<figure><img src="image_246.png" /><figcaption>TABLE 61 Vespid species in the USA and Europe</figcaption></figure>

<table><thead><tr><th>Genus</th><th>Species</th><th>Popular name</th><th>Species</th><th>Popular name</th></tr></thead><tbody><tr><td rowspan="3">Polistes</td><td>Europe</td><td>Europe</td><td>USA</td><td>USA</td></tr><tr><td>Gallicus</td><td rowspan="2">Wasp</td><td>Annularis</td><td rowspan="2">Paper wasp</td></tr><tr><td>Dominula</td><td>Fuscatus</td></tr><tr><td rowspan="3">Vespula</td><td>Vulgaris</td><td rowspan="3">Wasp</td><td>Exclamans</td><td rowspan="3">Yellow jacket</td></tr><tr><td>Germanica</td><td>Vulgaris</td></tr><tr><td>Rufa</td><td>Germanica</td></tr><tr><td rowspan="2">Dolichovespula</td><td>Media</td><td rowspan="2">Wasp</td><td>Maculifrons</td><td rowspan="2">Hornet</td></tr><tr><td>Saxonica</td><td>Maculata</td></tr><tr><td rowspan="3">Vespa</td><td>Crabro</td><td rowspan="3">European Hornet</td><td>Arenaria</td><td rowspan="3">Hornet</td></tr><tr><td>Orientalis</td><td>Crabro</td></tr><tr><td>Velutina nigrithorax</td><td></td></tr></tbody></table>

extensive cross-reactivity.²¹⁴ Additionally, DPPIV allergens of vespid venoms exhibit high cross-reactivity with HBV DPP IV (Api m 5) (Figure 136) and are therefore of no diagnostic value to discriminate between genuine VV and HBV sensitization. The same holds true for the 200 kDa vitellogenins Api m 12 and Ves v 6, that were described as novel pair of highly cross-reactive pan-allergens of HBV and VV¹²⁹³. Other less studied allergens such as serine protease (Pol d 4) of PDV might represent additional marker allergens,¹²⁹⁴ but clinical data supporting this claim are still missing. Moreover, a recent study elucidated the venomes of *P. dominula* and *Vespula spp.* (*V. germanica*, *V. vulgaris*) and identified new allergen candidates such as icarapin-like protein and phospholipase A2.¹²⁹⁵

### 3 – Sensitization to individual molecules and their clinical relevance

Reliable data on sensitization rate are available for many vespid allergens with different values (Table 63). In fact, these sensitization rates depend on many factors, like the test used for IgE detection, the inclusion criteria of the assessed patient population, an unambiguous identification of the allergy-eliciting insect by the patient as well as geographical differences. Moreover, differences can be observed in mono-sensitized (MS) and double-sensitized (DS) patients, as sensitization rates to individual HBV and YJV allergens are

lower in patients MS to the respective venoms compared with HBV/YJV-DS patients.⁸⁷

IgE sensitization to YJV Ves v 1 ranges between 39% and 66% in different populations of YJV-allergic patients,⁸⁷,¹²⁸³,¹²⁹⁶⁻¹²⁹⁸ and is higher in YJV/HBV-DS compared with YJV-MS patients.⁸⁷

Sensitization to Ves v 5 has been found in 82% to 98% of patients with a history of YJV allergy.⁸⁷,¹²¹³,¹²⁹⁶⁻¹³⁰²,¹³⁰⁴ Sensitization to Ves v 2 was reported only in 5-25% of YJV allergic patients and mostly directed against the cross-reactive carbohydrate determinants (CCD). The IgE protein reactivity was mostly directed against the enzymatically inactive isoform Ves v 2.0201.¹³⁰³ Sensitization to Ves v 3 and Ves v 6 is less investigated but was found in 57% and 39% of YJV allergic patients, respectively.¹²⁹³

Concerning diagnostic sensitivity, the addition of Ves v 1 to Ves v 5 increased sensitivity of CRD of YJV allergy in the range of 4% to 11% depending on the study populations.¹²⁹⁶⁻¹³⁰⁰, ¹³⁰²,¹³⁰⁴ Since both rVes v 5 and rVes v 1 are commercially available for diagnostic purposes, in YJV allergy CRD is valuable and can be used to exclude unspecific sensitization due to cross-reactive carbohydrate determinants (CCDs).

Nevertheless, rVes v 1 and 5 failed to diagnose 2-8% of subjects with established allergy¹³⁰⁰, thus probably indicating the need to add more allergens.

The sensitization rate to Pol d 1 was found to be 87% in a Spanish population of PDV/YJV-DS patients.¹²⁸⁹ More recently, Pol d 1 has
<figure><img src="image_252.png" /><figcaption>TABLE 61 Vespid species in the USA and Europe</figcaption></figure>

<table><thead><tr><th>Genus</th><th>Species</th><th>Popular name</th><th>Species</th><th>Popular name</th></tr></thead><tbody><tr><td rowspan="3">Polistes</td><td>Europe</td><td>Europe</td><td>USA</td><td>USA</td></tr><tr><td>Gallicus</td><td rowspan="2">Wasp</td><td>Annularis</td><td rowspan="2">Paper wasp</td></tr><tr><td>Dominula</td><td>Fuscatus</td></tr><tr><td rowspan="3">Vespula</td><td>Vulgaris</td><td rowspan="3">Wasp</td><td>Exclamans</td><td rowspan="3">Yellow jacket</td></tr><tr><td>Germanica</td><td>Vulgaris</td></tr><tr><td>Rufa</td><td>Germanica</td></tr><tr><td rowspan="2">Dolichovespula</td><td>Media</td><td rowspan="2">Wasp</td><td>Maculifrons</td><td rowspan="2">Hornet</td></tr><tr><td>Saxonica</td><td>Maculata</td></tr><tr><td rowspan="3">Vespa</td><td>Crabro</td><td rowspan="3">European Hornet</td><td>Arenaria</td><td rowspan="3">Hornet</td></tr><tr><td>Orientalis</td><td>Crabro</td></tr><tr><td>Velutina nigrithorax</td><td></td></tr></tbody></table>

extensive cross-reactivity.²¹⁴ Additionally, DPPIV allergens of vespid venoms exhibit high cross-reactivity with HBV DPP IV (Api m 5) (Figure 136) and are therefore of no diagnostic value to discriminate between genuine VV and HBV sensitization. The same holds true for the 200 kDa vitellogenins Api m 12 and Ves v 6, that were described as novel pair of highly cross-reactive pan-allergens of HBV and VV¹²⁹³. Other less studied allergens such as serine protease (Pol d 4) of PDV might represent additional marker allergens,¹²⁹⁴ but clinical data supporting this claim are still missing. Moreover, a recent study elucidated the venomes of *P. dominula* and *Vespula spp.* (V. germanica, V. vulgaris) and identified new allergen candidates such as icarapin-like protein and phospholipase A2.¹²⁹⁵

## 3 – Sensitization to individual molecules and their clinical relevance

Reliable data on sensitization rate are available for many vespid allergens with different values (Table 63). In fact, these sensitization rates depend on many factors, like the test used for IgE detection, the inclusion criteria of the assessed patient population, an unambiguous identification of the allergy-eliciting insect by the patient as well as geographical differences. Moreover, differences can be observed in mono-sensitized (MS) and double-sensitized (DS) patients, as sensitization rates to individual HBV and YJV allergens are

lower in patients MS to the respective venoms compared with HBV/ YJV-DS patients.⁸⁷

IgE sensitization to YJV Ves v 1 ranges between 39% and 66% in different populations of YJV-allergic patients,⁸⁷,¹²⁸³,¹²⁹⁶⁻¹²⁹⁸ and is higher in YJV/HBV-DS compared with YJV-MS patients.⁸⁷

Sensitization to Ves v 5 has been found in 82% to 98% of patients with a history of YJV allergy.⁸⁷,¹²¹³,¹²⁹⁶⁻¹³⁰²,¹³⁰⁴ Sensitization to Ves v 2 was reported only in 5-25% of YJV allergic patients and mostly directed against the cross-reactive carbohydrate determinants (CCD). The IgE protein reactivity was mostly directed against the enzymatically inactive isoform Ves v 2.0201.¹³⁰³ Sensitization to Ves v 3 and Ves v 6 is less investigated but was found in 57% and 39% of YJV allergic patients, respectively.¹²⁹³

Concerning diagnostic sensitivity, the addition of Ves v 1 to Ves v 5 increased sensitivity of CRD of YJV allergy in the range of 4% to 11% depending on the study populations.¹²⁹⁶⁻¹³⁰⁰, ¹³⁰²,¹³⁰⁴ Since both rVes v 5 and rVes v 1 are commercially available for diagnostic purposes, in YJV allergy CRD is valuable and can be used to exclude unspecific sensitization due to cross-reactive carbohydrate determinants (CCDs).

Nevertheless, rVes v 1 and 5 failed to diagnose 2-8% of subjects with established allergy¹³⁰⁰, thus probably indicating the need to add more allergens.

The sensitization rate to Pol d 1 was found to be 87% in a Spanish population of PDV/YJV-DS patients.¹²⁸⁹ More recently, Pol d 1 has
<table><caption>TABLE 62 Overview of the Vespoidea venom allergens, which are presently listed in the WHO/IUIS allergen nomenclature official database</caption><thead><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Molecular weight (kDa)</th></tr></thead><tbody><tr><td colspan="3">American paper wasps (Polistes annularis, P. exclamans, P. fuscatus, P. metricus)</td></tr><tr><td>Pol a 1, Pol e 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Pol a 2</td><td>Hyaluronidase</td><td>38</td></tr><tr><td>Pol e 4</td><td>Serine protease</td><td>33</td></tr><tr><td>Pol a 5, Pol e 5, Pol f 5, Pol m 5</td><td>Antigen 5</td><td>23</td></tr><tr><td colspan="3">Asian needle ant (Pachycondyla chinensis)</td></tr><tr><td>Pac c 3</td><td>Antigen 5</td><td>23</td></tr><tr><td colspan="3">Australian jumper ant (Myrmecia pilosula)</td></tr><tr><td>Myr p 1</td><td>Pilosulin-1</td><td>7.5/5.5</td></tr><tr><td>Myr p 2</td><td>Pilosulin-3</td><td>8.5/2-4</td></tr><tr><td>Myr p 3</td><td>Pilosulin-4.1</td><td>8.2</td></tr><tr><td colspan="3">European paper wasps (Polistes dominula, P. gallicus)</td></tr><tr><td>Pol d 1, Pol g 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Pol d 2</td><td>Hyaluronidase</td><td>50</td></tr><tr><td>Pol d 3</td><td>Dipeptidyl peptidase IV</td><td>100</td></tr><tr><td>Pol d 4</td><td>Serine protease</td><td>33</td></tr><tr><td>Pol d 5, Pol g 5</td><td>Antigen 5</td><td>23/24</td></tr><tr><td colspan="3">Fire ants (Solenopsis invicta, S. geminata, S. richteri, S. saevissima)</td></tr><tr><td>Sol i 1</td><td>Phospholipase A1</td><td>18</td></tr><tr><td>Sol i 2, Sol g 2, Sol r 2, Sol s 2</td><td>Unknown</td><td>14/13/13/13</td></tr><tr><td>Sol i 3, Sol r 3</td><td>Antigen 5</td><td>26/24</td></tr><tr><td>Sol g 3, Sol s 3</td><td>Unknown</td><td>24</td></tr><tr><td>Sol i 4, Sol g 4</td><td>Unknown</td><td>12</td></tr><tr><td colspan="3">Hornets (Vespa crabro, V. magnifica, V. mandarinia, V. velutina)</td></tr><tr><td>Vesp c 1, Vesp m 1, Vesp v 1</td><td>Phospholipase A1</td><td>34/34/36</td></tr><tr><td>Vesp ma 2</td><td>Hyaluronidase</td><td>35</td></tr><tr><td>Vesp c 5, Vesp ma 5; Vesp m 5, Vesp v 5</td><td>Antigen 5</td><td>23/25/23/23</td></tr><tr><td colspan="3">Polybia wasps (Polybia paulista, P. scutellaris)</td></tr><tr><td>Poly p 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Poly p 2</td><td>Hyaluronidase</td><td>33</td></tr><tr><td>Poly p 5, Poly s 5</td><td>Antigen 5</td><td>23/21</td></tr><tr><td colspan="3">Yellow hornet, White-faced hornet (Dolichovespula arenaria, D. maculata)</td></tr><tr><td>Dol m 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Dol m 2</td><td>Hyaluronidase</td><td>42</td></tr><tr><td>Dol a 5, Dol m 5</td><td>Antigen 5</td><td>23</td></tr><tr><td colspan="3">Yellow jackets (Vespula vulgaris, V. flavopilosa, V. germanica, V. maculifrons, V. pensylvanica, V. squamosa, V. vidua)</td></tr><tr><td>Ves v 1, Ves m 1, Ves s 1</td><td>Phospholipase</td><td>34</td></tr><tr><td>Ves v 2.0101, Ves m 2</td><td>A1 Hyaluronidase</td><td>45/46</td></tr><tr><td>Ves v 2.0201</td><td>Hyaluronidase (inactive)</td><td>45</td></tr><tr><td>Ves v 3</td><td>Dipeptidyl peptidase IV</td><td>23</td></tr><tr><td>Ves v 5, Ves f 5, Ves g 5, Ves m 5, Ves p 5, Ves s 5, Ves vi 5</td><td>Antigen 5</td><td>200</td></tr><tr><td>Ves v 6</td><td>Vitellogenin</td><td></td></tr></tbody></table>

been demonstrated as the most frequent Polistes allergen in Italian allergic patients. In fact, Pol d 1 sensitization was present in 97% (DS) to 100% (MS) of 128 PDV-positive patients. Moreover, it was frequently involved in case of positivity to a single PDV allergen

(48% in DS and 80% in MS patients), and it was positive in 95% of Pol d 5-negative subjects.¹³⁰⁵

Sensitization to Pol d 5 was found in 69-72% of Spanish PDV/YJV-DS patients, while it was observed in 53% and 20% in the DS
<table><caption>TABLE 62 Overview of the Vespoidea venom allergens, which are presently listed in the WHO/IUIS allergen nomenclature official database</caption><thead><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Molecular weight (kDa)</th></tr></thead><tbody><tr><td colspan="3">American paper wasps (Polistes annularis, P. exclamans, P. fuscatus, P. metricus)</td></tr><tr><td>Pol a 1, Pol e 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Pol a 2</td><td>Hyaluronidase</td><td>38</td></tr><tr><td>Pol e 4</td><td>Serine protease</td><td>33</td></tr><tr><td>Pol a 5, Pol e 5, Pol f 5, Pol m 5</td><td>Antigen 5</td><td>23</td></tr><tr><td colspan="3">Asian needle ant (Pachycondyla chinensis)</td></tr><tr><td>Pac c 3</td><td>Antigen 5</td><td>23</td></tr><tr><td colspan="3">Australian jumper ant (Myrmecia pilosula)</td></tr><tr><td>Myr p 1</td><td>Pilosulin-1</td><td>7.5/5.5</td></tr><tr><td>Myr p 2</td><td>Pilosulin-3</td><td>8.5/2-4</td></tr><tr><td>Myr p 3</td><td>Pilosulin-4.1</td><td>8.2</td></tr><tr><td colspan="3">European paper wasps (Polistes dominula, P. gallicus)</td></tr><tr><td>Pol d 1, Pol g 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Pol d 2</td><td>Hyaluronidase</td><td>50</td></tr><tr><td>Pol d 3</td><td>Dipeptidyl peptidase IV</td><td>100</td></tr><tr><td>Pol d 4</td><td>Serine protease</td><td>33</td></tr><tr><td>Pol d 5, Pol g 5</td><td>Antigen 5</td><td>23/24</td></tr><tr><td colspan="3">Fire ants (Solenopsis invicta, S. geminata, S. richteri, S. saevissima)</td></tr><tr><td>Sol i 1</td><td>Phospholipase A1</td><td>18</td></tr><tr><td>Sol i 2, Sol g 2, Sol r 2, Sol s 2</td><td>Unknown</td><td>14/13/13/13</td></tr><tr><td>Sol i 3, Sol r 3</td><td>Antigen 5</td><td>26/24</td></tr><tr><td>Sol g 3, Sol s 3</td><td>Unknown</td><td>24</td></tr><tr><td>Sol i 4, Sol g 4</td><td>Unknown</td><td>12</td></tr><tr><td colspan="3">Hornets (Vespa crabro, V. magnifica, V. mandarinia, V. velutina)</td></tr><tr><td>Vesp c 1, Vesp m 1, Vesp v 1</td><td>Phospholipase A1</td><td>34/34/36</td></tr><tr><td>Vesp ma 2</td><td>Hyaluronidase</td><td>35</td></tr><tr><td>Vesp c 5, Vesp ma 5; Vesp m 5, Vesp v 5</td><td>Antigen 5</td><td>23/25/23/23</td></tr><tr><td colspan="3">Polybia wasps (Polybia paulista, P. scutellaris)</td></tr><tr><td>Poly p 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Poly p 2</td><td>Hyaluronidase</td><td>33</td></tr><tr><td>Poly p 5, Poly s 5</td><td>Antigen 5</td><td>23/21</td></tr><tr><td colspan="3">Yellow hornet, White-faced hornet (Dolichovespula arenaria, D. maculata)</td></tr><tr><td>Dol m 1</td><td>Phospholipase A1</td><td>34</td></tr><tr><td>Dol m 2</td><td>Hyaluronidase</td><td>42</td></tr><tr><td>Dol a 5, Dol m 5</td><td>Antigen 5</td><td>23</td></tr><tr><td colspan="3">Yellow jackets (Vespula vulgaris, V. flavopilosa, V. germanica, V. maculifrons, V. pensylvanica, V. squamosa, V. vidua)</td></tr><tr><td>Ves v 1, Ves m 1, Ves s 1</td><td>Phospholipase</td><td>34</td></tr><tr><td>Ves v 2.0101, Ves m 2</td><td>A1 Hyaluronidase</td><td>45/46</td></tr><tr><td>Ves v 2.0201</td><td>Hyaluronidase (inactive)</td><td>45</td></tr><tr><td>Ves v 3</td><td>Dipeptidyl peptidase IV</td><td>23</td></tr><tr><td>Ves v 5, Ves f 5, Ves g 5, Ves m 5, Ves p 5, Ves s 5, Ves vi 5</td><td>Antigen 5</td><td>200</td></tr><tr><td>Ves v 6</td><td>Vitellogenin</td><td></td></tr></tbody></table>

been demonstrated as the most frequent Polistes allergen in Italian allergic patients. In fact, Pol d 1 sensitization was present in 97% (DS) to 100% (MS) of 128 PDV-positive patients. Moreover, it was frequently involved in case of positivity to a single PDV allergen

(48% in DS and 80% in MS patients), and it was positive in 95% of Pol d 5-negative subjects.¹³⁰⁵

Sensitization to Pol d 5 was found in 69-72% of Spanish PDV/YJV-DS patients, while it was observed in 53% and 20% in the DS
<figure><img src="image_247.png" /><figcaption>**FIGURE 135** Structures of selected vespid venom allergens. Structures were generated by either X-ray diffraction or structural modelling. α-helices, β-strands and coiled regions are shown in red, blue, and grey, respectively.</figcaption></figure>

and MS Italian group, respectively.<sup>1305</sup> Less is known about slgE sensitization to Pol d 2.

Preliminary unpublished data suggest a sensitization rate of approximately 25% in PDV-allergic patients.<sup>1306</sup> Primary sensitization to Pol d 2 may induce cross-reactivity with Api m 2 and Ves v 2.0201. However, only very few Api m 2-reactive patients show slgE to Pol d 2.

Sensitization to Pol d 3 is less investigated but was found in 66% of PDV-allergic patients, respectively.<sup>214</sup> In the South of Europe double sensitization to either Vespula or Polistes species is more frequent than that of Vespula and honeybee.<sup>1307,1308</sup> Although Polistes venom is devoid of CCDs,<sup>1309</sup> a definite discrimination may be difficult due to the high degree of cross-reactivity between the major

allergens of these venoms and to the absence of marker allergens available<sup>213</sup> (Figure 137).

Finally, no data are available on the correlation between certain molecular sensitization profiles to vespid allergens and severity of the sting reaction, increased risk of VIT failure (during or after its discontinuation) or of side effects.

## 4 - Clinical management

### Clinical diagnosis

The goal of vespid allergy diagnostics is to classify the type of reaction, to confirm an IgE-mediated pathogenesis, and to identify
<figure><img src="image_253.png" /><figcaption>**FIGURE 135** Structures of selected vespid venom allergens. Structures were generated by either X-ray diffraction or structural modelling. α-helices, β-strands and coiled regions are shown in red, blue, and grey, respectively.</figcaption></figure>

and MS Italian group, respectively.<sup>1305</sup> Less is known about slgE sensitization to Pol d 2.

Preliminary unpublished data suggest a sensitization rate of approximately 25% in PDV-allergic patients.<sup>1306</sup> Primary sensitization to Pol d 2 may induce cross-reactivity with Api m 2 and Ves v 2.0201. However, only very few Api m 2-reactive patients show slgE to Pol d 2.

Sensitization to Pol d 3 is less investigated but was found in 66% of PDV-allergic patients, respectively.<sup>214</sup> In the South of Europe double sensitization to either Vespula or Polistes species is more frequent than that of Vespula and honeybee.<sup>1307,1308</sup> Although Polistes venom is devoid of CCDs,<sup>1309</sup> a definite discrimination may be difficult due to the high degree of cross-reactivity between the major

allergens of these venoms and to the absence of marker allergens available<sup>213</sup> (Figure 137).

Finally, no data are available on the correlation between certain molecular sensitization profiles to vespid allergens and severity of the sting reaction, increased risk of VIT failure (during or after its discontinuation) or of side effects.

