<table><caption>TABLE 54 Peanut allergens (Only allergens approved by WHO/IUIS Allergen Nomenclature Sub-Committee; www.allergen.org, 08-09-2021)</caption><thead><tr><td>Allergen</td><td>Protein family</td><td>Other names</td><td>Molecular weight (kDa)</td><td>Heat stability</td></tr></thead><tbody><tr><td>Ara h 1</td><td>Vicillin</td><td>7S globulin</td><td>64</td><td>Yes</td></tr><tr><td>Ara h 2</td><td>2S albumin</td><td>Conglutin</td><td>17</td><td>Yes</td></tr><tr><td>Ara h 3</td><td>Legumin</td><td>Glycinin, 11S globulin</td><td>60, 37 (fragment)*</td><td>Yes</td></tr><tr><td>Ara h 4</td><td colspan="4">Renamed to Ara h 3.02, number not available for future submissions</td></tr><tr><td>Ara h 5</td><td>Profilin</td><td></td><td>15</td><td>No</td></tr><tr><td>Ara h 6</td><td>2S albumin</td><td>Conglutin</td><td>15</td><td>Yes</td></tr><tr><td>Ara h 7</td><td>2S albumin</td><td>Conglutin</td><td>15</td><td>Yes</td></tr><tr><td>Ara h 8</td><td>Bet v 1</td><td>Pathogenesis-related protein</td><td>17</td><td>No</td></tr><tr><td>Ara h 9</td><td>Non-specific lipid-transfer protein type 1</td><td>(PR)-10</td><td>9.8</td><td>Yes</td></tr><tr><td>Ara h 10</td><td>Oleosin</td><td></td><td>16</td><td>Yes</td></tr><tr><td>Ara h 11</td><td>Oleosin</td><td></td><td>14</td><td>Yes</td></tr><tr><td>Ara h 12</td><td>Defensin</td><td></td><td>8 (reducing)*, 12 (non-reducing)*, 5.184 (mass)*</td><td>To be expected</td></tr><tr><td>Ara h 13</td><td>Defensin</td><td></td><td>8 (reducing)*, 11 (non-reducing)*, 5.472 (mass)*</td><td>To be expected</td></tr><tr><td>Ara h 14</td><td>Oleosin</td><td></td><td>17.5</td><td>Yes</td></tr><tr><td>Ara h 15</td><td>Oleosin</td><td></td><td>17</td><td>Yes</td></tr><tr><td>Ara h 16</td><td>Non-specific lipid transfer protein 2</td><td></td><td>8.5</td><td>Yes</td></tr><tr><td>Ara h 17</td><td>Non-specific lipid transfer protein 1</td><td></td><td>11</td><td>Yes</td></tr><tr><td>Ara h 18</td><td>Cyclophilin - peptidyl-prolyl cis-trans isomerase</td><td></td><td>21</td><td>???</td></tr></tbody></table>

<figure><img src="image_229.png" /><figcaption>FIGURE 115 Identified peanut allergens</figcaption></figure>

were found to be major allergens for Western/Central and Southern Europeans.¹²³⁵

In patients with clinically relevant peanut allergy, it had been shown that 76–96% of peanut-allergic children and adolescents in the US and

Central and Northern Europe possess specific IgEs that respond to Ara h 2 and Ara h 6, compared with only 42% in Spain.¹⁶⁶⁴ The sensitization rates for Ara h 1 are between 63% and 80%. Those for Ara h 3 are somehow lower, while the rate for Ara h 7 is only 43%8. Ara h 9 is considered a secondary food allergen, particularly in Mediterranean countries.¹⁶⁶⁵ This secondary sensitization/ cross-reaction is likely due to other on-specific lipid transfer proteins (nsLTPs) (e.g., Pru p 3 in peach). Sensitizations to the Bet v 1-homologous PR-10 protein Ara h 8, the profilin Ara h 5, and glycoproteins (CCD) are usually caused by cross-reactions to pollen allergens. Sensitization rates vary depending on regional pollen exposure and on eating habits. The prevalence of sensitization to Ara h 10/11 and Ara h 14/15 is similar to that of Ara h 2 in a German cohort but still has to be investigated in larger groups of patients.¹²⁴¹ The fact that oleosins may be underrepresented or absent in aqueous peanut extracts represents a diagnostic gap hampering the identification of affected patients.¹²³⁶

## 5 – Clinical patterns of peanut allergy

Three distinct scenarios can prompt sensitizations and potential clinical symptoms depending on the different routes of exposure and physicochemical properties of the involved peanut proteins:
<table><caption>TABLE 54 Peanut allergens (Only allergens approved by WHO/IUIS Allergen Nomenclature Sub-Committee; www.allergen.org, 08-09-2021)</caption><thead><tr><td>Allergen</td><td>Protein family</td><td>Other names</td><td>Molecular weight (kDa)</td><td>Heat stability</td></tr></thead><tbody><tr><td>Ara h 1</td><td>Vicillin</td><td>7S globulin</td><td>64</td><td>Yes</td></tr><tr><td>Ara h 2</td><td>2S albumin</td><td>Conglutin</td><td>17</td><td>Yes</td></tr><tr><td>Ara h 3</td><td>Legumin</td><td>Glycinin, 11S globulin</td><td>60, 37 (fragment)*</td><td>Yes</td></tr><tr><td>Ara h 4</td><td colspan="4">Renamed to Ara h 3.02, number not available for future submissions</td></tr><tr><td>Ara h 5</td><td>Profilin</td><td></td><td>15</td><td>No</td></tr><tr><td>Ara h 6</td><td>2S albumin</td><td>Conglutin</td><td>15</td><td>Yes</td></tr><tr><td>Ara h 7</td><td>2S albumin</td><td>Conglutin</td><td>15</td><td>Yes</td></tr><tr><td>Ara h 8</td><td>Bet v 1</td><td>Pathogenesis-related protein</td><td>17</td><td>No</td></tr><tr><td>Ara h 9</td><td>Non-specific lipid-transfer protein type 1</td><td>(PR)-10</td><td>9.8</td><td>Yes</td></tr><tr><td>Ara h 10</td><td>Oleosin</td><td></td><td>16</td><td>Yes</td></tr><tr><td>Ara h 11</td><td>Oleosin</td><td></td><td>14</td><td>Yes</td></tr><tr><td>Ara h 12</td><td>Defensin</td><td></td><td>8 (reducing)*, 12 (non-reducing)*, 5.184 (mass)*</td><td>To be expected</td></tr><tr><td>Ara h 13</td><td>Defensin</td><td></td><td>8 (reducing)*, 11 (non-reducing)*, 5.472 (mass)*</td><td>To be expected</td></tr><tr><td>Ara h 14</td><td>Oleosin</td><td></td><td>17.5</td><td>Yes</td></tr><tr><td>Ara h 15</td><td>Oleosin</td><td></td><td>17</td><td>Yes</td></tr><tr><td>Ara h 16</td><td>Non-specific lipid transfer protein 2</td><td></td><td>8.5</td><td>Yes</td></tr><tr><td>Ara h 17</td><td>Non-specific lipid transfer protein 1</td><td></td><td>11</td><td>Yes</td></tr><tr><td>Ara h 18</td><td>Cyclophilin - peptidyl-prolyl cis-trans isomerase</td><td></td><td>21</td><td>???</td></tr></tbody></table>

<figure><img src="image_229.png" /><figcaption>FIGURE 115 Identified peanut allergens</figcaption></figure>

were found to be major allergens for Western/Central and Southern Europeans.¹²³⁵

In patients with clinically relevant peanut allergy, it had been shown that 76-96% of peanut-allergic children and adolescents in the US and

Central and Northern Europe possess specific IgEs that respond to Ara h 2 and Ara h 6, compared with only 42% in Spain.¹⁶⁶⁴ The sensitization rates for Ara h 1 are between 63% and 80%. Those for Ara h 3 are somehow lower, while the rate for Ara h 7 is only 43%8. Ara h 9 is considered a secondary food allergen, particularly in Mediterranean countries.¹⁶⁶⁵ This secondary sensitization/ cross-reaction is likely due to other on-specific lipid transfer proteins (nsLTPs) (e.g., Pru p 3 in peach). Sensitizations to the Bet v 1-homologous PR-10 protein Ara h 8, the profilin Ara h 5, and glycoproteins (CCD) are usually caused by cross-reactions to pollen allergens. Sensitization rates vary depending on regional pollen exposure and on eating habits. The prevalence of sensitization to Ara h 10/11 and Ara h 14/15 is similar to that of Ara h 2 in a German cohort but still has to be investigated in larger groups of patients.¹²⁴¹ The fact that oleosins may be underrepresented or absent in aqueous peanut extracts represents a diagnostic gap hampering the identification of affected patients.¹²³⁶

## 5 – Clinical patterns of peanut allergy

Three distinct scenarios can prompt sensitizations and potential clinical symptoms depending on the different routes of exposure and physicochemical properties of the involved peanut proteins:
A The sensitization to seed storage proteins (i.e., Ara h 1, 2, 3, 6, and 7), which are considered allergens predominantly in children with peanut allergies. Infants and young children with eczema are sensitized predominantly to seed storage proteins, while sensitization to pollen-related food allergens seems to be rare. Patients frequently react with immediate-type symptoms involving the skin (e.g., urticaria), the gastrointestinal tract (e.g., vomiting), the respiratory system (e.g., wheezing) and/or the cardiovascular system (e.g., drop in blood pressure). Ara h 2 and Ara h 6 are associated with a severe allergic reaction to peanut. This hazard is likely linked to the high stability of the allergens and their high proportion of the total protein content (Figure 116).

B The sensitizations to the Bet v 1-homologous PR-10 protein Ara h 8, the profilin Ara h 5, and CCD are usually caused by sensitization to pollen allergens. Birch pollen allergy is responsible for a considerable north-south gradient in Europe in terms of cross-reactions to Ara h 8; in regions of higher grass pollen exposure, increased cross-reactive IgE to Ara h 5 and CCD-containing peanut extracts can be expected. The involved proteins are largely labile to heat and digestion. Since peanuts are generally consumed roasted or cooked and not raw, mostly no or only mild and predominantly oropharyngeal symptoms develop, depending on the amount of allergen consumed.

C Ara h 9 is considered a secondary food allergen, particularly in Mediterranean countries. This secondary sensitization/cross-reaction is likely due to other nsLTP (e.g., Pru p 3 in peach).<sup>1237</sup> Since Ara h 9 possesses thermal and digestive stability, affected patients can develop systemic symptoms.<sup>1157</sup>

## 6 - Clinical diagnosis of peanut allergy

Specific questions, the proper interpretation of sensitization tests (i.e., SPT, IgE) and, if necessary, oral food challenges help to establish the diagnosis of peanut allergy.

<figure><img src="image_230.png" /><figcaption>FIGURE 116 Risk ramp for peanut allergens: increased risk for severe symptoms and anaphylactic reactions from left to right</figcaption></figure>

### Case history (anamnesis):

The patient is asked for her/his experiences with the following: previous reaction(s) to peanuts, symptoms, affected organ system(s), the onset and course (progression), co-factors (e.g., exercise, acute infection, emotional stress, disruption of routine, premenstrual status, medications, alcohol intake, previous reactions to other allergen sources (i.e., peach), additional (allergic) features (i.e., birch pollen allergy), additional atopic diseases (i.e., atopic eczema, asthma).

Working hypothesis: based on peanut-related clinical patterns A-C.

### SPT:

* Commercial peanut extract (reasonable results in case of highly abundant allergens; i.e., seed storage proteins)

* Prick-to-prick test with peanuts or offending peanut product (in case of severe anaphylactic reaction: primary IgE testing before SPT)

### IgE Testing:

Allergen-specific IgE:

* Peanut extract

* Ara h 2 and Ara h 6

* Ara h 8 and/or Bet v 1

* Ara h 9 and/or an LTP-representative (i.e., Pru p 3)

### Comments:

a. Ara h 2-specific IgE demonstrates the best diagnostic accuracy of peanut allergy in infants, children, and adults. Ara h 2-specific IgE at 0.35 kU/L is a useful cut-off value in the diagnostic approach for peanut allergy in children based on a systematic review and meta-analysis.<sup>1666</sup>

b. To predict a positive peanut challenge with 95% probability, the Ara h 2-specific IgE level has to exceed of 42 kU/L. To predict a negative peanut challenge with 90% probability, the Ara h 2-specific IgE level has to be <0.03 kU/L, with some discordant exceptions in German children based on a previous study<sup>150</sup> (Figure 117A,B).

c. An absolute (100%) prediction cannot be achieved with measurements of Ara h 2-specific IgE due to exceptions to the preceding. Subsequently, the individual clinical relevance of allergen-specific IgE concentrations (i.e., to single allergens of legumes) has to be determined on a case-by-case basis by the physician in charge.

d. As shown above, Ara h 2 has the greatest specificity; however, it has a lower sensitivity than do SPT and peanut-specific IgE. In a patient with a high prior probability, the clinician may use not only Ara h 2 but also SPT or peanut-specific IgE to confirm the diagnosis of peanut allergy.<sup>1238</sup>

**Stepwise approach.** The measurements of peanut-specific IgE and/or SPT are good screening parameters in patients at risk for peanut allergy. The absence of peanut-specific IgE has a high negative
A The sensitization to seed storage proteins (i.e., Ara h 1, 2, 3, 6, and 7), which are considered allergens predominantly in children with peanut allergies. Infants and young children with eczema are sensitized predominantly to seed storage proteins, while sensitization to pollen-related food allergens seems to be rare. Patients frequently react with immediate-type symptoms involving the skin (e.g., urticaria), the gastrointestinal tract (e.g., vomiting), the respiratory system (e.g., wheezing) and/or the cardiovascular system (e.g., drop in blood pressure). Ara h 2 and Ara h 6 are associated with a severe allergic reaction to peanut. This hazard is likely linked to the high stability of the allergens and their high proportion of the total protein content (Figure 116).

B The sensitizations to the Bet v 1-homologous PR-10 protein Ara h 8, the profilin Ara h 5, and CCD are usually caused by sensitization to pollen allergens. Birch pollen allergy is responsible for a considerable north-south gradient in Europe in terms of cross-reactions to Ara h 8; in regions of higher grass pollen exposure, increased cross-reactive IgE to Ara h 5 and CCD-containing peanut extracts can be expected. The involved proteins are largely labile to heat and digestion. Since peanuts are generally consumed roasted or cooked and not raw, mostly no or only mild and predominantly oropharyngeal symptoms develop, depending on the amount of allergen consumed.

C Ara h 9 is considered a secondary food allergen, particularly in Mediterranean countries. This secondary sensitization/cross-reaction is likely due to other nsLTP (e.g., Pru p 3 in peach).<sup>1237</sup> Since Ara h 9 possesses thermal and digestive stability, affected patients can develop systemic symptoms.<sup>1157</sup>

## 6 - Clinical diagnosis of peanut allergy

Specific questions, the proper interpretation of sensitization tests (i.e., SPT, IgE) and, if necessary, oral food challenges help to establish the diagnosis of peanut allergy.

<figure><img src="image_230.png" /><figcaption>FIGURE 116 Risk ramp for peanut allergens: increased risk for severe symptoms and anaphylactic reactions from left to right</figcaption></figure>

### Case history (anamnesis):

The patient is asked for her/his experiences with the following: previous reaction(s) to peanuts, symptoms, affected organ system(s), the onset and course (progression), co-factors (e.g., exercise, acute infection, emotional stress, disruption of routine, premenstrual status, medications, alcohol intake, previous reactions to other allergen sources (i.e., peach), additional (allergic) features (i.e., birch pollen allergy), additional atopic diseases (i.e., atopic eczema, asthma).

Working hypothesis: based on peanut-related clinical patterns A-C.

### SPT:

* Commercial peanut extract (reasonable results in case of highly abundant allergens; i.e., seed storage proteins)

* Prick-to-prick test with peanuts or offending peanut product (in case of severe anaphylactic reaction: primary IgE testing before SPT)

### IgE Testing:

Allergen-specific IgE:

* Peanut extract

* Ara h 2 and Ara h 6

* Ara h 8 and/or Bet v 1

* Ara h 9 and/or an LTP-representative (i.e., Pru p 3)

### Comments:

a. Ara h 2-specific IgE demonstrates the best diagnostic accuracy of peanut allergy in infants, children, and adults. Ara h 2-specific IgE at 0.35 kU/L is a useful cut-off value in the diagnostic approach for peanut allergy in children based on a systematic review and meta-analysis.<sup>1666</sup>

b. To predict a positive peanut challenge with 95% probability, the Ara h 2-specific IgE level has to exceed of 42 kU/L. To predict a negative peanut challenge with 90% probability, the Ara h 2-specific IgE level has to be <0.03 kU/L, with some discordant exceptions in German children based on a previous study<sup>150</sup> (Figure 117A,B).

c. An absolute (100%) prediction cannot be achieved with measurements of Ara h 2-specific IgE due to exceptions to the preceding. Subsequently, the individual clinical relevance of allergen-specific IgE concentrations (i.e., to single allergens of legumes) has to be determined on a case-by-case basis by the physician in charge.

d. As shown above, Ara h 2 has the greatest specificity; however, it has a lower sensitivity than do SPT and peanut-specific IgE. In a patient with a high prior probability, the clinician may use not only Ara h 2 but also SPT or peanut-specific IgE to confirm the diagnosis of peanut allergy.<sup>1238</sup>

**Stepwise approach.** The measurements of peanut-specific IgE and/or SPT are good screening parameters in patients at risk for peanut allergy. The absence of peanut-specific IgE has a high negative
predictive value. Positive peanut-specific IgE and/or SPT are only clinically relevant in the presence of corresponding symptoms.

In cases wherein the presence or absence of allergic reactions due to peanut consumption is not known, if the screening is positive, Ara h 2-specific IgE should be measured. Measurements of peanut- and Ara h 2-specific IgE play an essential role in case of suspected primary peanut allergy. A clear history of objective immediate allergic reactions to peanut and elevated Ara h 2-specific IgE are highly suggestive of a clinically relevant peanut allergy (Figure 118). An oral food challenge is often not needed. In patients with uncertain history of immediate reaction following peanut consumption, physicians can

proceed with the current diagnosis of peanut allergy according to (Figure 119). Peanut-specific IgE is sometimes detectable in children with severe atopic dermatitis and in the population. Therefore, unexpected findings of elevated IgE to peanuts are often seen in clinical practice. A stepwise approach takes the potential consequences into consideration. The most important initial question is related to the frequency (e.g., more than once a month) and time course (e.g., within the previous six weeks) of peanut consumption.

In general, the measurement of Ara h 1- and 3-specific IgE is often not necessary since mono-sensitizations to Ara h 1 and/or 3 are rare. In doubt, an oral food challenge test can clarify cases of a

<figure><img src="image_231.png" /><figcaption>FIGURE 117 Study results regarding the risk of peanut allergy associated with allergen-specific IgE to the 2S-albumin seed storage protein Ara h 2 (A) compared with whole peanut extract (B)</figcaption></figure>

<figure><img src="image_232.png" /><figcaption>FIGURE 118 Diagnostic algorithm for peanut allergy in clinical setting: Clear history of immediate reaction to peanut</figcaption></figure>
predictive value. Positive peanut-specific IgE and/or SPT are only clinically relevant in the presence of corresponding symptoms.

In cases wherein the presence or absence of allergic reactions due to peanut consumption is not known, if the screening is positive, Ara h 2-specific IgE should be measured. Measurements of peanut- and Ara h 2-specific IgE play an essential role in case of suspected primary peanut allergy. A clear history of objective immediate allergic reactions to peanut and elevated Ara h 2-specific IgE are highly suggestive of a clinically relevant peanut allergy (Figure 118). An oral food challenge is often not needed. In patients with uncertain history of immediate reaction following peanut consumption, physicians can

proceed with the current diagnosis of peanut allergy according to (Figure 119). Peanut-specific IgE is sometimes detectable in children with severe atopic dermatitis and in the population. Therefore, unexpected findings of elevated IgE to peanuts are often seen in clinical practice. A stepwise approach takes the potential consequences into consideration. The most important initial question is related to the frequency (e.g., more than once a month) and time course (e.g., within the previous six weeks) of peanut consumption.

In general, the measurement of Ara h 1- and 3-specific IgE is often not necessary since mono-sensitizations to Ara h 1 and/or 3 are rare. In doubt, an oral food challenge test can clarify cases of a

<figure><img src="image_231.png" /></figure>

<figure><img src="image_232.png" /><figcaption>FIGURE 117 Study results regarding the risk of peanut allergy associated with allergen-specific IgE to the 2S-albumin seed storage protein Ara h 2 (A) compared with whole peanut extract (B)</figcaption></figure>

<figure><img src="image_233.png" /><figcaption>FIGURE 118 Diagnostic algorithm for peanut allergy in clinical setting: Clear history of immediate reaction to peanut</figcaption></figure>
Incidental finding of peanut sensitisation

<figure><img src="image_233.png" /><figcaption>**FIGURE 119** Diagnostic algorithm for peanut allergy in clinical setting: Uncertain peanut allergy history</figcaption></figure>

negative or low IgE response to Ara h 2. If there is no specific IgE to any seed storage protein, a clinically relevant peanut allergy is unlikely, although it cannot be ruled out completely in the presence of sufficient clinical suspicion (diagnostic gap due to factors such as the lipophilic oleosins Ara h 10/11 and Ara h 14/15 missing in aqueous diagnostic extracts). Specific IgE to nsLTP Ara h 9 should be additionally determined in patients from the Mediterranean region. In patients with a typical clinical history of pollen food syndrome, specific IgE to PR-10 Ara h 8 and/or birch pollen measurement is considered (Figure 119).

