 5 is a food allergen, all other mammalian lipocalin allergens are respiratory allergens. They are present in saliva, danger, and urine. The crystal structures of several lipocalins have been resolved and their binding sites were analysed.<sup>469</sup> Mammalian lipocalin allergens belong to the categories of urinary proteins, odorant-binding proteins, salivary lipocalins or the von Ebner gland (VEG) proteins and some of them were shown to bind and release small volatile compounds, suggesting a role in chemical communication. However, the role of these ligands for allergenicity needs further investigations.

Arthropod lipocalins comprise the tick histamine-binding protein Arg r 1 and the cockroach allergens Bla g 4 and Per a 4. The binding of tyramine, a biogenic amine, to the cockroach allergen Bla g 4 was characterized by X-ray crystallography.<sup>470</sup> However, it is not yet
# A11 – Small molecules as immunomodulators and allergen ligands

Pierre Rougé, Christiane Hilger, Karin Hoffmann-Sommergruber, Claudia Traidl-Hoffmann

## Highlights

Ligand allergen interaction can induce conformational changes and affect:

* Protein stability against gastric, thermal, and lysosomal degradation

* Accessibility of IgE antibodies

* The sensitization process

Lipids (free lipids or lipid ligands) in conjunction with allergens can act as promoters or enhancers of inflammatory (allergic) responses.

## 1 – Introduction and overview

The knowledge about allergens, their structures, biological functions and interactions with immune cells has tremendously increased in the last two decades. However, there is still a lack of understanding how “harmless,” nontoxic proteins can initiate an allergic sensitization in predisposed individuals. Among other factors such as impaired epithelial barriers, small molecules and allergen ligands can contribute to the onset of an allergic sensitization as it has been shown by recent findings.

In this context, small molecules include lipids, glycosylated flavonoids and derivatives thereof, steroids, fatty acids, and plant hormones. These molecules can be part of the allergen-surrounding matrix such as pollen matrix, food matrix and components from animal or plant derived dust. For some of those components direct interaction with allergens (protein – ligand) has been shown, while for others co-localization was described. For a number of small molecules their interaction with the immune system, including both, the innate and the adaptive arm, was shown. At present, a number of molecular structures from allergens have been characterized. This detailed structural analysis allows to investigate protein ligand interactions. Binding of a ligand into the cavity of an allergenic protein can induce local conformational changes. In case this affects surface exposed areas that are part of an IgE epitope, this may lead to better accessibility of IgE antibodies and increased IgE-binding activity.

Furthermore, it has been shown that ligand binding can increase protein stability against gastric, thermal, and lysosomal degradation, leading to prolonged availability of the protein to interact with the immune system. In addition, the ligand itself can interact with

immune cells, such as binding and activating surface exposed receptors of the innate immune system, e.g., Toll-like receptors (TLRs). Lipid ligands can activate certain T cell subsets via CD1 presentation and thus contribute to allergic sensitization.

This chapter summarizes recent findings about allergens and their ligands and their role in an immune response. Specifically, these new findings show that ligands can play a relevant role in mounting an allergic immune response. It collates current findings about structural data and how these interactions can influence IgE-binding activity, protein stability, and oligomerization, and thus contribute to both, the onset of an allergic sensitization process and the allergic effector phase, respectively. Furthermore, for some allergens it became evident that additional matrix components are required to induce a Th2 type immune response.

### Lipocalins

The majority of mammalian allergens belong to the lipocalin protein family.<sup>345</sup> Lipocalins are a highly diverse protein family with many functions, and members of the family are also found in arthropods, plants, and bacteria. Lipocalins are characterized by a common tertiary structure composed of a central β-barrel formed by eight anti-parallel β-strands. The internal binding pocket carries a broad range of small hydrophobic molecules such as retinol, steroids, lipids, pheromones, and odorants (Chapter C07). So far, only a few natural ligands have been characterized in detail. Although lipocalins are important mammalian allergens, the mechanism of their allergenicity is still elusive.<sup>26</sup> Lipocalin allergens were found to elicit weak adaptive cellular immune responses, e.g., T cell epitopes of Bos d 2 and Can f 1 were only recognized suboptimally by human T cells. It is thus likely that a major contribution to allergenicity may be based on innate immunity, receptor binding, or their role in ligand binding.<sup>464</sup> The milk allergen Bos d 5, β-lactoglobulin, was shown to bind many ligands, mainly long-chain fatty acids, resulting in changes in structure and resistance to denaturation.<sup>465</sup> However, Bos d 5 also binds quercetin-iron complexes and in this case the ligand load seems to provide an immune-regulatory effect and protection against allergic sensitization to birch pollen allergens in mice.<sup>466-468</sup>