## 4 - Clinical management

### Clinical diagnosis

The goal of vespid allergy diagnostics is to classify the type of reaction, to confirm an IgE-mediated pathogenesis, and to identify
<figure><img src="image_248.png" /><figcaption>FIGURE 136 Cross-reactivity of vespid allergens and their homologues from honeybee venom. Potentially cross-reactive and marker allergens for the discrimination between primary vespid venom and honeybee venom allergy are shown in black and green, respectively. Of note, vespid phospholipases A1 (Ves v 1 and Pol d 1) and antigens 5 (Ves v 5 and Pol d 5) can serve as marker allergens to discriminate between vespid and honeybee venom allergy but are highly cross-reactive among each other. Black solid arrows: highly cross-reactive; black dotted arrows: limited cross-reactivity; grey arrows: cross-reactive, but detailed studies on its degree are missing.</figcaption></figure>

<table><caption>TABLE 63 Sensitisation rates to individual yellow jacket venom allergens and Polistes dominula/annularis venom allergens</caption><thead><tr><td>Allergenic molecule</td><td>Biochemical name</td><td>Sensitisation rate (%)</td><td>Molecular weight (kDa)</td></tr></thead><tbody><tr><td>Ves v 1</td><td>Phospholipase A1B</td><td>39-66 <sup>87, 1283, 1296-1298</sup></td><td>34</td></tr><tr><td>Ves v 2</td><td>Hyaluronidase</td><td>5-25 <sup>1303</sup></td><td>45</td></tr><tr><td>Ves v 3</td><td>Dipeptidyl peptidase IV</td><td>57 <sup>1293</sup></td><td>100</td></tr><tr><td>Ves v 5</td><td>Antigen 5</td><td>82-98 <sup>87, 213, 1296-1302, 1304</sup></td><td>23</td></tr><tr><td>Ves v 6</td><td>Vitellogenin</td><td>39 <sup>1293</sup></td><td>200</td></tr><tr><td>Pol d 1</td><td>Phospholipase A1</td><td>87 (nPol d 1) <sup>1289</sup> 97-100 (rPol d 1) <sup>1305</sup></td><td>34</td></tr><tr><td>Pol d 3</td><td>Dipeptidyl peptidase IV</td><td>66 (rPol d 3) <sup>214</sup></td><td>33</td></tr><tr><td>Pol d 5</td><td>Antigen 5</td><td>69-72 (nPol d 1) <sup>1289</sup> 20-53 (rPol d 1) <sup>1305</sup></td><td>23</td></tr><tr><td>Pol a 5</td><td>Antigen 5</td><td>44 (r Pol a 5) <sup>1289</sup></td><td></td></tr></tbody></table>

the culprit insect. Currently it is based on clinical history, skin tests and measurement of sIgE antibodies to vespid venom.¹²⁷⁵

As venom sensitization is found in up to 40% of history-negative persons, only those with a history of a previous systemic sting reaction (SR) are in general eligible for diagnostic testing.¹²⁷⁵

### Patient history:

The patient is asked to describe his/her symptoms and the course of the sting reaction, number of stings, clues as to the type of insect involved and individual risk factors for anaphylaxis.¹²⁸⁶ It may be useful to show the patient an entomological notice board to facilitate the identification of the stinging insect.

Vespinae and Polistinae subfamilies are very similar, with differences at the junction of the thorax and abdomen. Vespinae have a truncated junction while Polistinae are more oval in shape. Vespidae are almost hairless and have black and yellow striped abdomens.¹²⁸⁶

In patients with a history of severe SR dermatological evaluation is recommended to rule out a possible diagnosis of cutaneous mastocytosis. In patients with an unclear or suggestive psychosomatic

reaction, scrutiny of emergency room and ambulance records is recommended.¹²⁸⁶

### Skin tests:

The sensitivity of skin prick test (SPT) alone is estimated around 64%, while a combination of SPT and intradermal testing (IT) reaches a 94% sensitivity,¹²⁸⁶,¹²⁹⁸ hence it is recommended to perform both tests sequentially.¹²⁷⁵,¹³¹⁰

In case of negative skin tests but presence of a suggestive history of SR, cutaneous tests should be repeated after 1-2 months, along with serologic testing.

Standardized Hymenoptera venom products, including YJ and Polistes wasp venoms, are commercially available in many countries, being mixtures of the clinically relevant species for YJ (Vespula vulgaris, V. flavopilosa, V. germanica, V. maculifrons, V. pensylvanica, V. squamosa) and American Polistes (Polistes annularis, P. exclamans, P. fuscatus, P. metricus) venom extracts.

Contrary to the USA, dialyzed bee and yellow jacket venoms are used for diagnosis and therapy in some European countries, allowing different diagnostic accuracy with respect to the use of un-dialyzed
<figure><img src="image_254.png" /><figcaption>FIGURE 136 Cross-reactivity of vespid allergens and their homologues from honeybee venom. Potentially cross-reactive and marker allergens for the discrimination between primary vespid venom and honeybee venom allergy are shown in black and green, respectively. Of note, vespid phospholipases A1 (Ves v 1 and Pol d 1) and antigens 5 (Ves v 5 and Pol d 5) can serve as marker allergens to discriminate between vespid and honeybee venom allergy but are highly cross-reactive among each other. Black solid arrows: highly cross-reactive; black dotted arrows: limited cross-reactivity; grey arrows: cross-reactive, but detailed studies on its degree are missing.</figcaption></figure>

<table><caption>TABLE 63 Sensitisation rates to individual yellow jacket venom allergens and Polistes dominula/annularis venom allergens</caption><thead><tr><td>Allergenic molecule</td><td>Biochemical name</td><td>Sensitisation rate (%)</td><td>Molecular weight (kDa)</td></tr></thead><tbody><tr><td>Ves v 1</td><td>Phospholipase A1B</td><td>39-66 <sup>87, 1283, 1296-1298</sup></td><td>34</td></tr><tr><td>Ves v 2</td><td>Hyaluronidase</td><td>5-25 <sup>1303</sup></td><td>45</td></tr><tr><td>Ves v 3</td><td>Dipeptidyl peptidase IV</td><td>57 <sup>1293</sup></td><td>100</td></tr><tr><td>Ves v 5</td><td>Antigen 5</td><td>82-98 <sup>87, 213, 1296-1302, 1304</sup></td><td>23</td></tr><tr><td>Ves v 6</td><td>Vitellogenin</td><td>39 <sup>1293</sup></td><td>200</td></tr><tr><td>Pol d 1</td><td>Phospholipase A1</td><td>87 (nPol d 1) <sup>1289</sup> 97-100 (rPol d 1) <sup>1305</sup></td><td>34</td></tr><tr><td>Pol d 3</td><td>Dipeptidyl peptidase IV</td><td>66 (rPol d 3) <sup>214</sup></td><td>33</td></tr><tr><td>Pol d 5</td><td>Antigen 5</td><td>69-72 (nPol d 1) <sup>1289</sup> 20-53 (rPol d 1) <sup>1305</sup></td><td>23</td></tr><tr><td>Pol a 5</td><td>Antigen 5</td><td>44 (r Pol a 5) <sup>1289</sup></td><td></td></tr></tbody></table>

the culprit insect. Currently it is based on clinical history, skin tests and measurement of sIgE antibodies to vespid venom.¹²⁷⁵

As venom sensitization is found in up to 40% of history-negative persons, only those with a history of a previous systemic sting reaction (SR) are in general eligible for diagnostic testing.¹²⁷⁵

### Patient history:

The patient is asked to describe his/her symptoms and the course of the sting reaction, number of stings, clues as to the type of insect involved and individual risk factors for anaphylaxis.¹²⁸⁶ It may be useful to show the patient an entomological notice board to facilitate the identification of the stinging insect.

Vespinae and Polistinae subfamilies are very similar, with differences at the junction of the thorax and abdomen. Vespinae have a truncated junction while Polistinae are more oval in shape. Vespidae are almost hairless and have black and yellow striped abdomens.¹²⁸⁶

In patients with a history of severe SR dermatological evaluation is recommended to rule out a possible diagnosis of cutaneous mastocytosis. In patients with an unclear or suggestive psychosomatic

reaction, scrutiny of emergency room and ambulance records is recommended.¹²⁸⁶

### Skin tests:

The sensitivity of skin prick test (SPT) alone is estimated around 64%, while a combination of SPT and intradermal testing (IT) reaches a 94% sensitivity,¹²⁸⁶,¹²⁹⁸ hence it is recommended to perform both tests sequentially.¹²⁷⁵,¹³¹⁰

In case of negative skin tests but presence of a suggestive history of SR, cutaneous tests should be repeated after 1-2 months, along with serologic testing.

Standardized Hymenoptera venom products, including YJ and Polistes wasp venoms, are commercially available in many countries, being mixtures of the clinically relevant species for YJ (Vespula vulgaris, V. flavopilosa, V. germanica, V. maculifrons, V. pensylvanica, V. squamosa) and American Polistes (Polistes annularis, P. exclamans, P. fuscatus, P. metricus) venom extracts.

Contrary to the USA, dialyzed bee and yellow jacket venoms are used for diagnosis and therapy in some European countries, allowing different diagnostic accuracy with respect to the use of un-dialyzed
<figure><img src="image_249.png" /><figcaption>FIGURE 137 Diagnostic algorithm for component-resolved diagnostics of yellow jacket venom (YJV) and European paper wasp venom (PDV) allergy. A red minus indicates a negative and a green plus a positive test results. 1 Pol d 1 is currently only available for a selected multiplex slgE platform. Despite the potential of component-resolved diagnostics, clinical history, skin tests and the measurement of venoms IgE and serum tryptase build an indispensable basis for accurate diagnosis in Hymenoptera venom allergy. Moreover, cellular tests such as basophil activation test (BAT) and CAP inhibition assays may be helpful diagnostic tools in dissecting primary sensitization.</figcaption></figure>

extracts.<sup>1311</sup> In dialyzed venom, low molecular weight components (like histamine and components with histamine-releasing activity) smaller than 1000-3000 Dalton are removed. In view of lower cross-reactivity between venoms of the European and American species of Polistes<sup>1312</sup> commercial preparations of European Polistes dominula venom are now available.<sup>1286</sup> Even though a high cross-reactivity between Vespula species venom and V crabro has been confirmed, a commercial extract of Vespa crabro is also available in some countries.<sup>1313</sup>

No recombinant venom allergens are commercially available for skin testing.

### IgE testing:

**Total IgE** - Total IgE (tlgE) determination may be useful for appropriate interpretation of allergen-specific IgE, especially in the case of very low level of slgE (see also the chapter about bee venom allergy).

### Specific IgE to venom extracts and individual venom allergens:

Venom-slgE can be detected immediately after the sting, but the optimal time point will be 1-4 weeks later.<sup>1275</sup> The sensitivity of conventional YJV slgE assay using the whole extract ranges between 83.4% and 91%, the newly developed YJV solid-phase assay complemented with rVes v 5 having a higher sensitivity than the traditional YJV test.<sup>1296,1298</sup>

There is no correlation between the severity of sting reactions and the concentration of venom slgE to whole venom extracts<sup>1275,1310</sup> as some patients with minimal or absent venom-slgE antibodies can develop severe anaphylaxis.<sup>1310</sup> According to a recent study, the severity of sting reactions was not associated with results obtained by skin testing, venom-sslgE levels or even molecular slgE testing.<sup>1314</sup>

Of note, negative slgE and negative skin tests have been reported in the past in up to 15% of patients with systemic mastocytosis and history of a systemic reaction to insect stings,<sup>1315</sup> thus, restricting them from VIT. With the introduction of new methods including CRD and parameters of evaluation in the diagnostic work-up, this diagnostic gap has been solved and slgE can be detected in the vast majority of these patients.<sup>1297,1316</sup> According to some studies, slgE levels between 0.1 and 0.35 kUA/l should be considered relevant in patients with a clear clinical history and low levels of tlgE, irrespective of the presence of mast cell diseases.<sup>1297,1316</sup>

A double *in vitro* positivity to YJV and Polistes venom is common in Mediterranean countries, much more frequent than to YJV/PDV and HBV<sup>1307,1308</sup>. While CRD is able to adequately distinguish allergies to HBV and vespid venom (particularly YJV) (for more detailed information please refer to the bee venom chapter), this is not the case when a differentiation between allergies to various vespid venoms is required (Figure 135).

A previous study demonstrated that the measurement of relative levels of slgE to the phospholipases A1 (Ves v 1 and Pol d 1) and antigens 5 (Ves v 5 and Pol d 5) of YJV and PDV allowed the identification of the primary sensitizing venom in 67% of double-sensitized allergic patients, while Vespula hyaluronidase was shown to have no additional value as regards the specificity of the assay. A subsequent study of a very small patient cohort showed that the detection of slgE against the same four allergens could determine the correct venom for immunotherapy in the majority but not in all patients.<sup>1317</sup> Therefore, the additional availability of these and other (e.g., dipeptidyl peptidases IV) cross-reactive allergens from vespid venoms for CRD would represent an added value for advanced precision diagnostics in venom allergy.<sup>1318</sup>

According to some studies, the gold standard to resolve double sensitization in PDV and YJV allergy are CAP-inhibition assays with PDV and YJV.<sup>1319-1321</sup> Current limitations of the commercially available homologous allergens Pol d 5 and Ves v 5 to distinguish between YJV and PDV allergy in double-positive patients by CRD were demonstrated by the fact that a good accordance between Ag5-based CRD and CAP-inhibition assays can only be achieved when the value of slgE in kU/L to Ves v 5 is about twice of those to Pol d 5 and vice versa.<sup>1319,1320</sup> However, a later multicenter study did not
<figure><img src="image_255.png" /><figcaption>FIGURE 137 Diagnostic algorithm for component-resolved diagnostics of yellow jacket venom (YJV) and European paper wasp venom (PDV) allergy. A red minus indicates a negative and a green plus a positive test results. 1 Pol d 1 is currently only available for a selected multiplex slgE platform. Despite the potential of component-resolved diagnostics, clinical history, skin tests and the measurement of venoms IgE and serum tryptase build an indispensable basis for accurate diagnosis in Hymenoptera venom allergy. Moreover, cellular tests such as basophil activation test (BAT) and CAP inhibition assays may be helpful diagnostic tools in dissecting primary sensitization.</figcaption></figure>

extracts.<sup>1311</sup> In dialyzed venom, low molecular weight components (like histamine and components with histamine-releasing activity) smaller than 1000-3000 Dalton are removed. In view of lower cross-reactivity between venoms of the European and American species of Polistes<sup>1312</sup> commercial preparations of European Polistes dominula venom are now available.<sup>1286</sup> Even though a high cross-reactivity between Vespula species venom and V crabro has been confirmed, a commercial extract of Vespa crabro is also available in some countries.<sup>1313</sup>

No recombinant venom allergens are commercially available for skin testing.

### IgE testing:

**Total IgE** - Total IgE (tlgE) determination may be useful for appropriate interpretation of allergen-specific IgE, especially in the case of very low level of slgE (see also the chapter about bee venom allergy).

### Specific IgE to venom extracts and individual venom allergens:

Venom-slgE can be detected immediately after the sting, but the optimal time point will be 1-4 weeks later.<sup>1275</sup> The sensitivity of conventional YJV slgE assay using the whole extract ranges between 83.4% and 91%, the newly developed YJV solid-phase assay complemented with rVes v 5 having a higher sensitivity than the traditional YJV test.<sup>1296,1298</sup>

There is no correlation between the severity of sting reactions and the concentration of venom slgE to whole venom extracts<sup>1275,1310</sup> as some patients with minimal or absent venom-slgE antibodies can develop severe anaphylaxis.<sup>1310</sup> According to a recent study, the severity of sting reactions was not associated with results obtained by skin testing, venom-sslgE levels or even molecular slgE testing.<sup>1314</sup>

Of note, negative slgE and negative skin tests have been reported in the past in up to 15% of patients with systemic mastocytosis and history of a systemic reaction to insect stings,<sup>1315</sup> thus, restricting them from VIT. With the introduction of new methods including CRD and parameters of evaluation in the diagnostic work-up, this diagnostic gap has been solved and slgE can be detected in the vast majority of these patients.<sup>1297,1316</sup> According to some studies, slgE levels between 0.1 and 0.35 kUA/l should be considered relevant in patients with a clear clinical history and low levels of tlgE, irrespective of the presence of mast cell diseases.<sup>1297,1316</sup>

A double *in vitro* positivity to YJV and Polistes venom is common in Mediterranean countries, much more frequent than to YJV/PDV and HBV<sup>1307,1308</sup>. While CRD is able to adequately distinguish allergies to HBV and vespid venom (particularly YJV) (for more detailed information please refer to the bee venom chapter), this is not the case when a differentiation between allergies to various vespid venoms is required (Figure 135).

A previous study demonstrated that the measurement of relative levels of slgE to the phospholipases A1 (Ves v 1 and Pol d 1) and antigens 5 (Ves v 5 and Pol d 5) of YJV and PDV allowed the identification of the primary sensitizing venom in 67% of double-sensitized allergic patients, while Vespula hyaluronidase was shown to have no additional value as regards the specificity of the assay. A subsequent study of a very small patient cohort showed that the detection of slgE against the same four allergens could determine the correct venom for immunotherapy in the majority but not in all patients.<sup>1317</sup> Therefore, the additional availability of these and other (e.g., dipeptidyl peptidases IV) cross-reactive allergens from vespid venoms for CRD would represent an added value for advanced precision diagnostics in venom allergy.<sup>1318</sup>

According to some studies, the gold standard to resolve double sensitization in PDV and YJV allergy are CAP-inhibition assays with PDV and YJV.<sup>1319-1321</sup> Current limitations of the commercially available homologous allergens Pol d 5 and Ves v 5 to distinguish between YJV and PDV allergy in double-positive patients by CRD were demonstrated by the fact that a good accordance between Ag5-based CRD and CAP-inhibition assays can only be achieved when the value of slgE in kU/L to Ves v 5 is about twice of those to Pol d 5 and vice versa.<sup>1319,1320</sup> However, a later multicenter study did not
find any agreement between CAP-inhibition test results and double
slgE values of Ves v 5 over Pol d 5 or vice versa.<sup>1321</sup>

So far, only the major allergens Ves v 1 (phospholipase A1) and
Ves v 5 (antigen 5) of VV and Pol d 5 of PDV are available for routine
molecular diagnostics on most commonly used slgE singleplex assay
platform, while Pol d 1 is exclusively available for multiplex testing.

No individual (European) hornet allergens are available for rou-
tine diagnostics.

Finally, an incomplete cross-reactivity between European and American paper wasps was demonstrated<sup>1308,1312</sup> leading to the need to introduce, at least in Europe, the *Polistes gallicus* or *dominula* extract (the latter only being available in some European countries) into clinical practice for diagnostic and therapeutic purposes. The importance of the concept is also underlined by the possibility, as already reported, of lack of protection by immunotherapy with the American Polistes species venom mixture in European patients.<sup>1322</sup> Due to the increasing spread of Polistes dominula on several conti- nents, associated diagnostic and therapeutic problems are likely to gain importance in other areas of the world.

For the use of species-specific marker allergens that help to dif-
ferentiate between HBV and vespid venom allergy, please refer to
the chapter on bee venom allergy and the diagnostic algorithm de-
picted in (Figure 137).

IgE-inhibition test :

IgE-inhibition tests with whole venom extracts can be used
in particular cases to detect the primary sensitizing venom in pa-
tients double-positive to venoms without marker allergens, e.g.,
YJV and PDV.¹³¹⁹⁻¹³²¹ However, IgE-inhibition tests are costly, time-
consuming and results occasionally difficult to interpret.¹³²¹

Cellular tests :

Among cellular tests, basophil activation test (BAT) is the most
useful one and can be used as a diagnostic tool in some specific
cases, especially if skin tests and slgE antibodies to insect venom
extracts are negative.<sup>1296,1323</sup> BAT is also recommended in double-
positive patients with inconclusive recombinant or skin test double-
positive results, especially if the patient has had an anaphylactic
reaction to only one insect.<sup>1324</sup> BAT seems to be useful in monitor-
ing VIT, during the treatment and after its discontinuation.<sup>1276,1325</sup>
For more detailed information about cellular tests please refer to the
chapter about basophil activation testing.

Baseline serum tryptase :

It is recommended to determine the tryptase concentration in all patients with a history of a systemic reaction after a Hymenoptera sting. For more details refer to the bee venom chapter.

Sting challenge :

The aim of a sting challenge still remains to verify the induction
of tolerance during venom immunotherapy (VIT).<sup>1326</sup> The significant
improvement in health-related quality of life not only after initiation
of VIT but especially after a tolerated sting challenge may favor this
procedure, which should be performed exclusively under emergency
preparedness.

It should be noted that the outcome of sting challenge is influ-
enced by a number of factors, including insect biology, indicating
that bees yield more reliable sting challenge results than vespid.

**Prevention and Therapy**

Preventive measures :

A series of recommendations have been formulated aimed at
substantially minimizing the risk of field re-stings, although as yet no
evidence-based studies have been performed to support this.

In contrast to previous studies, a recent prospective, observa-
tional, multicenter trial, collecting 1,425 patients shows that taking
β-blockers or ACEI does not seem to aggravate the severity of insect
sting reactions in untreated patients.<sup>1327</sup>

Pharmacotherapy (emergency kit):

All patients with a previous SR due to a Hymenoptera sting should be prescribed an emergency kit (containing an adrenaline auto-injector, H1-antihistamines, and corticosteroids) depending on the severity of their previous reaction(s)) and advised to carry it, especially during the Hymenoptera season.<sup>1328</sup>

Self-injectable adrenaline should be considered for all patients with a history of a SR, particularly those who have experienced "moderate-severe" episodes or those with increased risk of future exposure to stings (beekeepers, gardeners, waste management workers etc.), underlying mast cell disorders or raised baseline serum tryptase or other co-morbidities.<sup>1328</sup>

Venom specific immunotherapy (VIT):

According to the European and American Guidelines, subcuta-
neous venom immunotherapy is the only treatment able to prevent
further systemic sting reactions.<sup>1310,1329</sup>

The effectiveness of honeybee and vespid VIT is different and ranges from 77 to 95% for HBV compared with 91 to 99% for vespid venom.¹³²⁹ The underlying reasons are still unclear. For more detailed information refer to the honeybee venom chapter.

Finally, VIT may benefit venom-allergic patients with mast cell dis-
eases, albeit to a lesser extent than patients without mastocytosis.<sup>1330</sup>

5 – Clinical cases

**Case 1 (original)**

**History:** Male beekeeper living in the Mediterranean area, 59 years old, frequently stung, sometimes with large local reaction. Stung by a probable vespid in the neck (no sting was found), within minutes he developed generalized itching and urticaria, dyspnea, nausea, vomiting, and generalized malaise. Care by an emergency doctor.

**Intradermal skin test:** HBV (0.001 µg/mL), YJV (1 µg/mL) and PDV (0.01 µg/mL) positive.

*In vitro testing (conventional)*: slgE 180 kU/L, slgE to HBV 20.6 kU/L, slgE to YJV 1.2 kU/L, PDV 18.9 kU/L, baseline tryptase 3.2 µg/L.

*In vitro testing (molecular)*: slgE to rApi m 1 18.2 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rVes v 5 0.15 kU/L, slgE to rPol d 5 16.5 kU/L, slgE to CCD (MUXF3) <0.1 kU/L (considering the clinical history, slgE to other HBV recombinant allergens were not measured).

REMA score: negative.

**Diagnosis:** PDV allergy (anaphylactic reaction), large local reac-
tion to HBV in beekeeper.
find any agreement between CAP-inhibition test results and double
slgE values of Ves v 5 over Pol d 5 or vice versa.<sup>1321</sup>

So far, only the major allergens Ves v 1 (phospholipase A1) and
Ves v 5 (antigen 5) of VV and Pol d 5 of PDV are available for routine
molecular diagnostics on most commonly used slgE singleplex assay
platform, while Pol d 1 is exclusively available for multiplex testing.

No individual (European) hornet allergens are available for rou-
tine diagnostics.

Finally, an incomplete cross-reactivity between European and American paper wasps was demonstrated<sup>1308,1312</sup> leading to the need to introduce, at least in Europe, the *Polistes gallicus* or *dominula* extract (the latter only being available in some European countries) into clinical practice for diagnostic and therapeutic purposes. The importance of the concept is also underlined by the possibility, as already reported, of lack of protection by immunotherapy with the American Polistes species venom mixture in European patients.<sup>1322</sup> Due to the increasing spread of Polistes dominula on several conti- nents, associated diagnostic and therapeutic problems are likely to gain importance in other areas of the world.