Oral food challenge tests

(In case of doubtful relationship between peanut ingestion and allergic reaction (see stepwise approach (Figures 118 and 119)): Depending on the patient history of titrated oral food challenges with peanuts (e.g., whole peanuts or lightly roasted peanut flour).

6 - Clinical management of peanut allergy

Advice and avoidance

Strict avoidance of all peanut-containing products is recom-
mended. Peanuts are required to be labeled in all prepacked and
non-prepacked food items. A great problem still exists with "may
contain" labels. sensitization to tree nuts is often experienced by
patients with peanut allergy, and avoidance of all species is com-
monly advised in case of suspicion of allergy to peanut or tree nut.
However, except for patients with concomitant tree nut allergies,
several tree nuts could potentially be ingested.<sup>1239</sup>

Pharmacotherapy for treatment of accidental allergic reactions

Due to the risk of severe reactions after the unintentional
ingestion of peanut-containing products, adrenaline for self-
administration should always be on-hand.

Allergen-specific immunotherapy

Commercial products for the allergen-specific immunotherapy for peanut allergy are available. The European Academy of Allergy and Clinical Immunology Guidelines on allergen¹²⁴⁰ immunotherapy state,²⁹⁵ “Oral immunotherapy is recommended as a treatment option to increase the threshold of reaction during treatment in children with peanut allergy from approximately four to five years of age.” However, it should only be undertaken in highly specialized clinical centers with expertise and facilities to safely deliver this therapy.

7 - Clinical cases

Case 1:

**History:** Boy, two years of age. He ate a peanut snack at a friend's house. He had never eaten peanut products prior to that incidence. After 30 minutes, he developed urticaria and coughing and after 40 minutes, wheezing (Figure 118).

In vitro testing: Peanut-specific IgE 5.2 kUA/L, Ara h 2-specific IgE 3.1 kUA/L.

Diagnosis: Peanut allergy

**Oral food challenge:** An oral food challenge was not necessary for the diagnosis.
Incidental finding of peanut sensitisation

<figure><img src="image_234.png" /><figcaption>**FIGURE 119** Diagnostic algorithm for peanut allergy in clinical setting: Uncertain peanut allergy history</figcaption></figure>

negative or low IgE response to Ara h 2. If there is no specific IgE to any seed storage protein, a clinically relevant peanut allergy is unlikely, although it cannot be ruled out completely in the presence of sufficient clinical suspicion (diagnostic gap due to factors such as the lipophilic oleosins Ara h 10/11 and Ara h 14/15 missing in aqueous diagnostic extracts). Specific IgE to nsLTP Ara h 9 should be additionally determined in patients from the Mediterranean region. In patients with a typical clinical history of pollen food syndrome, specific IgE to PR-10 Ara h 8 and/or birch pollen measurement is considered (Figure 119).

Oral food challenge tests

(In case of doubtful relationship between peanut ingestion and allergic reaction (see stepwise approach (Figures 118 and 119)): Depending on the patient history of titrated oral food challenges with peanuts (e.g., whole peanuts or lightly roasted peanut flour).

6 – Clinical management of peanut allergy

Advice and avoidance

Strict avoidance of all peanut-containing products is recom-
mended. Peanuts are required to be labeled in all prepacked and
non-prepacked food items. A great problem still exists with "may
contain" labels. sensitization to tree nuts is often experienced by
patients with peanut allergy, and avoidance of all species is com-
monly advised in case of suspicion of allergy to peanut or tree nut.
However, except for patients with concomitant tree nut allergies,
several tree nuts could potentially be ingested.<sup>1239</sup>

Pharmacotherapy for treatment of accidental allergic reactions

Due to the risk of severe reactions after the unintentional
ingestion of peanut-containing products, adrenaline for self-
administration should always be on-hand.

Allergen-specific immunotherapy

Commercial products for the allergen-specific immunotherapy for peanut allergy are available. The European Academy of Allergy and Clinical Immunology Guidelines on allergen¹²⁴⁰ immunotherapy state,²⁹⁵ “Oral immunotherapy is recommended as a treatment option to increase the threshold of reaction during treatment in children with peanut allergy from approximately four to five years of age.” However, it should only be undertaken in highly specialized clinical centers with expertise and facilities to safely deliver this therapy.

7 – Clinical cases

Case 1:

**History:** Boy, two years of age. He ate a peanut snack at a friend's house. He had never eaten peanut products prior to that incidence. After 30 minutes, he developed urticaria and coughing and after 40 minutes, wheezing (Figure 118).

*In vitro* testing: Peanut-specific IgE 5.2 kUA/L, Ara h 2-specific IgE 3.1 kUA/L.

Diagnosis: Peanut allergy

**Oral food challenge:** An oral food challenge was not necessary for the diagnosis.
**Recommendation:** Peanut avoidance, education from dietitian, emergency medication (including adrenaline autoinjector).

**Case 2:**

**History:** Girl, 5 years of age with hen's egg allergy. She developed urticaria after accidental ingestion to cake included egg and peanut. At the time of writing, she had never eaten peanuts or peanut products.

**In vitro testing:** Peanut-specific IgE 62.8 kU/L, Ara h 2-specific IgE 48.0 kU/L.

**Oral challenge:** A titrated peanut challenge was not recommended as the predicted probability for peanut allergy was 95% (Figure 117A,B).

**Diagnosis:** Peanut allergy likely (highly suggestive). Recommendation: Peanut avoidance, education from dietitian, emergency medication (including adrenaline autoinjector).

**Case 3:**

**History:** Boy, 14 yearsofage with allergic rhinoconjunctivitis during the spring. On a panel test, he showed sensitization to birch pollen and peanut. He had eaten peanuts in the past without allergic reactions, though his consumption was infrequent (Figure 119).

**In vitro testing:** Birch-specific IgE >100 kUA/L, peanut-specific IgE 20.1 kUA/L, Ara h 2-specific IgE 0.1 kUA/L. Oral challenge: An oral food challenge was not necessary for the diagnosis.

**Diagnosis:** Allergic rhinoconjunctivitis, relevant peanut allergy unlikely.

**Recommendation:** Consider regular consumption.

## 8 – Research and Future Perspectives

Unknown allergens other than those approved by WHO/ IUIS Allergen Nomenclature Sub-Committee might be still hidden in peanut proteins, especially the complex lipophilic matrix. To make a more accurate diagnosis and improve our understanding of cross-reactivity, these allergens related to clinical characteristics need to be examined. In addition, several studies have reported that IgE-binding epitopes may become a biomarker for characterizing numerous phenotypes of peanut allergy.¹⁴⁰,¹²⁴¹ Further research may be expected to determine a biomarker for the prediction of the prognosis and more effective products for allergen-immunotherapy.
**Recommendation:** Peanut avoidance, education from dietitian, emergency medication (including adrenaline autoinjector).

**Case 2:**

**History:** Girl, 5 years of age with hen's egg allergy. She developed urticaria after accidental ingestion to cake included egg and peanut. At the time of writing, she had never eaten peanuts or peanut products.

**In vitro testing:** Peanut-specific IgE 62.8 kU/L, Ara h 2-specific IgE 48.0 kU/L.

**Oral challenge:** A titrated peanut challenge was not recommended as the predicted probability for peanut allergy was 95% (Figure 117A,B).

**Diagnosis:** Peanut allergy likely (highly suggestive). Recommendation: Peanut avoidance, education from dietitian, emergency medication (including adrenaline autoinjector).

**Case 3:**

**History:** Boy, 14 yearsofage with allergic rhinoconjunctivitis during the spring. On a panel test, he showed sensitization to birch pollen and peanut. He had eaten peanuts in the past without allergic reactions, though his consumption was infrequent (Figure 119).

**In vitro testing:** Birch-specific IgE >100 kUA/L, peanut-specific IgE 20.1 kUA/L, Ara h 2-specific IgE 0.1 kUA/L. Oral challenge: An oral food challenge was not necessary for the diagnosis.

**Diagnosis:** Allergic rhinoconjunctivitis, relevant peanut allergy unlikely.

**Recommendation:** Consider regular consumption.

## 8 – Research and Future Perspectives

Unknown allergens other than those approved by WHO/ IUIS Allergen Nomenclature Sub-Committee might be still hidden in peanut proteins, especially the complex lipophilic matrix. To make a more accurate diagnosis and improve our understanding of cross-reactivity, these allergens related to clinical characteristics need to be examined. In addition, several studies have reported that IgE-binding epitopes may become a biomarker for characterizing numerous phenotypes of peanut allergy.¹⁴⁰,¹²⁴¹ Further research may be expected to determine a biomarker for the prediction of the prognosis and more effective products for allergen-immunotherapy.
# B19 - Tree nut and seed allergies

Anna M. Ehlers, Helen A. Brough, Gideon Lack, Edward F. Knol, Magnus Wickman, Suzana Rudolovic

## Highlights

* Sensitization to Bet v 1 homologues such as Cor a 1 and Jug r 5 occurs mainly in the adult population of the Northern hemisphere (birch pollen pandemic area) and it often results in no or mild symptoms.

* Sensitization to nsLTPS with peach Pru p 3 as a primary sensitizer occurs mainly in the Mediterranean area.

* Sensitization to hazelnut 2S albumin Cor a 14 is associated with severe allergic reactions and such associations may also be present for 2S albumins of other tree nuts and seeds (e.g., Jug r 1, Ana o 3, Ses i 1).

* Co-sensitization and *in vitro* cross-reactivity are often not clinically relevant, but *in vivo* cross-reactivity can occur.

* While clinically relevant sensitization to pistachio and pecan nut usually implies clinically relevant sensitization to cashew nut and walnut, respectively, the reverse does not always occur.

## 1 - Background

Tree nuts and seeds, but also legumes are in fact all seeds and allergens in these foods, are often shared. Such allergenic proteins belong to the families of storage proteins (2S albumins, vicillins or 7S globulins and legumins or 11S globulins), lipid transfer proteins (LTPs), pathogenesis-related (PR) 10 proteins (Bet v 1-homologues), profilins and oil-body associated oleosins. The degree of sequence homology of the same protein family between tree nuts and between seeds is largely dependent on the botanical relationship. Most allergic reactions are caused by consumption of tree nuts and seeds by individuals allergic to these foods. Severity of reactions varies from mild/moderate to severe or life-threatening and, although rarely, they can have fatal outcome.<sup>1242,1243</sup> Some nuts and seeds were uncommon in the Western diet, a variety of tree nuts and seeds have become increasingly popular over the past decades due to their favourable nutritional profile. Nuts and seeds are often eaten as single food items, such as snacks, or as ingredients in healthy foods and salads. As dietary practices have changed, dishes traditional to certain parts of the World are now commonly eaten worldwide. Dishes such as ragouts, Middle (e.g., hummus, tagine), Far Eastern (e.g., stir fry with cashew nuts, pad Thai) and Indian subcontinent dishes (e.g., curry, korma) and pastries, cakes, chocolate, and candies often contain seeds or various tree nuts as substantial ingredients (<u>Figure 120</u>).

The current pattern of increased exposure to and consumption of nuts and seeds is a potential explanation for the suggested increase in reported reactions to such food items in addition to the general increase in incidence of food allergies. This includes foods

such as flaxseed that appears as an emerging allergen<sup>1244</sup> and chia seeds.<sup>1245</sup> In this chapter, we will discuss allergens of hazelnut, almond, cashew/pistachio, walnut/pecan nut, and Brazil nut, as well as to a lesser extent allergens of macadamia, pine nut, and the seeds sesame, sunflower, pumpkin, poppy, mustard, flaxseed, chia seeds and buckwheat. Coconut is not considered as a tree nut in Europe; however, the approach in the United States of America is different. Although American College of Asthma, Allergy & Immunology does not recognise coconut as a tree nut,<sup>1246</sup> it is still a legal requirement to declare coconut as one of the tree nut allergens (Food and Drug Administration).

## 2 - Allergen sources

In Europe, Canada and the United States, tree nuts, peanuts and seeds are listed as priority food allergens. Tree nuts and seeds are found in many food products, as shown in (<u>Figure 120</u>). Today, many food items are also labelled with precautionary allergen labelling (PAL), for instance "may contain nuts/peanuts/seeds," which should provide consumers with information on unintentional presence of allergens caused by cross-contamination in food preparation and packaging. However, there is currently no explicit guidelines for PAL and food products are mostly labelled without proper risk assessment. This often causes substantial problems for tree nut or seed allergic individuals and leads to misinterpretation of labels. This practice is expected to change as a new guideline is in preparation by the European Union and its Member States (EUMS) in cooperation with Australia, United Kingdom and the United States of America (progress can be seen at https://ec.europa.eu/info/law/better-regulation/have-your-say/initiatives/12230-Food-safety-allergies-&-food-waste-new-EU-rules_en).<sup>1247</sup>

Allergic reactions have been described after exposure to all food items containing nuts or seeds. Oil made from these nuts and seeds have been found to contain potential allergenic proteins. However, the concentrations of these proteins are very low in refined oils and do not trigger allergic reactions in majority of allergic individuals.<sup>1248</sup> As most of tree nuts and seeds allergens, except for profilin and pathogenesis-related 10 (PR-10) allergens, are heat stable, processing does not have a great impact on the allergenicity of such food products.

## 3 - Major and relevant minor allergenic proteins of tree nuts and seeds

An overview of allergenic proteins from tree nuts and seeds is depicted in <u>Tables 55 and 56</u>. Tree nut allergens belong to a limited number of protein families. One important protein family comprises seed storage proteins (discussed in Chapter C08). Those proteins are known for their resistance to digestion and their heat stability. The most resistant family members are the 2S albumins with a molecular mass ranging from 10 to 16 kDa. Recent findings indicate that
# B19 - Tree nut and seed allergies

Anna M. Ehlers, Helen A. Brough, Gideon Lack, Edward F. Knol, Magnus Wickman, Suzana Rudolovic

## Highlights

* Sensitization to Bet v 1 homologues such as Cor a 1 and Jug r 5 occurs mainly in the adult population of the Northern hemisphere (birch pollen pandemic area) and it often results in no or mild symptoms.

* Sensitization to nsLTPS with peach Pru p 3 as a primary sensitizer occurs mainly in the Mediterranean area.

* Sensitization to hazelnut 2S albumin Cor a 14 is associated with severe allergic reactions and such associations may also be present for 2S albumins of other tree nuts and seeds (e.g., Jug r 1, Ana o 3, Ses i 1).

* Co-sensitization and *in vitro* cross-reactivity are often not clinically relevant, but *in vivo* cross-reactivity can occur.

* While clinically relevant sensitization to pistachio and pecan nut usually implies clinically relevant sensitization to cashew nut and walnut, respectively, the reverse does not always occur.

## 1 - Background

Tree nuts and seeds, but also legumes are in fact all seeds and allergens in these foods, are often shared. Such allergenic proteins belong to the families of storage proteins (2S albumins, vicillins or 7S globulins and legumins or 11S globulins), lipid transfer proteins (LTPs), pathogenesis-related (PR) 10 proteins (Bet v 1-homologues), profilins and oil-body associated oleosins. The degree of sequence homology of the same protein family between tree nuts and between seeds is largely dependent on the botanical relationship. Most allergic reactions are caused by consumption of tree nuts and seeds by individuals allergic to these foods. Severity of reactions varies from mild/moderate to severe or life-threatening and, although rarely, they can have fatal outcome.<sup>1242,1243</sup> Some nuts and seeds were uncommon in the Western diet, a variety of tree nuts and seeds have become increasingly popular over the past decades due to their favourable nutritional profile. Nuts and seeds are often eaten as single food items, such as snacks, or as ingredients in healthy foods and salads. As dietary practices have changed, dishes traditional to certain parts of the World are now commonly eaten worldwide. Dishes such as ragouts, Middle (e.g., hummus, tagine), Far Eastern (e.g., stir fry with cashew nuts, pad Thai) and Indian subcontinent dishes (e.g., curry, korma) and pastries, cakes, chocolate, and candies often contain seeds or various tree nuts as substantial ingredients (Figure 120).

The current pattern of increased exposure to and consumption of nuts and seeds is a potential explanation for the suggested increase in reported reactions to such food items in addition to the general increase in incidence of food allergies. This includes foods

such as flaxseed that appears as an emerging allergen<sup>1244</sup> and chia seeds.<sup>1245</sup> In this chapter, we will discuss allergens of hazelnut, almond, cashew/pistachio, walnut/pecan nut, and Brazil nut, as well as to a lesser extent allergens of macadamia, pine nut, and the seeds sesame, sunflower, pumpkin, poppy, mustard, flaxseed, chia seeds and buckwheat. Coconut is not considered as a tree nut in Europe; however, the approach in the United States of America is different. Although American College of Asthma, Allergy & Immunology does not recognise coconut as a tree nut,<sup>1246</sup> it is still a legal requirement to declare coconut as one of the tree nut allergens (Food and Drug Administration).

## 2 - Allergen sources

In Europe, Canada and the United States, tree nuts, peanuts and seeds are listed as priority food allergens. Tree nuts and seeds are found in many food products, as shown in (Figure 120). Today, many food items are also labelled with precautionary allergen labelling (PAL), for instance "may contain nuts/peanuts/seeds," which should provide consumers with information on unintentional presence of allergens caused by cross-contamination in food preparation and packaging. However, there is currently no explicit guidelines for PAL and food products are mostly labelled without proper risk assessment. This often causes substantial problems for tree nut or seed allergic individuals and leads to misinterpretation of labels. This practice is expected to change as a new guideline is in preparation by the European Union and its Member States (EUMS) in cooperation with Australia, United Kingdom and the United States of America (progress can be seen at https://ec.europa.eu/info/law/better-regulation/have-your-say/initiatives/12230-Food-safety-allergies-&-food-waste-new-EU-rules_en).<sup>1247</sup>

Allergic reactions have been described after exposure to all food items containing nuts or seeds. Oil made from these nuts and seeds have been found to contain potential allergenic proteins. However, the concentrations of these proteins are very low in refined oils and do not trigger allergic reactions in majority of allergic individuals.<sup>1248</sup> As most of tree nuts and seeds allergens, except for profilin and pathogenesis-related 10 (PR-10) allergens, are heat stable, processing does not have a great impact on the allergenicity of such food products.

## 3 - Major and relevant minor allergenic proteins of tree nuts and seeds

An overview of allergenic proteins from tree nuts and seeds is depicted in Tables 55 and 56. Tree nut allergens belong to a limited number of protein families. One important protein family comprises seed storage proteins (discussed in Chapter C08). Those proteins are known for their resistance to digestion and their heat stability. The most resistant family members are the 2S albumins with a molecular mass ranging from 10 to 16 kDa. Recent findings indicate that
Food and products that contain or
often contain tree nuts and seeds

Chocolate and pralines

Nougat (sugar paste made with nuts), for example, Torrone

Dishes such as almond chicken, pad thai, chili and trout amandine

Gianduja and giandula (chocolate blended with hazel nuts)

Marzipan (almond paste)

Almond milk

Nut milk

Tree nut oils

Spreads, for example, almond paste-based spreads, cheese spreads, chocolate nut spreads

Nutella (hazelnut)

Tahini, halva (sesame)

Vegetarian dishes

Various Indian curries and Asian dishes

Industrial made Pesto

Industrial made tomato sauce with cheese

Industrial made pasta dishes

Cereals

Other possible sources of tree nuts

Baked goods

Baking mixes, cereals, crackers and muesli

Barbecue and pesto sauces

Dressings and gravies

Flavoured coffees

Frozen desserts

Liqueurs, for example, amaretto,

Frangelico

Natural flavourings and extracts, for example, pure almond extract

Salads, for example, Waldorf salad

Snack foods, for example, trail mix

Non-food sources of tree nuts

Bean bags, kick sacks/hacky sacks

Bird seed

Cosmetics, hair care products, sun screens

Massage oils

Pet food

FIGURE 120 Foods and ingredients containing tree nuts, and seeds. Note: These lists are not complete and may change. Food and food products purchased from other countries, through mail-order or the internet, are not always produced using the same manufacturing old labeling standards as in Europe.

sensitization to 2S albumins, such as sensitization to hazelnut Cor a 14, might be related to rather severe allergic reactions.⁴³

Other prominent members of this family are 11S (legumins) and 7S globulins (vicilins), which have a molecular mass of around 50 and 70 kDa, respectively, and they form hexametric and trimeric aggregates, respectively. In children, sensitization to the hazelnut 11S globulin Cor a 9 has also been associated with the risk of an allergic response comparable to sensitization to the hazelnut 2S albumin Cor a 14.¹²⁴⁹

Pathogenesis-related (PR) proteins form an additional group of well-known tree nut and seed allergens: PR-10 proteins with a high homology to the major birch pollen allergen Bet v 1 and PR-14 proteins so-called nonspecific lipid transfer proteins (nsLTP). The most intensively studied Bet v 1 homologue in tree nuts is hazelnut Cor a 1 and sensitization to this allergen accounts for up to 90% of sensitization to hazelnut in Central and Northern Europe⁴⁴ [10]. Similarly, sensitization to walnut Jug r 5 was found in up to 90% of walnut allergic patients in the birch-pandemic area and sensitization to Jug r 5 correlated strongly with sensitization to Bet v 1.⁴⁶ By contrast, nonspecific LTPs are important allergens in the Mediterranean area, which has been described for hazelnut (Cor a 8), walnut (Jug r 3) and almond (Pru du 3). The primary sensitizer within the nsLTPs family is thought to be the peach nsLTPs Pru p 3. In the Northern hemisphere also the LTP from mugwort Art v 3 appears to play a role as primary sensitizer.¹²⁵⁰

Not yet well characterized allergens are lipophilic proteins, which belong to the group of oleosins (discussed in Chapter C-10). Specific IgE binding to this group has been shown in a subpopulation

of hazelnut allergic patients across Europe, but its clinical relevance has not yet been fully proven.