Whereas Bos d 5 is a food allergen, all other mammalian lipocalin allergens are respiratory allergens. They are present in saliva, danger, and urine. The crystal structures of several lipocalins have been resolved and their binding sites were analysed.<sup>469</sup> Mammalian lipocalin allergens belong to the categories of urinary proteins, odorant-binding proteins, salivary lipocalins or the von Ebner gland (VEG) proteins and some of them were shown to bind and release small volatile compounds, suggesting a role in chemical communication. However, the role of these ligands for allergenicity needs further investigations.

Arthropod lipocalins comprise the tick histamine-binding protein Arg r 1 and the cockroach allergens Bla g 4 and Per a 4. The binding of tyramine, a biogenic amine, to the cockroach allergen Bla g 4 was characterized by X-ray crystallography.<sup>470</sup> However, it is not yet
clear whether ligand binding has an effect on the allergenic activity of Bla g 4.

The so called mite group 13 allergens belong to the family of cytosolic fatty acid-binding proteins (cFABPs) and they are closely related to the lipocalins. cFABPs are intracellular highly conserved proteins, whereas almost all lipocalins are extracellular proteins. Lipocalins and cFABPs are members of the calcin protein super-family and they share similar β-barrel structures (see chapter A08). As other allergens, e.g. Der p 2 and Bla g 1, Der p 13 can accommodate lipid ligands in their hydrophobic cavity, but this accommodation is not suspected to provoke important conformational changes, due to the higher rigidity of the β-sheet (Der p 2, Der p 13) and α-helix (Bla g 1) structures surrounding the hydrophobic cavity (Figure 35).471-473

Group 13 mite allergens may contribute to the allergic sensitization process. Der p 13 was shown to selectively bind fatty acids and to initiate TLR2 dependent innate immune signalling.474 Furthermore, Der p 13 and Bla t 13 are sensed by an acute-phase protein, serum amyloid A1 (SAA1), that promotes pulmonary type 2 immunity.38

## 2 - Pathogenesis-related proteins (PR-10)

Allergens from the PR-10 family are major Fagales pollen allergens such as Bet v 1, food allergens from apple (Mal d 1), celeriac (Api g 1), hazelnut (Cor a 1), peanut (Ara h 8) and many more (see also Chapter B15). There is high sequence similarity among PR-10s from related species, which is reflected by high IgE cross-reactivity with and without clinical relevance.

Pathogenesis related proteins 10 share a conserved 3D structure including a hydrophobic cavity that can take up different ligands (Figure 36). So far, a number of different ligands have been identified for PR-10 proteins including flavonoids, cytokinins, steroids,

<figure><img src="image_118.png" /><figcaption>FIGURE 35 Cut sections of Der p 2 (left) and Bla g 1 (right), showing their core hydrophobic cavity. The large hydrophobic cavity of Bla g 1 can accommodate a variety of lipid molecules including fatty acids (palmitic, stearic, and oleic acids) and phospholipids (phosphatidylcholine, phosphatidylinositol, and phosphatidylserine).</figcaption></figure>

and derivates thereof (Table 9). This growing list of different ligands of PR-10 proteins indicates their different biological functions in the plant such as transport of small molecules, orchestrating germination and protection from environmental stress such as UV-radiation. For example, specific ligands such as the glycosylated flavonoid derivative quercetin-3-O-sophoride were identified for Bet v 1, resveratrol for Bet v 1 and Ara h 8, genistein for Ara h 8 and Bet v 1 and fatty acids for Bet v 1.469

In the case of Bet v 1, ligand binding did not result in an increased IgE-binding activity, although binding of phosphatidylcholine to Api g 1, Cor a 1, Mal d 1, and Pru p 1 induced conformational changes as shown by changed circular dichroism spectra. This provided protection from pepsinolysis to some degree, which resulted in basophil activation even with partly digested PR-10 proteins.33

<figure><img src="image_119.png" /><figcaption>FIGURE 36 Front face (A) and lateral face (B) of Bet v 1, the PR-10 of *Betula verrucosa*, showing the extent of the hydrophobic cavity (colored violet) harboring a single or several phenolic compounds through hydrophobic interactions (cartoon drawn with the ProteinsPlus server (https://proteins-plus/))</figcaption></figure>