For the use of species-specific marker allergens that help to dif-
ferentiate between HBV and vespid venom allergy, please refer to
the chapter on bee venom allergy and the diagnostic algorithm de-
picted in (Figure 137).

IgE-inhibition test:

IgE-inhibition tests with whole venom extracts can be used
in particular cases to detect the primary sensitizing venom in pa-
tients double-positive to venoms without marker allergens, e.g.,
YJV and PDV.<sup>1319-1321</sup> However, IgE-inhibition tests are costly, time-
consuming and results occasionally difficult to interpret.<sup>1321</sup>

Cellular tests:

Among cellular tests, basophil activation test (BAT) is the most
useful one and can be used as a diagnostic tool in some specific
cases, especially if skin tests and slgE antibodies to insect venom
extracts are negative.<sup>1296,1323</sup> BAT is also recommended in double-
positive patients with inconclusive recombinant or skin test double-
positive results, especially if the patient has had an anaphylactic
reaction to only one insect.<sup>1324</sup> BAT seems to be useful in monitor-
ing VIT, during the treatment and after its discontinuation.<sup>1276,1325</sup>
For more detailed information about cellular tests please refer to the
chapter about basophil activation testing.

Baseline serum tryptase:

It is recommended to determine the tryptase concentration in all patients with a history of a systemic reaction after a Hymenoptera sting. For more details refer to the bee venom chapter.

Sting challenge:

The aim of a sting challenge still remains to verify the induction
of tolerance during venom immunotherapy (VIT).<sup>1326</sup> The significant
improvement in health-related quality of life not only after initiation
of VIT but especially after a tolerated sting challenge may favor this
procedure, which should be performed exclusively under emergency
preparedness.

It should be noted that the outcome of sting challenge is influ-
enced by a number of factors, including insect biology, indicating
that bees yield more reliable sting challenge results than vespid.

**Prevention and Therapy**

Preventive measures:

A series of recommendations have been formulated aimed at
substantially minimizing the risk of field re-stings, although as yet no
evidence-based studies have been performed to support this.

In contrast to previous studies, a recent prospective, observa-
tional, multicenter trial, collecting 1,425 patients shows that taking
β-blockers or ACEI does not seem to aggravate the severity of insect
sting reactions in untreated patients.<sup>1327</sup>

Pharmacotherapy (emergency kit):

All patients with a previous SR due to a Hymenoptera sting should be prescribed an emergency kit (containing an adrenaline autoinjector, H1-antihistamines, and corticosteroids) depending on the severity of their previous reaction(s)) and advised to carry it, especially during the Hymenoptera season.<sup>1328</sup>

Self-injectable adrenaline should be considered for all patients with a history of a SR, particularly those who have experienced "moderate-severe" episodes or those with increased risk of future exposure to stings (beekeepers, gardeners, waste management workers etc.), underlying mast cell disorders or raised baseline serum tryptase or other co-morbidities.<sup>1328</sup>

Venom specific immunotherapy (VIT):

According to the European and American Guidelines, subcuta-
neous venom immunotherapy is the only treatment able to prevent
further systemic sting reactions.<sup>1310,1329</sup>

The effectiveness of honeybee and vespid VIT is different and ranges from 77 to 95% for HBV compared with 91 to 99% for vespid venom.<sup>1329</sup> The underlying reasons are still unclear. For more detailed information refer to the honeybee venom chapter.

Finally, VIT may benefit venom-allergic patients with mast cell dis-
eases, albeit to a lesser extent than patients without mastocytosis.<sup>1330</sup>

5 – Clinical cases

**Case 1 (original)**

**History:** Male beekeeper living in the Mediterranean area, 59 years old, frequently stung, sometimes with large local reaction. Stung by a probable vespid in the neck (no sting was found), within minutes he developed generalized itching and urticaria, dyspnea, nausea, vomiting, and generalized malaise. Care by an emergency doctor.

**Intradermal skin test:** HBV (0.001 µg/mL), YJV (1 µg/mL) and PDV (0.01 µg/mL) positive.

*In vitro* testing (conventional): tlgE 180 kU/L, slgE to HBV 20.6 kU/L, slgE to YJV 1.2 kU/L, PDV 18.9 kU/L, baseline tryptase 3.2 µg/L.

*In vitro* testing (molecular): slgE to rApi m 1 18.2 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rVes v 5 0.15 kU/L, slgE to rPol d 5 16.5 kU/L, slgE to CCD (MUXF3) <0.1 kU/L (considering the clinical history, slgE to other HBV recombinant allergens were not measured).

REMA score: negative.

**Diagnosis:** PDV allergy (anaphylactic reaction), large local reac-
tion to HBV in beekeeper.
**Recommendation:** VIT with PDV extract, emergency kit with adrenaline autoinjector.

### Case 2 (original)

**History:** Atopic female living in Mediterranean area, 61 years old, stung by an unidentified vespid in the hand with generalized urticaria and angioedema of the eyes and lips, nausea, dizziness; one year later after a sting by an unidentified vespid in the head, reaction with generalized urticaria, nausea, uterine cramps. Care by an emergency physician.

**Intradermal skin test:** HBV (negative), YJV (0.01 µg/mL) and PDV (0.01 µg/mL) positive.

**In vitro testing (conventional):** tlgE 850 kU/L, slgE to honeybee venom <0.1 kU/L, slgE to YJV 7.7 kU/L, PDV 7.4 kU/L, baseline tryptase 2.1 µg/L.

**In vitro testing (molecular):** slgE to rVes v 1 2.8 kU/L, slgE to rVes v 5 6.5 kU/L, PDV 6.1 kU/L. REMA score: negative.

**Diagnosis:** PDV and YJV allergy (anaphylaxis).

**Recommendation:** VIT with PDV and YJV extracts, emergency kit with adrenaline autoinjector.

### Case 3 (original)

**History:** Male living in Mediterranean area, 65 years old, history of 2 episodes of severe anaphylactic reaction (both after a vespid sting, in the head and the leg, respectively; in one case the patient family members identified the nest of a Polistes): both reactions characterized by hypotension and loss of consciousness without skin symptoms. Care by an emergency physician, in the second case hospitalization was required.

**Intradermal skin test:** HBV (negative); YJV (1 µg/mL) and PDV (1 µg/mL) positive.

**In vitro testing (conventional):** tlgE 35 kU/L, slgE to HBV <0.1 kU/L, slgE to YJV 1.5 kU/L, PDV 1.7 kU/L, baseline tryptase 7.5 µg/L.

**In vitro testing (molecular):** slgE to rVes v 5 1.2 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rPol d 5 1.1 kU/L. REMA score: positive.

**Diagnosis:** PDV and YJV allergy (anaphylaxis), mastocytosis.

**Recommendation:** VIT with PDV and YJV extracts, emergency kit with adrenaline autoinjector.

## 6 – Research and future perspectives

CRD is undoubtedly an innovative diagnostic method that leads to a more precise definition of the sensitization profile of the venom

allergic patient. The use of CRD is indicated in cases of a proven history of a previous SR and negative results in standard diagnostic tests and in patients with polysensitization to different venoms, as it may help the specialist to choose the most suitable venom for VIT (see also chapter on bee venom allergy).

Modern molecular allergology may pave the way towards novel future diagnostic and therapeutic techniques such as the use of recombinant allergens for skin testing or VIT, even though these options may not become available for clinical practice in the near future due to high regulatory demands for this kind of applications.

However, at present, while CRD makes it possible to distinguish between allergy to Apis mellifera and allergy to Vespula species venoms, the value of CRD is limited in cases of double positivity to Vespula-Polistes. Thus, new recombinant molecules are needed to improve the diagnosis of Polistes allergic patients, especially in the case of double-positivity to both Polistes spp. and Vespula spp. venom, in order to prevent unnecessary double VIT.

Other limitations of CRD are represented by the incidental detection, as observed for venom extracts, of slgE sensitization in patients without clinical history of a sting reaction, and by the inability to correlate slgE levels to venom components with the severity of the sting reaction. We hope that future studies using CRD may identify biomarkers able to distinguish between asymptomatic and symptomatic sensitization, and between different degrees of SR severity, as well as biomarkers for VIT efficacy, VIT tolerance and relapse after discontinuing the treatment.

### Advices for the use of molecular diagnostics for vespid venom allergy

* Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 are maker allergens to detect primary sensitization to vespid venom (to dissect honeybee and vespid venom allergy).

* Exceptions: No marker allergens are available to discriminate between primary sensitization to the different vespid species.

* slgE to Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 indicate primary vespid venom allergy.

* slgE to Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 is no reliable marker to dissect between primary sensitization to yellow jacket and/or European paper wasp venom.
**Recommendation:** VIT with PDV extract, emergency kit with adrenaline autoinjector.

### Case 2 (original)

**History:** Atopic female living in Mediterranean area, 61 years old, stung by an unidentified vespid in the hand with generalized urticaria and angioedema of the eyes and lips, nausea, dizziness; one year later after a sting by an unidentified vespid in the head, reaction with generalized urticaria, nausea, uterine cramps. Care by an emergency physician.

**Intradermal skin test:** HBV (negative), YJV (0.01 µg/mL) and PDV (0.01 µg/mL) positive.

**In vitro testing (conventional):** tlgE 850 kU/L, slgE to honeybee venom <0.1 kU/L, slgE to YJV 7.7 kU/L, PDV 7.4 kU/L, baseline tryptase 2.1 µg/L.

**In vitro testing (molecular):** slgE to rVes v 1 2.8 kU/L, slgE to rVes v 5 6.5 kU/L, PDV 6.1 kU/L. REMA score: negative.

**Diagnosis:** PDV and YJV allergy (anaphylaxis).

**Recommendation:** VIT with PDV and YJV extracts, emergency kit with adrenaline autoinjector.

### Case 3 (original)

**History:** Male living in Mediterranean area, 65 years old, history of 2 episodes of severe anaphylactic reaction (both after a vespid sting, in the head and the leg, respectively; in one case the patient family members identified the nest of a Polistes): both reactions characterized by hypotension and loss of consciousness without skin symptoms. Care by an emergency physician, in the second case hospitalization was required.

**Intradermal skin test:** HBV (negative); YJV (1 µg/mL) and PDV (1 µg/mL) positive.

**In vitro testing (conventional):** tlgE 35 kU/L, slgE to HBV <0.1 kU/L, slgE to YJV 1.5 kU/L, PDV 1.7 kU/L, baseline tryptase 7.5 µg/L.

**In vitro testing (molecular):** slgE to rVes v 5 1.2 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rPol d 5 1.1 kU/L. REMA score: positive.

**Diagnosis:** PDV and YJV allergy (anaphylaxis), mastocytosis.

**Recommendation:** VIT with PDV and YJV extracts, emergency kit with adrenaline autoinjector.

## 6 – Research and future perspectives

CRD is undoubtedly an innovative diagnostic method that leads to a more precise definition of the sensitization profile of the venom

allergic patient. The use of CRD is indicated in cases of a proven history of a previous SR and negative results in standard diagnostic tests and in patients with polysensitization to different venoms, as it may help the specialist to choose the most suitable venom for VIT (see also chapter on bee venom allergy).

Modern molecular allergology may pave the way towards novel future diagnostic and therapeutic techniques such as the use of recombinant allergens for skin testing or VIT, even though these options may not become available for clinical practice in the near future due to high regulatory demands for this kind of applications.

However, at present, while CRD makes it possible to distinguish between allergy to Apis mellifera and allergy to Vespula species venoms, the value of CRD is limited in cases of double positivity to Vespula-Polistes. Thus, new recombinant molecules are needed to improve the diagnosis of Polistes allergic patients, especially in the case of double-positivity to both Polistes spp. and Vespula spp. venom, in order to prevent unnecessary double VIT.

Other limitations of CRD are represented by the incidental detection, as observed for venom extracts, of slgE sensitization in patients without clinical history of a sting reaction, and by the inability to correlate slgE levels to venom components with the severity of the sting reaction. We hope that future studies using CRD may identify biomarkers able to distinguish between asymptomatic and symptomatic sensitization, and between different degrees of SR severity, as well as biomarkers for VIT efficacy, VIT tolerance and relapse after discontinuing the treatment.

### Advices for the use of molecular diagnostics for vespid venom allergy

* Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 are maker allergens to detect primary sensitization to vespid venom (to dissect honeybee and vespid venom allergy).

* Exceptions: No marker allergens are available to discriminate between primary sensitization to the different vespid species.

* slgE to Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 indicate primary vespid venom allergy.

* slgE to Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 is no reliable marker to dissect between primary sensitization to yellow jacket and/or European paper wasp venom.
# B22 - Occupational allergy

## Monika Raulf

### Highlights

* More than 400 occupational sensitizers are identified but only a limited number of them are characterized on the molecular level.

* Natural rubber latex (NRL) allergy is an excellent model for improving sIgE measurement with recombinant major allergens.

* IgE-sensitization profile in patients with baker's asthma showed great inter-individual variation.

* For diagnosis of wheat allergy due to baker's asthma extract-based diagnostic is still recommended.

* Including baking enzymes into the test panel is highly recommended for diagnosis of baker's asthma.

* Asp o 21, alpha-amylase produced in *Aspergillus oryzae*, is commercially available.

* Increasing the knowledge of occupational allergens and implement and evaluate standardized tools in clinical practice is necessary.

## 1 - The allergen sources and their clinical relevance

More than 400 occupational agents have been identified and documented as being potential "respiratory sensitizers".¹³³¹⁻¹³³³ They are triggers of occupational rhinitis (OR) and occupational asthma (OA). Development of OA is often preceded by allergic rhinitis. Both OR and OA are serious health problems in industrialized countries estimated to account for 5% to 15% of asthma cases in adults of working age, and the prevention, as well as diagnosis of these diseases is a challenge (Table 64).

OA is distinguished from work-enhanced asthma and reactive airway disease syndrome, which is caused by occupational exposure to airborne irritants. The "respiratory sensitizers" can be divided into high-molecular weight (HMW) and low-molecular weight (LMW)

substances.¹³³⁴⁻¹³³⁶ Typical LMW substances are isocyanates, acid anhydrides, metals, ammonium persulfate, fumes and vapours from detergents, bleaches and fixatives used by hairdressers, disinfectants and pharmaceuticals. In the case of an IgE-mediated mechanism, it is generally assumed that the allergenicity of these LMW or their metabolites is due to a mostly covalent interaction with some carrier proteins to form a hapten-carrier complex. The most common occupational HMW agents are proteins or glycoproteins derived from diverse plants, animals and micro-organisms. They are found in cereal flour, livestock and laboratory animals, mites, fish and seafood, fodder and detergent enzymes, mould (fungi), *Hevea brasiliensis* latex and wood dust. To date, only a few of the HMW agents have been biochemically and molecular characterized or are produced in recombinant form, because most of the respiratory sensitizing properties of the various occupational substances are only documented as individual case reports. Due to this lack of knowledge about allergen components and their allergenicity only a limited number of recombinant or native occupational relevant allergens are currently commercially available for the *in vitro* diagnosis. Crude extracts from the different allergen sources have traditionally been used for the detection of sensitization by specific IgE quantification or by skin prick tests, whereby the composition and amount of an allergenic extract very much influence the results. Unfortunately, standardized reagents are available for only a few occupational allergens.¹³³⁴⁻¹³³⁶

The focus here is on the presentation of examples with the possibilities and application of component-resolved diagnosis (CRD) with occupational allergens, their clinical relevance and their implementation into the *in vitro* diagnosis for occupational allergies.

### 1.1 Natural rubber latex (*Hevea brasiliensis*)

The milky sap of the rubber tree *Hevea brasiliensis* is the source for the production of commercial natural rubber latex (NRL) devices and represents also a source of potent allergenic proteins⁴,¹³³⁷⁻¹³⁴⁰,¹⁶⁹³ Most of *Hevea brasiliensis* grows commercially in a number of tropical countries, mainly in Thailand, Indochina, Malaysia and India. The milky sap is synthesized by specialized laticifer cells and collection of the latex is possible by scarifying the trunk of the *Hevea brasiliensis* tree. Ammonia treatment prevents coagulation resulting in

<table><caption>TABLE 64 Examples for typical occupational allergen sources at workplaces</caption><thead><tr><td>Workplace/ Trade and Industry</td><td>Alergen source</td></tr></thead><tbody><tr><td>Agriculture/farming</td><td>Cow dander, pollen, storage mites</td></tr><tr><td>Animal feed production</td><td>Soy, phytase</td></tr><tr><td>Bakery/Mills</td><td>Wheat flour, rye flour, different other grain flours, soy flour, α-amylase, xylanase, glucoamylase, storage mites, insects, moulds, spices</td></tr><tr><td>Food processing industry</td><td>Several cereals, plants, vegetables, fruits and spices, seeds, mushrooms, seafood (shellfish and fish), raw coffee beans, farm products (eggs), food additives, enzymes, food contaminants (e.g. mites, insects, moulds)</td></tr><tr><td>Health care facilities</td><td>Disinfectants, natural rubber latex</td></tr><tr><td>Laboratory animal facilities/Life science faculties of universities</td><td>Mouse, rat (urine, dander)</td></tr><tr><td>Laundry detergent industry</td><td>Enzymes: protease, cellulase, lipase, amylase</td></tr></tbody></table>
# B22 – Occupational allergy

## Monika Raulf

### Highlights

* More than 400 occupational sensitizers are identified but only a limited number of them are characterized on the molecular level.

* Natural rubber latex (NRL) allergy is an excellent model for improving sIgE measurement with recombinant major allergens.

* IgE-sensitization profile in patients with baker's asthma showed great inter-individual variation.

* For diagnosis of wheat allergy due to baker's asthma extract-based diagnostic is still recommended.

* Including baking enzymes into the test panel is highly recommended for diagnosis of baker's asthma.

* Asp o 21, alpha-amylase produced in *Aspergillus oryzae*, is commercially available.

* Increasing the knowledge of occupational allergens and implement and evaluate standardized tools in clinical practice is necessary.

## 1 – The allergen sources and their clinical relevance

More than 400 occupational agents have been identified and documented as being potential "respiratory sensitizers".¹³³¹⁻¹³³³ They are triggers of occupational rhinitis (OR) and occupational asthma (OA). Development of OA is often preceded by allergic rhinitis. Both OR and OA are serious health problems in industrialized countries estimated to account for 5% to 15% of asthma cases in adults of working age, and the prevention, as well as diagnosis of these diseases is a challenge (Table 64).

OA is distinguished from work-enhanced asthma and reactive airway disease syndrome, which is caused by occupational exposure to airborne irritants. The "respiratory sensitizers" can be divided into high-molecular weight (HMW) and low-molecular weight (LMW)

substances.¹³³⁴⁻¹³³⁶ Typical LMW substances are isocyanates, acid anhydrides, metals, ammonium persulfate, fumes and vapours from detergents, bleaches and fixatives used by hairdressers, disinfectants and pharmaceuticals. In the case of an IgE-mediated mechanism, it is generally assumed that the allergenicity of these LMW or their metabolites is due to a mostly covalent interaction with some carrier proteins to form a hapten-carrier complex. The most common occupational HMW agents are proteins or glycoproteins derived from diverse plants, animals and micro-organisms. They are found in cereal flour, livestock and laboratory animals, mites, fish and seafood, fodder and detergent enzymes, mould (fungi), *Hevea brasiliensis* latex and wood dust. To date, only a few of the HMW agents have been biochemically and molecular characterized or are produced in recombinant form, because most of the respiratory sensitizing properties of the various occupational substances are only documented as individual case reports. Due to this lack of knowledge about allergen components and their allergenicity only a limited number of recombinant or native occupational relevant allergens are currently commercially available for the *in vitro* diagnosis. Crude extracts from the different allergen sources have traditionally been used for the detection of sensitization by specific IgE quantification or by skin prick tests, whereby the composition and amount of an allergenic extract very much influence the results. Unfortunately, standardized reagents are available for only a few occupational allergens.¹³³⁴⁻¹³³⁶

The focus here is on the presentation of examples with the possibilities and application of component-resolved diagnosis (CRD) with occupational allergens, their clinical relevance and their implementation into the *in vitro* diagnosis for occupational allergies.

### 1.1 Natural rubber latex (*Hevea brasiliensis*)

The milky sap of the rubber tree *Hevea brasiliensis* is the source for the production of commercial natural rubber latex (NRL) devices and represents also a source of potent allergenic proteins⁴,¹³³⁷⁻¹³⁴⁰,¹⁶⁹³ Most of *Hevea brasiliensis* grows commercially in a number of tropical countries, mainly in Thailand, Indochina, Malaysia and India. The milky sap is synthesized by specialized laticifer cells and collection of the latex is possible by scarifying the trunk of the *Hevea brasiliensis* tree. Ammonia treatment prevents coagulation resulting in

<table><caption>TABLE 64 Examples for typical occupational allergen sources at workplaces</caption><thead><tr><td>Workplace/ Trade and Industry</td><td>Alergen source</td></tr></thead><tbody><tr><td>Agriculture/farming</td><td>Cow dander, pollen, storage mites</td></tr><tr><td>Animal feed production</td><td>Soy, phytase</td></tr><tr><td>Bakery/Mills</td><td>Wheat flour, rye flour, different other grain flours, soy flour, α-amylase, xylanase, glucoamylase, storage mites, insects, moulds, spices</td></tr><tr><td>Food processing industry</td><td>Several cereals, plants, vegetables, fruits and spices, seeds, mushrooms, seafood (shellfish and fish), raw coffee beans, farm products (eggs), food additives, enzymes, food contaminants (e.g. mites, insects, moulds)</td></tr><tr><td>Health care facilities</td><td>Disinfectants, natural rubber latex</td></tr><tr><td>Laboratory animal facilities/Life science faculties of universities</td><td>Mouse, rat (urine, dander)</td></tr><tr><td>Laundry detergent industry</td><td>Enzymes: protease, cellulase, lipase, amylase</td></tr></tbody></table>
hydrolysis of the latex proteins. The main constituent of Hevea latex is the polymeric hydrocarbon 1,4 cis-polyisoprene and only 1-2% of the fresh milky sap is made up of proteins. The proteins are heterogeneously distributed in the latex sap and they are involved in the biosynthesis of the polyisoprene, associated with the coagulation of latex and in the defense of the plant against various diseases. After ultra-centrifugation of the fresh latex sap basically three main fractions (rubber phase, the C-serum and the bottom fraction (B-serum)) are easily discerned (Figure 138).

In the 1980s and into the 1990s, cases of NRL allergy increased dramatically. One factor was the elevated hygiene standards in medicine in response to communicable infectious diseases (especially AIDS), which led to the increased use of NRL products, especially NRL gloves.¹³³⁷ The introduction of powder-free and/or gloves with a low allergen content, the reduction or even ban of powdered NRL gloves in some countries and public health campaigns on prevention have resulted in a significant decrease in NRL allergies, especially in the health care sector. Increasing awareness of the health risk posed by NRL products, particularly among health care workers and also among spina bifida patients undergoing surgery in their first days of life, initiated enhanced research on allergen characterization, quantification and improvement of allergy diagnosis. *Hevea brasiliensis*, the origin of NRL, is one of the best characterized occupational allergen sources.⁴,¹³³⁷ Up to now about 250 different NRL polypeptides were identified. About 60 are capable to bind human IgE and currently 15 allergens have been included in the latest nomenclature list of the International Nomenclature Committee of Allergens (IUIS) and assigned official numbers (Hev b 1-15). (www.allergen.org) (Table 65).