In tree nuts, most allergens have been described for hazelnut, which is most probably the direct result of more research for this food. For the seed allergens, the same protein families have been described like for tree nuts, with 2S albumins and 7S and 11S globulins as the most frequently described allergens. Consideration should be given to the fact that the absence of identified allergens in seeds belonging to certain protein families might be explained by the lack of research with many allergens still needing to be discovered.

4 – Sensitization to individual molecules and their clinical relevance

It is challenging to establish an overall prevalence of sensitization to tree nuts and seeds, particularly to an individual tree nut or seed allergen. The prevalence differs in different parts of the world and, except for sesame, allergy to seeds is quite rare in most of the countries. Studies investigating prevalence of tree nuts/ seeds sensitization vary in their methodology. Some studies define sensitization rates irrespective of sensitization to birch pollen, and other studies excluded individuals sensitized to birch pollen. This is important to note because sensitization to the pathogenesis-related 10 (PR-10) protein in birch pollen, Bet v 1, can lead to *in vitro* cross-reactivity with homologous proteins (e.g., Cor a 1),
Food and products that contain or
often contain tree nuts and seeds

Chocolate and pralines

Nougat (sugar paste made with nuts), for example, Torrone

Dishes such as almond chicken, pad thai, chili and trout amandine

Gianduja and giandula (chocolate blended with hazel nuts)

Marzipan (almond paste)

Almond milk

Nut milk

Tree nut oils

Spreads, for example, almond paste-based spreads, cheese spreads, chocolate nut spreads

Nutella (hazelnut)

Tahini, halva (sesame)

Vegetarian dishes

Various Indian curries and Asian dishes

Industrial made Pesto

Industrial made tomato sauce with cheese

Industrial made pasta dishes

Cereals

Other possible sources of tree nuts

Baked goods

Baking mixes, cereals, crackers and muesli

Barbecue and pesto sauces

Dressings and gravies

Flavoured coffees

Frozen desserts

Liqueurs, for example, amaretto,

Frangelico

Natural flavourings and extracts, for example, pure almond extract

Salads, for example, Waldorf salad

Snack foods, for example, trail mix

Non-food sources of tree nuts

Bean bags, kick sacks/hacky sacks

Bird seed

Cosmetics, hair care products, sun screens

Massage oils

Pet food

**FIGURE 120** Foods and ingredients containing tree nuts, and seeds. Note: These lists are not complete and may change. Food and food products purchased from other countries, through mail-order or the internet, are not always produced using the same manufacturing old labeling standards as in Europe.

sensitization to 2S albumins, such as sensitization to hazelnut Cor a 14, might be related to rather severe allergic reactions.⁴³

Other prominent members of this family are 11S (legumins) and 7S globulins (vicilins), which have a molecular mass of around 50 and 70 kDa, respectively, and they form hexametric and trimeric aggregates, respectively. In children, sensitization to the hazelnut 11S globulin Cor a 9 has also been associated with the risk of an allergic response comparable to sensitization to the hazelnut 2S albumin Cor a 14.¹²⁴⁹

Pathogenesis-related (PR) proteins form an additional group of well-known tree nut and seed allergens: PR-10 proteins with a high homology to the major birch pollen allergen Bet v 1 and PR-14 proteins so-called nonspecific lipid transfer proteins (nsLTP). The most intensively studied Bet v 1 homologue in tree nuts is hazelnut Cor a 1 and sensitization to this allergen accounts for up to 90% of sensitization to hazelnut in Central and Northern Europe⁴⁴ [10]. Similarly, sensitization to walnut Jug r 5 was found in up to 90% of walnut allergic patients in the birch-pandemic area and sensitization to Jug r 5 correlated strongly with sensitization to Bet v 1.⁴⁶ By contrast, nonspecific LTPs are important allergens in the Mediterranean area, which has been described for hazelnut (Cor a 8), walnut (Jug r 3) and almond (Pru du 3). The primary sensitizer within the nsLTPs family is thought to be the peach nsLTPs Pru p 3. In the Northern hemisphere also the LTP from mugwort Art v 3 appears to play a role as primary sensitizer.¹²⁵⁰

Not yet well characterized allergens are lipophilic proteins, which belong to the group of oleosins (discussed in Chapter C-10). Specific IgE binding to this group has been shown in a subpopulation

of hazelnut allergic patients across Europe, but its clinical relevance has not yet been fully proven.

In tree nuts, most allergens have been described for hazelnut, which is most probably the direct result of more research for this food. For the seed allergens, the same protein families have been described like for tree nuts, with 2S albumins and 7S and 11S globulins as the most frequently described allergens. Consideration should be given to the fact that the absence of identified allergens in seeds belonging to certain protein families might be explained by the lack of research with many allergens still needing to be discovered.

4 – Sensitization to individual molecules and their clinical relevance

It is challenging to establish an overall prevalence of sensitization to tree nuts and seeds, particularly to an individual tree nut or seed allergen. The prevalence differs in different parts of the world and, except for sesame, allergy to seeds is quite rare in most of the countries. Studies investigating prevalence of tree nuts/ seeds sensitization vary in their methodology. Some studies define sensitization rates irrespective of sensitization to birch pollen, and other studies excluded individuals sensitized to birch pollen. This is important to note because sensitization to the pathogenesis-related 10 (PR-10) protein in birch pollen, Bet v 1, can lead to *in vitro* cross-reactivity with homologous proteins (e.g., Cor a 1),
<table><caption>TABLE 55 Identified tree nut allergens. Overview of the different tree nut allergens divided by protein family</caption><thead><tr><th rowspan="2">Source</th><th colspan="3">Seed storage proteins</th><th colspan="2">Pathogenesis-related proteins</th><th rowspan="2">Profilins</th><th rowspan="2">Oleosins</th><th rowspan="2">Others</th></tr><tr><th>2S albumins</th><th>7S globulins</th><th>11S globulins</th><th>PR-10 proteins<br>Bet v 1-homologue</th><th>PR-14 proteins<br>nsLTPs</th></tr></thead><tbody><tr><td>Hazelnut<br><i>Corylus avellana</i></td><td>Cor a 14</td><td>Cor a 11</td><td>Cor a 9</td><td>Cor a 1</td><td>Cor a 8</td><td>Cor a 2</td><td>Cor a 12<br>Cor a 13<br>Cor a 15</td><td></td></tr><tr><td>Almond<br><i>Prunus dulcis</i></td><td>Pru du 2S<br>albumin<br>Pru du AP<sup>4</sup></td><td>Pru du 8<sup>3</sup></td><td>Pru du 6</td><td>Pru du 1</td><td>Pru du 3</td><td>Pru du 4</td><td>Pru du 2<sup>1</sup><br>Pru du 5<sup>2</sup><br>Pis v 4<sup>3</sup></td><td></td></tr><tr><td>Cashew nut<br><i>Anacardium<br>occidentale</i></td><td>Ana o 3</td><td>Ana o 1</td><td>Ana o 2</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pistachio<br><i>Pistacia vera</i></td><td>Pis v 1</td><td>Pis v 3</td><td>Pis v 2<br>Pis v 5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Walnut<br><i>Juglans regia</i></td><td>Jug r 1</td><td>Jug r 2<br>Jug r 6</td><td>Jug r 4</td><td>Jug r 5</td><td>Jug r 3<br>Jug r 8</td><td>Jug r 7</td><td></td><td></td></tr><tr><td>Pecan nut<br><i>Carya illinoinensis</i></td><td>Car i 1</td><td>Car i 2</td><td>Car i 4</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Brazil nut<br><i>Bertholletia excelsa</i></td><td>Ber e 1</td><td></td><td>Ber e 2</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Macadamia nut<br><i>Macadamia<br>integrifolia</i></td><td></td><td></td><td>Mac i 1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pine nut<br><i>Pinus pinea</i></td><td>Pin p 1</td><td></td><td>Pin p vi-cilin</td><td></td><td></td><td></td><td>Pin p 17<br>kDa<sup>6</sup></td><td></td></tr><tr><td>Coconut<br><i>Cocos nucifera</i></td><td></td><td>Coc n 1</td><td>Coc n 4</td><td></td><td></td><td></td><td>Con n 5</td><td></td></tr></tbody></table>

"Bold font indicates the availability on commercial diagnostic platforms; nsLTP: non-specific lipid transfer protein, PR10: pathogenesis-related protein 10; 1: Thaumatin, 2: Ribosomal protein P2, 3: Antimicrobial seed storage protein, 4: conglutin, 5: Mn superoxide dismutase, 6: not known yet"

Bold font indicates the availability on commercial diagnostic platforms; nsLTP: non-specific lipid transfer protein, PR10: pathogenesis-related protein 10; 1: Thaumatin, 2: Ribosomal protein P2, 3: Antimicrobial seed storage protein, 4: conglutin, 5: Mn superoxide dismutase, 6: not known yet

which accounts for a substantial part of sensitization to tree nuts and seeds. Those IgE cross-reactivities are mostly clinically irrelevant or cause only mild reactions to particular foods while sensitization to seed storage proteins is less common but can cause severe allergic reactions.

The EuroPrevall study reported significant differences in rates of sensitization to tree nuts in children between different study centres across Europe, with hazelnut sensitization rates varying from 1.87% in Reykjavik to 8.63% in Madrid and 9.52% in Utrecht and to 14.35% in Zurich (Table 57). Similar pattern was observed in terms of walnut sensitization with 1.37% in Reykjavik, 7.45% in Madrid and 9.52% in Zurich. Amongst seeds, sesame sensitization was the most common across most of the centres, with the lowest rates in Reykjavik at 2.86% and Vilnius at 3.03% and higher rates in Madrid (11.90%) and Zurich (12.10%). In Madrid, for example, sesame was one of the three foods to which sensitization was most common. Nevertheless, the patterns of probable food allergy were different. Probable food allergy (defined as slgE and reported hypersensitivity-related

symptoms within 2 hours after ingestion) to hazelnut was the most observed in Vilnius at 2.15%, followed by Zurich at 0.81%. Probable food allergy to walnut was the most reported in Athens (0.56%). The rates of probable food allergy to seeds were <0.01% for most centres.¹⁰²⁸ Results published by EuroPrevall-INCO Study Team in China, India, and Russia also showed variations in the prevalence of IgE sensitization (defined as specific IgE >0.7 kU/L) and probable allergy to tree nuts and seeds in children in the three participating countries. Interestingly, despite quite high rates of sensitization to nuts and seeds, particularly in India, rates of probable allergy are low.¹²⁵¹

Sensitization patterns also differ between EuroPrevall study centres in the adult population using the same methodology (Table 58). Whilst the rate of probable allergy to hazelnut was quite low in Athens, at 0.06%, it reached 2.57% in Zurich. Probable allergy to walnut was the most reported in Madrid (0.71%) and Zurich (0.58%) and the least commonly in Reykjavik (0.05%). Despite low rate of probable allergy in Athens at 0.29%, walnut was one of the
<table><caption>TABLE 55 Identified tree nut allergens. Overview of the different tree nut allergens divided by protein family</caption><thead><tr><th rowspan="2">Source</th><th colspan="3">Seed storage proteins</th><th colspan="2">Pathogenesis-related proteins</th><th rowspan="2">Profilins</th><th rowspan="2">Oleosins</th><th rowspan="2">Others</th></tr><tr><th>2S albumins</th><th>7S globulins</th><th>11S globulins</th><th>PR-10 proteins<br>Bet v 1-homologue</th><th>PR-14 proteins<br>nsLTPs</th></tr></thead><tbody><tr><td>Hazelnut<br><i>Corylus avellana</i></td><td>Cor a 14</td><td>Cor a 11</td><td>Cor a 9</td><td>Cor a 1</td><td>Cor a 8</td><td>Cor a 2</td><td>Cor a 12<br>Cor a 13<br>Cor a 15</td><td></td></tr><tr><td>Almond<br><i>Prunus dulcis</i></td><td>Pru du 2S<br>albumin<br>Pru du AP<sup>4</sup></td><td>Pru du 8<sup>3</sup></td><td>Pru du 6</td><td>Pru du 1</td><td>Pru du 3</td><td>Pru du 4</td><td>Pru du 2<sup>1</sup><br>Pru du 5<sup>2</sup><br>Pis v 4<sup>3</sup></td><td></td></tr><tr><td>Cashew nut<br><i>Anacardium<br>occidentale</i></td><td>Ana o 3</td><td>Ana o 1</td><td>Ana o 2</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pistachio<br><i>Pistacia vera</i></td><td>Pis v 1</td><td>Pis v 3</td><td>Pis v 2<br>Pis v 5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Walnut<br><i>Juglans regia</i></td><td>Jug r 1</td><td>Jug r 2<br>Jug r 6</td><td>Jug r 4</td><td>Jug r 5</td><td>Jug r 3<br>Jug r 8</td><td>Jug r 7</td><td></td><td></td></tr><tr><td>Pecan nut<br><i>Carya illinoinensis</i></td><td>Car i 1</td><td>Car i 2</td><td>Car i 4</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Brazil nut<br><i>Bertholletia excelsa</i></td><td>Ber e 1</td><td></td><td>Ber e 2</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Macadamia nut<br><i>Macadamia<br>integrifolia</i></td><td></td><td></td><td>Mac i 1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pine nut<br><i>Pinus pinea</i></td><td>Pin p 1</td><td></td><td>Pin p vi-cilin</td><td></td><td></td><td></td><td>Pin p 17<br>kDa<sup>6</sup></td><td></td></tr><tr><td>Coconut<br><i>Cocos nucifera</i></td><td></td><td>Coc n 1</td><td>Coc n 4</td><td></td><td></td><td></td><td>Con n 5</td><td></td></tr></tbody></table>

"Bold font indicates the availability on commercial diagnostic platforms; nsLTP: non-specific lipid transfer protein, PR10: pathogenesis-related protein 10; 1: Thaumatin, 2: Ribosomal protein P2, 3: Antimicrobial seed storage protein, 4: conglutin, 5: Mn superoxide dismutase, 6: not known yet"

Bold font indicates the availability on commercial diagnostic platforms; nsLTP: non-specific lipid transfer protein, PR10: pathogenesis-related protein 10; 1: Thaumatin, 2: Ribosomal protein P2, 3: Antimicrobial seed storage protein, 4: conglutin, 5: Mn superoxide dismutase, 6: not known yet

which accounts for a substantial part of sensitization to tree nuts and seeds. Those IgE cross-reactivities are mostly clinically irrelevant or cause only mild reactions to particular foods while sensitization to seed storage proteins is less common but can cause severe allergic reactions.

The EuroPrevall study reported significant differences in rates of sensitization to tree nuts in children between different study centres across Europe, with hazelnut sensitization rates varying from 1.87% in Reykjavik to 8.63% in Madrid and 9.52% in Utrecht and to 14.35% in Zurich (Table 57). Similar pattern was observed in terms of walnut sensitization with 1.37% in Reykjavik, 7.45% in Madrid and 9.52% in Zurich. Amongst seeds, sesame sensitization was the most common across most of the centres, with the lowest rates in Reykjavik at 2.86% and Vilnius at 3.03% and higher rates in Madrid (11.90%) and Zurich (12.10%). In Madrid, for example, sesame was one of the three foods to which sensitization was most common. Nevertheless, the patterns of probable food allergy were different. Probable food allergy (defined as slgE and reported hypersensitivity-related

symptoms within 2 hours after ingestion) to hazelnut was the most observed in Vilnius at 2.15%, followed by Zurich at 0.81%. Probable food allergy to walnut was the most reported in Athens (0.56%). The rates of probable food allergy to seeds were <0.01% for most centres.1028 Results published by EuroPrevall-INCO Study Team in China, India, and Russia also showed variations in the prevalence of IgE sensitization (defined as specific IgE >0.7 kU/L) and probable allergy to tree nuts and seeds in children in the three participating countries. Interestingly, despite quite high rates of sensitization to nuts and seeds, particularly in India, rates of probable allergy are low.1251

Sensitization patterns also differ between EuroPrevall study centres in the adult population using the same methodology (Table 58). Whilst the rate of probable allergy to hazelnut was quite low in Athens, at 0.06%, it reached 2.57% in Zurich. Probable allergy to walnut was the most reported in Madrid (0.71%) and Zurich (0.58%) and the least commonly in Reykjavik (0.05%). Despite low rate of probable allergy in Athens at 0.29%, walnut was one of the
<table><caption>TABLE 56 Identified seed allergens. Overview of the different seed allergens divided by protein family</caption><thead><tr><th rowspan="2">Source</th><th colspan="3">Seed storage proteins</th><th colspan="2">Pathogenesis-related proteins</th><th rowspan="2">Profilins</th><th rowspan="2">Oleosins</th><th rowspan="2">Others</th></tr><tr><th>2S albumins</th><th>7S globulins</th><th>11S globulins</th><th>PR-10 proteins<br>Bet v 1-homologue</th><th>PR-14 proteins<br>nsLTPs</th></tr></thead><tbody><tr><td>Sesame</td><td>Ses i 1</td><td>Ses i 3</td><td>Ses i 6</td><td></td><td></td><td>Ses i 8</td><td>Ses i 4</td><td></td></tr><tr><td>Sesamum indicum</td><td>Ses i 2</td><td></td><td>Ses i 7</td><td></td><td></td><td>Sin a 4</td><td>Ses i 5</td><td></td></tr><tr><td>Mustard seed</td><td>Sin a 1</td><td></td><td>Sin a 2</td><td></td><td>Sin a 3</td><td></td><td></td><td></td></tr><tr><td>Sinapis alba</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sunflower seed</td><td>Hel a 2S</td><td></td><td></td><td></td><td>Hel a 3</td><td>Hel a 2</td><td></td><td>Hel a 4<sup>a</sup></td></tr><tr><td>Helianthus annuus</td><td>albumin</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pumpkin seed</td><td>Cuc ma 5</td><td></td><td>Cuc ma 4</td><td></td><td></td><td>Cuc ma 2</td><td></td><td></td></tr><tr><td>Cucurbita maxima</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Poppy seed</td><td></td><td></td><td></td><td>Pap s 1</td><td></td><td>Pap s 2</td><td></td><td>Pap s</td></tr><tr><td>Papaver somniferum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>34kDa<sup>b</sup></td></tr><tr><td>Buckwheat</td><td>Fag e 2</td><td>Fag e 3</td><td>Fag e 1<sup>a</sup></td><td></td><td></td><td></td><td></td><td>Fag e 10</td></tr><tr><td>Fagopyrum esculentum</td><td></td><td>Fag e 4</td><td>Fag e 5</td><td></td><td></td><td></td><td></td><td>Fag e T<sup>b</sup></td></tr><tr><td>Flaxseed</td><td>Lin u 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Linum usitatissimum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Bold font indicates the availability on commercial diagnostic platforms; nsLTP: non-specific lipid transfer protein, PR10: pathogenesis-related protein 10; 1: Defensin, 2: Alpha-amylase inhibitor, 3: Trypsin inhibitor, 4: 13S globulin, legumin-like protein, 5: not known yet. Chia seed not available.