<table><caption>TABLE 9 Structures of allergenic PR-10 Bet v 1-like proteins in complexes with natural compounds or their derivatives, available in the RCSB Protein Data Bank (PDB) (http://www.rcsb.org).</caption><thead><tr><th>PR-10 protein</th><th>compound</th><th>PDB code</th></tr></thead><tbody><tr><td>Bet v 1</td><td>desoxycholate</td><td>4A83</td></tr><tr><td>Bet v 1</td><td>kinetin</td><td>4A85</td></tr><tr><td>Bet v 1</td><td>naringenin</td><td>4A87</td></tr><tr><td>Fra a 1</td><td>catechin</td><td>4C94</td></tr><tr><td>Vig r 6</td><td>gibberellic acid</td><td>4PSB,</td></tr><tr><td>Ara h 8</td><td>epicatechin</td><td>6AX0 4MA6, 6AWV,<br>6AWT</td></tr><tr><td>Ara h 8</td><td>caffeic acid</td><td>6AWU</td></tr><tr><td>Ara h 8</td><td>3-hydroxy-2-naphthoic acid</td><td>6AWW</td></tr><tr><td>Ara h 8</td><td>quercetin</td><td>6AWS, 6B1D</td></tr></tbody></table>
clear whether ligand binding has an effect on the allergenic activity of Bla g 4.

The so called mite group 13 allergens belong to the family of cytosolic fatty acid-binding proteins (cFABPs) and they are closely related to the lipocalins. cFABPs are intracellular highly conserved proteins, whereas almost all lipocalins are extracellular proteins. Lipocalins and cFABPs are members of the calcin protein super-family and they share similar β-barrel structures (see chapter A08). As other allergens, e.g. Der p 2 and Bla g 1, Der p 13 can accommodate lipid ligands in their hydrophobic cavity, but this accommodation is not suspected to provoke important conformational changes, due to the higher rigidity of the β-sheet (Der p 2, Der p 13) and α-helix (Bla g 1) structures surrounding the hydrophobic cavity (Figure 35).471-473

Group 13 mite allergens may contribute to the allergic sensitization process. Der p 13 was shown to selectively bind fatty acids and to initiate TLR2 dependent innate immune signalling.474 Furthermore, Der p 13 and Bla t 13 are sensed by an acute-phase protein, serum amyloid A1 (SAA1), that promotes pulmonary type 2 immunity.38

## 2 – Pathogenesis-related proteins (PR-10)

Allergens from the PR-10 family are major Fagales pollen allergens such as Bet v 1, food allergens from apple (Mal d 1), celeriac (Api g 1), hazelnut (Cor a 1), peanut (Ara h 8) and many more (see also Chapter B15). There is high sequence similarity among PR-10s from related species, which is reflected by high IgE cross-reactivity with and without clinical relevance.

Pathogenesis related proteins 10 share a conserved 3D structure including a hydrophobic cavity that can take up different ligands (Figure 36). So far, a number of different ligands have been identified for PR-10 proteins including flavonoids, cytokinins, steroids,

<figure><img src="image_117.png" /><figcaption>FIGURE 35 Cut sections of Der p 2 (left) and Bla g 1 (right), showing their core hydrophobic cavity. The large hydrophobic cavity of Bla g 1 can accommodate a variety of lipid molecules including fatty acids (palmitic, stearic, and oleic acids) and phospholipids (phosphatidylcholine, phosphatidylinositol, and phosphatidylserine).</figcaption></figure>

and derivates thereof (Table 9). This growing list of different ligands of PR-10 proteins indicates their different biological functions in the plant such as transport of small molecules, orchestrating germination and protection from environmental stress such as UV-radiation. For example, specific ligands such as the glycosylated flavonoid derivative quercetin-3-O-sophoride were identified for Bet v 1, resveratrol for Bet v 1 and Ara h 8, genistein for Ara h 8 and Bet v 1 and fatty acids for Bet v 1.469

In the case of Bet v 1, ligand binding did not result in an increased IgE-binding activity, although binding of phosphatidylcholine to Api g 1, Cor a 1, Mal d 1, and Pru p 1 induced conformational changes as shown by changed circular dichroism spectra. This provided protection from pepsinolysis to some degree, which resulted in basophil activation even with partly digested PR-10 proteins.33