Hev b 1 together with Hev b 3 are rubber particle proteins. Hev b 5 is an acidic (pI 3.5) and heat-stable 16-24 kDa protein, rich of glutamic acid and of proline residues. The first recombinant Hev b 5 (rHev b 5) was described by Slater et al.¹³⁴¹ Prohevein, Hev b 6.01, was isolated from the B-serum and posttranslational cleavage

proceeds two further proteins, the 4.7 kDa hevein (Hev b 6.02) and the 14 kDa C-terminal domain Hev b 6.03.¹³⁴¹ All three allergens additionally exist in the plant and the ratio between Hev b 6.01 and Hev b 6.03 is about 30:1. Hev b 6.02 (hevein) comprises the most important part of IgE-binding epitopes in the prohevein molecule. In addition, hevein shows homology to several chitin-binding lectin domains¹³⁴² and may be responsible for certain cross-reactivities to several other plants and food. Most of the Hev b proteins have been cloned and expressed as recombinant proteins. Sequencing demonstrated both unique epitopes and sequences commonly found in other plant proteins. Sequence homology helps to explain the cross-reactivity to a variety of foods experienced by latex allergic individuals.

Studies demonstrated that various risk groups like patients with spina bifida (SB) and occupational latex exposed health care workers (HCWs) are sensitized by different NRL allergens¹³⁴³⁻¹³⁴⁵ based on the different route of exposure (direct blood contact versus inhalation) or as also shown in differences in the allergen levels measured between internal and external surfaces of NRL gloves.¹³⁴⁶ In the case of health care workers suffering from occupational latex allergy the most important NRL allergens are Hev b 5 and Hev 6.01 or Hev b 6.02, respectively. Other NRL allergens like Hev b 1 or Hev b 3 often recognized by specific IgE of spina bifida patients are only minor allergens in latex allergic health care workers.¹³⁴³ About 30-50% of latex-allergic patients show allergic symptoms to plant-derived foods, especially fresh fruits.¹¹⁶⁹ The association was called latex-fruit syndrome (review in¹³⁴⁷) and huge amounts of relevant fruits, constantly increasing, are described and the most commonly involved are avocado, banana, chestnut, and kiwi. Several latex allergens were discussed as responsible for the latex-fruit cross-reactivity (Figure 139), such as Hev b 2,¹³⁴⁸ Hev b 6.02,¹³⁴⁹⁻¹³⁵¹ Hev b 7,¹³⁵² Hev b 8 and Hev b 12.¹³⁵³

In some cases, the use of recombinant single latex allergens for NRL-specific IgE mapping was helpful to discriminate between cross-reactivity and co-sensitization of latex and fruits.¹³⁵⁴,¹³⁵⁵ Especially in plant allergens like NRL, grass pollen¹³⁵⁰ or wood allergens¹³⁵⁶ and also in insect venoms⁴⁵⁹ the presence of cross-reactive carbohydrate determinants (CCDs) can negatively influence the specificity of the *in vitro* diagnostic test. Therefore, it is necessary to exclude glyco-epitopes (with low clinical relevance) responsible for IgE binding. Corresponding CCD screening tools (e.g., horseradish peroxidase, bromelain, ascorbate oxidase) and/or inhibition testing can be performed to clarify the origin of the IgE binding to latex (protein epitopes versus glyco-peptides). Attention should be paid also in false-positive results with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.¹³⁵⁷ A serological work-up including at least one CCD screening tool and the recombinant allergens rHev b 1, rHev b 3, rHev b 5 and rHev b 6.01 is highly recommended and might support diagnosis in patients with suspected IgE-mediated NRL type I-allergy⁴,¹³³⁸,¹³³⁹ (Figure 140).

The starting point to evaluate sensitization to NRL is the ImmunoCAP specific IgE test with the rHev b 5-amplified latex extract (k82 "spiked" with rHev b 5), which showed superior sensitivity

<figure><img src="image_250.png" /><figcaption>FIGURE 138 Separation of latex sap after centrifugation and distribution of the latex allergens</figcaption></figure>
hydrolysis of the latex proteins. The main constituent of Hevea latex is the polymeric hydrocarbon 1,4 cis-polyisoprene and only 1-2% of the fresh milky sap is made up of proteins. The proteins are heterogeneously distributed in the latex sap and they are involved in the biosynthesis of the polyisoprene, associated with the coagulation of latex and in the defense of the plant against various diseases. After ultra-centrifugation of the fresh latex sap basically three main fractions (rubber phase, the C-serum and the bottom fraction (B-serum)) are easily discerned (Figure 138).

In the 1980s and into the 1990s, cases of NRL allergy increased dramatically. One factor was the elevated hygiene standards in medicine in response to communicable infectious diseases (especially AIDS), which led to the increased use of NRL products, especially NRL gloves.¹³³⁷ The introduction of powder-free and/or gloves with a low allergen content, the reduction or even ban of powdered NRL gloves in some countries and public health campaigns on prevention have resulted in a significant decrease in NRL allergies, especially in the health care sector. Increasing awareness of the health risk posed by NRL products, particularly among health care workers and also among spina bifida patients undergoing surgery in their first days of life, initiated enhanced research on allergen characterization, quantification and improvement of allergy diagnosis. *Hevea brasiliensis*, the origin of NRL, is one of the best characterized occupational allergen sources.⁴,¹³³⁷ Up to now about 250 different NRL polypeptides were identified. About 60 are capable to bind human IgE and currently 15 allergens have been included in the latest nomenclature list of the International Nomenclature Committee of Allergens (IUIS) and assigned official numbers (Hev b 1-15). (www.allergen.org) (Table 65).

Hev b 1 together with Hev b 3 are rubber particle proteins. Hev b 5 is an acidic (pI 3.5) and heat-stable 16-24 kDa protein, rich of glutamic acid and of proline residues. The first recombinant Hev b 5 (rHev b 5) was described by Slater et al.¹³⁴¹ Prohevein, Hev b 6.01, was isolated from the B-serum and posttranslational cleavage

proceeds two further proteins, the 4.7 kDa hevein (Hev b 6.02) and the 14 kDa C-terminal domain Hev b 6.03.¹³⁴¹ All three allergens additionally exist in the plant and the ratio between Hev b 6.01 and Hev b 6.03 is about 30:1. Hev b 6.02 (hevein) comprises the most important part of IgE-binding epitopes in the prohevein molecule. In addition, hevein shows homology to several chitin-binding lectin domains¹³⁴² and may be responsible for certain cross-reactivities to several other plants and food. Most of the Hev b proteins have been cloned and expressed as recombinant proteins. Sequencing demonstrated both unique epitopes and sequences commonly found in other plant proteins. Sequence homology helps to explain the cross-reactivity to a variety of foods experienced by latex allergic individuals.

Studies demonstrated that various risk groups like patients with spina bifida (SB) and occupational latex exposed health care workers (HCWs) are sensitized by different NRL allergens¹³⁴³⁻¹³⁴⁵ based on the different route of exposure (direct blood contact versus inhalation) or as also shown in differences in the allergen levels measured between internal and external surfaces of NRL gloves.¹³⁴⁶ In the case of health care workers suffering from occupational latex allergy the most important NRL allergens are Hev b 5 and Hev 6.01 or Hev b 6.02, respectively. Other NRL allergens like Hev b 1 or Hev b 3 often recognized by specific IgE of spina bifida patients are only minor allergens in latex allergic health care workers.¹³⁴³ About 30-50% of latex-allergic patients show allergic symptoms to plant-derived foods, especially fresh fruits.¹¹⁶⁹ The association was called latex-fruit syndrome (review in¹³⁴⁷) and huge amounts of relevant fruits, constantly increasing, are described and the most commonly involved are avocado, banana, chestnut, and kiwi. Several latex allergens were discussed as responsible for the latex-fruit cross-reactivity (Figure 139), such as Hev b 2,¹³⁴⁸ Hev b 6.02,¹³⁴⁹⁻¹³⁵¹ Hev b 7,¹³⁵² Hev b 8 and Hev b 12.¹³⁵³

In some cases, the use of recombinant single latex allergens for NRL-specific IgE mapping was helpful to discriminate between cross-reactivity and co-sensitization of latex and fruits.¹³⁵⁴,¹³⁵⁵ Especially in plant allergens like NRL, grass pollen¹³⁵⁰ or wood allergens¹³⁵⁶ and also in insect venoms⁴⁵⁹ the presence of cross-reactive carbohydrate determinants (CCDs) can negatively influence the specificity of the *in vitro* diagnostic test. Therefore, it is necessary to exclude glyco-epitopes (with low clinical relevance) responsible for IgE binding. Corresponding CCD screening tools (e.g., horseradish peroxidase, bromelain, ascorbate oxidase) and/or inhibition testing can be performed to clarify the origin of the IgE binding to latex (protein epitopes versus glyco-peptides). Attention should be paid also in false-positive results with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.¹³⁵⁷ A serological work-up including at least one CCD screening tool and the recombinant allergens rHev b 1, rHev b 3, rHev b 5 and rHev b 6.01 is highly recommended and might support diagnosis in patients with suspected IgE-mediated NRL type I-allergy⁴,¹³³⁸,¹³³⁹ (Figure 140).

The starting point to evaluate sensitization to NRL is the ImmunoCAP specific IgE test with the rHev b 5-amplified latex extract (k82 "spiked" with rHev b 5), which showed superior sensitivity

<figure><img src="image_256.png" /><figcaption>FIGURE 138 Separation of latex sap after centrifugation and distribution of the latex allergens</figcaption></figure>
<table><caption>TABLE 65 Allergens of *Hevea brasiliensis* (para rubber tree latex) according to WHO/IUIS Allergen Nomenclature Sub-Committee</caption><thead><tr><th>Allergen</th><th>Hevea brasiliensis protein (and molecular weight (kDa))</th><th>Clinical relevance</th></tr></thead><tbody><tr><td>Hev b 1*</td><td>Rubber elongation factor (14 kDa)</td><td>Major allergen in SB</td></tr><tr><td>Hev b 2</td><td>β-1,3-Glucanase (34 kDa)</td><td>Relevance under discussion†</td></tr><tr><td>Hev b 3*</td><td>Small rubber particle proteins (24 kDa)</td><td>Major allergen in SB</td></tr><tr><td>Hev b 4</td><td>Lecithinase homologue (53-55 kDa)</td><td>Minor allergen†</td></tr><tr><td>Hev b 5*</td><td>Acidic structural protein (16 kDa)</td><td>Major allergen in HCW and important in SB</td></tr><tr><td>Hev b 6.01*</td><td>Prohevein (20 kDa) (precursor of hevein<br>Hev b 6.02, the major IgE binding domain)</td><td>Major allergen in HCW</td></tr><tr><td>Hev b 7</td><td>Patatin-like protein (esterase) from latex-B- and C-serum (44 kDa)<br>(two isoforms: Hev b 7.01 and Hev b 7.02)</td><td>Minor allergen</td></tr><tr><td>Hev b 8</td><td>Profilin (actin-binding protein) (14 kDa) (several isoforms and variants)</td><td>Minor allergen</td></tr><tr><td>Hev b 9</td><td>Enolase (51 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 10</td><td>Manganese superoxide dismutase (MnSOD) (26 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 11</td><td>Class I chitinase (30 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 12</td><td>Non-specific lipid transfer protein type 1 (nsLTP1) (9 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 13</td><td>Esterase (42 kDa)</td><td>Relevance under discussion†</td></tr><tr><td>Hev b 14</td><td>Hevamine (30 kDa)</td><td>Minor allergen†</td></tr><tr><td>Hev b 15</td><td>Serine protease inhibitor (7.5 kDa)</td><td>Minor allergen</td></tr></tbody></table>

Hev b: *Hevea brasiliensis*; SB: spina bifida patients, HCW: health care worker; * recommended for specific IgE antibody testing to verify clinical relevance of latex sensitisation according to⁴, 1336, 1337; #not available in recombinant form; adapted from⁴, 1337

<figure><img src="image_251.png" /><figcaption>FIGURE 139 Molecular background and association of latex-fruit/vegetable syndrome—allergens with potential importance for cross-reactivity</figcaption></figure>
<table><caption>TABLE 65 Allergens of *Hevea brasiliensis* (para rubber tree latex) according to WHO/IUIS Allergen Nomenclature Sub-Committee</caption><thead><tr><th>Allergen</th><th>Hevea brasiliensis protein (and molecular weight (kDa))</th><th>Clinical relevance</th></tr></thead><tbody><tr><td>Hev b 1*</td><td>Rubber elongation factor (14 kDa)</td><td>Major allergen in SB</td></tr><tr><td>Hev b 2</td><td>β-1,3-Glucanase (34 kDa)</td><td>Relevance under discussion†</td></tr><tr><td>Hev b 3*</td><td>Small rubber particle proteins (24 kDa)</td><td>Major allergen in SB</td></tr><tr><td>Hev b 4</td><td>Lecithinase homologue (53-55 kDa)</td><td>Minor allergen†</td></tr><tr><td>Hev b 5*</td><td>Acidic structural protein (16 kDa)</td><td>Major allergen in HCW and important in SB</td></tr><tr><td>Hev b 6.01*</td><td>Prohevein (20 kDa) (precursor of hevein<br>Hev b 6.02, the major IgE binding domain)</td><td>Major allergen in HCW</td></tr><tr><td>Hev b 7</td><td>Patatin-like protein (esterase) from latex-B- and C-serum (44 kDa)<br>(two isoforms: Hev b 7.01 and Hev b 7.02)</td><td>Minor allergen</td></tr><tr><td>Hev b 8</td><td>Profilin (actin-binding protein) (14 kDa) (several isoforms and variants)</td><td>Minor allergen</td></tr><tr><td>Hev b 9</td><td>Enolase (51 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 10</td><td>Manganese superoxide dismutase (MnSOD) (26 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 11</td><td>Class I chitinase (30 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 12</td><td>Non-specific lipid transfer protein type 1 (nsLTP1) (9 kDa)</td><td>Minor allergen</td></tr><tr><td>Hev b 13</td><td>Esterase (42 kDa)</td><td>Relevance under discussion†</td></tr><tr><td>Hev b 14</td><td>Hevamine (30 kDa)</td><td>Minor allergen†</td></tr><tr><td>Hev b 15</td><td>Serine protease inhibitor (7.5 kDa)</td><td>Minor allergen</td></tr></tbody></table>

Hev b: *Hevea brasiliensis*; SB: spina bifida patients, HCW: health care worker; * recommended for specific IgE antibody testing to verify clinical relevance of latex sensitisation according to⁴, 1336, 1337; #not available in recombinant form; adapted from⁴, 1337

<figure><img src="image_257.png" /><figcaption>FIGURE 139 Molecular background and association of latex-fruit/vegetable syndrome—allergens with potential importance for cross-reactivity</figcaption></figure>
<figure><img src="image_252.png" /><figcaption>FIGURE 140 Diagnostic algorithm for natural rubber latex (type I allergy)</figcaption></figure>

compared with the results of previously tested negative sera. This procedure of "spiking" is in general useful if relevant allergens are labile to survive all the steps required for the production of a standardized allergen preparation.¹³⁵⁸ A retrospective study of Vandenplas et al.¹³⁴⁴ demonstrated that high levels of sIgE to rHev b 5 plus rHev b 6.01 or rHev b 6.02 are the most accurate predictors of a positive response in an inhalation challenge test, showing better diagnostic efficiency than the NRL (k82)-ImmunoCAP specific IgE test. On the other hand, none of the subjects in this study with a positive inhalation challenge with NRL gloves and a negative NRL-sIgE result showed reactivity to any of the 12 tested recombinant NRL allergens. Accordingly, the determination of sIgE to available recombinant NRL allergens failed to improve the negative predictive values of the NRL-sIgE test.

Especially for NRL allergy, the *in vitro* diagnostic tools are gaining importance, since in Europe the “classical” diagnostic tools such as latex extracts for skin prick test (different manufacturers withdrawn latex extracts from the market) and powdered gloves for workplace-related bronchial challenge tests are no longer commercially available. This leads to a deficit in the diagnostic procedure and to the need for validated substitutes. In the case of NRL, the recombinant available Hev b-allergens in combination with CCDs tools could be useful in the diagnosis of NRL allergy.

## 1.2 Wheat allergy in baker's asthma

"Baker's asthma," which is the generally used term of asthma in bakers and bakery workers, is one of the oldest recognized occupational diseases described by Ramazzini in about 1700. It is one of the most frequently occurring forms of OA. Most studies indicate that wheat (*Triticum aestivum*) flour proteins are allergens for 60-70% of symptomatic bakers,¹³⁵⁹ although other cereals like rye (*Secale cereale*), barley (*Hordeum vulgare*), oats (*Avena sativa*), and corn, and noncereal sources, enzymes, and insects, may be involved because bakeries are complex environments¹³⁶⁰ (Figure 141).

Focusing on wheat, which is in many parts of the world a major crop, and is immensely diverse, with over 25.000 different cultivars.³²² The wheat seeds are composed of endosperm (85%), husk (13%) and a germ (2%). During milling, endosperm was separated from husk and germ and the size of the endosperm was reduced. Wheat flour, which is mainly made from endosperm, is composed of starch (about 70-75%), and four groups of proteins, namely glutenins, gliadins, globulins and water/salt-soluble albumins. In addition, nonstarch polysaccharides (about 2-3%), in particular arabinoxylans, and lipids (2%) are minor but important constituents. Wheat, as a complex allergenic mixture, contains more vegetable proteins than the other two globally important cereals, corn and rice; more than 100 different protein spots can be detected as IgE binding in wheat
<figure><img src="image_258.png" /><figcaption>FIGURE 140 Diagnostic algorithm for natural rubber latex (type I allergy)</figcaption></figure>

compared with the results of previously tested negative sera. This procedure of “spiking” is in general useful if relevant allergens are labile to survive all the steps required for the production of a standardized allergen preparation.¹³⁵⁸ A retrospective study of Vandenplas et al.¹³⁴⁴ demonstrated that high levels of slgE to rHev b 5 plus rHev b 6.01 or rHev b 6.02 are the most accurate predictors of a positive response in an inhalation challenge test, showing better diagnostic efficiency than the NRL (k82)-ImmunoCAP specific IgE test. On the other hand, none of the subjects in this study with a positive inhalation challenge with NRL gloves and a negative NRL-slgE result showed reactivity to any of the 12 tested recombinant NRL allergens. Accordingly, the determination of slgE to available recombinant NRL allergens failed to improve the negative predictive values of the NRL-slgE test.

Especially for NRL allergy, the *in vitro* diagnostic tools are gaining importance, since in Europe the “classical” diagnostic tools such as latex extracts for skin prick test (different manufacturers withdrawn latex extracts from the market) and powdered gloves for workplace-related bronchial challenge tests are no longer commercially available. This leads to a deficit in the diagnostic procedure and to the need for validated substitutes. In the case of NRL, the recombinant available Hev b-allergens in combination with CCDs tools could be useful in the diagnosis of NRL allergy.

## 1.2 Wheat allergy in baker's asthma

"Baker's asthma," which is the generally used term of asthma in bakers and bakery workers, is one of the oldest recognized occupational diseases described by Ramazzini in about 1700. It is one of the most frequently occurring forms of OA. Most studies indicate that wheat (*Triticum aestivum*) flour proteins are allergens for 60-70% of symptomatic bakers,¹³⁵⁹ although other cereals like rye (*Secale cereale*), barley (*Hordeum vulgare*), oats (*Avena sativa*), and corn, and noncereal sources, enzymes, and insects, may be involved because bakeries are complex environments¹³⁶⁰ (Figure 141).