<table><caption>TABLE 57 Prevalence of food sensitization in comparison to probable food allergy in children across Europe</caption><thead><tr><th></th><th>Zurich</th><th>Madrid</th><th>Athens</th><th>Utrecht</th><th>Vilnius</th><th>Lodz</th><th>Reykjavik</th><th>Hong Kong</th><th>Guangzhou</th><th>Shaoquan</th><th>Tomsk</th><th>India</th></tr></thead><tbody><tr><td>Hazelnut</td><td>14.35<br>0.81</td><td>8.63<br>0.53</td><td>3.63<br>0.28</td><td>9.52<br>0.74</td><td>6.82<br>2.15</td><td>7.56<br>0.78</td><td>1.87<br>0.07</td><td>1.6<br>&lt;0.01</td><td>&lt;0.01<br>&lt;0.01</td><td>2.0<br>&lt;0.01</td><td>3.2<br>0.05</td><td>3.8<br>&lt;0.01</td></tr><tr><td>Walnut</td><td>9.52<br>0.27</td><td>7.45<br>0.53</td><td>5.33<br>0.56</td><td>3.55<br>0.53</td><td>2.28<br>&lt;0.01</td><td>3.58<br>0.48</td><td>1.37<br>&lt;0.01</td><td>1.0<br>&lt;0.01</td><td>&lt;0.01<br>&lt;0.01</td><td>2.1<br>&lt;0.01</td><td>0.7<br>0.03</td><td>4.2<br>&lt;0.01</td></tr><tr><td>Sesame</td><td>12.10<br>&lt;0.01</td><td>11.90<br>&lt;0.01</td><td>5.82<br>&lt;0.01</td><td>6.50<br>&lt;0.01</td><td>3.03<br>&lt;0.01</td><td>5.50<br>&lt;0.01</td><td>2.86<br>0.15</td><td>2.2<br>-</td><td>&lt;0.01<br>-</td><td>3.3<br>-</td><td>2.3<br>-</td><td>8.0<br>-</td></tr><tr><td>Mustard</td><td>4.92<br>&lt;0.01</td><td>3.33<br>&lt;0.01</td><td>2.18<br>&lt;0.01</td><td>1.72<br>&lt;0.01</td><td>3.04<br>&lt;0.01</td><td>2.48<br>&lt;0.01</td><td>0.37<br>&lt;0.01</td><td>0.1<br>-</td><td>&lt;0.01<br>-</td><td>1.0<br>-</td><td>0.9<br>-</td><td>3.3<br>-</td></tr><tr><td>Sunflower</td><td>8.89<br>&lt;0.01</td><td>6.16<br>0.53</td><td>4.85<br>&lt;0.01</td><td>4.67<br>&lt;0.01</td><td>3.41<br>&lt;0.01</td><td>4.54<br>&lt;0.01</td><td>1.37<br>&lt;0.01</td><td>1.6<br>&lt;0.01</td><td>&lt;0.01<br>&lt;0.01</td><td>2.0<br>&lt;0.01</td><td>1.0<br>0.03</td><td>5.5<br>&lt;0.01</td></tr><tr><td>Poppy seed</td><td>8.50<br>&lt;0.01</td><td>6.32<br>&lt;0.01</td><td>3.15<br>&lt;0.01</td><td>4.88<br>&lt;0.01</td><td>2.28<br>&lt;0.01</td><td>3.99<br>&lt;0.01</td><td>0.75<br>&lt;0.01</td><td>0.1<br>-</td><td>&lt;0.01<br>-</td><td>2.0<br>-</td><td>1.1<br>-</td><td>5.9<br>-</td></tr><tr><td>Buckwheat</td><td>8.89<br>0.14</td><td>7.55<br>&lt;0.01</td><td>5.58<br>&lt;0.01</td><td>4.77<br>&lt;0.01</td><td>2.65<br>&lt;0.01</td><td>3.99<br>&lt;0.01</td><td>1.37<br>0.07</td><td>2.0<br>-</td><td>&lt;0.01<br>-</td><td>2.8<br>-</td><td>0.9<br>-</td><td>7.5<br>-</td></tr></tbody></table>

Table is adapted from Lyons and co-workers 2020 [ref:1028] and Li and co-workers 2019 [ref:1252] – weighted samples of children (7-10 years) with and without allergic symptoms completed a questionnaire and donated serum for IgE analysis; probable food allergy was defined as sIgE and reported hypersensitivity-related symptoms within 2 hours after ingestion"

three foods to which the most symptoms of probable allergy were reported. Seed allergy was much less commonly reported. Despite low prevalence of sunflower seeds allergy in Athens, at 0.07%, this still appears as one of the foods with the most reported probable food allergy in this centre.¹⁰²⁹

In a combined population of children and adults who reported allergic reactions to hazelnut (70% had a confirmed hazelnut allergy by DBPCFCs or history of severe allergy), sensitization to Cor a 1 was most prevalent (74.3%), followed at distance by Cor a 2 (19.6%), and up to 10 times higher than sensitization to other hazelnut components. While sensitization to Cor a 14 was highly associated with sensitization to Cor a 9 with a higher prevalence in children

(42.0% vs 5.8%), sensitization to Cor a 11 was overall below 10%. Geographical differences were shown for Cor a 1 (more prevalent in Northern and Central Europe, ≥60%), closely related to sensitization to birch pollen, and for Cor a 8 (more prevalent in the Mediterranean area, ranging from 36% to 83%). Sensitization to oleosin Cor a 12 was observed all over Europe in up to 25% of the patients with a higher rate in children (11.4% vs 34%) [10]. Comparable to hazelnut, sensitization to the PR-10 protein Jug r 5 in walnut dominated in the birch-pandemic area with high correlation to birch pollen sensitization whilst sensitization to the LTP Jug r 3 dominated in Southern Europe. Notably, sensitization to walnut seed storage proteins was found in up to 10% of enrolled subjects.⁴⁶
<table><caption>TABLE 56 Identified seed allergens. Overview of the different seed allergens divided by protein family</caption><thead><tr><th rowspan="2">Source</th><th colspan="3">Seed storage proteins</th><th colspan="2">Pathogenesis-related proteins</th><th rowspan="2">Profilins</th><th rowspan="2">Oleosins</th><th rowspan="2">Others</th></tr><tr><th>2S albumins</th><th>7S globulins</th><th>11S globulins</th><th>PR-10 proteins<br>Bet v 1-homologue</th><th>PR-14 proteins<br>nsLTPs</th></tr></thead><tbody><tr><td>Sesame</td><td>Ses i 1</td><td>Ses i 3</td><td>Ses i 6</td><td></td><td></td><td>Ses i 8</td><td>Ses i 4</td><td></td></tr><tr><td>Sesamum indicum</td><td>Ses i 2</td><td></td><td>Ses i 7</td><td></td><td></td><td>Sin a 4</td><td>Ses i 5</td><td></td></tr><tr><td>Mustard seed</td><td>Sin a 1</td><td></td><td>Sin a 2</td><td></td><td>Sin a 3</td><td></td><td></td><td></td></tr><tr><td>Sinapis alba</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sunflower seed</td><td>Hel a 2S</td><td></td><td></td><td></td><td>Hel a 3</td><td>Hel a 2</td><td></td><td>Hel a 4<sup>a</sup></td></tr><tr><td>Helianthus annuus</td><td>albumin</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pumpkin seed</td><td>Cuc ma 5</td><td></td><td>Cuc ma 4</td><td></td><td></td><td>Cuc ma 2</td><td></td><td></td></tr><tr><td>Cucurbita maxima</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Poppy seed</td><td></td><td></td><td></td><td>Pap s 1</td><td></td><td>Pap s 2</td><td></td><td>Pap s</td></tr><tr><td>Papaver somniferum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>34kDa<sup>b</sup></td></tr><tr><td>Buckwheat</td><td>Fag e 2</td><td>Fag e 3</td><td>Fag e 1<sup>a</sup></td><td></td><td></td><td></td><td></td><td>Fag e 10</td></tr><tr><td>Fagopyrum esculentum</td><td></td><td>Fag e 4</td><td>Fag e 5</td><td></td><td></td><td></td><td></td><td>Fag e T<sup>1</sup></td></tr><tr><td>Flaxseed</td><td>Lin u 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Linum usitatissimum</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Bold font indicates the availability on commercial diagnostic platforms; nsLTP: non-specific lipid transfer protein, PR10: pathogenesis-related protein 10; 1: Defensin, 2: Alpha-amylase inhibitor, 3: Trypsin inhibitor, 4: 13S globulin, legumin-like protein, 5: not known yet. Chia seed not available.

<table><caption>TABLE 57 Prevalence of food sensitization in comparison to probable food allergy in children across Europe</caption><thead><tr><th></th><th>Zurich</th><th>Madrid</th><th>Athens</th><th>Utrecht</th><th>Vilnius</th><th>Lodz</th><th>Reykjavik</th><th>Hong Kong</th><th>Guangzhou</th><th>Shaoquan</th><th>Tomsk</th><th>India</th></tr></thead><tbody><tr><td>Hazelnut</td><td>14.35<br>0.81</td><td>8.63<br>0.53</td><td>3.63<br>0.28</td><td>9.52<br>0.74</td><td>6.82<br>2.15</td><td>7.56<br>0.78</td><td>1.87<br>0.07</td><td>1.6<br>&lt;0.01</td><td>&lt;0.01<br>&lt;0.01</td><td>2.0<br>&lt;0.01</td><td>3.2<br>0.05</td><td>3.8<br>&lt;0.01</td></tr><tr><td>Walnut</td><td>9.52<br>0.27</td><td>7.45<br>0.53</td><td>5.33<br>0.56</td><td>3.55<br>0.53</td><td>2.28<br>&lt;0.01</td><td>3.58<br>0.48</td><td>1.37<br>&lt;0.01</td><td>1.0<br>&lt;0.01</td><td>&lt;0.01<br>&lt;0.01</td><td>2.1<br>&lt;0.01</td><td>0.7<br>0.03</td><td>4.2<br>&lt;0.01</td></tr><tr><td>Sesame</td><td>12.10<br>&lt;0.01</td><td>11.90<br>&lt;0.01</td><td>5.82<br>&lt;0.01</td><td>6.50<br>&lt;0.01</td><td>3.03<br>&lt;0.01</td><td>5.50<br>&lt;0.01</td><td>2.86<br>0.15</td><td>2.2<br>-</td><td>&lt;0.01<br>-</td><td>3.3<br>-</td><td>2.3<br>-</td><td>8.0<br>-</td></tr><tr><td>Mustard</td><td>4.92<br>&lt;0.01</td><td>3.33<br>&lt;0.01</td><td>2.18<br>&lt;0.01</td><td>1.72<br>&lt;0.01</td><td>3.04<br>&lt;0.01</td><td>2.48<br>&lt;0.01</td><td>0.37<br>&lt;0.01</td><td>0.1<br>-</td><td>&lt;0.01<br>-</td><td>1.0<br>-</td><td>0.9<br>-</td><td>3.3<br>-</td></tr><tr><td>Sunflower</td><td>8.89<br>&lt;0.01</td><td>6.16<br>0.53</td><td>4.85<br>&lt;0.01</td><td>4.67<br>&lt;0.01</td><td>3.41<br>&lt;0.01</td><td>4.54<br>&lt;0.01</td><td>1.37<br>&lt;0.01</td><td>1.6<br>&lt;0.01</td><td>&lt;0.01<br>&lt;0.01</td><td>2.0<br>&lt;0.01</td><td>1.0<br>0.03</td><td>5.5<br>&lt;0.01</td></tr><tr><td>Poppy seed</td><td>8.50<br>&lt;0.01</td><td>6.32<br>&lt;0.01</td><td>3.15<br>&lt;0.01</td><td>4.88<br>&lt;0.01</td><td>2.28<br>&lt;0.01</td><td>3.99<br>&lt;0.01</td><td>0.75<br>&lt;0.01</td><td>0.1<br>-</td><td>&lt;0.01<br>-</td><td>2.0<br>-</td><td>1.1<br>-</td><td>5.9<br>-</td></tr><tr><td>Buckwheat</td><td>8.89<br>0.14</td><td>7.55<br>&lt;0.01</td><td>5.58<br>&lt;0.01</td><td>4.77<br>&lt;0.01</td><td>2.65<br>&lt;0.01</td><td>3.99<br>&lt;0.01</td><td>1.37<br>0.07</td><td>2.0<br>-</td><td>&lt;0.01<br>-</td><td>2.8<br>-</td><td>0.9<br>-</td><td>7.5<br>-</td></tr></tbody></table>

Table is adapted from Lyons and co-workers 2020 [ref:1028] and Li and co-workers 2019 [ref:1252] – weighted samples of children (7-10 years) with and without allergic symptoms completed a questionnaire and donated serum for IgE analysis; probable food allergy was defined as sIgE and reported hypersensitivity-related symptoms within 2 hours after ingestion"

three foods to which the most symptoms of probable allergy were reported. Seed allergy was much less commonly reported. Despite low prevalence of sunflower seeds allergy in Athens, at 0.07%, this still appears as one of the foods with the most reported probable food allergy in this centre.¹⁰²⁹

In a combined population of children and adults who reported allergic reactions to hazelnut (70% had a confirmed hazelnut allergy by DBPCFCs or history of severe allergy), sensitization to Cor a 1 was most prevalent (74.3%), followed at distance by Cor a 2 (19.6%), and up to 10 times higher than sensitization to other hazelnut components. While sensitization to Cor a 14 was highly associated with sensitization to Cor a 9 with a higher prevalence in children

(42.0% vs 5.8%), sensitization to Cor a 11 was overall below 10%. Geographical differences were shown for Cor a 1 (more prevalent in Northern and Central Europe, ≥60%), closely related to sensitization to birch pollen, and for Cor a 8 (more prevalent in the Mediterranean area, ranging from 36% to 83%). Sensitization to oleosin Cor a 12 was observed all over Europe in up to 25% of the patients with a higher rate in children (11.4% vs 34%) [10]. Comparable to hazelnut, sensitization to the PR-10 protein Jug r 5 in walnut dominated in the birch-pandemic area with high correlation to birch pollen sensitization whilst sensitization to the LTP Jug r 3 dominated in Southern Europe. Notably, sensitization to walnut seed storage proteins was found in up to 10% of enrolled subjects.⁴⁶
<table><caption>TABLE 58 Prevalence of food sensitization in comparison to probable food allergy in adults across Europe</caption><thead><tr><th></th><th>Zurich</th><th>Madrid</th><th>Utrecht</th><th>Lodz</th><th>Sofia</th><th>Reykjavik</th><th>India</th></tr></thead><tbody><tr><td rowspan="2">Hazelnut</td><td>17.79</td><td>6.00</td><td>11.95</td><td>6.54</td><td>6.27</td><td>1.27</td><td>7.27</td></tr><tr><td>2.57</td><td>0.76</td><td>0.93</td><td>1.33</td><td>-</td><td>0.10</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Walnut</td><td>5.59</td><td>7.65</td><td>1.86</td><td>3.57</td><td>2.69</td><td>0.07</td><td>6.21</td></tr><tr><td>0.58</td><td>0.71</td><td>0.10</td><td>0.15</td><td>-</td><td>0.05</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Sesame</td><td>7.61</td><td>10.24</td><td>3.24</td><td>4.26</td><td>4.48</td><td>1.27</td><td>13.97</td></tr><tr><td>0.03</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>0.02</td></tr><tr><td rowspan="2">Mustard</td><td>3.59</td><td>2.71</td><td>0.41</td><td>1.71</td><td>2.69</td><td>0.37</td><td>4.68</td></tr><tr><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>0.03</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Sunflower</td><td>5.81</td><td>8.24</td><td>1.86</td><td>3.49</td><td>2.69</td><td>0.75</td><td>8.20</td></tr><tr><td>0.05</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Poppy seed</td><td>5.44</td><td>7.77</td><td>1.38</td><td>3.12</td><td>3.58</td><td>0.97</td><td>8.71</td></tr><tr><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Buckwheat</td><td>6.18</td><td>7.06</td><td>0.89</td><td>3.05</td><td>3.58</td><td>0.74</td><td>12.41</td></tr><tr><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr></tbody></table>

Table is adapted from Burney and co-workers 2014 [1144], Lyons and co-workers 2019 [1031] and Mahesh boonand co-workers 2016 [1677] – weighted samples of adults with and without allergic symptoms completed a questionnaire and donated serum for IgE analysis; probable food allergy was defined as sIgE and reported hypersensitivity-related symptoms within 2 hours after ingestion.

Australian data from the SchoolNuts study published in 2018 reported the prevalence of clinic-defined tree nut allergy to be 2.3% and sesame allergy to be 0.2% in adolescents. Amongst tree nuts, the most prevalent allergies were cashew nut allergy at 2.3% and pistachio nut allergy at 1.6%. Other tree nut allergies were less common and varied from 0.1% to almond and Brazil nut to 0.7% to walnut and hazelnut.¹²⁵² The patterns of sensitization through childhood reported by the SchoolNuts Study team were interesting. Preceding results reported by the same team had shown very low rate of tree nut allergies in Australian infants, with 0.1% of infants having an allergic reaction by the age of one, but sensitization to tested tree nuts was detected in 31% of infants who had other food allergies, particularly peanut and hen's egg allergy. At age 6 years, prevalence of sensitization to tree nuts was 7.3% and tree nut allergy was 3.3%, with cashew nut allergy being the most common at 2.7%, followed by hazelnut at 0.9% and almond at 0.3%.¹²⁵³ In an American study from 2010, self-reported tree nut allergy in 1997, 2002 and 2008 was found in 0.5%, 0.7% and 0.6% respectively, of the population. By contrast, only 0.1% reported sesame allergy.¹²⁵⁴

During the last decade, several papers have appeared on allergic reactions to cashew nut. In a recent review on cashew nut allergy, the authors concluded that an increase of sensitization in cashew nut allergy in recent decades could not be clearly documented, despite the impression that this has been the case, particularly as exposure to cashew nut has increased in the population.¹²⁵⁵ Cashew nut is now used in industrial food as a replacement for the more

expensive pine nuts and for its properties of improving texture and displaying a more reduced rancidity. In a recent population-based study of children admitted to emergency rooms due to reaction to foods in Stockholm during 2007, 5% had reacted to cashew nut, 3% to hazelnut, 2% to almond, walnut, or pistachio, whereas 0.3-0.5% reported reactions to pecan nuts, Brazils nut or coconut.¹²⁵⁶

## 5 – Clinical relevance of sensitization to individual allergenic proteins

The clinical relevance of sensitization to individual allergens is most studied in hazelnut, followed by walnut, cashew nut and sesame seed. In order to diagnose a hazelnut allergy in children, the area under the curve (AUC) for sIgE is the largest for Cor a 14 (0.87) and Cor a 9 (0.81) whilst sIgE to Cor a 1 (0.55) or Cor a 8 (0.59) have no additional value in diagnosing hazelnut allergy compared with hazelnut extract.¹²⁴⁹ It has to be considered that the hazelnut extract might be spiked with Cor a 1 (known for the ImmunoCAP platform¹²⁵⁷). Notably, as described in section 2, the prevalence of sensitization (Cor a 1, 2 and 8) seems to inversely correlate with the risk of an allergic reaction to hazelnut and sensitization to Cor a 9 and 14, as shown in (Figure 121). In adults, the AUC for Cor a 9 and Cor a 14 is decreased to 0.66 and 0.67, respectively, as described by Masthoff and co-workers,⁴³ probably due to the increased prevalence of sensitization to Cor a 1 in the adult population.⁴⁴ Data on sensitization to walnut, cashew nut, and
<table><caption>TABLE 58 Prevalence of food sensitization in comparison to probable food allergy in adults across Europe</caption><thead><tr><th></th><th>Zurich</th><th>Madrid</th><th>Utrecht</th><th>Lodz</th><th>Sofia</th><th>Reykjavik</th><th>India</th></tr></thead><tbody><tr><td rowspan="2">Hazelnut</td><td>17.79</td><td>6.00</td><td>11.95</td><td>6.54</td><td>6.27</td><td>1.27</td><td>7.27</td></tr><tr><td>2.57</td><td>0.76</td><td>0.93</td><td>1.33</td><td>-</td><td>0.10</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Walnut</td><td>5.59</td><td>7.65</td><td>1.86</td><td>3.57</td><td>2.69</td><td>0.07</td><td>6.21</td></tr><tr><td>0.58</td><td>0.71</td><td>0.10</td><td>0.15</td><td>-</td><td>0.05</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Sesame</td><td>7.61</td><td>10.24</td><td>3.24</td><td>4.26</td><td>4.48</td><td>1.27</td><td>13.97</td></tr><tr><td>0.03</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>0.02</td></tr><tr><td rowspan="2">Mustard</td><td>3.59</td><td>2.71</td><td>0.41</td><td>1.71</td><td>2.69</td><td>0.37</td><td>4.68</td></tr><tr><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>0.03</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Sunflower</td><td>5.81</td><td>8.24</td><td>1.86</td><td>3.49</td><td>2.69</td><td>0.75</td><td>8.20</td></tr><tr><td>0.05</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Poppy seed</td><td>5.44</td><td>7.77</td><td>1.38</td><td>3.12</td><td>3.58</td><td>0.97</td><td>8.71</td></tr><tr><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td rowspan="2">Buckwheat</td><td>6.18</td><td>7.06</td><td>0.89</td><td>3.05</td><td>3.58</td><td>0.74</td><td>12.41</td></tr><tr><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>&lt;0.01</td><td>-</td><td>&lt;0.01</td><td>&lt;0.01</td></tr></tbody></table>

Table is adapted from Burney and co-workers 2014 [1144], Lyons and co-workers 2019 [1031] and Mahesh boonand co-workers 2016 [1677] – weighted samples of adults with and without allergic symptoms completed a questionnaire and donated serum for IgE analysis; probable food allergy was defined as sIgE and reported hypersensitivity-related symptoms within 2 hours after ingestion.