<figure><img src="image_118.png" /><figcaption>FIGURE 36 Front face (A) and lateral face (B) of Bet v 1, the PR-10 of *Betula verrucosa*, showing the extent of the hydrophobic cavity (colored violet) harboring a single or several phenolic compounds through hydrophobic interactions (cartoon drawn with the ProteinsPlus server (https://proteins-plus/))</figcaption></figure>

<table><caption>TABLE 9 Structures of allergenic PR-10 Bet v 1-like proteins in complexes with natural compounds or their derivatives, available in the RCSB Protein Data Bank (PDB) (http://www.rcsb.org).</caption><thead><tr><th>PR-10<br>protein</th><th>compound</th><th>PDB<br>code</th></tr></thead><tbody><tr><td>Bet v 1</td><td>desoxycholate</td><td>4A83</td></tr><tr><td>Bet v 1</td><td>kinetin</td><td>4A85</td></tr><tr><td>Bet v 1</td><td>naringenin</td><td>4A87</td></tr><tr><td>Fra a 1</td><td>catechin</td><td>4C94</td></tr><tr><td>Vig r 6</td><td>gibberellic acid</td><td>4PSB,</td></tr><tr><td>Ara h 8</td><td>epicatechin</td><td>6AX0 4MA6, 6AWV,<br>6AWT</td></tr><tr><td>Ara h 8</td><td>caffeic acid</td><td>6AWU</td></tr><tr><td>Ara h 8</td><td>3-hydroxy-2-<br>naphthoic acid</td><td>6AWW</td></tr><tr><td>Ara h 8</td><td>quercetin</td><td>6AWS, 6B1D</td></tr></tbody></table>
However, these conformational changes seem to be more limited, as compared to those observed in nsLTP allergens. In fact, small molecule ligands enter the ligand-binding pocket of PR10 Bet v 1-like allergens but still remain far from the molecular surface area that contains the major B-cell epitope identified in Bet v 1 (Figure 36).<sup>475</sup> This B-cell epitope is well conserved in other closely related PR10 allergens from strawberry (Fra a 1 from *Fragaria ananassa*), and non-allergenic PR-10 proteins, like St. John's wort (*Hypericum perforatum*), and LIPR-10.2B from lupine yellow seed (*Lupinus luteus*), respectively (Figure 37).

Another *in vitro* study identified phytoprostanes E1, derived from alpha-linolenic acid and present in pollen, as a ligand of Bet v 1. This specific ligand interaction conferred increased stability of Bet v 1 against proteolytic degradation by inhibiting cathepsin protease activity, which is relevant for lysosomal degradation. This prolonged proteolytic processing causes low loading and reduced number of class II MHC-peptide formation in antigen presenting cells, a process that is supposed to favor a Th2 polarized immune response.<sup>476</sup>

### 3 – Serum albumins

Serum albumins are highly conserved large globular proteins of mammals and birds. They are abundant in blood, but they are also present in milk, saliva, dander and meat, representing clinically relevant respiratory and food allergens (Chapter C04).<sup>477</sup> Serum albumins transport a multitude of metabolites, nutrients, drugs, and other molecules. Their structure allows to adopt multiple conformations and simultaneous binding of various ligands.<sup>469</sup> Due to their highly conserved function, it is conceivable that animal serum albumins transport biologically active ligands that are also recognized by the human organism with an impact on the immune response. However, there is

no information to date available whether bound ligands have an effect on the allergic immune response, neither mediated by the ligands nor by potential ligand binding-induced conformational changes.

### 4 – Niemann-Pick protein type C2 (NPC2) family

More than 30 allergens have been identified in both American and European house dust mites (HDMs) species (Chapter B04). Out of those, group 2 mite allergens belong to the Niemann Pick protein type C2 (NPC2) family. NPC2 proteins are carriers of cholesterol<sup>478</sup> in vertebrates, but they are also found in arthropods. They contain a large internal hydrophobic cavity and are able to bind lipid ligands including LPS.<sup>469,479</sup> Der p 2 was shown to have functional homology to MD-2, the LPS-binding component of Toll-like receptor 4 (TLR4) complex.<sup>317</sup> LPS binding by Der p 2 resulted in enhanced signalling by TLR4 and is considered to result in a Th2 airway inflammation. Group 2 mite allergens were also identified from storage mites, e.g., Blo t 2, Gly d 2, and Lep d 2. These data highlight the role of TLR activation and their potential contribution to the allergic immune response.