Focusing on wheat, which is in many parts of the world a major crop, and is immensely diverse, with over 25.000 different cultivars.³²² The wheat seeds are composed of endosperm (85%), husk (13%) and a germ (2%). During milling, endosperm was separated from husk and germ and the size of the endosperm was reduced. Wheat flour, which is mainly made from endosperm, is composed of starch (about 70-75%), and four groups of proteins, namely glutenins, gliadins, globulins and water/salt-soluble albumins. In addition, nonstarch polysaccharides (about 2-3%), in particular arabinoxylans, and lipids (2%) are minor but important constituents. Wheat, as a complex allergenic mixture, contains more vegetable proteins than the other two globally important cereals, corn and rice; more than 100 different protein spots can be detected as IgE binding in wheat
<figure><img src="image_253.png" /><figcaption>**FIGURE 141** Different types of IgE-mediated wheat allergy (food allergy versus respiratory allergy)</figcaption></figure>

flour by means of high resolution 2-dimensional gel electrophoresis and immunoblotting.<sup>1361</sup> Twenty-eight wheat allergens are listed so far in the WHO/IUIS Allergen Nomenclature database (www.allergen.org), from the wheat profilin (Tri a 12) up to Tri a 45.<sup>1336</sup> These allergens are not only characterized with respect to baker's asthma; most are also ingested food allergens. The most relevant allergenic wheat fractions for baker's asthma are the water-/ salt-soluble albumins and globulins (Figure 142, Table 66). Diagnosis is based on a consistent clinical history, skin prick testing and/or specific IgE antibody tests and inhalation wheat challenges. Nonetheless this allergic disease is often misdiagnosed, with significant legal, economic and health consequences for the affected worker. Although specific inhalation challenge with wheat flour is considered as gold standard, it is often difficult to perform. Additionally, wheat and rye skin prick test extracts are not well-characterized and demonstrate a low diagnostic sensitivity.<sup>1362,1363</sup>

Several wheat allergens isolated as native allergen or produced in recombinant form have been used in IgE assays in different systems (e.g., singleplex, multiplex, ELISA, immunoblotting) and with different groups of bakers; in many cases, the IgE reactivity of these allergens has been determined only in single studies and their clinical relevance is unclear. The highly varying results may reflect differences in populations or in the different approaches to identify IgE-reactive proteins, making comparisons difficult.<sup>322,1364-1366</sup> One of the best characterized and commercially available single wheat allergen is the omega-5-gliadin (Tri a 19), a 65 kDa seed storage protein, which is involved in wheat-dependent exercise-induced anaphylaxis (WDEIA) and also important for the early childhood type I-wheat allergy. Tri a 19 is not relevant for diagnosis of baker's asthma.<sup>1191</sup> In the study of Sander et al.<sup>1191</sup> a panel of 19 recombinant wheat flour allergens and two cross-reactive carbohydrate determinants (CCD) was investigated using the singleplex technology for specific IgE quantification (CAP-FEIA system) in the sera of 101 bakers with occupational allergy from Germany, Spain and the Netherlands and of 29 pollen-sensitized control subjects without occupational exposure but with wheat-specific IgE. The results indicate that different α-amylase inhibitors are important allergens for baker's asthma, but none of the single allergens reached major allergen status. The geographical origin of the bakers and control subjects was not a significant determinant of the sensitization pattern, and each baker

<figure><img src="image_254.png" /><figcaption>**FIGURE 142** Wheat allergens with relevance for wheat-allergic bakers (according to 1191)</figcaption></figure>

showed an individual IgE-binding profile with large interindividual variability. The highest frequencies of IgE binding were found for thiol reductase (Tri a 27) and the wheat dimeric alpha-amylase inhibitor (Tri a 28). Tri a 19, Tri a 26 and Tri a 36, relevant wheat allergens in food-allergic patients, are irrelevant in the diagnostic of baker's asthma. In addition, two isoforms of Tri a 14 (Tri a 14.0101, ns LTP 9.1 and Tri a 14.0201, nsLTP 9.7) were tested and both were classified as minor allergens with 11% and 5% positive IgE response, respectively in all bakers. No cross-reactivity to grass pollen using inhibition experiments was found for Tri a 15, Tri a 30 (both are alpha-amylase inhibitors), Tri a 21 (apha-beta-gliadin) and Tri a 31 (serpin), whereas nsLTP (Tri a 14) and Tri a 25 (thioredoxin) share epitopes with grass pollen allergens. Although a combination of IgE tests to five components (Tri a 27, Tri a 28, tetrameric alpha-amylase inhibitor CM2 (Tri a 29.02), serine protease inhibitor-like allergen (Tri a 39), and 1-cys-peroxiredoxin (Tri a 32), produced the highest diagnostic efficiency in receiver operating characteristic analyses, but this was still lower than the determination of sIgE antibodies against the whole
<figure><img src="image_259.png" /><figcaption>**FIGURE 141** Different types of IgE-mediated wheat allergy (food allergy versus respiratory allergy)</figcaption></figure>

flour by means of high resolution 2-dimensional gel electrophoresis and immunoblotting.<sup>1361</sup> Twenty-eight wheat allergens are listed so far in the WHO/IUIS Allergen Nomenclature database (www.allergen.org), from the wheat profilin (Tri a 12) up to Tri a 45.<sup>1336</sup> These allergens are not only characterized with respect to baker's asthma; most are also ingested food allergens. The most relevant allergenic wheat fractions for baker's asthma are the water-/ salt-soluble albumins and globulins (Figure 142, Table 66). Diagnosis is based on a consistent clinical history, skin prick testing and/or specific IgE antibody tests and inhalation wheat challenges. Nonetheless this allergic disease is often misdiagnosed, with significant legal, economic and health consequences for the affected worker. Although specific inhalation challenge with wheat flour is considered as gold standard, it is often difficult to perform. Additionally, wheat and rye skin prick test extracts are not well-characterized and demonstrate a low diagnostic sensitivity.<sup>1362,1363</sup>

Several wheat allergens isolated as native allergen or produced in recombinant form have been used in IgE assays in different systems (e.g., singleplex, multiplex, ELISA, immunoblotting) and with different groups of bakers; in many cases, the IgE reactivity of these allergens has been determined only in single studies and their clinical relevance is unclear. The highly varying results may reflect differences in populations or in the different approaches to identify IgE-reactive proteins, making comparisons difficult.<sup>322,1364-1366</sup> One of the best characterized and commercially available single wheat allergen is the omega-5-gliadin (Tri a 19), a 65 kDa seed storage protein, which is involved in wheat-dependent exercise-induced anaphylaxis (WDEIA) and also important for the early childhood type I-wheat allergy. Tri a 19 is not relevant for diagnosis of baker's asthma.<sup>1191</sup> In the study of Sander et al.<sup>1191</sup> a panel of 19 recombinant wheat flour allergens and two cross-reactive carbohydrate determinants (CCD) was investigated using the singleplex technology for specific IgE quantification (CAP-FEIA system) in the sera of 101 bakers with occupational allergy from Germany, Spain and the Netherlands and of 29 pollen-sensitized control subjects without occupational exposure but with wheat-specific IgE. The results indicate that different α-amylase inhibitors are important allergens for baker's asthma, but none of the single allergens reached major allergen status. The geographical origin of the bakers and control subjects was not a significant determinant of the sensitization pattern, and each baker

<figure><img src="image_260.png" /><figcaption>**FIGURE 142** Wheat allergens with relevance for wheat-allergic bakers (according to 1191)</figcaption></figure>

showed an individual IgE-binding profile with large interindividual variability. The highest frequencies of IgE binding were found for thiol reductase (Tri a 27) and the wheat dimeric alpha-amylase inhibitor (Tri a 28). Tri a 19, Tri a 26 and Tri a 36, relevant wheat allergens in food-allergic patients, are irrelevant in the diagnostic of baker's asthma. In addition, two isoforms of Tri a 14 (Tri a 14.0101, ns LTP 9.1 and Tri a 14.0201, nsLTP 9.7) were tested and both were classified as minor allergens with 11% and 5% positive IgE response, respectively in all bakers. No cross-reactivity to grass pollen using inhibition experiments was found for Tri a 15, Tri a 30 (both are alpha-amylase inhibitors), Tri a 21 (apha-beta-gliadin) and Tri a 31 (serpin), whereas nsLTP (Tri a 14) and Tri a 25 (thioredoxin) share epitopes with grass pollen allergens. Although a combination of IgE tests to five components (Tri a 27, Tri a 28, tetrameric alpha-amylase inhibitor CM2 (Tri a 29.02), serine protease inhibitor-like allergen (Tri a 39), and 1-cys-peroxiredoxin (Tri a 32), produced the highest diagnostic efficiency in receiver operating characteristic analyses, but this was still lower than the determination of sIgE antibodies against the whole
<table><caption>TABLE 66 Relevant airborne wheat (Triticum aestivum) allergens according to WHO/IUIS Allergen Nomenclature Sub-Committee</caption><thead><tr><td>Allergen</td><td>Biochemical name</td><td>Notes</td></tr></thead><tbody><tr><td>Tri a 15</td><td>Wheat monomeric α-amylase inhibitor 0.28 (WMA-1-0.28)</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; 9 of 101 (9%) bakers have a positive IgE-response <sup>1191</sup></td></tr><tr><td>Tri a 25</td><td>Thioredoxin</td><td>not exclusive for baker's asthma; 18 of 101 (18%) bakers and 5 of 29 (17%) patients with grass pollen allergy have a positive IgE-response <sup>1191</sup></td></tr><tr><td>Tri a 27</td><td>Thiol reductase homologue</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; recognized by 27% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 28</td><td>Dimeric α-amylase inhibitor 0.19</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; recognized by 24% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 29 -<br>Tri a 29.0101 -<br>Tri a 29.0201</td><td>Tetrameric α-amylase inhibitor<br>CM1<br>CM2</td><td>in contrast to Tri a 29.0201, Tri a 29.0101 is not exclusively recognized in patients with baker's allergy</td></tr><tr><td>Tri a 30</td><td>Tetrameric<br>α-amylase inhibitor CM3</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; recognized by 10% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 31</td><td>Triosephosphate-isomerase<br>(TPIS)</td><td>not exclusively recognized by specific IgE from bakers</td></tr><tr><td>Tri a 32</td><td>1-cys-peroxiredoxin</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE</td></tr><tr><td>Tri a 33</td><td>Serpin</td><td>recognized by only 8% of patients with baker's asthma and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 34</td><td>Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)</td><td>recognized by only 5% of patients with baker's asthma and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 35</td><td>Dehydrin</td><td>recognized by only 2% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 39</td><td>Serine protease inhibitor-like protein (SPILA)</td><td>recognized by 18% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 40</td><td>WTAI-CM1 17 protein (α-amylase inhibitor)</td><td>recognized by 8% of IgE-positive bakers and 15% of IgE-positive grass-pollen allergic patients; addition of Tri a 40.0101 to the panel of recombinant allergen components had only minimal influence on diagnostic sensitivity and could not improve specificity <sup>1366</sup></td></tr></tbody></table>

http://www.allergen.org, 1CM= Chloroform/methanol-soluble, Bold = the combination of these five allergens in recombinant form coupled to ImmunoCAP produced the best diagnostic performance according to <sup>1191</sup>

wheat flour extracts. Additional testing with Tri a 40.0101, a further wheat alpha-amylase inhibitor in the same group of bakers and controls had only minimal influence on diagnostic sensitivity and failed to improve specificity.<sup>1366</sup> Due to the superior diagnostic sensitivity of slgE antibodies testing against the whole wheat flour extracts, the authors concluded that this is mandatory for the *in vitro* diagnostic procedure of baker's asthma. Nevertheless, the component-resolved diagnostics might help to distinguish between sensitization caused by occupational respiratory flour exposure (baker's asthma), wheat-induced food allergy and wheat seropositivity based on cross-reactivity to grass pollen, but further single wheat allergens should be made commercially available for this purpose.

### 1.3. Examples of other occupationally relevant plant allergens

*Cannabis sativa* (hemp) is the most commonly used psychoactive drug worldwide. In recent years, access to cannabis for both medical and nonmedical purposes have expanded. In addition to the use of cannabis as a medicine and intoxicant, there are numerous other uses of the hemp plant, including as edible oils and increasingly as in form of so-called lifestyle products. This is also marked by an increasing number of people working in this growing industry. Increasingly, exposure to cannabis in these workplaces is causing health problems, including allergic complaints in particular.<sup>1367</sup> Due to the increasing legalization, an expansion of the cannabis producing and processing but also the distributing industries is to be expected. In 2020,
<table><caption>TABLE 66 Relevant airborne wheat (Triticum aestivum) allergens according to WHO/IUIS Allergen Nomenclature Sub-Committee</caption><thead><tr><td>Allergen</td><td>Biochemical name</td><td>Notes</td></tr></thead><tbody><tr><td>Tri a 15</td><td>Wheat monomeric α-amylase inhibitor 0.28 (WMA-1-0.28)</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; 9 of 101 (9%) bakers have a positive IgE-response <sup>1191</sup></td></tr><tr><td>Tri a 25</td><td>Thioredoxin</td><td>not exclusive for baker's asthma; 18 of 101 (18%) bakers and 5 of 29 (17%) patients with grass pollen allergy have a positive IgE-response <sup>1191</sup></td></tr><tr><td>Tri a 27</td><td>Thiol reductase homologue</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; recognized by 27% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 28</td><td>Dimeric α-amylase inhibitor 0.19</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; recognized by 24% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 29 -<br>Tri a 29.0101 -<br>Tri a 29.0201</td><td>Tetrameric α-amylase inhibitor<br>CM1<br>CM2</td><td>in contrast to Tri a 29.0201, Tri a 29.0101 is not exclusively recognized in patients with baker's allergy</td></tr><tr><td>Tri a 30</td><td>Tetrameric<br>α-amylase inhibitor CM3</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE; recognized by 10% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 31</td><td>Triosephosphate-isomerase<br>(TPIS)</td><td>not exclusively recognized by specific IgE from bakers</td></tr><tr><td>Tri a 32</td><td>1-cys-peroxiredoxin</td><td>relevant in patients with baker's allergy, but not relevant for those with grass pollen allergy with wheat specific IgE</td></tr><tr><td>Tri a 33</td><td>Serpin</td><td>recognized by only 8% of patients with baker's asthma and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 34</td><td>Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)</td><td>recognized by only 5% of patients with baker's asthma and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 35</td><td>Dehydrin</td><td>recognized by only 2% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 39</td><td>Serine protease inhibitor-like protein (SPILA)</td><td>recognized by 18% of patients with baker's allergy and 0% of grass-pollen allergic patients <sup>1191</sup></td></tr><tr><td>Tri a 40</td><td>WTAI-CM1 17 protein (α-amylase inhibitor)</td><td>recognized by 8% of IgE-positive bakers and 15% of IgE-positive grass-pollen allergic patients; addition of Tri a 40.0101 to the panel of recombinant allergen components had only minimal influence on diagnostic sensitivity and could not improve specificity <sup>1366</sup></td></tr></tbody></table>

http://www.allergen.org, 1CM= Chloroform/methanol-soluble, Bold = the combination of these five allergens in recombinant form coupled to ImmunoCAP produced the best diagnostic performance according to <sup>1191</sup>

wheat flour extracts. Additional testing with Tri a 40.0101, a further wheat alpha-amylase inhibitor in the same group of bakers and controls had only minimal influence on diagnostic sensitivity and failed to improve specificity.<sup>1366</sup> Due to the superior diagnostic sensitivity of slgE antibodies testing against the whole wheat flour extracts, the authors concluded that this is mandatory for the *in vitro* diagnostic procedure of baker's asthma. Nevertheless, the component-resolved diagnostics might help to distinguish between sensitization caused by occupational respiratory flour exposure (baker's asthma), wheat-induced food allergy and wheat seropositivity based on cross-reactivity to grass pollen, but further single wheat allergens should be made commercially available for this purpose.

### 1.3. Examples of other occupationally relevant plant allergens

*Cannabis sativa* (hemp) is the most commonly used psychoactive drug worldwide. In recent years, access to cannabis for both medical and nonmedical purposes have expanded. In addition to the use of cannabis as a medicine and intoxicant, there are numerous other uses of the hemp plant, including as edible oils and increasingly as in form of so-called lifestyle products. This is also marked by an increasing number of people working in this growing industry. Increasingly, exposure to cannabis in these workplaces is causing health problems, including allergic complaints in particular.<sup>1367</sup> Due to the increasing legalization, an expansion of the cannabis producing and processing but also the distributing industries is to be expected. In 2020,
around 150,000 people were employed in cultivation, harvesting, processing and distribution. Under these conditions, one can expect an exponential growth of this industry. Workers involved in cannabis production and processing are at risk of inhaling the organic dust. There is a risk of inhaling organic dust from the cannabis plant parts as well as contaminants including bacterial and fungal components. Both the duration and the type of occupational exposure contribute to the different symptoms and courses of disease. Cannabis allergy has been described in cannabis growers, bird breeders, factory workers and laboratory personnel, who have shown both skin and/or respiratory symptoms based on exposure. According to these reports, allergic reaction was caused by cannabis pollen, leaves, hemp seeds and/or flower tops.<sup>1368</sup> It can be assumed that even among heavy cannabis users, personal exposures do not reach the level of occupational exposures, where contact may last for a longer period of time. Studies in recent years have shown a high prevalence of respiratory problems among hemp workers. According to Decuyper et al.<sup>1368</sup> 42% of the participants reported respiratory and/or cutaneous symptoms on occupational cannabis exposure. In addition, many hemp workers were found to have high levels of hemp-specific IgE. So far, four allergens are listed in WHO/IUIS Allergen Nomenclature database (www.allergen.org) (Table 67).

According to the publication of Decuyper et al.<sup>1368</sup> the most effective and practical tests to confirm cannabis allergy are the skin prick tests with an nCan s 3-rich extract and the slgE rCan s 3. Can s 3-sensitization carries the risk of systemic reactions to plant-derived foods and cofactor-mediated reactions. However, there is still a lack of knowledge on further allergens in cannabis, which could be particularly important for the diagnosis of occupationally induced cannabis sensitization.

Soybean (Glycine max) is not only a major food allergen, but inhalation of soy flour is also associated with occupational and environmental allergies<sup>1336</sup> Bakers in particular are exposed, as soy flour is often used as an additive in bread. Therefore, it is not surprising that soybeans are associated with baker's asthma.<sup>322</sup> Additionally, sensitization in baker's is also possible for lupine (probably cross-reactivity also to soy) and peanut. The soybean allergens involved in baker's asthma are predominantly high-MW proteins found both in soybean hull and flour.<sup>1370</sup> By contrast, the relatively low-MW proteins concentrated in the soybean hull Gly m 1 (7 kDa, the hydropathic protein from soybean with two isoforms Gly m 1.0101 and Gly m 1.0102) and Gly m 2 (8 kDa, the defensin) are responsible for the asthma attacks during unloading of soybean at the in the Spanish seaports. Eight soy allergens are listed in WHO/IUIS Allergen Nomenclature database (www.allergen.org). Gly m 4, 5 and 6 are available as CCD-free recombinant soy allergens, but further validation is needed if these allergens are relevant occupational allergens and useful to implement them in diagnosis for occupational asthma in exposed workers.

Green coffee bean dust is known to be a relevant cause of occupational allergic diseases in coffee industry workers, therefore coffee bean proteins may play a role as occupational allergens.<sup>1336</sup> The first allergen isolated from *Coffea arabica* was a class III-chitinase

with a molecular weight of 32 kDa, listed in the WHO/IUIS database as Cof a 1. Cof a 2 and Cof a 3 (9 and 7 kDa, respectively) two cysteine-rich metallothioneins were identified as further coffee allergens.<sup>1694</sup> Peters et al.<sup>1371</sup> showed that the only commercially available diagnostic tests based on native extracts of green coffee beans are not sensitive enough to correctly diagnose a substantial number of affected coffee workers. Their results suggest that the natural allergen extracts do not contain sufficient amounts of the Cof a 1, 2 and 3.<sup>1371</sup> Therefore, the authors suggested the production and application of recombinant coffee allergens for the development of standardized and sensitive diagnostic tools and/or the spiking the natural extract with recombinant coffee allergens to improve the diagnostics of coffee allergy.

IgE-mediated sensitization to some wood dusts has been described in case reports<sup>1372</sup> and obeche (*Triplochiton scleroxylon*) wood dust is one of the known causes of these immunological OA. An endochitinase 38 kDa was characterized as an allergen and included in the nomenclature list of the International Nomenclature Committee of Allergens (IUIS) and assigned with the official name Trip s 1.<sup>1373</sup> To date, no further wood dust allergens are listed (http://www.allergen.org) and no single wood allergen is commercially available. Specific IgE measurement was possible with an obeche extract (k212) and skin prick testing as described by Hannu et al.<sup>1374</sup> Aranda et al.<sup>1375</sup> described two new proteins as allergen (24 kDa identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin) tested in 12 subjects with confirmed OA/OR, 40 asymptomatic exposed and 10 control subjects. 82% of the cases showed also IgE reactivity to cross-reactive carbohydrate determinants (CCDs). Therefore, testing with CCD tools is strongly recommended for diagnostic specificity, as mentioned previously.<sup>1356</sup>

### 1.4. Occupational relevant allergens of animal origin

Laboratory animal allergy (LAA) is an important occupational disease and is commonly seen in technicians, animal caretakers, physicians, and scientists working in the pharmaceutical industries, university laboratories, and animal breeding facilities<sup>4,785,1336,1376</sup> (Table 68).

Rodents like mice and rats, which are often used in animal research, are the most common causes of LAA. Urine is the main source of allergenic proteins in both mice and rats, but allergens can also be found in dander, hair, saliva and serum. As with most mammalian inhalant allergens, the major allergens in mice and rats are lipocalins (Mus m 1 and Rat n 1, respectively). In the case of LAA, determination of slgE antibodies is based on extracts prepared from epithelia, serum-/urine protein as mixture or alone. Only Mus m 1, the major mouse allergen, is available as a single component on the multiplex test system. In addition to skin prick tests and ImmunoCAP Specific IgE test to determine slgE levels for urine and epithelia allergens, Caballero et al.<sup>1377</sup> studied 20 of 75 workers using multiallergen IgE immunoblotting. This system can be useful in providing the sensitization profile for each allergic worker and therefore it is one step forward in the molecular diagnosis of LAA. An additional important source of occupational animal allergen exposure are stables of cattle farmers. Therefore, allergen from cow dander was responsible for
around 150,000 people were employed in cultivation, harvesting, processing and distribution. Under these conditions, one can expect an exponential growth of this industry. Workers involved in cannabis production and processing are at risk of inhaling the organic dust. There is a risk of inhaling organic dust from the cannabis plant parts as well as contaminants including bacterial and fungal components. Both the duration and the type of occupational exposure contribute to the different symptoms and courses of disease. Cannabis allergy has been described in cannabis growers, bird breeders, factory workers and laboratory personnel, who have shown both skin and/or respiratory symptoms based on exposure. According to these reports, allergic reaction was caused by cannabis pollen, leaves, hemp seeds and/or flower tops.<sup>1368</sup> It can be assumed that even among heavy cannabis users, personal exposures do not reach the level of occupational exposures, where contact may last for a longer period of time. Studies in recent years have shown a high prevalence of respiratory problems among hemp workers. According to Decuyper et al.<sup>1368</sup> 42% of the participants reported respiratory and/or cutaneous symptoms on occupational cannabis exposure. In addition, many hemp workers were found to have high levels of hemp-specific IgE. So far, four allergens are listed in WHO/IUIS Allergen Nomenclature database (www.allergen.org) (Table 67).

According to the publication of Decuyper et al.<sup>1368</sup> the most effective and practical tests to confirm cannabis allergy are the skin prick tests with an nCan s 3-rich extract and the slgE rCan s 3. Can s 3-sensitization carries the risk of systemic reactions to plant-derived foods and cofactor-mediated reactions. However, there is still a lack of knowledge on further allergens in cannabis, which could be particularly important for the diagnosis of occupationally induced cannabis sensitization.

Soybean (Glycine max) is not only a major food allergen, but inhalation of soy flour is also associated with occupational and environmental allergies<sup>1336</sup> Bakers in particular are exposed, as soy flour is often used as an additive in bread. Therefore, it is not surprising that soybeans are associated with baker's asthma.<sup>322</sup> Additionally, sensitization in baker's is also possible for lupine (probably cross-reactivity also to soy) and peanut. The soybean allergens involved in baker's asthma are predominantly high-MW proteins found both in soybean hull and flour.<sup>1370</sup> By contrast, the relatively low-MW proteins concentrated in the soybean hull Gly m 1 (7 kDa, the hydropathic protein from soybean with two isoforms Gly m 1.0101 and Gly m 1.0102) and Gly m 2 (8 kDa, the defensin) are responsible for the asthma attacks during unloading of soybean at the in the Spanish seaports. Eight soy allergens are listed in WHO/IUIS Allergen Nomenclature database (www.allergen.org). Gly m 4, 5 and 6 are available as CCD-free recombinant soy allergens, but further validation is needed if these allergens are relevant occupational allergens and useful to implement them in diagnosis for occupational asthma in exposed workers.

Green coffee bean dust is known to be a relevant cause of occupational allergic diseases in coffee industry workers, therefore coffee bean proteins may play a role as occupational allergens.<sup>1336</sup> The first allergen isolated from *Coffea arabica* was a class III-chitinase

with a molecular weight of 32 kDa, listed in the WHO/IUIS database as Cof a 1. Cof a 2 and Cof a 3 (9 and 7 kDa, respectively) two cysteine-rich metallothioneins were identified as further coffee allergens.<sup>1694</sup> Peters et al.<sup>1371</sup> showed that the only commercially available diagnostic tests based on native extracts of green coffee beans are not sensitive enough to correctly diagnose a substantial number of affected coffee workers. Their results suggest that the natural allergen extracts do not contain sufficient amounts of the Cof a 1, 2 and 3.<sup>1371</sup> Therefore, the authors suggested the production and application of recombinant coffee allergens for the development of standardized and sensitive diagnostic tools and/or the spiking the natural extract with recombinant coffee allergens to improve the diagnostics of coffee allergy.