Australian data from the SchoolNuts study published in 2018 reported the prevalence of clinic-defined tree nut allergy to be 2.3% and sesame allergy to be 0.2% in adolescents. Amongst tree nuts, the most prevalent allergies were cashew nut allergy at 2.3% and pistachio nut allergy at 1.6%. Other tree nut allergies were less common and varied from 0.1% to almond and Brazil nut to 0.7% to walnut and hazelnut.1252 The patterns of sensitization through childhood reported by the SchoolNuts Study team were interesting. Preceding results reported by the same team had shown very low rate of tree nut allergies in Australian infants, with 0.1% of infants having an allergic reaction by the age of one, but sensitization to tested tree nuts was detected in 31% of infants who had other food allergies, particularly peanut and hen's egg allergy. At age 6 years, prevalence of sensitization to tree nuts was 7.3% and tree nut allergy was 3.3%, with cashew nut allergy being the most common at 2.7%, followed by hazelnut at 0.9% and almond at 0.3%.1253 In an American study from 2010, self-reported tree nut allergy in 1997, 2002 and 2008 was found in 0.5%, 0.7% and 0.6% respectively, of the population. By contrast, only 0.1% reported sesame allergy.1254

During the last decade, several papers have appeared on allergic reactions to cashew nut. In a recent review on cashew nut allergy, the authors concluded that an increase of sensitization in cashew nut allergy in recent decades could not be clearly documented, despite the impression that this has been the case, particularly as exposure to cashew nut has increased in the population.1255 Cashew nut is now used in industrial food as a replacement for the more

expensive pine nuts and for its properties of improving texture and displaying a more reduced rancidity. In a recent population-based study of children admitted to emergency rooms due to reaction to foods in Stockholm during 2007, 5% had reacted to cashew nut, 3% to hazelnut, 2% to almond, walnut, or pistachio, whereas 0.3-0.5% reported reactions to pecan nuts, Brazils nut or coconut.1256

## 5 – Clinical relevance of sensitization to individual allergenic proteins

The clinical relevance of sensitization to individual allergens is most studied in hazelnut, followed by walnut, cashew nut and sesame seed. In order to diagnose a hazelnut allergy in children, the area under the curve (AUC) for sIgE is the largest for Cor a 14 (0.87) and Cor a 9 (0.81) whilst sIgE to Cor a 1 (0.55) or Cor a 8 (0.59) have no additional value in diagnosing hazelnut allergy compared with hazelnut extract.1249 It has to be considered that the hazelnut extract might be spiked with Cor a 1 (known for the ImmunoCAP platform1257). Notably, as described in section 2, the prevalence of sensitization (Cor a 1, 2 and 8) seems to inversely correlate with the risk of an allergic reaction to hazelnut and sensitization to Cor a 9 and 14, as shown in (Figure 121). In adults, the AUC for Cor a 9 and Cor a 14 is decreased to 0.66 and 0.67, respectively, as described by Masthoff and co-workers,43 probably due to the increased prevalence of sensitization to Cor a 1 in the adult population.44 Data on sensitization to walnut, cashew nut, and
sesame seed support the clinical relevance of slgE to 2S albumins of different sources.⁴⁶,¹²⁵⁵,¹²⁵⁸,¹²⁵⁹ However, robust data on severity of an allergic reaction to those nuts and sensitization to individual allergens, as shown for peanut allergens, are lacking.

### Cross-reactivity across tree nuts and seed allergens

Seed storage proteins such as 2S albumins, 7S globulins, and 11S globulins, share parts of their amino acid sequences across tree nuts and seeds (Figures 122–124). Due to this homology, they also share common IgE-binding epitopes, which leads to IgE cross-reactivity in vitro and potentially to clinically relevant cross-reactivity. However, as antibodies may bind to specific epitopes residing in regions that are more conserved (less variable) than the entire protein, cross-reactivity may also occur to a higher extent than indicated by the overall percentage sequence identity. Accessibility of epitopes, especially for 11S globulins, which consist of dimer of homotrimers, also has to be considered. This has been shown for nonhomologous peanut allergens Ara h 1, Ara h 2 and Ara h 3 (see section C-10).

Such homology is particularly high between 2S albumins of phylogenetically related plants such as cashew nut and pistachio (Anacardiaceae family) and between walnut and pecan nut (Juglandaceae family). Cross-reactivity between those tree nut combinations has not only been shown in vitro but also its clinical relevance has been proven. While clinically relevant sensitization to pistachio and pecan nut always implies clinically relevant sensitization to cashew nut and walnut, respectively, the reverse does not always occur. Hence, cashew nut Ana o 3 and walnut Jug r 1 are considered to be the respective primary sensitizers.¹²⁶⁰ As shown in (Figure 121), Cor a 14 and Jug r 1 share a lower but still significant sequence identity of 66%. Such high sequence identity can lead to clinically relevant IgE cross-reactivity. The ProNut study confirmed clinically relevant cross-reactivity between walnut, pecan nut, hazelnut, and macadamia in descending order.¹²⁶¹ Other sequence identities of this protein family range from 24% to 49%.

Regarding 7S globulins, the highest sequence identities are also shared between cashew nut Ana o 1 and pistachio Pis v 3 (81%) and

walnut and pecan nut (93% for Jug r 2 and 44% for Jug r 6), whereas hazelnut Cor a 11 and walnut Jug r 2 only share a sequence identity of 49%. However, Jug r 2 carries cross-reactive carbohydrate determinants (CCD), which makes it difficult to draw firm conclusions on any associations. The newly identified walnut 7S globulin Jug r 6 shares higher homology with Cor a 11 (76%), Ana o 1 (59%) and Pis v 3 (62%). The sequence homology of 7S globulin between other tree nuts and seeds, ranged from 27% to 58%.

For the 11S globulins, the sequence homology between the different nuts and legumes shows a slightly different picture with higher homology in general. Again, the rather high homology between the 7S globulins of cashew and pistachio, as well as hazelnut and walnut are evident. High homology of the hazelnut Cor a 9 with the corresponding 11S globulins from poppy seed and buckwheat (Fag e 1) points to a potential for both IgE and clinical cross-reactivity.¹²⁶² Notably, clinically relevant cross-reactivity has been described between buckwheat and latex. However, the implicated allergen has not yet been identified.¹²⁶³

## 6 – Tree nut and seed allergy diagnosis and management

### Clinical pattern and relevance

Five clear patterns of clinical relevance have been described amongst individuals sensitized or allergic to tree nuts and seeds.

A Primary sensitization to one tree nut or seed allergen;

B Co-sensitization to at least two primary tree nut and/or seed allergens;

C Primary sensitization and allergy to at least one tree nut or seed and cross-reactive IgE to another phylogenetically related tree nut or seed (high degree of sequence homology);

D Primary sensitization and allergy to at least one tree nut or seed and cross-reactive IgE to another phylogenetically not closely related tree nut or seed (low to moderate degree of sequence homology);

E Primary sensitization to pollen and cross-reactive IgE between PR-10 and LTP allergen proteins in tree nuts and seeds.

**Example for pattern A.** The patient is sensitized to only one tree nut or seed out of several tested. Irrespective of symptoms the IgE is relatively low. This patient is in general of younger age. At a very low age, IgE levels below 0.35 kU/L can be found in children reacting to nuts. This patient should avoid the culprit nut or seed but no other nuts and seed if they have tested negative to them.

**Example for pattern B.** This patient is often polysensitized to nuts and/or seeds with relatively high slgE levels to all tested relevant allergens. A patient with tree nut or seed allergy and polysensitization with generally high slgE is recommended to avoid all tree nuts with total restriction and to avoid any seeds that are causing symptoms.

<figure><img src="image_234.png" /><figcaption>**FIGURE 121** Risk ramp for hazelnut allergens: increased risk for allergic symptoms from the left to the right and increased rate of sensitization from the right to the left. In case of known clinical relevance, the ramp was supplemented with the known risk of other seed allergens. Green: Pollen-related highly cross-reactive allergens, Yellow: Food allergens with increased thermal stability and digestive resistance; 1: common in Northern and Central Europe, hazelnut extract is spiked with the PR-10 protein, 2: increased sensitization in the Mediterranean area</figcaption></figure>
sesame seed support the clinical relevance of slgE to 2S albumins of different sources.⁴⁶,¹²⁵⁵,¹²⁵⁸,¹²⁵⁹ However, robust data on severity of an allergic reaction to those nuts and sensitization to individual allergens, as shown for peanut allergens, are lacking.

### Cross-reactivity across tree nuts and seed allergens

Seed storage proteins such as 2S albumins, 7S globulins, and 11S globulins, share parts of their amino acid sequences across tree nuts and seeds (Figures 122–124). Due to this homology, they also share common IgE-binding epitopes, which leads to IgE cross-reactivity in vitro and potentially to clinically relevant cross-reactivity. However, as antibodies may bind to specific epitopes residing in regions that are more conserved (less variable) than the entire protein, cross-reactivity may also occur to a higher extent than indicated by the overall percentage sequence identity. Accessibility of epitopes, especially for 11S globulins, which consist of dimer of homotrimers, also has to be considered. This has been shown for nonhomologous peanut allergens Ara h 1, Ara h 2 and Ara h 3 (see section C-10).

Such homology is particularly high between 2S albumins of phylogenetically related plants such as cashew nut and pistachio (Anacardiaceae family) and between walnut and pecan nut (Juglandaceae family). Cross-reactivity between those tree nut combinations has not only been shown in vitro but also its clinical relevance has been proven. While clinically relevant sensitization to pistachio and pecan nut always implies clinically relevant sensitization to cashew nut and walnut, respectively, the reverse does not always occur. Hence, cashew nut Ana o 3 and walnut Jug r 1 are considered to be the respective primary sensitizers.¹²⁶⁰ As shown in (Figure 121), Cor a 14 and Jug r 1 share a lower but still significant sequence identity of 66%. Such high sequence identity can lead to clinically relevant IgE cross-reactivity. The ProNut study confirmed clinically relevant cross-reactivity between walnut, pecan nut, hazelnut, and macadamia in descending order.¹²⁶¹ Other sequence identities of this protein family range from 24% to 49%.

Regarding 7S globulins, the highest sequence identities are also shared between cashew nut Ana o 1 and pistachio Pis v 3 (81%) and

walnut and pecan nut (93% for Jug r 2 and 44% for Jug r 6), whereas hazelnut Cor a 11 and walnut Jug r 2 only share a sequence identity of 49%. However, Jug r 2 carries cross-reactive carbohydrate determinants (CCD), which makes it difficult to draw firm conclusions on any associations. The newly identified walnut 7S globulin Jug r 6 shares higher homology with Cor a 11 (76%), Ana o 1 (59%) and Pis v 3 (62%). The sequence homology of 7S globulin between other tree nuts and seeds, ranged from 27% to 58%.

For the 11S globulins, the sequence homology between the different nuts and legumes shows a slightly different picture with higher homology in general. Again, the rather high homology between the 7S globulins of cashew and pistachio, as well as hazelnut and walnut are evident. High homology of the hazelnut Cor a 9 with the corresponding 11S globulins from poppy seed and buckwheat (Fag e 1) points to a potential for both IgE and clinical cross-reactivity.¹²⁶² Notably, clinically relevant cross-reactivity has been described between buckwheat and latex. However, the implicated allergen has not yet been identified.¹²⁶³

## 6 – Tree nut and seed allergy diagnosis and management

### Clinical pattern and relevance

Five clear patterns of clinical relevance have been described amongst individuals sensitized or allergic to tree nuts and seeds.

A Primary sensitization to one tree nut or seed allergen;

B Co-sensitization to at least two primary tree nut and/or seed allergens;

C Primary sensitization and allergy to at least one tree nut or seed and cross-reactive IgE to another phylogenetically related tree nut or seed (high degree of sequence homology);

D Primary sensitization and allergy to at least one tree nut or seed and cross-reactive IgE to another phylogenetically not closely related tree nut or seed (low to moderate degree of sequence homology);

E Primary sensitization to pollen and cross-reactive IgE between PR-10 and LTP allergen proteins in tree nuts and seeds.

**Example for pattern A.** The patient is sensitized to only one tree nut or seed out of several tested. Irrespective of symptoms the IgE is relatively low. This patient is in general of younger age. At a very low age, IgE levels below 0.35 kU/L can be found in children reacting to nuts. This patient should avoid the culprit nut or seed but no other nuts and seed if they have tested negative to them.

**Example for pattern B.** This patient is often polysensitized to nuts and/or seeds with relatively high slgE levels to all tested relevant allergens. A patient with tree nut or seed allergy and polysensitization with generally high slgE is recommended to avoid all tree nuts with total restriction and to avoid any seeds that are causing symptoms.

<figure><img src="image_235.png" /><figcaption>**FIGURE 121** Risk ramp for hazelnut allergens: increased risk for allergic symptoms from the left to the right and increased rate of sensitization from the right to the left. In case of known clinical relevance, the ramp was supplemented with the known risk of other seed allergens. Green: Pollen-related highly cross-reactive allergens, Yellow: Food allergens with increased thermal stability and digestive resistance; 1: common in Northern and Central Europe, hazelnut extract is spiked with the PR-10 protein, 2: increased sensitization in the Mediterranean area</figcaption></figure>
<figure><img src="image_235.png" /><figcaption>FIGURE 122 Amino acid sequence identity among 2S albumins between different tree nuts and seeds (without signal sequence; bold >50%) Cora 14: D0PWG2; Jug r 1: P93198; Car i 1: Q84XA9; Ana o 3: Q8H2B8; Pis v 1: B7P072; Ber e 1: P04403; Pin p 1: A0A0K3AVY3; Ses i 1: Q9AUD1; Sin a 1: P15322; Cuc ma 5: Q39649; Fag e 2: Q2PS07; Lin u 1: Q8LPD3</figcaption></figure>

<figure><img src="image_236.png" /><figcaption>FIGURE 123 Amino acid sequence identity among 7S globulins between different tree nuts and seeds Cora 11: Q8S4P9; Jug r 6: A0A214E5L6; Car i 2: B3STU4; Ana o 1: Q8L5L5; Pis v 3: B4X640; Pru du 8: A0A516F3L2; Mac i 1: Q9SPL3; Coc n 1: A0A0S3B0K0; Ses i 3: Q9AUD0; Fag e 3: A5HIX6 (only fragment available), *Homology between Jug r 2 and Car i 2 is 93%</figcaption></figure>

<figure><img src="image_237.png" /><figcaption>FIGURE 124 Amino acid sequence identity among 11S globulins between different tree nuts and seeds Cora 9: Q8W1C2; Jug r 4: Q2TPW5; Car i 4: B5KVH4; Ana o 2: Q8GZP6; Pis v 5: B7SLJ1; Pru du 6: E3SH28; Ber e 2: Q84ND2; Ses i 7: Q9AUD2; Sin a 2: Q2TLW0; Cuc ma 4: P13744; Fag e 1: K4PY05</figcaption></figure>

**Example for pattern C.** The patient is sensitized to cashew nut and pistachio, or to walnut and pecan nut with rather equal slgE levels between the botanically related groups of tree nuts. Patients sensitized to only cashew nut and pistachio should only avoid those tree nuts. No other restrictions should be made. The same recommendation would be appropriate for patients sensitized and allergic to only walnut and pecan nut. Testing for other tree nut allergies is required prior to considering introduction. For patients sensitized to walnut, clinical cross-reaction to hazelnut and macadamia may occur.¹²⁶¹

**Example for pattern D.** This is the most common pattern in patients with tree nut allergy: allergic to several nuts with high slgE levels to those tree nuts, but much lower slgE levels to other tree

nuts. Usually, those patients tolerate other nuts well, but this needs to be confirmed by oral food challenge (OFC). In the ProNuts study, children allergic to at least one tree nut were, on average able to tolerate 9 other tree nuts or sesame seed by undergoing sequential OFCs.¹²⁶¹

**Example for pattern E.** This patient is most likely birch pollen allergic (Bet v 1) if s/he is resident in Northern Europe. If resident in Southern Europe, positive IgE to Artemisia, Paretaria or plane tree (LTP) may be present. Approximately 80% of the population with concurrent birch pollen allergy will experience "birch pollen related food allergy" to other PR-10 allergens. The PR-10 allergen Cora 1 is unstable to gastric digestion and is heat labile. Symptoms in the oral cavity may be unpleasant but will not cause systemic
<figure><img src="image_236.png" /><figcaption>FIGURE 122 Amino acid sequence identity among 2S albumins between different tree nuts and seeds (without signal sequence; bold >50%) Cora 14: D0PWG2; Jug r 1: P93198; Car i 1: Q84XA9; Ana o 3: Q8H2B8; Pis v 1: B7P072; Ber e 1: P04403; Pin p 1: A0A0K3AVY3; Ses i 1: Q9AUD1; Sin a 1: P15322; Cuc ma 5: Q39649; Fag e 2: Q2PS07; Lin u 1: Q8LPD3</figcaption></figure>

<figure><img src="image_237.png" /><figcaption>FIGURE 123 Amino acid sequence identity among 7S globulins between different tree nuts and seeds Cora 11: Q8S4P9; Jug r 6: A0A214E5L6; Car i 2: B3STU4; Ana o 1: Q8L5L5; Pis v 3: B4X640; Pru du 8: A0A516F3L2; Mac i 1: Q9SPL3; Coc n 1: A0A0S3B0K0; Ses i 3: Q9AUD0; Fag e 3: A5HIX6 (only fragment available), *Homology between Jug r 2 and Car i 2 is 93%</figcaption></figure>

<figure><img src="image_238.png" /><figcaption>FIGURE 124 Amino acid sequence identity among 11S globulins between different tree nuts and seeds Cora 9: Q8W1C2; Jug r 4: Q2TPW5; Car i 4: B5KVH4; Ana o 2: Q8GZP6; Pis v 5: B7SLJ1; Pru du 6: E3SH28; Ber e 2: Q84ND2; Ses i 7: Q9AUD2; Sin a 2: Q2TLW0; Cuc ma 4: P13744; Fag e 1: K4PY05</figcaption></figure>

**Example for pattern C.** The patient is sensitized to cashew nut and pistachio, or to walnut and pecan nut with rather equal slgE levels between the botanically related groups of tree nuts. Patients sensitized to only cashew nut and pistachio should only avoid those tree nuts. No other restrictions should be made. The same recommendation would be appropriate for patients sensitized and allergic to only walnut and pecan nut. Testing for other tree nut allergies is required prior to considering introduction. For patients sensitized to walnut, clinical cross-reaction to hazelnut and macadamia may occur.¹²⁶¹

**Example for pattern D.** This is the most common pattern in patients with tree nut allergy: allergic to several nuts with high slgE levels to those tree nuts, but much lower slgE levels to other tree

nuts. Usually, those patients tolerate other nuts well, but this needs to be confirmed by oral food challenge (OFC). In the ProNuts study, children allergic to at least one tree nut were, on average able to tolerate 9 other tree nuts or sesame seed by undergoing sequential OFCs.¹²⁶¹

**Example for pattern E.** This patient is most likely birch pollen allergic (Bet v 1) if s/he is resident in Northern Europe. If resident in Southern Europe, positive IgE to Artemisia, Paretaria or plane tree (LTP) may be present. Approximately 80% of the population with concurrent birch pollen allergy will experience "birch pollen related food allergy" to other PR-10 allergens. The PR-10 allergen Cora 1 is unstable to gastric digestion and is heat labile. Symptoms in the oral cavity may be unpleasant but will not cause systemic
reactions under normal circumstances. In most tree nuts and seeds, PR-10 proteins are likely to be present, most notably in Fagales trees (plant order that include: *Betulaceae* (Birch, Alder, Hazel tree, Hornbeam), *Fagaceae* (chestnut, trigonobalanus, beech tree, oak tree), *Juglandaceae* (walnut, hickory, wingnuts tree), *Myricaceae* (bay-berry), *Nothofagaceae* (southern beech), *Casuarinaceae* (she-oak), and *Ticodendracae* family).

**Clinical diagnosis**

Specific questions, appropriate interpretation of sensitization test results and, under certain conditions, open or blinded OFCs will help to establish the diagnosis of tree nut and seed allergy and the grade of severity. The following work-up might facilitate an accurate diagnosis, which will be beneficial for the patient (Figure 125).

**Detailed patient history:**

The following are examples of questions that will help establish a detailed patient history:

* Were there previous reaction(s) to tree nuts and/or seeds or is this the first reaction?

* Did the patient previously tolerate the offending food?

* What were the symptoms, and which were the affected organs?

* Were multiple foods ingested or multiple nuts?

* What was the approximate dose causing the symptoms?

* What was the time for onset of symptoms after ingestion?

* What is the estimated time until the administration of the adrenaline autoinjector (AAI)?

* What was the response to injection of adrenaline?

* Were one, two or more AAI used?

* Did the patient engage in concomitant exercise, take nonsteroidal anti-inflammatories (NSAID), consume alcohol, or other potentially aggravating factors?

* Is the patient birch pollen allergic or allergic to plants with pollen containing LTPs?

**Appropriate interpretation of sensitization results:**

**Skin prick test (SPT)**

Commercial extracts of tree nuts and seeds or prick-to-prick test with fresh tree nuts or seeds have a limited value due to false positive responses in case of cross-reactive labile tree nut/seed aller- genic proteins, such as Cor a 1. However, they are an important first step in the diagnostic algorithm for evaluation of tree nut and seed allergies.

<figure><img src="image_238.png" /></figure>

**FIGURE 125** Diagnostic work-up in tree nut, peanut and/or seed-related allergic reactions. Arrows indicate potential diagnostic steps; dashed arrows indicate that mild and severe reactions can be associated with different clinical features (based on information from the detailed patient history).
reactions under normal circumstances. In most tree nuts and seeds, PR-10 proteins are likely to be present, most notably in Fagales trees (plant order that include: *Betulaceae* (Birch, Alder, Hazel tree, Hornbeam), *Fagaceae* (chestnut, trigonobalanus, beech tree, oak tree), *Juglandaceae* (walnut, hickory, wingnuts tree), *Myricaceae* (bay-berry), *Nothofagaceae* (southern beech), *Casuarinaceae* (she-oak), and *Ticodendracae* family).

**Clinical diagnosis**

Specific questions, appropriate interpretation of sensitization test results and, under certain conditions, open or blinded OFCs will help to establish the diagnosis of tree nut and seed allergy and the grade of severity. The following work-up might facilitate an accurate diagnosis, which will be beneficial for the patient (Figure 125).