### 5 – Nonspecific lipid transfer proteins (nsLTPs)

Although the interaction of nsLTPs with various hydrophobic ligands has been known for a long time, only recently the involvement of lipid ligands in the allergenicity of these proteins has been questioned and further clarified. However, due to the extreme diversity of either natural or foreign ligands susceptible to be accommodated more or less specifically by the large tunnel-like hydrophobic cavity occurring in the core structure of these proteins (Figure 38), the identification of relevant ligands is a challenging task.<sup>480</sup>

<figure><img src="image_120.png" /></figure>

**FIGURE 37** A-D. Cut sections (A-D) and complete three-dimensional structures shown with transparent surfaces (E-H) of Bet v 1 in complex with naringenin, PDB:4A87 (A, E), Fra a 1 in complex with catechin, PDB: 4C94 (B, F), LIPR-10.2B from yellow lupine (*Lupinus luteus*) with zeatin, PDB 2QIM (C, G), and PR-10 Bet v 1-like protein of St. John's wort (*Hypericum perforatum*) with melatonin, PDB 5IUF (D, H). Ligands are colored red and areas corresponding to the major B-cell epitope identified in Bet v 1, and the corresponding conserved regions of other proteins are colored green.
However, these conformational changes seem to be more limited, as compared to those observed in nsLTP allergens. In fact, small molecule ligands enter the ligand-binding pocket of PR10 Bet v 1-like allergens but still remain far from the molecular surface area that contains the major B-cell epitope identified in Bet v 1 (Figure 36).<sup>475</sup> This B-cell epitope is well conserved in other closely related PR10 allergens from strawberry (Fra a 1 from *Fragaria ananassa*), and non-allergenic PR-10 proteins, like St. John's wort (*Hypericum perforatum*), and LIPR-10.2B from lupine yellow seed (*Lupinus luteus*), respectively (Figure 37).

Another *in vitro* study identified phytoprostanes E1, derived from alpha-linolenic acid and present in pollen, as a ligand of Bet v 1. This specific ligand interaction conferred increased stability of Bet v 1 against proteolytic degradation by inhibiting cathepsin protease activity, which is relevant for lysosomal degradation. This prolonged proteolytic processing causes low loading and reduced number of class II MHC-peptide formation in antigen presenting cells, a process that is supposed to favor a Th2 polarized immune response.<sup>476</sup>

### 3 – Serum albumins

Serum albumins are highly conserved large globular proteins of mammals and birds. They are abundant in blood, but they are also present in milk, saliva, dander and meat, representing clinically relevant respiratory and food allergens (Chapter C04).<sup>477</sup> Serum albumins transport a multitude of metabolites, nutrients, drugs, and other molecules. Their structure allows to adopt multiple conformations and simultaneous binding of various ligands.<sup>469</sup> Due to their highly conserved function, it is conceivable that animal serum albumins transport biologically active ligands that are also recognized by the human organism with an impact on the immune response. However, there is

no information to date available whether bound ligands have an effect on the allergic immune response, neither mediated by the ligands nor by potential ligand binding-induced conformational changes.

### 4 – Niemann-Pick protein type C2 (NPC2) family

More than 30 allergens have been identified in both American and European house dust mites (HDMs) species (Chapter B04). Out of those, group 2 mite allergens belong to the Niemann Pick protein type C2 (NPC2) family. NPC2 proteins are carriers of cholesterol<sup>478</sup> in vertebrates, but they are also found in arthropods. They contain a large internal hydrophobic cavity and are able to bind lipid ligands including LPS.<sup>469,479</sup> Der p 2 was shown to have functional homology to MD-2, the LPS-binding component of Toll-like receptor 4 (TLR4) complex.<sup>317</sup> LPS binding by Der p 2 resulted in enhanced signalling by TLR4 and is considered to result in a Th2 airway inflammation. Group 2 mite allergens were also identified from storage mites, e.g., Blo t 2, Gly d 2, and Lep d 2. These data highlight the role of TLR activation and their potential contribution to the allergic immune response.