IgE-mediated sensitization to some wood dusts has been described in case reports<sup>1372</sup> and obeche (*Triplochiton scleroxylon*) wood dust is one of the known causes of these immunological OA. An endochitinase 38 kDa was characterized as an allergen and included in the nomenclature list of the International Nomenclature Committee of Allergens (IUIS) and assigned with the official name Trip s 1.<sup>1373</sup> To date, no further wood dust allergens are listed (http://www.allergen.org) and no single wood allergen is commercially available. Specific IgE measurement was possible with an obeche extract (k212) and skin prick testing as described by Hannu et al.<sup>1374</sup> Aranda et al.<sup>1375</sup> described two new proteins as allergen (24 kDa identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin) tested in 12 subjects with confirmed OA/OR, 40 asymptomatic exposed and 10 control subjects. 82% of the cases showed also IgE reactivity to cross-reactive carbohydrate determinants (CCDs). Therefore, testing with CCD tools is strongly recommended for diagnostic specificity, as mentioned previously.<sup>1356</sup>

## 1.4. Occupational relevant allergens of animal origin

Laboratory animal allergy (LAA) is an important occupational disease and is commonly seen in technicians, animal caretakers, physicians, and scientists working in the pharmaceutical industries, university laboratories, and animal breeding facilities<sup>4,785,1336,1376</sup> (Table 68).

Rodents like mice and rats, which are often used in animal research, are the most common causes of LAA. Urine is the main source of allergenic proteins in both mice and rats, but allergens can also be found in dander, hair, saliva and serum. As with most mammalian inhalant allergens, the major allergens in mice and rats are lipocalins (Mus m 1 and Rat n 1, respectively). In the case of LAA, determination of slgE antibodies is based on extracts prepared from epithelia, serum-/urine protein as mixture or alone. Only Mus m 1, the major mouse allergen, is available as a single component on the multiplex test system. In addition to skin prick tests and ImmunoCAP Specific IgE test to determine slgE levels for urine and epithelia allergens, Caballero et al.<sup>1377</sup> studied 20 of 75 workers using multiallergen IgE immunoblotting. This system can be useful in providing the sensitization profile for each allergic worker and therefore it is one step forward in the molecular diagnosis of LAA. An additional important source of occupational animal allergen exposure are stables of cattle farmers. Therefore, allergen from cow dander was responsible for
<table><caption>TABLE 67 Relevant *Cannabis sativa* allergens (Indian hemp) according to WHO/IUIS Allergen Nomenclature Sub-Committee</caption><thead><tr><td>Allergen</td><td>Biochemical name</td><td>Notes</td></tr></thead><tbody><tr><td>Can s 2</td><td>Profilin</td><td>14 kDa; mostly inhalation, but multiple exposure routes possible, minor allergen (5 of 32 patients were positive in IgE binding tests (www.allergen.org))</td></tr><tr><td>Can s 3</td><td>Non-specific lipid transfer protein type 1</td><td>9 kDa; major allergen responsible for likely-anaphylaxis to cannabis; inhalation; (Up to 72% of 25 Cannabis allergic patients reporting likely-anaphylaxis to Cannabis are Can s 3 sensitised testing by three diagnostic methods (BAT, sIgE, and SPT). (www.allergen.org))</td></tr><tr><td>Can s 4</td><td>Oxygen evolving Enhancer Protein 2</td><td>27.3 kDa; minor allergen</td></tr><tr><td>Can s 5</td><td>Pathogenesis related protein 10 homologue</td><td>17.7 kDa; homologue of the major birch pollen allergen; (35 of 45 subjects with allergy to Cannabis (n=25) and/or birch pollen (n=20) were positive in IgE-binding tested by cytometric bead assay using recombinant protein as the target. (www.allergen.org))</td></tr></tbody></table>

<table><caption>TABLE 68 Mammalian allergens involved in occupational OR/OA¹³³⁵</caption><thead><tr><td>Allergen</td><td>Major allergens</td><td>Protein family</td><td>Main source</td><td>Exposed workers</td></tr></thead><tbody><tr><td rowspan="4">Cow<br/>(Bos domesticus)</td><td>Bos d 2</td><td>Lipocalin</td><td>Dander</td><td>Dairy farmers</td></tr><tr><td>nBos d 4*</td><td>Alpha-lactalbumin</td><td>Milk</td><td>Candy and pastry workers</td></tr><tr><td>nBos d 6*</td><td>Albumin</td><td>Serum</td><td>Lab workers</td></tr><tr><td>nBos d 8*</td><td>Casein</td><td>Milk</td><td>Leather tanning</td></tr><tr><td>Mouse<br/>(Mus musculus)</td><td>Mus m 1 **</td><td>Lipocalin</td><td>Urine</td><td>Dermatological powder use</td></tr><tr><td>Rat<br/>(Rattus norvegicus)</td><td>Rat n 1</td><td>Lipocalin</td><td>Urine</td><td>Laboratory animal workers</td></tr><tr><td rowspan="2">Guinea pig<br/>(Cavia porcellus)</td><td>Cav p 1**</td><td>Lipocalin</td><td>Dander, saliva</td><td>Laboratory animal workers</td></tr><tr><td>Cav p 2</td><td>Lipocalin</td><td>Saliva, dander</td><td>Laboratory animal workers</td></tr></tbody></table>

most cases of OA in Finland in the last century.²⁸⁸,⁷⁸⁵ The lipocalin is Bos d 2 (20 kDa) is the predominant allergen in cow dander and responsible for respiratory allergy in cattle farmers. Twelve allergens from *Bos domesticus* (*Bos taurus*/*domestic cattle*) are listed in WHO/IUIS Allergen Nomenclature database (www.allergen.org), but most of them are described as an important allergen group in patients with food allergy induced by milk or meat. Some of them are available as single allergens for CRD (nBos d 6 (bovine serum albumin), nBos d 4 (α-lactalbumin), nBos d 8 (casein). Only few of them are occupationally relevant. Laboratory workers may be exposed to airborne Bos d 6 (BSA) as it is widely used in biochemical and immunological assays and two cases of OA have been attributed to inhalation of serum albumin powder (Bos d 6) in laboratory workers.¹³⁷⁸,¹³⁷⁹ In addition, in candy and pastry workers Bos d 4¹³⁷⁹,¹³⁸⁰ or in leather tanning the casein Bos d 8¹³⁸¹ appear to play a role.

Occupational exposure to seafood during processing of fish and shellfish may induce OR and OA. The prevalence of occupational rhinitis associated with seafood in epidemiological studies is estimated to be 5-24% and occupational asthma is more commonly associated with shellfish (4-36%) than bony fish (2-8%).¹⁰⁵⁴,¹³⁸² Several allergenic proteins have been identified in these different groups, 29 fish allergens and 34 allergens from various crustacean and

mollusk species are listed in the WHO/IUIS database (www.allergen.org). The availability of individual seafood allergens for sIgE testing is still limited, but two important allergens parvalbumin (rCyp c 1 from *Cyprinus carpio* and rCad c 1 from *Gadus morhua*) and shrimp tropomyosin (rPen a 1 from *Penaeus aztecus* and nPen m 1 from *Penaeus monodon*) as well as prawn arginine kinase (nPen m 2) and sarcoplasmic calcium-binding protein (nPen m 4) are available as singleplex assays and/or on multiplex platforms. Further studies are necessary to prove if these recombinant allergens may be relevant for the diagnosis of respiratory allergies in the occupational setting where sensitization results from inhalation exposure. Tropomyosin appears to be important and was recognized as relevant in a case report¹³⁸³ in which a cook's mate was diagnosed with a clinically and occupationally relevant type I allergy to squid with cross-reaction to tropomyosin of other invertebrates and therefore recognized as an occupational disease. Beekeepers, gardeners, farmers, truck drivers, and masons are the professionals most frequently involved in occupational hymenoptera venom allergy.¹³⁸⁴ Relevant allergens are described in Chapters B20 and B21.

## 1.5 Microbial-derived occupational allergens

Enzymes have been used widely as additives to improve industrial processes. In several workplaces like enzyme production and

<footnote>* Commercially available for component resolved IgE-diagnosis (**) only on an Allergen Microchip)</footnote>
<table><caption>TABLE 67 Relevant *Cannabis sativa* allergens (Indian hemp) according to WHO/IUIS Allergen Nomenclature Sub-Committee</caption><thead><tr><td>Allergen</td><td>Biochemical name</td><td>Notes</td></tr></thead><tbody><tr><td>Can s 2</td><td>Profilin</td><td>14 kDa; mostly inhalation, but multiple exposure routes possible, minor allergen (5 of 32 patients were positive in IgE binding tests (www.allergen.org))</td></tr><tr><td>Can s 3</td><td>Non-specific lipid transfer protein type 1</td><td>9 kDa; major allergen responsible for likely-anaphylaxis to cannabis; inhalation; (Up to 72% of 25 Cannabis allergic patients reporting likely-anaphylaxis to Cannabis are Can s 3 sensitised testing by three diagnostic methods (BAT, sIgE, and SPT). (www.allergen.org))</td></tr><tr><td>Can s 4</td><td>Oxygen evolving Enhancer Protein 2</td><td>27.3 kDa; minor allergen</td></tr><tr><td>Can s 5</td><td>Pathogenesis related protein 10 homologue</td><td>17.7 kDa; homologue of the major birch pollen allergen; (35 of 45 subjects with allergy to Cannabis (n=25) and/or birch pollen (n=20) were positive in IgE-binding tested by cytometric bead assay using recombinant protein as the target. (www.allergen.org))</td></tr></tbody></table>

<table><caption>TABLE 68 Mammalian allergens involved in occupational OR/OA¹³³⁵</caption><thead><tr><td>Allergen</td><td>Major allergens</td><td>Protein family</td><td>Main source</td><td>Exposed workers</td></tr></thead><tbody><tr><td rowspan="4">Cow<br>(Bos domesticus)</td><td>Bos d 2</td><td>Lipocalin</td><td>Dander</td><td>Dairy farmers</td></tr><tr><td>nBos d 4*</td><td>Alpha-lactalbumin</td><td>Milk</td><td>Candy and pastry workers</td></tr><tr><td>nBos d 6*</td><td>Albumin</td><td>Serum</td><td>Lab workers</td></tr><tr><td>nBos d 8*</td><td>Casein</td><td>Milk</td><td>Leather tanning</td></tr><tr><td>Mouse<br>(Mus musculus)</td><td>Mus m 1 **</td><td>Lipocalin</td><td>Urine</td><td>Dermatological powder use</td></tr><tr><td>Rat<br>(Rattus norvegicus)</td><td>Rat n 1</td><td>Lipocalin</td><td>Urine</td><td>Laboratory animal workers</td></tr><tr><td rowspan="2">Guinea pig<br>(Cavia porcellus)</td><td>Cav p 1**</td><td>Lipocalin</td><td>Dander, saliva</td><td>Laboratory animal workers</td></tr><tr><td>Cav p 2</td><td>Lipocalin</td><td>Saliva, dander</td><td>Laboratory animal workers</td></tr></tbody></table>

most cases of OA in Finland in the last century.²⁸⁸,⁷⁸⁵ The lipocalin is Bos d 2 (20 kDa) is the predominant allergen in cow dander and responsible for respiratory allergy in cattle farmers. Twelve allergens from *Bos domesticus* (*Bos taurus*/*domestic cattle*) are listed in WHO/IUIS Allergen Nomenclature database (www.allergen.org), but most of them are described as an important allergen group in patients with food allergy induced by milk or meat. Some of them are available as single allergens for CRD (nBos d 6 (bovine serum albumin), nBos d 4 (α-lactalbumin), nBos d 8 (casein). Only few of them are occupationally relevant. Laboratory workers may be exposed to airborne Bos d 6 (BSA) as it is widely used in biochemical and immunological assays and two cases of OA have been attributed to inhalation of serum albumin powder (Bos d 6) in laboratory workers.¹³⁷⁸,¹³⁷⁹ In addition, in candy and pastry workers Bos d 4¹³⁷⁹,¹³⁸⁰ or in leather tanning the casein Bos d 8¹³⁸¹ appear to play a role.

Occupational exposure to seafood during processing of fish and shellfish may induce OR and OA. The prevalence of occupational rhinitis associated with seafood in epidemiological studies is estimated to be 5-24% and occupational asthma is more commonly associated with shellfish (4-36%) than bony fish (2-8%).¹⁰⁵⁴,¹³⁸² Several allergenic proteins have been identified in these different groups, 29 fish allergens and 34 allergens from various crustacean and

mollusk species are listed in the WHO/IUIS database (www.allergen.org). The availability of individual seafood allergens for sIgE testing is still limited, but two important allergens parvalbumin (rCyp c 1 from *Cyprinus carpio* and rCad c 1 from *Gadus morhua*) and shrimp tropomyosin (rPen a 1 from *Penaeus aztecus* and nPen m 1 from *Penaeus monodon*) as well as prawn arginine kinase (nPen m 2) and sarcoplasmic calcium-binding protein (nPen m 4) are available as singleplex assays and/or on multiplex platforms. Further studies are necessary to prove if these recombinant allergens may be relevant for the diagnosis of respiratory allergies in the occupational setting where sensitization results from inhalation exposure. Tropomyosin appears to be important and was recognized as relevant in a case report¹³⁸³ in which a cook's mate was diagnosed with a clinically and occupationally relevant type I allergy to squid with cross-reaction to tropomyosin of other invertebrates and therefore recognized as an occupational disease. Beekeepers, gardeners, farmers, truck drivers, and masons are the professionals most frequently involved in occupational hymenoptera venom allergy.¹³⁸⁴ Relevant allergens are described in Chapters B20 and B21.

## 1.5 Microbial-derived occupational allergens

Enzymes have been used widely as additives to improve industrial processes. In several workplaces like enzyme production and

<footnote>* Commercially available for component resolved IgE-diagnosis (**) only on an Allergen Microchip)</footnote>
refinement, bakeries, food processing, laundry detergent production, animal feeding etc., they act as airborne sensitizers and the prevalence of occupational allergies is increasing.<sup>1335,1336,1695</sup> Most enzymes are derived from microbes usually produced in bacterial microorganisms belonging to Bacillus sp. and Pseudomonas sp. and fungal organisms such as *Aspergillus* spp., Streptomyces spp. and Trichoderma spp. In addition to Bacillus-derived proteases, like alcalase and maxatase and savinase, an important diagnostic tool for slgE testing is alpha-amylase, an allergen relevant to baker's asthma and produced in *Aspergillus* oryzae. This alpha-amylase is listed in the nomenclature list of WHO/ IUIS as Asp o 21. Enzymes derived from *Aspergillus niger* glucoamylase and also cellulase are also relevant in baker's asthma and available for slgE antibody testing. In bakery workers, a clear relationship between exposure to alpha-amylase, derived from *Aspergillus oryzae*, and IgE production has been reported. The prevalence of sensitization to alpha-amylase and glucoamylase ranged between 5% and 24% among symptomatic workers without sensitization to cereal flour. In the modern baking industry, sensitization to glucoamylase (28%) and cellulase (16%) appears to be most common. Furthermore, xylanolytic enzymes can also cause occupational asthma and occupational rhinitis in bakers. The major determinant of sensitization to enzymes is the level of exposure and its ability to become airborne (summarized in.<sup>1054</sup> It is important to realize that enzymes from other species may be cloned into, e.g., *bacillus* and *aspergillus*, which are then used as production organism of the enzymes for industrial purposes. Thus, when dealing with a suspected enzyme allergy, it is important to test the preparation to which the patient has been exposed and not solely rely on extracts or allergens from the production organisms. This may necessitate access to custom-made diagnostics for SPT or IgE measurements.<sup>1385</sup>

Diagnosis of mould allergy is complicated because of the heterogeneity of the test materials and the decrease in the number of commercially available mould extracts for SPT.<sup>1386,1387</sup> Currently only eight single mould allergens from three mould genera are available for molecular diagnosis: rAlt a 1 and rAlt a 6 from *Alternaria alternata*, rAsp f 1, 2, 3, 4, 6 from *Aspergillus fumigatus* and rCla h 8 *Cladosporium herbarum*. Occupational exposure to mould has been reported especially in waste collectors and composting workers. Therefore, e.g., allergic bronchopulmonary aspergillosis (ABPA), an intense inflammatory reaction induced by exposure to *Aspergillus fumigatus* has been reported in garden waste (compost) or garbage collectors.<sup>1388,1389</sup> For diagnosis to mould see Chapter B07.

## 2 – Clinical management

### Clinical diagnosis

Diagnosis of occupational respiratory allergy is made by a combination of medical history, physical examination, positive methacholine challenge result or bronchodilator responsiveness, determination of IgE-mediated sensitization to HMW allergens (by skin

prick testing and/ or serologically specific IgE measurement, and possibly basophil activation testing to LMW chemicals and HMW allergens). Based on the fact that occupational respiratory allergy especially occupational asthma should be suspected in every adult with new-onset asthma, the question about the occurrence of the respiratory symptoms in relation to the workplace is important. If the patient with asthma-like symptoms is not at work the specific inhalation challenge (SIC) in the laboratory under controlled conditions to the suspected occupational agents is considered the gold standard.<sup>1390</sup> The accuracy of the diagnosis can be improved by the measurement of sputum eosinophils before and after challenge. Additional measurement of the fractional exhaled nitric oxide (FeNO) should be regarded as an additional criterion for the interpretation of SIC with occupational agents, because an increase in FeNO after SIC is highly predictive of occupational asthma. If specific inhalation challenge in the laboratory and/or PEF

monitoring at work are not possible and occupational asthma is strongly suspected from history, a combination of objective evidence of asthma plus a positive skin test or the verification of specific IgE by serological tests to the suspected agent has a high predictive value for occupational asthma<sup>1391</sup> (summarized in). Skin prick tests are often taken as the method of choice for the determination of sensitization in practice because results are available immediately and the procedure is cost effective. Unfortunately, only very few skin prick test extracts for the diagnosis of occupational allergy are commercially available. Additionally, there is a lack of standardization and validation for most available extracts of occupational agents and the allergenic potency of SPT extracts may vary significantly among manufacturers.<sup>1362</sup> Therefore, testing of specific IgE with extracts—if available—is in most cases the best choice. Especially in the cases of natural rubber latex allergy recombinant allergens are available and should be used. The binding to CCD should also be checked, especially in the case of plant allergen sources, in order to be able to exclude a probable clinically relevant sensitization.

## 3 – Clinical cases

### Case 1 (published in<sup>1351</sup>)

**Clinical history:** A 37 year-old man, developed urticaria with skin redness, itching, dyspnea and tachycardia 5 minutes after drinking a glass of apple juice supplemented with acerola (*Malpighia glabra*; Barbados cherry); no allergy to apple and apple juice was well tolerated; in the past seasonal hay fever symptoms caused by grass pollen and wild herbal pollen; since childhood, a significant contact urticaria induced by natural rubber latex products was well known; OAS after ingesting avocado, celery, walnut, and curry during pollen season.

**SPT:** wheal size same as for histamine—grass pollen mixture, latex, rye; weak reactions to plantain, hazel, birch pollen; limited reaction to mugwort and ragweed pollen and to curry; intracutaneous skin reaction with acerola pulp and with acerola-containing apple juice; apple juice without acerola negative.
refinement, bakeries, food processing, laundry detergent production, animal feeding etc., they act as airborne sensitizers and the prevalence of occupational allergies is increasing.<sup>1335,1336,1695</sup> Most enzymes are derived from microbes usually produced in bacterial microorganisms belonging to Bacillus sp. and Pseudomonas sp. and fungal organisms such as *Aspergillus* spp., Streptomyces spp. and Trichoderma spp. In addition to Bacillus-derived proteases, like alcalase and maxatase and savinase, an important diagnostic tool for slgE testing is alpha-amylase, an allergen relevant to baker's asthma and produced in *Aspergillus* oryzae. This alpha-amylase is listed in the nomenclature list of WHO/ IUIS as Asp o 21. Enzymes derived from *Aspergillus niger* glucoamylase and also cellulase are also relevant in baker's asthma and available for slgE antibody testing. In bakery workers, a clear relationship between exposure to alpha-amylase, derived from *Aspergillus oryzae*, and IgE production has been reported. The prevalence of sensitization to alpha-amylase and glucoamylase ranged between 5% and 24% among symptomatic workers without sensitization to cereal flour. In the modern baking industry, sensitization to glucoamylase (28%) and cellulase (16%) appears to be most common. Furthermore, xylanolytic enzymes can also cause occupational asthma and occupational rhinitis in bakers. The major determinant of sensitization to enzymes is the level of exposure and its ability to become airborne (summarized in.<sup>1054</sup> It is important to realize that enzymes from other species may be cloned into, e.g., *bacillus* and *aspergillus*, which are then used as production organism of the enzymes for industrial purposes. Thus, when dealing with a suspected enzyme allergy, it is important to test the preparation to which the patient has been exposed and not solely rely on extracts or allergens from the production organisms. This may necessitate access to custom-made diagnostics for SPT or IgE measurements.<sup>1385</sup>

Diagnosis of mould allergy is complicated because of the heterogeneity of the test materials and the decrease in the number of commercially available mould extracts for SPT.<sup>1386,1387</sup> Currently only eight single mould allergens from three mould genera are available for molecular diagnosis: rAlt a 1 and rAlt a 6 from *Alternaria alternata*, rAsp f 1, 2, 3, 4, 6 from *Aspergillus fumigatus* and rCla h 8 *Cladosporium herbarum*. Occupational exposure to mould has been reported especially in waste collectors and composting workers. Therefore, e.g., allergic bronchopulmonary aspergillosis (ABPA), an intense inflammatory reaction induced by exposure to *Aspergillus fumigatus* has been reported in garden waste (compost) or garbage collectors.<sup>1388,1389</sup> For diagnosis to mould see Chapter B07.

## 2 – Clinical management

### Clinical diagnosis

Diagnosis of occupational respiratory allergy is made by a combination of medical history, physical examination, positive methacholine challenge result or bronchodilator responsiveness, determination of IgE-mediated sensitization to HMW allergens (by skin

prick testing and/ or serologically specific IgE measurement, and possibly basophil activation testing to LMW chemicals and HMW allergens). Based on the fact that occupational respiratory allergy especially occupational asthma should be suspected in every adult with new-onset asthma, the question about the occurrence of the respiratory symptoms in relation to the workplace is important. If the patient with asthma-like symptoms is not at work the specific inhalation challenge (SIC) in the laboratory under controlled conditions to the suspected occupational agents is considered the gold standard.<sup>1390</sup> The accuracy of the diagnosis can be improved by the measurement of sputum eosinophils before and after challenge. Additional measurement of the fractional exhaled nitric oxide (FeNO) should be regarded as an additional criterion for the interpretation of SIC with occupational agents, because an increase in FeNO after SIC is highly predictive of occupational asthma. If specific inhalation challenge in the laboratory and/or PEF

monitoring at work are not possible and occupational asthma is strongly suspected from history, a combination of objective evidence of asthma plus a positive skin test or the verification of specific IgE by serological tests to the suspected agent has a high predictive value for occupational asthma<sup>1391</sup> (summarized in). Skin prick tests are often taken as the method of choice for the determination of sensitization in practice because results are available immediately and the procedure is cost effective. Unfortunately, only very few skin prick test extracts for the diagnosis of occupational allergy are commercially available. Additionally, there is a lack of standardization and validation for most available extracts of occupational agents and the allergenic potency of SPT extracts may vary significantly among manufacturers.<sup>1362</sup> Therefore, testing of specific IgE with extracts—if available—is in most cases the best choice. Especially in the cases of natural rubber latex allergy recombinant allergens are available and should be used. The binding to CCD should also be checked, especially in the case of plant allergen sources, in order to be able to exclude a probable clinically relevant sensitization.