**Detailed patient history:**

The following are examples of questions that will help establish a detailed patient history:

* Were there previous reaction(s) to tree nuts and/or seeds or is this the first reaction?

* Did the patient previously tolerate the offending food?

* What were the symptoms, and which were the affected organs?

* Were multiple foods ingested or multiple nuts?

* What was the approximate dose causing the symptoms?

* What was the time for onset of symptoms after ingestion?

* What is the estimated time until the administration of the adrenaline autoinjector (AAI)?

* What was the response to injection of adrenaline?

* Were one, two or more AAI used?

* Did the patient engage in concomitant exercise, take nonsteroidal anti-inflammatories (NSAID), consume alcohol, or other potentially aggravating factors?

* Is the patient birch pollen allergic or allergic to plants with pollen containing LTPs?

**Appropriate interpretation of sensitization results:**

**Skin prick test (SPT)**

Commercial extracts of tree nuts and seeds or prick-to-prick test with fresh tree nuts or seeds have a limited value due to false positive responses in case of cross-reactive labile tree nut/seed aller- genic proteins, such as Cor a 1. However, they are an important first step in the diagnostic algorithm for evaluation of tree nut and seed allergies.

<figure><img src="image_239.png" /></figure>

**FIGURE 125** Diagnostic work-up in tree nut, peanut and/or seed-related allergic reactions. Arrows indicate potential diagnostic steps; dashed arrows indicate that mild and severe reactions can be associated with different clinical features (based on information from the detailed patient history).
## IgE Testing

Total IgE measurements do not aid in the diagnosis of specific tree nut or seed allergies. It may, however, explain polysensitization to multiple nuts and seeds in patients with a highly raised total IgE who may only react clinically to one particular food.

In clear cases with exposure to a single tree nut or seed followed by a systemic reaction one could question whether slgE testing is needed. However, the rationale for performing slgE testing is to confirm the presence of allergen-specific IgE and examine the possibility of co-sensitization or cross-reactive IgE to other tree nuts or seeds and to assess the risk of a reaction at exposure:

* In hazelnut or walnut allergic individuals, slgE to Cor a 14 and Jug r 1, with a sequence homology of 66%, could be tested (clinical pattern A, B or D). In patients with a history of allergic reactions to hazelnut, slgE to Cor a 14 and Cor a 9 can be measured to disentangle primary allergy from sensitization to solely Cor a 1 (if tree nut-related clinical pattern A-D),

* slgE to Cor a 1 and/or Bet v 1 or Cor a 8 and LTP containing pollens could confirm the pollen food syndrome (if hazelnut-related clinical pattern E),

* For tree nut or seed extracts containing PR-10 protein (hazelnut extract is spiked with Cor a 1, known for the ImmunoCAP platform [21]) or LTPs: risk of clinically irrelevant false positive results (clinical pattern E),

* For other tree nuts and seeds allergen components, there is to date little experience in clinical practice; however, sensitization to 2S albumins of cashew nut (Ana o 3), pistachio (Pis v 1) and sesame (Ses i 1) seem to be a reliable marker for clinically relevant sensitization (pattern A, B and D)

For interpretation of sensitization test results see also (Figure 125). Results are only clinically relevant in case of corresponding symptoms after tree nut or seed ingestion.

Basophil activation testing (BAT) can also be performed with tree nuts and seeds allergens, such as from hazelnut.¹²⁶⁴ In contrast to peanut allergy, their diagnostic implications are not yet clear.

## Oral food challenges

Oral food challenges (open or blinded) should be performed in cases of doubtful relationship between reported symptoms following ingestion and slgE test results and in patients who have avoided certain tree nuts, legumes/peanuts or seeds due to a previous reaction to this kind of foods and sensitization to that food can be demonstrated:

Depending on the clinical pattern (A – E), an oral food challenge with standardized increasing doses of the offending food may be performed. It is important to not stop at a too low dose: PRACTALL guidelines of 4.43 g of tree nut protein (2 grams top dose).¹²⁶⁵

Low dose challenge in tree nut/seed allergic individuals to reduce fear of products labelled with "may contain" may play a role in improving quality of life and dietary restrictions.

Airborne challenges for those with a fear being in an environment where nuts/seeds are present (restaurants, cafés, parties, travelling by air) may be helpful in certain specific cases.

### Clinical management

#### Allergen avoidance

Balanced avoidance of the offending tree nut, and/or seeds. If a patient is likely to experience a systemic reaction on a minor dose, a high degree of precaution is required. If reactions due to PR-10 allergy, it is up to the patient to decide on amount of exposure.

#### Pharmacotherapy (emergency kit)

In patients with previously anaphylaxis or systemic reactions on a minor dose, emergency medication including AAI for self-administration is required accompanied with a personalized emergency treatment plan. As reactions to tree nuts and seeds may be variable, some centres will prescribe AAI even when mild allergic reactions to these foods have been experienced¹²⁶⁶ [30].

Nonsedating antihistamines are useful for the management of mild to moderate reactions, but adrenaline is the first line treatment for anaphylaxis and antihistamines are relegated to a second or third-line treatment.

Steroids are no longer part of standard treatment for allergic reactions unless asthma exacerbation may have contributed to severity of anaphylaxis or in case of refractory anaphylaxis. Additional symptomatic treatment as required.

#### Allergen-specific immunotherapy

At present, commercially available products for allergen-specific immunotherapy of tree nut or seed allergies are not available. Research into tree nut and sesame desensitization is ongoing in certain US centres and commercial companies are working on walnut and cashew nut desensitization.

Birch pollen immunotherapy has also been considered as a treatment option in patients with respiratory allergy and pollen food syndrome to variety of fruits, vegetables, and nuts. Next to birch pollen allergy, the patients suffered from pollen food syndrome mainly to apple and hazelnut. Unfortunately, results from those studies are not very consistent. While some studies reported significant reduction and even complete resolution of pollen food syndrome symptoms in some participants,¹¹⁷²,¹²⁶⁶ other studies were not able to confirm such an effect.¹¹⁷³,¹²⁶⁷ In addition, studies investigating the role of immunotherapy in patients with pollen food syndrome were primarily designed to investigate the efficacy of immunotherapy in allergic rhinitis, they usually included small numbers of participants, and they were necessarily not double-blind placebo controlled. Nevertheless, immunotherapy remains as a treatment of interest, particularly in patients with severe symptoms of pollen food syndrome, that requires further research.
## IgE Testing

Total IgE measurements do not aid in the diagnosis of specific tree nut or seed allergies. It may, however, explain polysensitization to multiple nuts and seeds in patients with a highly raised total IgE who may only react clinically to one particular food.

In clear cases with exposure to a single tree nut or seed followed by a systemic reaction one could question whether slgE testing is needed. However, the rationale for performing slgE testing is to confirm the presence of allergen-specific IgE and examine the possibility of co-sensitization or cross-reactive IgE to other tree nuts or seeds and to assess the risk of a reaction at exposure:

* In hazelnut or walnut allergic individuals, slgE to Cor a 14 and Jug r 1, with a sequence homology of 66%, could be tested (clinical pattern A, B or D). In patients with a history of allergic reactions to hazelnut, slgE to Cor a 14 and Cor a 9 can be measured to disentangle primary allergy from sensitization to solely Cor a 1 (if tree nut-related clinical pattern A-D),

* slgE to Cor a 1 and/or Bet v 1 or Cor a 8 and LTP containing pollens could confirm the pollen food syndrome (if hazelnut-related clinical pattern E),

* For tree nut or seed extracts containing PR-10 protein (hazelnut extract is spiked with Cor a 1, known for the ImmunoCAP platform [21]) or LTPs: risk of clinically irrelevant false positive results (clinical pattern E),

* For other tree nuts and seeds allergen components, there is to date little experience in clinical practice; however, sensitization to 2S albumins of cashew nut (Ana o 3), pistachio (Pis v 1) and sesame (Ses i 1) seem to be a reliable marker for clinically relevant sensitization (pattern A, B and D)

For interpretation of sensitization test results see also (Figure 125). Results are only clinically relevant in case of corresponding symptoms after tree nut or seed ingestion.

Basophil activation testing (BAT) can also be performed with tree nuts and seeds allergens, such as from hazelnut.¹²⁶⁴ In contrast to peanut allergy, their diagnostic implications are not yet clear.

## Oral food challenges

Oral food challenges (open or blinded) should be performed in cases of doubtful relationship between reported symptoms following ingestion and slgE test results and in patients who have avoided certain tree nuts, legumes/peanuts or seeds due to a previous reaction to this kind of foods and sensitization to that food can be demonstrated:

Depending on the clinical pattern (A – E), an oral food challenge with standardized increasing doses of the offending food may be performed. It is important to not stop at a too low dose: PRACTALL guidelines of 4.43 g of tree nut protein (2 grams top dose).¹²⁶⁵

Low dose challenge in tree nut/seed allergic individuals to reduce fear of products labelled with "may contain" may play a role in improving quality of life and dietary restrictions.

Airborne challenges for those with a fear being in an environment where nuts/seeds are present (restaurants, cafés, parties, travelling by air) may be helpful in certain specific cases.

## Clinical management

### Allergen avoidance

Balanced avoidance of the offending tree nut, and/or seeds. If a patient is likely to experience a systemic reaction on a minor dose, a high degree of precaution is required. If reactions due to PR-10 allergy, it is up to the patient to decide on amount of exposure.

### Pharmacotherapy (emergency kit)

In patients with previously anaphylaxis or systemic reactions on a minor dose, emergency medication including AAI for self-administration is required accompanied with a personalized emergency treatment plan. As reactions to tree nuts and seeds may be variable, some centres will prescribe AAI even when mild allergic reactions to these foods have been experienced¹²⁶⁶ [30].

Nonsedating antihistamines are useful for the management of mild to moderate reactions, but adrenaline is the first line treatment for anaphylaxis and antihistamines are relegated to a second or third-line treatment.

Steroids are no longer part of standard treatment for allergic reactions unless asthma exacerbation may have contributed to severity of anaphylaxis or in case of refractory anaphylaxis. Additional symptomatic treatment as required.

### Allergen-specific immunotherapy

At present, commercially available products for allergen-specific immunotherapy of tree nut or seed allergies are not available. Research into tree nut and sesame desensitization is ongoing in certain US centres and commercial companies are working on walnut and cashew nut desensitization.

Birch pollen immunotherapy has also been considered as a treatment option in patients with respiratory allergy and pollen food syndrome to variety of fruits, vegetables, and nuts. Next to birch pollen allergy, the patients suffered from pollen food syndrome mainly to apple and hazelnut. Unfortunately, results from those studies are not very consistent. While some studies reported significant reduction and even complete resolution of pollen food syndrome symptoms in some participants,¹¹⁷²,¹²⁶⁶ other studies were not able to confirm such an effect.¹¹⁷³,¹²⁶⁷ In addition, studies investigating the role of immunotherapy in patients with pollen food syndrome were primarily designed to investigate the efficacy of immunotherapy in allergic rhinitis, they usually included small numbers of participants, and they were necessarily not double-blind placebo controlled. Nevertheless, immunotherapy remains as a treatment of interest, particularly in patients with severe symptoms of pollen food syndrome, that requires further research.
# B20 - Bee venom allergy

Simon Blank, Maria Beatrice Bilò, Thilo Jakob, Markus Ollert

## Highlights

* In honeybee venom allergy, CRD is a valuable tool since CCD-free Api m 1, Api m 3, Api m 4 and Api m 10 are marker allergens for detection of specific IgE sensitization to honeybee venom.

* The differentiating marker allergens Api m 1, Api m 3, Api m 4 and Api m 10 allow discrimination between primary sensitization to honeybee and vespid venom.

* There are no marker allergens available that allow discrimination between primary honeybee and bumblebee venom sensitization.

* It is recommended to determine the baseline serum tryptase level in all patients with a history of a systemic sting reaction.

* Venom-specific immunotherapy is an effective treatment to protect against future severe sting reactions.

## 1 – The allergen sources

Hymenoptera venom allergy is one of the most serious IgE-mediated hypersensitivities due to the high risk of severe and even fatal anaphylaxis. In adults (> 18 years), 48.2% of cases of severe anaphylaxis are caused by Hymenoptera stings (20.2% in children).<sup>1273</sup> Bees (Family Apidae) are flying insects of the order Hymenoptera with more than 5.700 known species (Figure 126). The most common elicitors of bee venom allergy are honeybees (Apis spp.), which are known for their outstanding role in pollination and for producing honey and beeswax.

Only two honeybee species have been truly domesticated, whereby *Apis mellifera* (European, western or common honeybee) (Figure 127) is the most common domesticated species and shows a world-wide distribution due to human-induced global dispersal. The other domesticated species *Apis cerana* (eastern honeybee) is native to southern and eastern Asia. Primary allergy to bumblebee (*Bombus spp.*) venom (BBV) is rare and primarily workplace-associated as bumblebees (Figure 127) are increasingly used for pollination in greenhouses.

Sensitization to bee venom occurs after a sting, whereby honeybees are the only stinging Hymenoptera that nearly always leave their stinger with adherent venom sac in the skin of the victim. Meanwhile the venom is continuously pumped into the skin until the venom sac is exhausted or the stinger removed.

## 2 – Allergen families and allergenic molecules

Bee venom is a complex mixture of low molecular weight substances such as biogenic amines and basic peptides and of higher molecular weight proteins, many of them with enzymatic activity. The venom of the honeybee *Apis mellifera* is one of the best characterized Hymenoptera venoms. In addition to well-established honeybee

venom (HBV) allergens, several new relevant allergens of low abundance were identified in the last decade mainly by proteomic approaches (Figure 128). Currently, 12 different HBV allergens are listed in the WHO/IUIS allergen nomenclature database. By contrast, BBV is less well investigated and only two allergen families (phospholipases A2, Bom t 1 and Bom p 1; protease, Bom t 4 and Bom p 4) are listed in the WHO/IUIS allergen nomenclature database (Figure 128).

Many allergens of bee venoms are proteins, which exhibit direct toxicity in the stung victim. Others have functions in the venom sac such as activation of toxic proteins, thereby, protecting the surrounding tissue of the venom gland. The venom allergens of different honeybee species are highly similar and also bumblebee venom closely resembles honeybee venom. Both venoms are reported to be highly cross-reactive.<sup>1274</sup> The IgE-binding capacity of most of the bee venom allergens seems to depend on correct three-dimensional folding of the molecules (Figure 129).

Both HBV and BBV contain phospholipases A2 (PLA2) (Api m 1, Api c 1, Api d 1, Bom t 1, Bom p 1) as relevant major allergens. PLAs2 are hydrolases that cleave fatty acids from phospholipids in cell membranes at the sn-2 position. This enzymatic activity leads to direct toxic effects such as cell lysis, pore formation and release of pro-inflammatory mediators. Additionally PLAs2 mediate catalytic-independent neurotoxicity by binding to N-type receptors. PLA2 makes up for up to 16% of the venoms dry weight. Although phospholipases A1 of vespid venoms catalyze a related enzymatic reaction, they share neither sequence identity nor structural similarity with PLAs2 of bee venom. The resulting lack of cross-reactivity renders PLAs ideal marker allergens to discriminate between bee and vespid sensitization.

The main component of HBV (50% of dry weight) is melittin (Api m 4), a cytotoxic 26 amino acid peptide that as a tetramer integrates into cell membranes and induces cell death, destruction of mast cells and vascular dilation. Moreover, melittin is the main pain-inducing substance of HBV mediated by the activation of nociceptors.

Together, Api m 1 and Api m 4 account for more than 60% of the HBVs' dry weight. By contrast, all other allergens are present in comparably low amounts. Nevertheless, several of them are of high relevance in allergy diagnosis (Figure 128, Table 59)

Hyaluronidases are common components of Hymenoptera venoms and have been annotated as allergens for eight species, including the honeybee Apis mellifera (Api m 2). So far, no allergenic hyaluronidase is annotated for BBV. Hyaluronidases cleave hyaluronan, a main component in vertebrates' extracellular matrix. Therefore, they promote the spreading of the venom at the injection site. Api m 2 shares 44-53% sequence identity and extended structural similarity with the hyaluronidases of yellow jacket (Ves v 2) and European paper wasp (Pol d 2) venom. However, cross-reactivity independent of cross-reactive carbohydrate determinants (CCDs) seems to be limited.

So far, acid phosphatase was only annotated as allergen for HBV (Api m 3). However, also BBV contains an allergenic acid phosphatase that shows moderate cross-reactivity with Api m 3. The function of acid phosphatases in Hymenoptera venoms so far remains
# B20 - Bee venom allergy

Simon Blank, Maria Beatrice Bilò, Thilo Jakob, Markus Ollert

## Highlights

* In honeybee venom allergy, CRD is a valuable tool since CCD-free Api m 1, Api m 3, Api m 4 and Api m 10 are marker allergens for detection of specific IgE sensitization to honeybee venom.

* The differentiating marker allergens Api m 1, Api m 3, Api m 4 and Api m 10 allow discrimination between primary sensitization to honeybee and vespid venom.

* There are no marker allergens available that allow discrimination between primary honeybee and bumblebee venom sensitization.

* It is recommended to determine the baseline serum tryptase level in all patients with a history of a systemic sting reaction.

* Venom-specific immunotherapy is an effective treatment to protect against future severe sting reactions.

## 1 – The allergen sources

Hymenoptera venom allergy is one of the most serious IgE-mediated hypersensitivities due to the high risk of severe and even fatal anaphylaxis. In adults (> 18 years), 48.2% of cases of severe anaphylaxis are caused by Hymenoptera stings (20.2% in children).<sup>1273</sup> Bees (Family Apidae) are flying insects of the order Hymenoptera with more than 5.700 known species (Figure 126). The most common elicitors of bee venom allergy are honeybees (Apis spp.), which are known for their outstanding role in pollination and for producing honey and beeswax.

Only two honeybee species have been truly domesticated, whereby *Apis mellifera* (European, western or common honeybee) (Figure 127) is the most common domesticated species and shows a world-wide distribution due to human-induced global dispersal. The other domesticated species *Apis cerana* (eastern honeybee) is native to southern and eastern Asia. Primary allergy to bumblebee (*Bombus spp.*) venom (BBV) is rare and primarily workplace-associated as bumblebees (Figure 127) are increasingly used for pollination in greenhouses.

Sensitization to bee venom occurs after a sting, whereby honeybees are the only stinging Hymenoptera that nearly always leave their stinger with adherent venom sac in the skin of the victim. Meanwhile the venom is continuously pumped into the skin until the venom sac is exhausted or the stinger removed.

## 2 – Allergen families and allergenic molecules

Bee venom is a complex mixture of low molecular weight substances such as biogenic amines and basic peptides and of higher molecular weight proteins, many of them with enzymatic activity. The venom of the honeybee *Apis mellifera* is one of the best characterized Hymenoptera venoms. In addition to well-established honeybee

venom (HBV) allergens, several new relevant allergens of low abundance were identified in the last decade mainly by proteomic approaches (Figure 128). Currently, 12 different HBV allergens are listed in the WHO/IUIS allergen nomenclature database. By contrast, BBV is less well investigated and only two allergen families (phospholipases A2, Bom t 1 and Bom p 1; protease, Bom t 4 and Bom p 4) are listed in the WHO/IUIS allergen nomenclature database (Figure 128).

Many allergens of bee venoms are proteins, which exhibit direct toxicity in the stung victim. Others have functions in the venom sac such as activation of toxic proteins, thereby, protecting the surrounding tissue of the venom gland. The venom allergens of different honeybee species are highly similar and also bumblebee venom closely resembles honeybee venom. Both venoms are reported to be highly cross-reactive.<sup>1274</sup> The IgE-binding capacity of most of the bee venom allergens seems to depend on correct three-dimensional folding of the molecules (Figure 129).

Both HBV and BBV contain phospholipases A2 (PLA2) (Api m 1, Api c 1, Api d 1, Bom t 1, Bom p 1) as relevant major allergens. PLAs2 are hydrolases that cleave fatty acids from phospholipids in cell membranes at the sn-2 position. This enzymatic activity leads to direct toxic effects such as cell lysis, pore formation and release of pro-inflammatory mediators. Additionally PLAs2 mediate catalytic-independent neurotoxicity by binding to N-type receptors. PLA2 makes up for up to 16% of the venoms dry weight. Although phospholipases A1 of vespid venoms catalyze a related enzymatic reaction, they share neither sequence identity nor structural similarity with PLAs2 of bee venom. The resulting lack of cross-reactivity renders PLAs ideal marker allergens to discriminate between bee and vespid sensitization.

The main component of HBV (50% of dry weight) is melittin (Api m 4), a cytotoxic 26 amino acid peptide that as a tetramer integrates into cell membranes and induces cell death, destruction of mast cells and vascular dilation. Moreover, melittin is the main pain-inducing substance of HBV mediated by the activation of nociceptors.

Together, Api m 1 and Api m 4 account for more than 60% of the HBVs' dry weight. By contrast, all other allergens are present in comparably low amounts. Nevertheless, several of them are of high relevance in allergy diagnosis (Figure 128, Table 59)

Hyaluronidases are common components of Hymenoptera venoms and have been annotated as allergens for eight species, including the honeybee Apis mellifera (Api m 2). So far, no allergenic hyaluronidase is annotated for BBV. Hyaluronidases cleave hyaluronan, a main component in vertebrates' extracellular matrix. Therefore, they promote the spreading of the venom at the injection site. Api m 2 shares 44-53% sequence identity and extended structural similarity with the hyaluronidases of yellow jacket (Ves v 2) and European paper wasp (Pol d 2) venom. However, cross-reactivity independent of cross-reactive carbohydrate determinants (CCDs) seems to be limited.