### 5 – Nonspecific lipid transfer proteins (nsLTPs)

Although the interaction of nsLTPs with various hydrophobic ligands has been known for a long time, only recently the involvement of lipid ligands in the allergenicity of these proteins has been questioned and further clarified. However, due to the extreme diversity of either natural or foreign ligands susceptible to be accommodated more or less specifically by the large tunnel-like hydrophobic cavity occurring in the core structure of these proteins (Figure 38), the identification of relevant ligands is a challenging task.<sup>480</sup>

<figure><img src="image_119.png" /></figure>

**FIGURE 37** A-D. Cut sections (A-D) and complete three-dimensional structures shown with transparent surfaces (E-H) of Bet v 1 in complex with naringenin, PDB:4A87 (A, E), Fra a 1 in complex with catechin, PDB: 4C94 (B, F), LIPR-10.2B from yellow lupine (*Lupinus luteus*) with zeatin, PDB 2QIM (C, G), and PR-10 Bet v 1-like protein of St. John's wort (*Hypericum perforatum*) with melatonin, PDB 5IUF (D, H). Ligands are colored red and areas corresponding to the major B-cell epitope identified in Bet v 1, and the corresponding conserved regions of other proteins are colored green.
<figure><img src="image_121.png" /><figcaption>**FIGURE 38** Cartoon showing the extent of the hydrophobic cavity (colored violet) of Pru p 3, the peach nsLTP (cartoon drawn with the Proteins Plus server (https://proteins-plus/)), and some apolar ligands for the nsLTP</figcaption></figure>

As PR-14 proteins, nsLTP are involved in the defense of plants against abiotic and biotic stress.⁴⁸¹ They transport apolar molecules, which are used as building blocks to elaborate and reinforce the cellular surfaces protecting the plant, e.g., from infection by phytopathogenic fungi or bacteria. They also participate in the biogenesis of cell membranes and some nsLTPs also display antimicrobial activity resulting from the permeabilization of the phytopathogen's cell membrane. Accordingly, a huge number of hydrophobic ligands can be accommodated by nsLTPs, including fatty acids, phospholipids, prostaglandins, and jasmonic acid (a plant hormone).

Pru p 3, the nsLTP from peach (Prunus persica), offers a nice example of a surface molecule located in the fuzz covering the fruit, which is continuously exposed to environmental factors, and is involved in the transport of lipid ligands such as oleic acid.⁴⁸²

The interaction of Pru p 3 with oleic acid was reported to enhance the IgE-binding capacity of the nsLTP.⁴⁸² As a possible explanation, a specific conformational change of the extended C-terminal loop of Pru p 3 resulting from the contact with the tail of the inserted oleic acid was determined. This has been suspected to modify the topographical distribution of amino acid residues from the discontinuous epitope #3, which coincides in part with the C-terminal loop, explaining the increased antibody-binding activity (Figure 39)¹⁶⁵⁵. By contrast, binding to oleic acid did not induce any conformational change in epitopes #1 and #2, which occur in more rigid alpha helical segments of Pru p 3.⁴⁸³

The replacement of oleic acid by stearic acid, a saturated C18 fatty acid exhibiting a trans-conformation different from the cis-conformation of oleic acid, failed to induce enhancement of IgE binding, thus indicating that a specific spatial localization of the fatty acid within the hydrophobic cavity is a prerequisite for the Pru p 3-ligand complex to induce a conformational change. A similar conformational change has been invoked to account for the enhanced IgE binding of Jug r 3, the nsLTP from walnut (Juglans regia), observed upon binding of oleic acid to the hydrophobic cavity.¹⁶⁵⁶

In addition to these direct effects on both, the stability and allergenicity of nsLTPs, the indirect effects of lipid ligands on the mechanism and regulation of the allergic response have been deeply investigated, using nsLTP, e.g., Pru p 3, and their natural ligands, e.g., the alkaloid camptothecin associated to phytosphingosine, as models.⁴⁸³⁻⁴⁸⁵ As a result, phytosphingosine was identified as a foreign ligand susceptible to contribute to the activation and regulation of

<figure><img src="image_122.png" /><figcaption>**FIGURE 39** A. Cut section of Pru p 3 in complex with oleic acid (OLA) inserted in the hydrophobic cavity. B. Cartoon showing how oleic acid inserts into the hydrophobic cavity of Pru p 3 in the vicinity of the long C-terminal loop (colored red) of the nsLTP. C. Surface occupied by the C-terminal loop (colored red) of Pru p 3 in contact with OLA. D. Surface occupied by the continuous IgE-binding epitope 3 (colored red) of Pru p 3. The yellow line delineates the region of epitope 3 impacted by binding of oleic acid to the hydrophobic cavity of Pru p 3.</figcaption></figure>

the allergenic response via signaling pathways common to innate immunity and allergic responses (see below).

## 6 – Other allergens families

For Der p 5, a member of the group 5 mite allergen family, lipid binding has been shown to activate TLR2 signalling in airway epithelial cells.