## 3 – Clinical cases

### Case 1 (published in<sup>1351</sup>)

**Clinical history:** A 37 year-old man, developed urticaria with skin redness, itching, dyspnea and tachycardia 5 minutes after drinking a glass of apple juice supplemented with acerola (*Malpighia glabra*; Barbados cherry); no allergy to apple and apple juice was well tolerated; in the past seasonal hay fever symptoms caused by grass pollen and wild herbal pollen; since childhood, a significant contact urticaria induced by natural rubber latex products was well known; OAS after ingesting avocado, celery, walnut, and curry during pollen season.

**SPT:** wheal size same as for histamine—grass pollen mixture, latex, rye; weak reactions to plantain, hazel, birch pollen; limited reaction to mugwort and ragweed pollen and to curry; intracutaneous skin reaction with acerola pulp and with acerola-containing apple juice; apple juice without acerola negative.
**In vitro** testing: Total IgE 145 kU/L, specific IgE to latex 24.7 kU/L; acerola (EAST) 1.5 kU/L; CAP class 3 to grass pollen, CAP class 2 to plantain, peanut, tomato soy bean, CAP class 1 to hazelnut pollen, ragweed pollen, banana, green apple, herbal mixture and nut mixture; single recombinant latex allergens: Hev b 6.01 17.1 kU/L, Hev b 6.02 18.7 kU/L, Hev b 8 0.7 kU/L, Hev b 1, 3, 5, 10 <0.35 kU/L. Inhibition studies showed that IgE binding to acerola allergens was completely inhibited by latex and acerola extract, whereas preincubation of the patient's serum with acerola showed nearly complete inhibition to latex (79%) and rHev b 6.01 (85%). rHev b 8 as a solid-phase allergen and acerola as an inhibitor revealed no significant inhibition.

**Oral challenge:** open oral challenge tests with apple juice with and without acerola and diluted acerola pulp; negative for apple juice; exposure to acerola pulp with a latency period of 5 minutes induced itching and swelling lips

**Diagnosis:** Latex-acerola cross-reactivity based on Hev b 6.01/ Hev b 6.02; Primary sensitization to latex led to cross-reactivity to acerola; (Pro-)hevein is the important allergen and responsible for the cross-reactivity;

Allergologists should include acerola on the growing list of latex-cross-reactive food allergens and inform latex-allergic patients about cross-reacting allergens as the traces of acerola in apple juice.

### Case 2 (theoretical) Baker's asthma

**Clinical history:** A 55-year old baker, ex-smoker, starting his apprenticeship at the age of 14 years, health problems at his workplace started 5 years later: sneezing, running nose, nose blocked, watering eyes, stinging eyes, cough, chest tightness asthma diagnosed 7 years later. The complaints got better at weekends and during holidays. **Questions:** What are the causes of the workplace-related symptoms? Are the complaints based on sensitization to environmental allergens?

**SPT:** wheat flour, rye flour and grass pollen positive comparable wheal size than histamine.

**Specific IgE:** total IgE 110 kU/L, wheat flour 54.2 kU/L, rye flour 15.4 kU/L, grass pollen 5.6 kU/L and alpha-amylase 1.2 kU/L. Workplace-specific challenge test with rye: the patient develops symptoms. Is there a relation between wheat/rye sensitization and grass pollen sensitization? **Approach to find the answer:** Cross-inhibition testing with wheat and rye, respectively on the solid phase and grass pollen as liquid inhibitor and vice versa. **Results:** 12% IgE inhibition when rye flour as solid phase and grass pollen as inhibitor; 10% IgE inhibition when wheat flour as solid phase and grass pollen as inhibitor; 87% IgE inhibition when grass pollen as solid phase and rye flour as inhibitor; 90% IgE inhibition when grass pollen as solid phase and wheat flour as inhibitor.

**Conclusion:** Based on the results of the inhibition experiments, the sensitization to the allergens at the workplace (wheat and rye) are independent of the sensitization to the (possible) cross-reactive allergen (grass pollen). The primary source of the sensitization are wheat flour and rye flour. The patient is also sensitized to the enzyme α-amylase. The clear clinical history, the workplace-relates symptoms, the positive SPT and high concentration of flour-specific IgE are good predictors for a positive challenge test. According to the recommendation in¹³⁹² a workplace-related challenge test can be avoided in highly sensitized bakers.

## 4 – Summary and perspective

For occupational type I-allergy with a huge variety of different sensitizers, only limited numbers of allergens are characterized on the molecular level so far and assessment of slgE reactivity to single allergen components has only been studied in detail for NRL and wheat allergy in the case of baker's asthma. Only few of allergen components are available for the routine diagnostics. Natural rubber latex is an excellent model for the useful application of recombinant single allergens for improvement of routine diagnostics (via spiking of the latex extracts with rHev b 5) and the use of individual allergens is possible to determine the risk of severe allergic reactions, therefore recommendations to avoid latex products and cross-reactive foods can be given to the patient. The characterization of the allergic components responsible for wheat allergy in bakers has been the focus of several research groups for many years. Since the individual sensitization profile of bakers to wheat proteins is very heterogeneous, no main allergen could be identified. However, the group of alpha-amylase inhibitors seem to be important allergens for wheat allergy in bakers, but these allergens are not commercially available. Therefore, using the wheat extract is the best *in vitro* diagnostic option. Wheat ω5-gliadin (Tri a 19), the major sensitizing allergen WDEIA, and other typical wheat allergens in food allergic individuals (e.g., Tri a 26 or Tri a 36) are irrelevant for the diagnosis of baker's asthma. Further characterization of occupational sensitizers for IgE-mediated diseases (occupational rhinitis/ asthma, dermatitis) is urgently needed, with candidate or promising markers to be evaluated in multi-center studies to improve *in vitro* diagnostics for occupational allergic diseases. This is important as many skin prick test extracts are at risk of being withdrawn from the market (especially rare allergen preparations such as occupational allergens), which could lead to a lack of diagnostic tools. Therefore, increased efforts should be made to apply the knowledge of allergen characterization to the field of occupational allergy diagnostics.
**In vitro** testing: Total IgE 145 kU/L, specific IgE to latex 24.7 kU/L; acerola (EAST) 1.5 kU/L; CAP class 3 to grass pollen, CAP class 2 to plantain, peanut, tomato soy bean, CAP class 1 to hazelnut pollen, ragweed pollen, banana, green apple, herbal mixture and nut mixture; single recombinant latex allergens: Hev b 6.01 17.1 kU/L, Hev b 6.02 18.7 kU/L, Hev b 8 0.7 kU/L, Hev b 1, 3, 5, 10 <0.35 kU/L. Inhibition studies showed that IgE binding to acerola allergens was completely inhibited by latex and acerola extract, whereas preincubation of the patient's serum with acerola showed nearly complete inhibition to latex (79%) and rHev b 6.01 (85%). rHev b 8 as a solid-phase allergen and acerola as an inhibitor revealed no significant inhibition.

**Oral challenge:** open oral challenge tests with apple juice with and without acerola and diluted acerola pulp; negative for apple juice; exposure to acerola pulp with a latency period of 5 minutes induced itching and swelling lips

**Diagnosis:** Latex-acerola cross-reactivity based on Hev b 6.01/ Hev b 6.02; Primary sensitization to latex led to cross-reactivity to acerola; (Pro-)hevein is the important allergen and responsible for the cross-reactivity;

Allergologists should include acerola on the growing list of latex-cross-reactive food allergens and inform latex-allergic patients about cross-reacting allergens as the traces of acerola in apple juice.

### Case 2 (theoretical) Baker's asthma

**Clinical history:** A 55-year old baker, ex-smoker, starting his apprenticeship at the age of 14 years, health problems at his workplace started 5 years later: sneezing, running nose, nose blocked, watering eyes, stinging eyes, cough, chest tightness asthma diagnosed 7 years later. The complaints got better at weekends and during holidays. **Questions:** What are the causes of the workplace-related symptoms? Are the complaints based on sensitization to environmental allergens?

**SPT:** wheat flour, rye flour and grass pollen positive comparable wheal size than histamine.

**Specific IgE:** total IgE 110 kU/L, wheat flour 54.2 kU/L, rye flour 15.4 kU/L, grass pollen 5.6 kU/L and alpha-amylase 1.2 kU/L. Workplace-specific challenge test with rye: the patient develops symptoms. Is there a relation between wheat/rye sensitization and grass pollen sensitization? **Approach to find the answer:** Cross-inhibition testing with wheat and rye, respectively on the solid phase and grass pollen as liquid inhibitor and vice versa. **Results:** 12% IgE inhibition when rye flour as solid phase and grass pollen as inhibitor; 10% IgE inhibition when wheat flour as solid phase and grass pollen as inhibitor; 87% IgE inhibition when grass pollen as solid phase and rye flour as inhibitor; 90% IgE inhibition when grass pollen as solid phase and wheat flour as inhibitor.

**Conclusion:** Based on the results of the inhibition experiments, the sensitization to the allergens at the workplace (wheat and rye) are independent of the sensitization to the (possible) cross-reactive allergen (grass pollen). The primary source of the sensitization are wheat flour and rye flour. The patient is also sensitized to the enzyme α-amylase. The clear clinical history, the workplace-relates symptoms, the positive SPT and high concentration of flour-specific IgE are good predictors for a positive challenge test. According to the recommendation in¹³⁹² a workplace-related challenge test can be avoided in highly sensitized bakers.

## 4 – Summary and perspective

For occupational type I-allergy with a huge variety of different sensitizers, only limited numbers of allergens are characterized on the molecular level so far and assessment of slgE reactivity to single allergen components has only been studied in detail for NRL and wheat allergy in the case of baker's asthma. Only few of allergen components are available for the routine diagnostics. Natural rubber latex is an excellent model for the useful application of recombinant single allergens for improvement of routine diagnostics (via spiking of the latex extracts with rHev b 5) and the use of individual allergens is possible to determine the risk of severe allergic reactions, therefore recommendations to avoid latex products and cross-reactive foods can be given to the patient. The characterization of the allergic components responsible for wheat allergy in bakers has been the focus of several research groups for many years. Since the individual sensitization profile of bakers to wheat proteins is very heterogeneous, no main allergen could be identified. However, the group of alpha-amylase inhibitors seem to be important allergens for wheat allergy in bakers, but these allergens are not commercially available. Therefore, using the wheat extract is the best *in vitro* diagnostic option. Wheat ω5-gliadin (Tri a 19), the major sensitizing allergen WDEIA, and other typical wheat allergens in food allergic individuals (e.g., Tri a 26 or Tri a 36) are irrelevant for the diagnosis of baker's asthma. Further characterization of occupational sensitizers for IgE-mediated diseases (occupational rhinitis/ asthma, dermatitis) is urgently needed, with candidate or promising markers to be evaluated in multi-center studies to improve *in vitro* diagnostics for occupational allergic diseases. This is important as many skin prick test extracts are at risk of being withdrawn from the market (especially rare allergen preparations such as occupational allergens), which could lead to a lack of diagnostic tools. Therefore, increased efforts should be made to apply the knowledge of allergen characterization to the field of occupational allergy diagnostics.
# C01 - Profilins

Riccardo Asero, Domingo Barber, Giorgio Celi

## Highlights

* Up to 50% of pollen allergic patients are sensitized to profilin.

* Sensitization virtually always follows primary sensitization to a specific pollen source.

* On SPT most pollen sources score positive.

* Clinical relevance is variable but potentially present.

* Up to 50% of sensitized patients may have food allergy, oral allergy syndrome in most cases.

* Clinical reactivity to raw tomato, melon, watermelon, and/or citrus fruits is typically associated with profilin hypersensitivity

* Patients tolerate processed foods.

* The spectrum of offending plant foods is sometimes very large.

## 1 - The protein

Profilin is a protein of 12-15 kDa in size present in all eukaryotic cells and involved in the organization of cytoskeleton as well as in signal transduction. Although it can form oligomeric assemblies (mostly as a consequence of protein purification and storage processes) profilin is a monomeric actin-binding protein and a key regulator of actin-filament dynamics during processes such as cell movement, cytokinesis, and signalling¹³⁹³ (Figure 143, Table 69). In higher plants, it is identified as an allergen in monocot and dicot angiosperms.

## 2 - The protein family

As mentioned above, profilins are present in all eukaryotic cells, and in effect there is an officially registered profilin (Tyr p 36) originating from the storage mite, *Tyrophagus putrescentiae*. Nonetheless, as plant profilins represent the only clinically relevant allergens of this family described so far, the present chapter will deal specifically with plant derived profilins. Profilins from higher plants constitute a family of highly conserved proteins showing sequence identities of at least 75% even between members from distantly related organisms (Tables 70 and 71). In view of the high sequence homology, cross-reactivity between profilins is extremely common and involves virtually every plant source. (Figure 144). Thus, profilin can be considered the archetypal pan-allergen.¹³⁸⁹

## 3 - Clinical relevance of profilin

### A) As an airborne allergen (Table 70)

Profilins are able to elicit IgE responses in 10-60% of pollen-allergic patients¹³⁹⁴,¹³⁹⁵; however, the sensitization prevalence seems on the rise, as more and more allergic patients seen at allergy departments show sensitization to a large number of botanically

<figure><img src="image_255.png" /><figcaption>FIGURE 143 Three dimensional structures of Phl p 12 (grass pollen) and Cuc m 2 (melon). Identical amino acid residues are in red, conservative changes in orange and unrelated amino acids are in blue. The structure is highly conserved in all profilins, consisting of a central 6-stranded antiparallel β-sheet and two α-helices situated at the N- and C-terminal sides. The figure was generated using PyMOL Molecular Graphics System v1.6.</figcaption></figure>

<table><caption>TABLE 69 Basic protein characteristics of Phl p 12</caption><tr><td>Allergen source</td><td>Grass pollen</td></tr><tr><td>Protein family</td><td>Profilin</td></tr><tr><td>Biological function</td><td>Actin binding protein</td></tr><tr><td>Molecular structure</td><td>Central 6-stranded β sheet and two α-helices</td></tr><tr><td>Theoretical MW</td><td>14 kDa</td></tr><tr><td>Length</td><td>131 amino acids</td></tr><tr><td>Ligand binding</td><td>Actin, poly-L proline</td></tr><tr><td>Glycosylation</td><td>None</td></tr><tr><td>Distribution</td><td>Every plant cell</td></tr></table>

unrelated plants¹³⁹⁵. As a rule, profilin sensitization follows sensitization to a primary, major allergenic pollen source. In most cases, grass pollen is responsible for profilin hypersensitivity but, depending on geographical differences, also birch pollen, ragweed pollen, and mugwort pollen may act as primary sensitizers.⁶¹⁰,¹³⁹⁵ Being a minor pollen allergen, profilin sensitization is almost always associated with the sensitization to major pollen allergens. Assessing the clinical relevance of profilin as an airborne allergen is quite complicated, and in effect, it has been seldom investigated. In a Spanish study, only profilin-sensitized, pollen-allergic patients scored positive on a conjunctival provocation test with date palm profilin, thus suggesting that profilin may act as an aeroallergen.¹³⁹⁶ Another study based on nasal/bronchial provocation with date palm profilin in sensitized subjects confirmed this finding.¹³⁹⁷ However, in a field
# C01 - Profilins

Riccardo Asero, Domingo Barber, Giorgio Celi

## Highlights

* Up to 50% of pollen allergic patients are sensitized to profilin.

* Sensitization virtually always follows primary sensitization to a specific pollen source.

* On SPT most pollen sources score positive.

* Clinical relevance is variable but potentially present.

* Up to 50% of sensitized patients may have food allergy, oral allergy syndrome in most cases.

* Clinical reactivity to raw tomato, melon, watermelon, and/or citrus fruits is typically associated with profilin hypersensitivity

* Patients tolerate processed foods.

* The spectrum of offending plant foods is sometimes very large.

## 1 - The protein

Profilin is a protein of 12-15 kDa in size present in all eukaryotic cells and involved in the organization of cytoskeleton as well as in signal transduction. Although it can form oligomeric assemblies (mostly as a consequence of protein purification and storage processes) profilin is a monomeric actin-binding protein and a key regulator of actin-filament dynamics during processes such as cell movement, cytokinesis, and signalling¹³⁹³ (Figure 143, Table 69). In higher plants, it is identified as an allergen in monocot and dicot angiosperms.

## 2 - The protein family

As mentioned above, profilins are present in all eukaryotic cells, and in effect there is an officially registered profilin (Tyr p 36) originating from the storage mite, *Tyrophagus putrescentiae*. Nonetheless, as plant profilins represent the only clinically relevant allergens of this family described so far, the present chapter will deal specifically with plant derived profilins. Profilins from higher plants constitute a family of highly conserved proteins showing sequence identities of at least 75% even between members from distantly related organisms (Tables 70 and 71). In view of the high sequence homology, cross-reactivity between profilins is extremely common and involves virtually every plant source. (Figure 144). Thus, profilin can be considered the archetypal pan-allergen.¹³⁸⁹

## 3 - Clinical relevance of profilin

### A) As an airborne allergen (Table 70)

Profilins are able to elicit IgE responses in 10-60% of pollen-allergic patients¹³⁹⁴,¹³⁹⁵; however, the sensitization prevalence seems on the rise, as more and more allergic patients seen at allergy departments show sensitization to a large number of botanically

<figure><img src="image_261.png" /><figcaption>FIGURE 143 Three dimensional structures of Phl p 12 (grass pollen) and Cuc m 2 (melon). Identical amino acid residues are in red, conservative changes in orange and unrelated amino acids are in blue. The structure is highly conserved in all profilins, consisting of a central 6-stranded antiparallel β-sheet and two α-helices situated at the N- and C-terminal sides. The figure was generated using PyMOL Molecular Graphics System v1.6.</figcaption></figure>

<table><caption>TABLE 69 Basic protein characteristics of Phl p 12</caption><tr><td>Allergen source</td><td>Grass pollen</td></tr><tr><td>Protein family</td><td>Profilin</td></tr><tr><td>Biological function</td><td>Actin binding protein</td></tr><tr><td>Molecular structure</td><td>Central 6-stranded β sheet and two α-helices</td></tr><tr><td>Theoretical MW</td><td>14 kDa</td></tr><tr><td>Length</td><td>131 amino acids</td></tr><tr><td>Ligand binding</td><td>Actin, poly-L proline</td></tr><tr><td>Glycosylation</td><td>None</td></tr><tr><td>Distribution</td><td>Every plant cell</td></tr></table>

unrelated plants¹³⁹⁵. As a rule, profilin sensitization follows sensitization to a primary, major allergenic pollen source. In most cases, grass pollen is responsible for profilin hypersensitivity but, depending on geographical differences, also birch pollen, ragweed pollen, and mugwort pollen may act as primary sensitizers.⁶¹⁰,¹³⁹⁵ Being a minor pollen allergen, profilin sensitization is almost always associated with the sensitization to major pollen allergens. Assessing the clinical relevance of profilin as an airborne allergen is quite complicated, and in effect, it has been seldom investigated. In a Spanish study, only profilin-sensitized, pollen-allergic patients scored positive on a conjunctival provocation test with date palm profilin, thus suggesting that profilin may act as an aeroallergen.¹³⁹⁶ Another study based on nasal/bronchial provocation with date palm profilin in sensitized subjects confirmed this finding.¹³⁹⁷ However, in a field
<table><caption>TABLE 70 Selection of profilins from different pollen sources</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="7">Fagales</td><td>Birch (Betula pendula)</td><td>Bet v 2</td></tr><tr><td>Hazel tree (Corylus avellana)</td><td>Cor a 2</td></tr><tr><td>Alder (Alnus glutinosa)</td><td>*Aln g 2</td></tr><tr><td>Hornbeam (Carpinus betulus)</td><td>*Car b 2</td></tr><tr><td>Oak (Quercus alba)</td><td>*Que a 2</td></tr><tr><td>Beech (Fagus sylvatica)</td><td>*Fag s 2</td></tr><tr><td>Graminae</td><td>Timothy (Phleum pratense)</td><td>Phl p 12</td></tr><tr><td rowspan="2">Asteraceae</td><td>All other grass pollen species</td><td>"Allergen" 12</td></tr><tr><td>Mugwort (Artemisia vulgaris)</td><td>Art v 4</td></tr><tr><td rowspan="2">Urticaceae</td><td>Ragweed (Ambrosia artemisiifolia)</td><td>Amb a 8</td></tr><tr><td>Pellitory (Parietaria judaica)</td><td>Par j 3</td></tr><tr><td rowspan="2">Oleaceae</td><td>Olive (Olea europaea)</td><td>Ole e 2</td></tr><tr><td>Ash (Fraxinus excelsior)</td><td>*Fra e 2</td></tr><tr><td>Cupressaceae</td><td>Cypress (Cupressus sempervirens)</td><td>*Cup s 8</td></tr><tr><td rowspan="2">Euphorbiaceae</td><td>Annual mercury (Mercurialis annua)</td><td>Mer a 1</td></tr><tr><td>Date palm (Phoenix dactylifera)</td><td>Pho d 2</td></tr></tbody></table>

*additional allergens listed in allergome

study, the clinical impact of profilin hypersensitivity turned out to be rather limited, as most sensitized patients reported symptoms only in the specific season of the primary sensitizing pollen source.⁵⁷⁹ Nonetheless, the only one case of primary sensitization to profilin reported so far suffered from long lasting seasonal symptoms.¹³⁹⁸ Further, recent studies showed that in certain geographic areas profilin sensitization represents a marker of more severe respiratory allergy in patients with pollen-mediated rhino-conjunctivitis and asthma, probably because in most cases it occurs in patients with multiple primary sensitizations to different sources.¹³⁹⁹

### Clinical relevance of profilin as an airborne allergen

* Up to 50% of pollen allergic patients are sensitized to profilin.

* Sensitization virtually always follows primary sensitization to a specific pollen source.

* On SPT most pollen sources score positive.

* Clinical relevance is variable but potentially present.