So far, acid phosphatase was only annotated as allergen for HBV (Api m 3). However, also BBV contains an allergenic acid phosphatase that shows moderate cross-reactivity with Api m 3. The function of acid phosphatases in Hymenoptera venoms so far remains
<figure><img src="image_239.png" /></figure>

**FIGURE 126** Taxonomy of allergy-relevant bee species. As the taxonomy of the order Hymenoptera is highly complex, only a selection of allergy-relevant taxa is shown. Only those species are included that are listed in the WHO/IUIS allergen nomenclature database. For taxonomic overview of allergy-relevant vespids (Vespidae) refer to the vespid venom chapter. The family Formicidae (ants) also contains species with relevance for Hymenoptera venom allergy.

<figure><img src="image_240.png" /></figure>

**FIGURE 127** The honeybee and the bumblebee

elusive. They may catalyze the liberation of purines, mainly adenosine, which act as multitoxins.

Another protein family found in several Hymenoptera venoms is the family of dipeptidyl peptidases IV (DPPIV).

The DPPIV of HBV (Api m 5) catalyzes the conversion from promelittin to melittin by liberating dipeptides from the N-terminus. By only activating melittin in the venom sac, the honeybees probably protect themselves against its toxic effects. HBV Api m 5 exhibits extensive cross-reactivity with the homologous allergens of yellow jacket and European paper wasp venom Ves v 3 and Pol d 3, respectively.

Icarapin (Api m 10) is a protein of so far unknown function in HBV that contains no known functional domains. Nevertheless, it is a conserved protein, as icarapin-like proteins were identified in various species of the phylogenic class Insecta. Structure predictions reveal that large parts of the protein seem to be disordered (Figure 129). So far no homologous allergens have been described in any other species.

Other less investigated bee venom allergens include protease inhibitor (Api m 6), CUB serine protease (Api m 7), carboxylesterase (Api m 8), serine carboxypeptidase (Api m 9), major royal jelly protein
<figure><img src="image_240.png" /></figure>

**FIGURE 126** Taxonomy of allergy-relevant bee species. As the taxonomy of the order Hymenoptera is highly complex, only a selection of allergy-relevant taxa is shown. Only those species are included that are listed in the WHO/IUIS allergen nomenclature database. For taxonomic overview of allergy-relevant vespids (Vespidae) refer to the vespid venom chapter. The family Formicidae (ants) also contains species with relevance for Hymenoptera venom allergy.

<figure><img src="image_241.png" /></figure>

**FIGURE 127** The honeybee and the bumblebee

elusive. They may catalyze the liberation of purines, mainly adenosine, which act as multitoxins.

Another protein family found in several Hymenoptera venoms is the family of dipeptidyl peptidases IV (DPPIV).

The DPPIV of HBV (Api m 5) catalyzes the conversion from promelittin to melittin by liberating dipeptides from the N-terminus. By only activating melittin in the venom sac, the honeybees probably protect themselves against its toxic effects. HBV Api m 5 exhibits extensive cross-reactivity with the homologous allergens of yellow jacket and European paper wasp venom Ves v 3 and Pol d 3, respectively.

Icarapin (Api m 10) is a protein of so far unknown function in HBV that contains no known functional domains. Nevertheless, it is a conserved protein, as icarapin-like proteins were identified in various species of the phylogenic class Insecta. Structure predictions reveal that large parts of the protein seem to be disordered (Figure 129). So far no homologous allergens have been described in any other species.

Other less investigated bee venom allergens include protease inhibitor (Api m 6), CUB serine protease (Api m 7), carboxylesterase (Api m 8), serine carboxypeptidase (Api m 9), major royal jelly protein
<figure><img src="image_241.png" /><figcaption>FIGURE 128 Annotated allergens of honeybee (A. mellifera) and bumblebee (B. terrestris) venom. For HBV, only selected allergens with proven high relevance are depicted. Allergens from the same protein family are shown in identical colors.</figcaption></figure>

<figure><img src="image_242.png" /><figcaption>FIGURE 129 Structures of selected relevant honeybee venom allergens. α-helices, β-strands and coiled regions are shown in red, blue and grey, respectively. The structures of Api m 1 (PDB: 1POC) and Api m 2 (PDB: 1FCU) were solved by crystallography. All other structures were generated by structural modeling.</figcaption></figure>

<table><caption>TABLE 59 Most relevant allergens of honeybee (A. mellifera) venom</caption><thead><tr><td>Allergenic molecule</td><td>Biochemical name</td><td>Prevalence among patients (%)</td><td>Molecular weight (kDa)</td></tr></thead><tbody><tr><td>Api m 1</td><td>Phospholipase A2</td><td>57-97 <sup>87,101,1301,1302,1304,1681,1682,1683,1684,1685,1686,1687,1688,1689</sup></td><td>16</td></tr><tr><td>Api m 2</td><td>Hyaluronidase</td><td>28-60 <sup>87,101,1302,1304,1681,1683,1688,1689</sup></td><td>39</td></tr><tr><td>Api m 3</td><td>Acid phosphatase</td><td>28-63 <sup>87,101,1681,1689</sup></td><td>43</td></tr><tr><td>Api m 4</td><td>Melittin</td><td>17-54 <sup>87,101,1281,1688</sup></td><td>3</td></tr><tr><td>Api m 5</td><td>Dipeptidyl peptidase IV</td><td>16-70 <sup>87,101,1681,1688</sup></td><td>100</td></tr><tr><td>Api m 10</td><td>Icarapin</td><td>35-73 <sup>87,101,99,1681,1688</sup></td><td>50-55</td></tr></tbody></table>

8/9 (Api m 11), vitellogenin (Api m 12) and protease (Bom t 4, Bom p 4). An overview of the honeybee and bumblebee venom allergens, which are presently listed in the WHO/IUIS allergen nomenclature official database, is given in (Table 60).

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

Approximately 9 to 42% of the adult population shows a sensitization to Hymenoptera venom (including bees and vespids) without previous history of a sting reaction.<sup>1275,1690</sup> As the presence of specific IgE (sIgE) does not necessarily imply clinically relevant venom allergy, for most of these patients it is likely that the sensitization is asymptomatic. A recent study showed that baseline sIgE levels to bee venom, vespid venom, rApi m 1, and rVes v 5 did not differ between asymptotically sensitized subjects, allergic patients, and VIT-treated patients.<sup>1276</sup>

The prevalence of systemic reactions to Hymenoptera stings among adults ranges between 0.3 and 7.5%.<sup>1275,1277</sup> The prevalence of sensitization to HBV is related to the degree of exposure. Thus, the frequency of honeybee venom allergy is higher in rural than in urban populations and especially beekeepers and their family members are at a higher risk for honeybee venom allergy.<sup>1278</sup>

Previous analyses of the sensitization to individual allergens using native purified allergens or immunoblots with venom extracts are only partially reliable due to the interference of clinically irrelevant IgE antibodies directed against CCDs, N-linked glycan structures present on several Hymenoptera venom allergens. Nowadays, advanced recombinant strategies allow the production of correctly folded allergens, devoid of carbohydrate-based cross-reactivity, which allow the elucidation of the role of particular allergens beyond clinically irrelevant cross-reactivity.<sup>1279</sup> Thus, reliable data on sensitization rates are available for several HBV allergens (Table 59).

Detailed data on sensitization rates in HBV-allergic individuals are available for Api m 1 (57-97%), Api m 2 (28-60%), Api m 3
<figure><img src="image_242.png" /><figcaption>FIGURE 128 Annotated allergens of honeybee (A. mellifera) and bumblebee (B. terrestris) venom. For HBV, only selected allergens with proven high relevance are depicted. Allergens from the same protein family are shown in identical colors.</figcaption></figure>

<figure><img src="image_243.png" /></figure>

<figure><img src="image_244.png" /></figure>

<figure><img src="image_245.png" /></figure>

<figure><img src="image_246.png" /></figure>

<figure><img src="image_247.png" /></figure>

<figure><img src="image_248.png" /><figcaption>FIGURE 129 Structures of selected relevant honeybee venom allergens. α-helices, β-strands and coiled regions are shown in red, blue and grey, respectively. The structures of Api m 1 (PDB: 1POC) and Api m 2 (PDB: 1FCU) were solved by crystallography. All other structures were generated by structural modeling.</figcaption></figure>

<table><caption>TABLE 59 Most relevant allergens of honeybee (A. mellifera) venom</caption><thead><tr><td>Allergenic molecule</td><td>Biochemical name</td><td>Prevalence among patients (%)</td><td>Molecular weight (kDa)</td></tr></thead><tbody><tr><td>Api m 1</td><td>Phospholipase A2</td><td>57-97 <sup>87,101,1301,1302,1304,1681,1682,1683,1684,1685,1686,1687,1688,1689</sup></td><td>16</td></tr><tr><td>Api m 2</td><td>Hyaluronidase</td><td>28-60 <sup>87,101,1302,1304,1681,1683,1688,1689</sup></td><td>39</td></tr><tr><td>Api m 3</td><td>Acid phosphatase</td><td>28-63 <sup>87,101,1681,1689</sup></td><td>43</td></tr><tr><td>Api m 4</td><td>Melittin</td><td>17-54 <sup>87,101,1281,1688</sup></td><td>3</td></tr><tr><td>Api m 5</td><td>Dipeptidyl peptidase IV</td><td>16-70 <sup>87,101,1681,1688</sup></td><td>100</td></tr><tr><td>Api m 10</td><td>Icarapin</td><td>35-73 <sup>87,101,99,1681,1688</sup></td><td>50-55</td></tr></tbody></table>

8/9 (Api m 11), vitellogenin (Api m 12) and protease (Bom t 4, Bom p 4). An overview of the honeybee and bumblebee venom allergens, which are presently listed in the WHO/IUIS allergen nomenclature official database, is given in (Table 60).

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

Approximately 9 to 42% of the adult population shows a sensitization to Hymenoptera venom (including bees and vespids) without previous history of a sting reaction.<sup>1275,1690</sup> As the presence of specific IgE (sIgE) does not necessarily imply clinically relevant venom allergy, for most of these patients it is likely that the sensitization is asymptomatic. A recent study showed that baseline sIgE levels to bee venom, vespid venom, rApi m 1, and rVes v 5 did not differ between asymptomatically sensitized subjects, allergic patients, and VIT-treated patients.<sup>1276</sup>

The prevalence of systemic reactions to Hymenoptera stings among adults ranges between 0.3 and 7.5%.<sup>1275,1277</sup> The prevalence of sensitization to HBV is related to the degree of exposure. Thus, the frequency of honeybee venom allergy is higher in rural than in urban populations and especially beekeepers and their family members are at a higher risk for honeybee venom allergy.<sup>1278</sup>

Previous analyses of the sensitization to individual allergens using native purified allergens or immunoblots with venom extracts are only partially reliable due to the interference of clinically irrelevant IgE antibodies directed against CCDs, N-linked glycan structures present on several Hymenoptera venom allergens. Nowadays, advanced recombinant strategies allow the production of correctly folded allergens, devoid of carbohydrate-based cross-reactivity, which allow the elucidation of the role of particular allergens beyond clinically irrelevant cross-reactivity.<sup>1279</sup> Thus, reliable data on sensitization rates are available for several HBV allergens (Table 59).

Detailed data on sensitization rates in HBV-allergic individuals are available for Api m 1 (57-97%), Api m 2 (28-60%), Api m 3
<table><caption>TABLE 60 Overview of the bee 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">Bumblebees (Bombus pensylvanicus, B. terrestris)</td></tr><tr><td>Bom p 1, Bom t 1</td><td>Phospholipase A2</td><td>16</td></tr><tr><td>Bom p 4, Bom t 4</td><td>Protease</td><td>27</td></tr><tr><td colspan="3">Honeybees (Apis mellifera, A. cerana, A. dorsata)</td></tr><tr><td>Api m 1, Api c 1, Api d 1</td><td>Phospholipase A2</td><td>16</td></tr><tr><td>Api m 2</td><td>Hyaluronidase</td><td>39</td></tr><tr><td>Api m 3</td><td>Acid phosphatase</td><td>43</td></tr><tr><td>Api m 4</td><td>Melittin</td><td>3</td></tr><tr><td>Api m 5</td><td>Dipeptidyl peptidase IV</td><td>100</td></tr><tr><td>Api m 6</td><td>Protease inhibitor</td><td>8</td></tr><tr><td>Api m 7</td><td>CUB serine protease</td><td>39</td></tr><tr><td>Api m 8</td><td>Carboxylesterase</td><td>70</td></tr><tr><td>Api m 9</td><td>Serine carboxypeptidase</td><td>60</td></tr><tr><td>Api m 10</td><td>Icarapin</td><td>50-55</td></tr><tr><td>Api m 11.0101</td><td>Major royal jelly protein 8</td><td>45</td></tr><tr><td>Api m 11.0201</td><td>Major royal jelly protein 9</td><td>46</td></tr><tr><td>Api m 12</td><td>Vitellogenin</td><td>200</td></tr></tbody></table>

(28-63%), Api m 4 (17-54%), Api m 5 (16-70%), and Api m 10 (35-73%), which seem to be the most relevant allergens of HBV (Table 59). Of note, the obtained sensitization rates may vary strongly based on the inclusion criteria of the patient population (e.g., mono-sensitized patients versus patients sensitized to different venoms, geographical differences or the method used for sIgE detection)¹²⁸⁰. Other allergens of HBV are less well characterized. However, preliminary, partially unpublished data suggest rather a role as minor allergens. Nevertheless, such allergens may be of special relevance for selected patients. HBV-allergic patients exhibit a wide variety of sensitization profiles to the different HBV allergens.⁸⁷

To date, available studies do not allow a final conclusion if particular sensitization profiles might correlate with the severity of the disease. One prospective study found that Api m 4 sensitization (sIgE > 0.98 kU/L) may be a risk factor for systemic reactions during the initiation phase of venom-specific immunotherapy (VIT) and for more severe systemic reactions after a honeybee sting.¹²⁸¹ Another allergen that is an interesting candidate as marker for personalized risk assessment in VIT is Api m 10. A retrospective multicenter study of VIT-treated HBV-allergic patients showed that a dominant Api m 10 sensitization (defined as >50% of sIgE to whole HBV) represents a relevant risk factor for treatment failure.¹⁰¹ Although the role of particular allergens in allergic reactions and tolerance induction is not finally understood, the knowledge of sensitization profiles in the future may allow a better risk stratification in VIT and thus personalized treatment.

The HBV allergens Api m 1, Api m 3, Api m 4 and Api m 10 can be considered as marker allergens, which in combination with vespid phospholipases A1 (Ves v 1/ Pol d 1) and antigens 5 (Ves v 5/ Pol d 5), allow to discriminate between HBV and yellow jacket / wasp venom allergy

(Figure 130). Component-resolved diagnostics (CRD) is highly valuable for adequate diagnosis, particularly in double-sensitized patients or in those who were not able to identify the stinging Hymenoptera species. While the hyaluronidase Api m 2 is a major allergen of HBV, the homologous allergens of yellow jacket venom (YJV) (Ves v 2) and European paper wasp (Polistes dominula) venom (PDV) (Pol d 2) seem to be of minor allergologic relevance in vespid venom allergy. Moreover, cross-reactivity beyond CCDs between Api m 2 and its vespid homologues is limited (Figure 130). Hence CCD-free Api m 2 may contribute as marker allergen to detect primary HBV sensitization. By contrast, the HBV DPPIV Api m 5 exhibits pronounced cross-reactivity with its homologues of YJV and PDV (Ves v 3 and Pol d 3). Additionally vitellogenins (Api m 12 and Ves v 6) contribute to cross-reactivity between HBV and YJV (Figure 130).

## 4 – Clinical management

### Clinical diagnosis

The diagnosis of bee venom allergy comprises the patient history of a systemic sting reaction, a positive skin test response and/or the detection of venom-sIgE antibodies.

#### Patient history:

One focus of taking the patient history should be the identification of the culprit insect. An important factor for the identification of honeybees is that they are the only stinging Hymenoptera species that nearly always leaves their stinger with adherent venom sac in the skin of the victim. However, several patients are not able to discriminate between honeybee and vespid stings, so that the results of patient's history often remain inconclusive.
<table><caption>TABLE 60 Overview of the bee 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"><em>Bumblebees (Bombus pensylvanicus, B. terrestris)</em></td></tr><tr><td>Bom p 1, Bom t 1</td><td>Phospholipase A2</td><td>16</td></tr><tr><td>Bom p 4, Bom t 4</td><td>Protease</td><td>27</td></tr><tr><td colspan="3"><em>Honeybees (Apis mellifera, A. cerana, A. dorsata)</em></td></tr><tr><td>Api m 1, Api c 1, Api d 1</td><td>Phospholipase A2</td><td>16</td></tr><tr><td>Api m 2</td><td>Hyaluronidase</td><td>39</td></tr><tr><td>Api m 3</td><td>Acid phosphatase</td><td>43</td></tr><tr><td>Api m 4</td><td>Melittin</td><td>3</td></tr><tr><td>Api m 5</td><td>Dipeptidyl peptidase IV</td><td>100</td></tr><tr><td>Api m 6</td><td>Protease inhibitor</td><td>8</td></tr><tr><td>Api m 7</td><td>CUB serine protease</td><td>39</td></tr><tr><td>Api m 8</td><td>Carboxylesterase</td><td>70</td></tr><tr><td>Api m 9</td><td>Serine carboxypeptidase</td><td>60</td></tr><tr><td>Api m 10</td><td>Icarapin</td><td>50-55</td></tr><tr><td>Api m 11.0101</td><td>Major royal jelly protein 8</td><td>45</td></tr><tr><td>Api m 11.0201</td><td>Major royal jelly protein 9</td><td>46</td></tr><tr><td>Api m 12</td><td>Vitellogenin</td><td>200</td></tr></tbody></table>

(28-63%), Api m 4 (17-54%), Api m 5 (16-70%), and Api m 10 (35-73%), which seem to be the most relevant allergens of HBV (Table 59). Of note, the obtained sensitization rates may vary strongly based on the inclusion criteria of the patient population (e.g., mono-sensitized patients versus patients sensitized to different venoms, geographical differences or the method used for sIgE detection)¹²⁸⁰. Other allergens of HBV are less well characterized. However, preliminary, partially unpublished data suggest rather a role as minor allergens. Nevertheless, such allergens may be of special relevance for selected patients. HBV-allergic patients exhibit a wide variety of sensitization profiles to the different HBV allergens.⁸⁷

To date, available studies do not allow a final conclusion if particular sensitization profiles might correlate with the severity of the disease. One prospective study found that Api m 4 sensitization (sIgE > 0.98 kU/L) may be a risk factor for systemic reactions during the initiation phase of venom-specific immunotherapy (VIT) and for more severe systemic reactions after a honeybee sting.¹²⁸¹ Another allergen that is an interesting candidate as marker for personalized risk assessment in VIT is Api m 10. A retrospective multicenter study of VIT-treated HBV-allergic patients showed that a dominant Api m 10 sensitization (defined as >50% of sIgE to whole HBV) represents a relevant risk factor for treatment failure.¹⁰¹ Although the role of particular allergens in allergic reactions and tolerance induction is not finally understood, the knowledge of sensitization profiles in the future may allow a better risk stratification in VIT and thus personalized treatment.

The HBV allergens Api m 1, Api m 3, Api m 4 and Api m 10 can be considered as marker allergens, which in combination with vespid phospholipases A1 (Ves v 1/ Pol d 1) and antigens 5 (Ves v 5/ Pol d 5), allow to discriminate between HBV and yellow jacket / wasp venom allergy

(Figure 130). Component-resolved diagnostics (CRD) is highly valuable for adequate diagnosis, particularly in double-sensitized patients or in those who were not able to identify the stinging Hymenoptera species. While the hyaluronidase Api m 2 is a major allergen of HBV, the homologous allergens of yellow jacket venom (YJV) (Ves v 2) and European paper wasp (Polistes dominula) venom (PDV) (Pol d 2) seem to be of minor allergologic relevance in vespid venom allergy. Moreover, cross-reactivity beyond CCDs between Api m 2 and its vespid homologues is limited (Figure 130). Hence CCD-free Api m 2 may contribute as marker allergen to detect primary HBV sensitization. By contrast, the HBV DPPIV Api m 5 exhibits pronounced cross-reactivity with its homologues of YJV and PDV (Ves v 3 and Pol d 3). Additionally vitellogenins (Api m 12 and Ves v 6) contribute to cross-reactivity between HBV and YJV (Figure 130).

## 4 – Clinical management

### Clinical diagnosis

The diagnosis of bee venom allergy comprises the patient history of a systemic sting reaction, a positive skin test response and/or the detection of venom-sIgE antibodies.