### B) As a plant food allergen (Table 71)

Although profilin is present in every plant-derived food, its relevance as a food allergen has long been underestimated.⁵²,¹⁴⁰⁰ Nonetheless, its role as a plant food allergen in about 50% of sensitized subjects has recently emerged¹¹³⁵, with clinical allergy to certain foods such as melon, watermelon, citrus fruits, banana, pineapple, persimmon, zucchini, and tomato being characteristically associated with profilin hypersensitivity (see Chapter B15).⁶⁹⁴,¹¹³⁵,¹⁴⁰¹⁻¹⁴⁰⁷ In view of the high pepsin sensitivity of this

<table><caption>TABLE 71 Profilins from some plant foods</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="3">Rosaceae</td><td>Apple (Malus domestica)</td><td>Mal d 4</td></tr><tr><td>Peach (Prunus persica)</td><td>Pru p 4</td></tr><tr><td>Pear (Pyrus communis)</td><td>Pyr p 4</td></tr><tr><td>Cucurbitaceae</td><td>Melon (Cucumis melo)</td><td>Cuc m 2</td></tr><tr><td>Actinidiaceae</td><td>Kiwi (Actinidia deliciosa)</td><td>Act d 9</td></tr><tr><td rowspan="2">Apiaceae</td><td>Celery (Apium graveolens)</td><td>Api g 4</td></tr><tr><td>Carrot (Daucus carota)</td><td>Dau c 4</td></tr><tr><td>Rutaceae</td><td>Orange (Citrus sinensis)</td><td>Cit s 2</td></tr><tr><td rowspan="2">Leguminosae</td><td>Peanut (Arachis hypogaea)</td><td>Ara h 5</td></tr><tr><td>Soybean (Glycine maxima)</td><td>Gly m 3</td></tr><tr><td>Solanaceae</td><td>Tomato (Solanum lycopersicum)</td><td>Sola l 1</td></tr><tr><td>Bromeliaceae</td><td>Pineapple (Ananas comosus)</td><td>Ana c 1</td></tr><tr><td>Corylaceae</td><td>Hazelnut (Corylus avellana)</td><td>Cor a 2</td></tr><tr><td>Brassicaceae</td><td>Yellow mustard (Sinapis alba)</td><td>Sin a 4</td></tr><tr><td>Asteraceae</td><td>Sunflower (Helianthus annuus)</td><td>Hel a 2</td></tr><tr><td>Moraceae</td><td>Fig (Ficus carica)</td><td>*Fic c 4</td></tr></tbody></table>

*additional allergens listed in allergome

protein¹⁴⁰², the clinical expression of profilin-induced food allergy is in most cases the oral allergy syndrome. However, cases of systemic allergic reactions induced by profilin have been reported in specific areas in Spain where the levels of grass pollen allergy are extremely high (Figure 145). Oral provocation of food allergic patients with low doses of purified profilin has proven to induce severe reactions in grass pollen (GP) allergic patients resident in areas with high grass pollen exposure.¹⁴⁰⁸ This fact should be taken into account when evaluating severe food allergic reactions in areas where GP allergy is dominant. Further, recent studies showed that under certain conditions (i.e., in the presence of certain specific co-factors) labile plant food allergens, including profilin, are able to induce systemic allergic reactions in patients not reactive to stable allergens.³³⁸ Finally, one study showed that plant food-induced allergic reactions in profilin-hypersensitive individuals are associated with significant damage to the epithelial barrier of the oral mucosa. Such damage favours profilin penetration into the oral mucosa with subsequent local inflammation³³⁹. Another physical feature that may contribute to the "reduced allergenicity" of profilins is their low thermal stability.¹⁴⁰⁹

### Clinical relevance of profilin as a plant food allergen

* Up to 50% of sensitized patients may have food allergy

* Oral allergy syndrome in most cases

* Raw tomato, melon, watermelon, and citrus fruits are typically associated with profilin sensitization

* Patients tolerate processed foods

* Spectrum of offending plant foods is sometimes very large
<table><caption>TABLE 70 Selection of profilins from different pollen sources</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="7">Fagales</td><td>Birch (Betula pendula)</td><td>Bet v 2</td></tr><tr><td>Hazel tree (Corylus avellana)</td><td>Cor a 2</td></tr><tr><td>Alder (Alnus glutinosa)</td><td>*Aln g 2</td></tr><tr><td>Hornbeam (Carpinus betulus)</td><td>*Car b 2</td></tr><tr><td>Oak (Quercus alba)</td><td>*Que a 2</td></tr><tr><td>Beech (Fagus sylvatica)</td><td>*Fag s 2</td></tr><tr><td>Graminae</td><td>Timothy (Phleum pratense)</td><td>Phl p 12</td></tr><tr><td rowspan="2">Asteraceae</td><td>All other grass pollen species</td><td>"Allergen" 12</td></tr><tr><td>Mugwort (Artemisia vulgaris)</td><td>Art v 4</td></tr><tr><td rowspan="2">Urticaceae</td><td>Ragweed (Ambrosia artemisiifolia)</td><td>Amb a 8</td></tr><tr><td>Pellitory (Parietaria judaica)</td><td>Par j 3</td></tr><tr><td rowspan="2">Oleaceae</td><td>Olive (Olea europaea)</td><td>Ole e 2</td></tr><tr><td>Ash (Fraxinus excelsior)</td><td>*Fra e 2</td></tr><tr><td>Cupressaceae</td><td>Cypress (Cupressus sempervirens)</td><td>*Cup s 8</td></tr><tr><td rowspan="2">Euphorbiaceae</td><td>Annual mercury (Mercurialis annua)</td><td>Mer a 1</td></tr><tr><td>Date palm (Phoenix dactylifera)</td><td>Pho d 2</td></tr></tbody></table>

*additional allergens listed in allergome

study, the clinical impact of profilin hypersensitivity turned out to be rather limited, as most sensitized patients reported symptoms only in the specific season of the primary sensitizing pollen source.⁵⁷⁹ Nonetheless, the only one case of primary sensitization to profilin reported so far suffered from long lasting seasonal symptoms.¹³⁹⁸ Further, recent studies showed that in certain geographic areas profilin sensitization represents a marker of more severe respiratory allergy in patients with pollen-mediated rhino-conjunctivitis and asthma, probably because in most cases it occurs in patients with multiple primary sensitizations to different sources.¹³⁹⁹

### Clinical relevance of profilin as an airborne allergen

* Up to 50% of pollen allergic patients are sensitized to profilin.

* Sensitization virtually always follows primary sensitization to a specific pollen source.

* On SPT most pollen sources score positive.

* Clinical relevance is variable but potentially present.

### B) As a plant food allergen (Table 71)

Although profilin is present in every plant-derived food, its relevance as a food allergen has long been underestimated.⁵²,¹⁴⁰⁰ Nonetheless, its role as a plant food allergen in about 50% of sensitized subjects has recently emerged¹¹³⁵, with clinical allergy to certain foods such as melon, watermelon, citrus fruits, banana, pineapple, persimmon, zucchini, and tomato being characteristically associated with profilin hypersensitivity (see Chapter B15).⁶⁹⁴,¹¹³⁵,¹⁴⁰¹⁻¹⁴⁰⁷ In view of the high pepsin sensitivity of this

<table><caption>TABLE 71 Profilins from some plant foods</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="3">Rosaceae</td><td>Apple (Malus domestica)</td><td>Mal d 4</td></tr><tr><td>Peach (Prunus persica)</td><td>Pru p 4</td></tr><tr><td>Pear (Pyrus communis)</td><td>Pyr p 4</td></tr><tr><td>Cucurbitaceae</td><td>Melon (Cucumis melo)</td><td>Cuc m 2</td></tr><tr><td>Actinidiaceae</td><td>Kiwi (Actinidia deliciosa)</td><td>Act d 9</td></tr><tr><td rowspan="2">Apiaceae</td><td>Celery (Apium graveolens)</td><td>Api g 4</td></tr><tr><td>Carrot (Daucus carota)</td><td>Dau c 4</td></tr><tr><td>Rutaceae</td><td>Orange (Citrus sinensis)</td><td>Cit s 2</td></tr><tr><td rowspan="2">Leguminosae</td><td>Peanut (Arachis hypogaea)</td><td>Ara h 5</td></tr><tr><td>Soybean (Glycine maxima)</td><td>Gly m 3</td></tr><tr><td>Solanaceae</td><td>Tomato (Solanum lycopersicum)</td><td>Sola l 1</td></tr><tr><td>Bromeliaceae</td><td>Pineapple (Ananas comosus)</td><td>Ana c 1</td></tr><tr><td>Corylaceae</td><td>Hazelnut (Corylus avellana)</td><td>Cor a 2</td></tr><tr><td>Brassicaceae</td><td>Yellow mustard (Sinapis alba)</td><td>Sin a 4</td></tr><tr><td>Asteraceae</td><td>Sunflower (Helianthus annuus)</td><td>Hel a 2</td></tr><tr><td>Moraceae</td><td>Fig (Ficus carica)</td><td>*Fic c 4</td></tr></tbody></table>

*additional allergens listed in allergome

protein¹⁴⁰², the clinical expression of profilin-induced food allergy is in most cases the oral allergy syndrome. However, cases of systemic allergic reactions induced by profilin have been reported in specific areas in Spain where the levels of grass pollen allergy are extremely high (Figure 145). Oral provocation of food allergic patients with low doses of purified profilin has proven to induce severe reactions in grass pollen (GP) allergic patients resident in areas with high grass pollen exposure.¹⁴⁰⁸ This fact should be taken into account when evaluating severe food allergic reactions in areas where GP allergy is dominant. Further, recent studies showed that under certain conditions (i.e., in the presence of certain specific co-factors) labile plant food allergens, including profilin, are able to induce systemic allergic reactions in patients not reactive to stable allergens.³³⁸ Finally, one study showed that plant food-induced allergic reactions in profilin-hypersensitive individuals are associated with significant damage to the epithelial barrier of the oral mucosa. Such damage favours profilin penetration into the oral mucosa with subsequent local inflammation³³⁹. Another physical feature that may contribute to the "reduced allergenicity" of profilins is their low thermal stability.¹⁴⁰⁹

### Clinical relevance of profilin as a plant food allergen

* Up to 50% of sensitized patients may have food allergy

* Oral allergy syndrome in most cases

* Raw tomato, melon, watermelon, and citrus fruits are typically associated with profilin sensitization

* Patients tolerate processed foods

* Spectrum of offending plant foods is sometimes very large
<figure><img src="image_256.png" /><figcaption>FIGURE 144 Cross-reactivity between profilins from different pollen sources and plant foods</figcaption></figure>

<figure><img src="image_257.png" /><figcaption>FIGURE 145 Areas of Spain characterized by a high prevalence of profilin sensitization (dots) where some cases of severe profilin-induced food allergy have been recorded.</figcaption></figure>

### C) Profilin and natural rubber latex allergy

The end of the last century and the beginning of the current one have been characterized by an impressive increase in the prevalence of allergy to natural rubber latex. NRL contains many allergenic proteins, including profilin (Hev b 8). Therefore, the crude extract of *Hevea brasiliensis* latex scores often positive in patients with multiple pollen sensitization. This has frequently caused concern for the risk of intra-operative anaphylactic reactions. However, most latex products have been replaced by synthetic products and therefore the risk of allergic reactions due to latex allergens including profilin is no longer a health issue. Furthermore, patients who show uniquely IgE reactivity to profilin in NRL can undergo surgery and other medical procedures without any risk.¹⁴¹⁰,¹⁴¹¹

## 4 - Clinical management

Profilin hypersensitivity can be diagnosed *in-vivo* by SPT using a commercial profilin-enriched date palm pollen extract that has been

available only in Italy, Spain, and Austria ²⁸⁰; such extract for skin testing shows a sensitivity and specificity that is very close to that of the recombinant grass pollen profilin for *in-vitro* use (Phl p 12).¹⁴¹² Unfortunately, due to problems in registration at national regulatory agencies this product is currently no longer being commercialized in certain countries. Several recombinant profilins are currently available for the routine *in-vitro* diagnosis of IgE hypersensitivity. In the ImmunoCAP Specific IgE test (Thermo Fisher Scientific) Phl p 12, Bet v 2, Pru p 4, and Hev b 8, the profilins from grass pollen, birch pollen, peach, and natural rubber latex are the 4 profilins currently available. In the ImmunoCAP ISAC multiplex assay, Mer a 1 (from annual mercury) is present as well (in a former version of the assay Ole e 2, the olive pollen profilin, has also been present but is currently withdrawn due to its low sensitivity¹⁴¹³). A second multi-allergen panel commercially available, namely the ALEX 2, shows sensitivities to the profilins Phl p 12, Bet v 2, Hev b 8, Pho d 2 (date palm), Mer a 1 (annual mercury), and Cuc m 2 (melon). Regarding the clinical significance of profilin-specific IgE levels, their value in predicting an oral allergy syndrome seems extremely limited or absent.¹⁴¹⁴ Since profilins are extremely well conserved and cross-reacting and show an
<figure><img src="image_262.png" /><figcaption>FIGURE 144 Cross-reactivity between profilins from different pollen sources and plant foods</figcaption></figure>

<figure><img src="image_263.png" /><figcaption>FIGURE 145 Areas of Spain characterized by a high prevalence of profilin sensitization (dots) where some cases of severe profilin-induced food allergy have been recorded.</figcaption></figure>

### C) Profilin and natural rubber latex allergy

The end of the last century and the beginning of the current one have been characterized by an impressive increase in the prevalence of allergy to natural rubber latex. NRL contains many allergenic proteins, including profilin (Hev b 8). Therefore, the crude extract of *Hevea brasiliensis* latex scores often positive in patients with multiple pollen sensitization. This has frequently caused concern for the risk of intra-operative anaphylactic reactions. However, most latex products have been replaced by synthetic products and therefore the risk of allergic reactions due to latex allergens including profilin is no longer a health issue. Furthermore, patients who show uniquely IgE reactivity to profilin in NRL can undergo surgery and other medical procedures without any risk.¹⁴¹⁰,¹⁴¹¹

## 4 - Clinical management

Profilin hypersensitivity can be diagnosed *in-vivo* by SPT using a commercial profilin-enriched date palm pollen extract that has been

available only in Italy, Spain, and Austria ²⁸⁰; such extract for skin testing shows a sensitivity and specificity that is very close to that of the recombinant grass pollen profilin for *in-vitro* use (Phl p 12).¹⁴¹² Unfortunately, due to problems in registration at national regulatory agencies this product is currently no longer being commercialized in certain countries. Several recombinant profilins are currently available for the routine *in-vitro* diagnosis of IgE hypersensitivity. In the ImmunoCAP Specific IgE test (Thermo Fisher Scientific) Phl p 12, Bet v 2, Pru p 4, and Hev b 8, the profilins from grass pollen, birch pollen, peach, and natural rubber latex are the 4 profilins currently available. In the ImmunoCAP ISAC multiplex assay, Mer a 1 (from annual mercury) is present as well (in a former version of the assay Ole e 2, the olive pollen profilin, has also been present but is currently withdrawn due to its low sensitivity¹⁴¹³). A second multi-allergen panel commercially available, namely the ALEX 2, shows sensitivities to the profilins Phl p 12, Bet v 2, Hev b 8, Pho d 2 (date palm), Mer a 1 (annual mercury), and Cuc m 2 (melon). Regarding the clinical significance of profilin-specific IgE levels, their value in predicting an oral allergy syndrome seems extremely limited or absent.¹⁴¹⁴ Since profilins are extremely well conserved and cross-reacting and show an
equivalent IgE-binding potency<sup>1415,1416</sup> one wonders if a representative marker allergen is sufficient to detect hypersensitive patients. In this sense, one study showed that one single marker protein is sufficient to diagnose or exclude sensitization.<sup>1413</sup> As for the homology between profilins from different sources, extracts from two pollen sources, namely Parietaria and cypress, score frequently negative in profilin hypersensitive patients.<sup>694</sup> Whether this depends on a more limited cross-reactivity of profilins from these two plants with the other profilins.<sup>1417,1623</sup> or on a lower concentration of the protein in the extract<sup>1418</sup> is still a matter of debate; further studies are needed to clarify this point. Profilin hypersensitivity<sup>610</sup> does not normally require special clinical measures. Although most pollen sources are probably able to induce profilin hypersensitivity, from a respiratory point of view the clinical impact of the inhaled protein is in most cases quite limited.<sup>579</sup> Interestingly enough, profilin co-sensitization is associated with less severe reactions to foods in patients hypersensitive to lipid transfer proteins or seed storage proteins as well as, in certain geographic areas, with less severe respiratory allergy in patients sensitized to airborne allergens.<sup>1419</sup> Some studies have demonstrated the disappearance of profilin-associated food allergy in subjects submitted to injection immunotherapy with different pollen extracts.<sup>613,1420</sup> Regarding allergen immunotherapy, one study showed that commercial extracts for allergen immunotherapy of most pollens (except Parietaria) are rich in profilin and hence potentially able to desensitize to this allergen,<sup>1421</sup> although grass pollen extracts have the highest relative content.<sup>1397</sup> Profilin strong reactors constitute a good model to understand the link between respiratory and food allergy and to understand the causes underlying severe allergic phenotypes. Multiomic analysis of grass pollen allergic, profilin sensitized patients, suffering from severe profilin-mediated food reactions, allowed to conclude that uncontrolled T cell proliferation induced by exposure to profilin might explain these severe phenotypes.<sup>1422</sup> In fact, differential profilin induced T cell proliferation in profilin sensitized subjects has been described comparing patients from Denmark and Spain.<sup>1423</sup> This model, together with other sharing similar severe allergic phenotypes, could offer new clues to understand allergic disease evolution.<sup>1424</sup> In conclusion, the data coming from the most recent studies support the need of a change in the way we have been looking at this allergen.<sup>693</sup>

# 5 - Clinical cases

## Case 1

**Clinical History:** A 32-year-old woman resident in Extremadura, a heavily grass-exposed area, with pollen allergy and clinical history of oral allergy syndrome, urticaria, and asthma following the ingestion of melon, watermelon, banana, peach and orange. Open challenge with melon was positive.

**Tests with extracts:** On SPT, with exception of cypress, all pollen scored positive, and no reactivity to peach LTP was detected.

**Tests with molecules:** ImmunoCAP ISAC (ISU-E) microarray scored positive for: Cyn d 1: 76.7, Phl p 1: 75, Phl p 2: 21, Phl p 4: 1.0, Phl p 5: 51.5, Phl p 6: 2.6, Phl p 11: 0.73, Bet

v 2: 6.39, Hev b 8: 5.14, Mer a 1: 8.94, Phl p 12: 1.64, Mux F3: 0.67, Ole e 1: 0.37.

**Oral challenges:** On DBPCFC, the administration of 74 µg of pure date palm pollen profilin induced OAS and FEV1 decline of 20%, that were treated with antihistamine and bronchodilators.

**Diagnosis:** Grass pollen allergy with severe profilin-mediated food allergy was eventually diagnosed.

## Case 2

**Clinical history:** A 38-year-old man living in the surroundings of Milan, Italy. At the age of 32 starts having severe rhino-conjunctivitis from mid-August to the end of September when he returns home from the summer holidays at the sea (where he is well). After 3 years, oral itching following the ingestion of melon, watermelon, tomato, banana, orange, and peach appears.

**Tests with extracts:** SPT with pollen allergens score positive for grass, mugwort, ragweed, plantain, birch, hazel, plane, and olive, and negative for Parietaria and cypress. A SPT with profilin-enriched date palm pollen extracts scores intensely positive.

**Tests with molecules:** The ImmunoCAP Specific IgE test scores strongly positive for Amb a 1 and Phl p 12, and negative for Phl p 1, Phl p 5, Phl p 7, Art v 1, Pla a 1, Pla l 1, Bet v 1, Cor a 1, Ole e 1, Par j 2, and Pru p 3.

**Diagnosis:** Respiratory allergy to ragweed and profilin-induced food allergy is eventually diagnosed.
equivalent IgE-binding potency<sup>1415,1416</sup> one wonders if a representative marker allergen is sufficient to detect hypersensitive patients. In this sense, one study showed that one single marker protein is sufficient to diagnose or exclude sensitization.<sup>1413</sup> As for the homology between profilins from different sources, extracts from two pollen sources, namely Parietaria and cypress, score frequently negative in profilin hypersensitive patients.<sup>694</sup> Whether this depends on a more limited cross-reactivity of profilins from these two plants with the other profilins.<sup>1417,1623</sup> or on a lower concentration of the protein in the extract<sup>1418</sup> is still a matter of debate; further studies are needed to clarify this point. Profilin hypersensitivity<sup>610</sup> does not normally require special clinical measures. Although most pollen sources are probably able to induce profilin hypersensitivity, from a respiratory point of view the clinical impact of the inhaled protein is in most cases quite limited.<sup>579</sup> Interestingly enough, profilin co-sensitization is associated with less severe reactions to foods in patients hypersensitive to lipid transfer proteins or seed storage proteins as well as, in certain geographic areas, with less severe respiratory allergy in patients sensitized to airborne allergens.<sup>1419</sup> Some studies have demonstrated the disappearance of profilin-associated food allergy in subjects submitted to injection immunotherapy with different pollen extracts.<sup>613,1420</sup> Regarding allergen immunotherapy, one study showed that commercial extracts for allergen immunotherapy of most pollens (except Parietaria) are rich in profilin and hence potentially able to desensitize to this allergen,<sup>1421</sup> although grass pollen extracts have the highest relative content.<sup>1397</sup> Profilin strong reactors constitute a good model to understand the link between respiratory and food allergy and to understand the causes underlying severe allergic phenotypes. Multiomic analysis of grass pollen allergic, profilin sensitized patients, suffering from severe profilin-mediated food reactions, allowed to conclude that uncontrolled T cell proliferation induced by exposure to profilin might explain these severe phenotypes.<sup>1422</sup> In fact, differential profilin induced T cell proliferation in profilin sensitized subjects has been described comparing patients from Denmark and Spain.<sup>1423</sup> This model, together with other sharing similar severe allergic phenotypes, could offer new clues to understand allergic disease evolution.<sup>1424</sup> In conclusion, the data coming from the most recent studies support the need of a change in the way we have been looking at this allergen.<sup>693</sup>

# 5 – Clinical cases

## Case 1

**Clinical History:** A 32-year-old woman resident in Extremadura, a heavily grass-exposed area, with pollen allergy and clinical history of oral allergy syndrome, urticaria, and asthma following the ingestion of melon, watermelon, banana, peach and orange. Open challenge with melon was positive.

**Tests with extracts:** On SPT, with exception of cypress, all pollen scored positive, and no reactivity to peach LTP was detected.

**Tests with molecules:** ImmunoCAP ISAC (ISU-E) microarray scored positive for: Cyn d 1: 76.7, Phl p 1: 75, Phl p 2: 21, Phl p 4: 1.0, Phl p 5: 51.5, Phl p 6: 2.6, Phl p 11: 0.73, Bet

v 2: 6.39, Hev b 8: 5.14, Mer a 1: 8.94, Phl p 12: 1.64, Mux F3: 0.67, Ole e 1: 0.37.

**Oral challenges:** On DBPCFC, the administration of 74 µg of pure date palm pollen profilin induced OAS and FEV1 decline of 20%, that were treated with antihistamine and bronchodilators.

**Diagnosis:** Grass pollen allergy with severe profilin-mediated food allergy was eventually diagnosed.

## Case 2

**Clinical history:** A 38-year-old man living in the surroundings of Milan, Italy. At the age of 32 starts having severe rhino-conjunctivitis from mid-August to the end of September when he returns home from the summer holidays at the sea (where he is well). After 3 years, oral itching following the ingestion of melon, watermelon, tomato, banana, orange, and peach appears.

**Tests with extracts:** SPT with pollen allergens score positive for grass, mugwort, ragweed, plantain, birch, hazel, plane, and olive, and negative for Parietaria and cypress. A SPT with profilin-enriched date palm pollen extracts scores intensely positive.

**Tests with molecules:** The ImmunoCAP Specific IgE test scores strongly positive for Amb a 1 and Phl p 12, and negative for Phl p 1, Phl p 5, Phl p 7, Art v 1, Pla a 1, Pla l 1, Bet v 1, Cor a 1, Ole e 1, Par j 2, and Pru p 3.

**Diagnosis:** Respiratory allergy to ragweed and profilin-induced food allergy is eventually diagnosed.