#### Patient history:

One focus of taking the patient history should be the identification of the culprit insect. An important factor for the identification of honeybees is that they are the only stinging Hymenoptera species that nearly always leaves their stinger with adherent venom sac in the skin of the victim. However, several patients are not able to discriminate between honeybee and vespid stings, so that the results of patient's history often remain inconclusive.
<figure><img src="image_243.png" /><figcaption>FIGURE 130 Cross-reactivity of honeybee venom allergens and their homologues from vespid venom. Potentially cross-reactive and marker allergens for the discrimination between primary honeybee and vespid venom sensitization are shown in black and green, respectively. Of note, vespid phosholipases A1 (Ves v 1 and Pol d 1) and antigens 5 (Ves v 5 and Pol d 5) can serve as marker allergens to descriminate between vespid and honeybee venom allergy but are highly cross-reactive among each other (green arrows). 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>

Moreover, patient history should consider information on num-
ber and date of sting reactions, severity of symptoms and the time
between sting and the onset of symptoms as well as the assessment
of potential risk factors such as medication, cardiovascular risks and
other diseases such as mast cell disorders.

Skin tests:

Skin tests are performed as prick test and/or intradermal test with commercial honeybee venom and vespid venom extract at least 2 weeks after the sting reaction to avoid possible false-negative results during the refractory period. For more detailed information please refer to the vespid venom chapter.

Baseline serum tryptase:

It is recommended to determine the baseline tryptase level in all patients with a history of a systemic reaction after a Hymenoptera sting to identify patients at higher risk of developing severe reactions due to undiagnosed clonal mast cell disorders. Adult patients with mast cell disorders and/or elevated baseline serum tryptase are not only at risk of more severe reactions following stings but in some studies are also considered a risk population during VIT for a lower clinical efficacy and/or a greater occurrence of side effects.

It is noteworthy that even in the presence of normal tryptase
level, patients with severe anaphylaxis (and absence of urticaria
or angioedema) due to stings may suffer from clonal mast cell dis-
orders. Vice versa, high tryptase levels can also be found in other
conditions (e.g., hematologic malignancies, parasitic infections, end-
stage chronic renal disease, aneurysms of the abdominal aorta, he-
reditary alpha-tryptasemia).

IgE testing:

**Total IgE:** Although it is not generally recommended in the guidelines, the measurement of total IgE (tIgE) levels in combination with sIgE test results can be useful to improve and simplify interpretation.

This is particularly relevant in connection with very low sIgE lev-
els, since each sIgE level has a different relevance if produced in an
environment with high or low tIgE values. In 54% of Hymenoptera
venom-sensitized individuals, the ratio of sIgE/tIgE was >4%.¹²⁸²
Thus, in the clinical management of bee venom allergy, the measure-
ment of tIgE can provide guidance to the clinician in the context of
the ratio sIgE/tIgE.

Specific IgE to venom extracts and individual venom allergens:

Specific IgE measurements to honeybee and vespid venom ex-
tracts might show multiple positive test results due to sensitization
to multiple venoms or to the presence of sIgE to CCDs or homolo-
gous allergens present in different venoms. Results might be neg-
ative due to the underrepresentation of particular allergens in the
extract, or a higher sensitivity of individual components. While this
has convincingly been demonstrated for Ves v 5 and YJV extract (see
chapter on vespid venom allergy), conflicting results were reported
in two studies that have addressed this issue in HBV allergy.¹²⁸³,¹²⁸⁴

Specific IgE detection to BBV could be useful in patients heav-
ily exposed to bumblebee stings. Although major allergens of BBV
and HBV are cross-reactive, additional species-specific epitopes are
present due to an incomplete sequence identity.

*Specific IgE measurements to CCD marker molecules* such as MUXF can be used to confirm the presence of CCD-sIgE antibodies as reason of multiple positive test results. However, since CCD-sIgE and allergen-sIgE might be present, the detection of CCD-sIgE alone does not allow the exclusion of sensitization to protein epitopes of multiple venoms.

CRD using CCD-free individual allergens is recommended: (A) In
case of multiple positive test results with different venoms to dis-
criminate between true sensitization and cross-reactivity. (B) For
diagnosis in patients with inconclusive patient history to identify the
<figure><img src="image_249.png" /><figcaption>FIGURE 130 Cross-reactivity of honeybee venom allergens and their homologues from vespid venom. Potentially cross-reactive and marker allergens for the discrimination between primary honeybee and vespid venom sensitization are shown in black and green, respectively. Of note, vespid phosholipases A1 (Ves v 1 and Pol d 1) and antigens 5 (Ves v 5 and Pol d 5) can serve as marker allergens to descriminate between vespid and honeybee venom allergy but are highly cross-reactive among each other (green arrows). 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>

Moreover, patient history should consider information on num-
ber and date of sting reactions, severity of symptoms and the time
between sting and the onset of symptoms as well as the assessment
of potential risk factors such as medication, cardiovascular risks and
other diseases such as mast cell disorders.

Skin tests:

Skin tests are performed as prick test and/or intradermal test with commercial honeybee venom and vespid venom extract at least 2 weeks after the sting reaction to avoid possible false-negative results during the refractory period. For more detailed information please refer to the vespid venom chapter.

Baseline serum tryptase:

It is recommended to determine the baseline tryptase level in all patients with a history of a systemic reaction after a Hymenoptera sting to identify patients at higher risk of developing severe reactions due to undiagnosed clonal mast cell disorders. Adult patients with mast cell disorders and/or elevated baseline serum tryptase are not only at risk of more severe reactions following stings but in some studies are also considered a risk population during VIT for a lower clinical efficacy and/or a greater occurrence of side effects.

It is noteworthy that even in the presence of normal tryptase
level, patients with severe anaphylaxis (and absence of urticaria
or angioedema) due to stings may suffer from clonal mast cell dis-
orders. Vice versa, high tryptase levels can also be found in other
conditions (e.g., hematologic malignancies, parasitic infections, end-
stage chronic renal disease, aneurysms of the abdominal aorta, he-
reditary alpha-tryptasemia).

IgE testing:

**Total IgE:** Although it is not generally recommended in the guidelines, the measurement of total IgE (tIgE) levels in combination with sIgE test results can be useful to improve and simplify interpretation.

This is particularly relevant in connection with very low sIgE lev-
els, since each sIgE level has a different relevance if produced in an
environment with high or low tIgE values. In 54% of Hymenoptera
venom-sensitized individuals, the ratio of sIgE/tIgE was >4%.¹²⁸²
Thus, in the clinical management of bee venom allergy, the measure-
ment of tIgE can provide guidance to the clinician in the context of
the ratio sIgE/tIgE.

Specific IgE to venom extracts and individual venom allergens:

Specific IgE measurements to honeybee and vespid venom ex-
tracts might show multiple positive test results due to sensitization
to multiple venoms or to the presence of sIgE to CCDs or homolo-
gous allergens present in different venoms. Results might be neg-
ative due to the underrepresentation of particular allergens in the
extract, or a higher sensitivity of individual components. While this
has convincingly been demonstrated for Ves v 5 and YJV extract (see
chapter on vespid venom allergy), conflicting results were reported
in two studies that have addressed this issue in HBV allergy.¹²⁸³,¹²⁸⁴

Specific IgE detection to BBV could be useful in patients heav-
ily exposed to bumblebee stings. Although major allergens of BBV
and HBV are cross-reactive, additional species-specific epitopes are
present due to an incomplete sequence identity.

*Specific IgE measurements to CCD marker molecules* such as MUXF can be used to confirm the presence of CCD-sIgE antibodies as reason of multiple positive test results. However, since CCD-sIgE and allergen-sIgE might be present, the detection of CCD-sIgE alone does not allow the exclusion of sensitization to protein epitopes of multiple venoms.

CRD using CCD-free individual allergens is recommended: (A) In
case of multiple positive test results with different venoms to dis-
criminate between true sensitization and cross-reactivity. (B) For
diagnosis in patients with inconclusive patient history to identify the
culprit insect(s). (C) In case of negative test results with different venoms despite a convincing clinical history due to potentially enhanced sensitivity of the CRD.

The allergens Api m 1, Api m 3, Api m 4 and Api m 10 can be considered as marker allergens for genuine HBV sensitization. Together with marker allergens for vespid venom sensitization Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 they are useful tools to elucidate primary sensitization, particularly in patients with double-positive test results who were not able to identify the culprit insect or in cases of inconsistent clinical history and test results with venom extracts. Due to the limited cross-reactivity between CCD-free recombinant Api m 2 and its vespid homologues, Api m 2 may contribute as marker allergen to detect primary HBV sensitization. However, as cross-reactivity and, hence, primary sensitization to vespid venom cannot be excluded with absolute certainty, Api m 2-sIgE has to be interpreted with care and seen in the context of clinical history. This is of particular importance as the vespid homologues and thus comparative slgE measurements are not available for clinical routine diagnostics. Due to the extended cross-reactivity between Api m 5 and its vespid homologues, slgE to Api m 5 cannot be considered as reliable marker for primary HBV allergy. However, slgE to Api m 5 can be a confirmatory marker in patients with double-positive skin tests and/ or slgE to HBV and YJV/ PDV, who have an indicative history of HBV allergy. Again, the vespid homologues are not available for comparative measurements in clinical routine. A diagnostic algorithm for the interpretation of slgE test results in CRD to discriminate between primary HBV and vespid venom allergy or to prove true primary sensitization to different venoms is given in (Figure 131). To date, no individual BBV allergens are available for routine diagnostics.

Cellular tests:

When skin tests and slgE measurements yield negative results in patients with a systemic anaphylactic reaction, additional cellular tests, such as basophil activation, are recommended and have shown additional benefits when used together with allergen components (for more detailed information about cellular tests please refer to the chapters about vespid venom allergy and basophil activation testing).

Sting challenge:

A sting challenge with a living insect is not recommended as diagnostic tool in untreated patients and should serve only as control of success of venom immunotherapy. For more detailed information refer to the vespid venom chapter.

Prevention and Therapy

Avoidance of bee stings:

Although some behavioral rules exist that might contribute to minimizing the existing risk, avoiding stings completely is challenging. These rules include: avoidance of perfumes and floral or bright colored clothing, careful outdoor eating and drinking, wearing shoes outside, avoidance of swatting to bees, keeping windows of the vehicle closed and staying away from beehives.

Pharmacotherapy (emergency kit):

Due to the risk of severe reactions, patients allergic to bee venom should carry an emergency kit including an adrenaline autoinjector

<figure><img src="image_244.png" /></figure>

FIGURE 131 Diagnostic algorithm for component-resolved diagnostics of honeybee venom (HBV) and yellow jacket venom (YJV) allergy. This algorithm can also be used to discriminate between HBV and P. dominula venom (PDV) allergy using the PDV homologues of Ves v 1 and Ves v 5, Pol d 1 and Pol d 5. A plus indicates a positive and a minus a negative test result. 1 The HBV allergens Api m 2 and Api m 5 show potential cross-reactivity to homologous allergens of YJV and PDV that are not commercially available, so that a positive test result does not necessarily exclude YJV or PDV allergy. Despite the potential of component-resolved diagnostics, clinical history, skin tests and the measurement of venom-sIgE and serum tryptase build an indispensable basis for accurate diagnosis in Hymenoptera venom allergy. Moreover, cellular tests such as basophil activation test (BAT) may be helpful in dissecting double-positive or double-negative test results.

for self-administration, especially during the bee season. Although, this is a highly debated issue, according to current guidelines, also patients with previous severe allergic reactions who have successfully undergone immunotherapy are recommended to carry an emergency kit to eliminate a remaining risk.

Specific immunotherapy:

HBV VIT is recommended in children and adults with documented sensitization to HBV either by skin test and/ or slgE tests and a history of a systemic sting reaction exceeding general skin symptoms as well as in adults with generalized skin symptoms if quality of life is impaired. VIT is not indicated when neither skin testing nor slgE indicate a sensitization nor for patients with large local
culprit insect(s). (C) In case of negative test results with different venoms despite a convincing clinical history due to potentially enhanced sensitivity of the CRD.

The allergens Api m 1, Api m 3, Api m 4 and Api m 10 can be considered as marker allergens for genuine HBV sensitization. Together with marker allergens for vespid venom sensitization Ves v 1 / Pol d 1 and Ves v 5 / Pol d 5 they are useful tools to elucidate primary sensitization, particularly in patients with double-positive test results who were not able to identify the culprit insect or in cases of inconsistent clinical history and test results with venom extracts. Due to the limited cross-reactivity between CCD-free recombinant Api m 2 and its vespid homologues, Api m 2 may contribute as marker allergen to detect primary HBV sensitization. However, as cross-reactivity and, hence, primary sensitization to vespid venom cannot be excluded with absolute certainty, Api m 2-sIgE has to be interpreted with care and seen in the context of clinical history. This is of particular importance as the vespid homologues and thus comparative slgE measurements are not available for clinical routine diagnostics. Due to the extended cross-reactivity between Api m 5 and its vespid homologues, slgE to Api m 5 cannot be considered as reliable marker for primary HBV allergy. However, slgE to Api m 5 can be a confirmatory marker in patients with double-positive skin tests and/ or slgE to HBV and YJV/ PDV, who have an indicative history of HBV allergy. Again, the vespid homologues are not available for comparative measurements in clinical routine. A diagnostic algorithm for the interpretation of slgE test results in CRD to discriminate between primary HBV and vespid venom allergy or to prove true primary sensitization to different venoms is given in (Figure 131). To date, no individual BBV allergens are available for routine diagnostics.

Cellular tests:

When skin tests and slgE measurements yield negative results in patients with a systemic anaphylactic reaction, additional cellular tests, such as basophil activation, are recommended and have shown additional benefits when used together with allergen components (for more detailed information about cellular tests please refer to the chapters about vespid venom allergy and basophil activation testing).

Sting challenge:

A sting challenge with a living insect is not recommended as diagnostic tool in untreated patients and should serve only as control of success of venom immunotherapy. For more detailed information refer to the vespid venom chapter.

Prevention and Therapy

Avoidance of bee stings:

Although some behavioral rules exist that might contribute to minimizing the existing risk, avoiding stings completely is challenging. These rules include: avoidance of perfumes and floral or bright colored clothing, careful outdoor eating and drinking, wearing shoes outside, avoidance of swatting to bees, keeping windows of the vehicle closed and staying away from beehives.

Pharmacotherapy (emergency kit):

Due to the risk of severe reactions, patients allergic to bee venom should carry an emergency kit including an adrenaline autoinjector

<figure><img src="image_250.png" /></figure>

FIGURE 131 Diagnostic algorithm for component-resolved diagnostics of honeybee venom (HBV) and yellow jacket venom (YJV) allergy. This algorithm can also be used to discriminate between HBV and P. dominula venom (PDV) allergy using the PDV homologues of Ves v 1 and Ves v 5, Pol d 1 and Pol d 5. A plus indicates a positive and a minus a negative test result. 1 The HBV allergens Api m 2 and Api m 5 show potential cross-reactivity to homologous allergens of YJV and PDV that are not commercially available, so that a positive test result does not necessarily exclude YJV or PDV allergy. Despite the potential of component-resolved diagnostics, clinical history, skin tests and the measurement of venom-sIgE and serum tryptase build an indispensable basis for accurate diagnosis in Hymenoptera venom allergy. Moreover, cellular tests such as basophil activation test (BAT) may be helpful in dissecting double-positive or double-negative test results.

for self-administration, especially during the bee season. Although, this is a highly debated issue, according to current guidelines, also patients with previous severe allergic reactions who have successfully undergone immunotherapy are recommended to carry an emergency kit to eliminate a remaining risk.

Specific immunotherapy:

HBV VIT is recommended in children and adults with documented sensitization to HBV either by skin test and/ or slgE tests and a history of a systemic sting reaction exceeding general skin symptoms as well as in adults with generalized skin symptoms if quality of life is impaired. VIT is not indicated when neither skin testing nor slgE indicate a sensitization nor for patients with large local
or unusual reactions.¹²⁸⁵ In addition, incidental positive slgE results in a multiplex test are not an indication for VIT without relevant clinical history of a systemic sting reaction.

HBV also seems to be sufficient in nonprofessionally exposed BBV-allergic patients who most likely react on the basis of cross-reactivity and a primary sensitization to HBV. By contrast, in heavily exposed greenhouse workers who are frequently stung by bumblebees a VIT with BBV would be preferable.¹²⁷⁴ However, BBV routine therapeutic intervention is commercially not generally available and such approaches have only been reported in case reports. The success of specific immunotherapy may be monitored by a sting challenge test with a live insect (see also chapter B21).

## Tips for the use of molecular diagnostics for honeybee venom allergy

* Api m 1, Api m 3, Api m 4 and Api m 10 are marker allergens to detect primary sensitization to honeybee venom (to dissect honeybee and vespid venom allergy).

* **Exceptions:** No marker allergens are available to discriminate between primary honeybee and bumblebee venom allergy.

* slgE to Api m 1, Api m 3, Api m 4 and Api m 10 indicates primary bee venom allergy.

* slgE to Api m 2 may be a helpful marker to detect primary bee venom allergy. Interpret results with care in the context of clinical history.

* slgE to Api m 5 does not exclude primary vespid venom allergy.

# 5 - Clinical cases

## Case 1 (original)

**History:** Female, 40 years old, stung by unidentified insect in tip of the middle finger of the right hand. Within minutes generalized itching and urticaria, dyspnea and a feeling of tightness in the throat. Care by an emergency physician.

**Skin prick test:** HBV (100 µg/mL) and YJV venom (300 µg/mL) positive.

**In vitro testing (conventional):** tIgE 18.6 kU/L, slgE to HBV 6.9 kU/L, slgE to YJV 1.3 kU/L, baseline tryptase 6.3 µg/L.

**In vitro testing (molecular):** slgE to rApi m 1 5.7 kU/L, slgE to rApi m 2 <0.1 kU/L, slgE to Api m 3 <0.1 kU/L, slgE to Api m 10 1.7 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rVes v 5 <0.1 kU/L, slgE to CCD (MUXF3) 2.4 kU/L.

**Diagnosis:** Sting anaphylaxis with HBV sensitization, cross-reactivity to YJV likely due to CCD-sIgE.

**Recommendation:** VIT with HBV extract, emergency kit with adrenaline autoinjector.

## Case 2 (original)

**History:** Male, 52 years old, history of 3 episodes of severeaphylactic reactions (1 after a honeybee sting and 2 after yellow jacket stings).

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

**In vitro testing (conventional):** tIgE 15 kU/L, slgE to HBV <0.1 kU/L, slgE to YJV 1.1 kU/L, baseline tryptase 18.9 µg/L.

**In vitro testing (molecular):** slgE to rApi m 1 <0.1 kU/L, slgE to rApi m 2 <0.1 kU/L, slgE to rApi m 3 1.28 kU/L, slgE to rApi m 10 0.37 kU/L, slgE to rVes v 1 1.99 kU/L, slgE to rVes v 5 1.53 kU/L, slgE to CCD (MUXF3) <0.1 kU/L.

**Diagnosis:** Sting anaphylaxis with HBV and YJV sensitization, elevated baseline serum tryptase.

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

## Case 3 (original):

**History:** Male, 45 years old, stung by an unidentified insect in the neck, within minutes generalized itching, dyspnea, loss of consciousness. Care by an emergency physician.

**Skin prick test:** HBV (100 µg/mL) and YJV (100 µg/mL) positive.

**In vitro testing (conventional):** tIgE 360 kU/L, slgE to HBV 23.6 kU/L, slgE to YJV 4.3 kU/L, baseline tryptase 3.1 µg/L.

**In vitro testing (molecular):** slgE to rApi m 1 10.9 kU/L, slgE to rApi m 2 2.51 kU/L, slgE to Api m 3 <0.1 kU/L, slgE to Api m 5 2.31 kU/L, slgE to Api m 10 <0.1 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rVes v 5 7.4 kU/L, slgE to CCD (MUXF3) <0.1 kU/L.

**Diagnosis:** Sting anaphylaxis with HBV and YJV sensitization.

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

# 6 - Research and future perspectives

The increasing knowledge of the identity of relevant Hymenoptera venom allergens and the availability of their recombinant CCD-free counterparts has led to the development of an advanced CRD in venom allergy. The currently available CRD is a valuable tool to resolve cross-reactivity and primary sensitization; particularly to discriminate between HBV and vespid venom allergy. At this stage, a limitation of CRD in clinical routine is the unavailability of homologous allergen pairs from HBV and vespid venoms that would allow comparative slgE measurements facilitating the evaluation of obtained test results.

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.

Additionally, there is some evidence that some allergens and patients' sensitization profiles may act as biomarkers to identify
or unusual reactions.¹²⁸⁵ In addition, incidental positive slgE results in a multiplex test are not an indication for VIT without relevant clinical history of a systemic sting reaction.

HBV also seems to be sufficient in nonprofessionally exposed BBV-allergic patients who most likely react on the basis of cross-reactivity and a primary sensitization to HBV. By contrast, in heavily exposed greenhouse workers who are frequently stung by bumblebees a VIT with BBV would be preferable.¹²⁷⁴ However, BBV routine therapeutic intervention is commercially not generally available and such approaches have only been reported in case reports. The success of specific immunotherapy may be monitored by a sting challenge test with a live insect (see also chapter B21).

## Tips for the use of molecular diagnostics for honeybee venom allergy

* Api m 1, Api m 3, Api m 4 and Api m 10 are marker allergens to detect primary sensitization to honeybee venom (to dissect honeybee and vespid venom allergy).

* **Exceptions:** No marker allergens are available to discriminate between primary honeybee and bumblebee venom allergy.

* slgE to Api m 1, Api m 3, Api m 4 and Api m 10 indicates primary bee venom allergy.

* slgE to Api m 2 may be a helpful marker to detect primary bee venom allergy. Interpret results with care in the context of clinical history.

* slgE to Api m 5 does not exclude primary vespid venom allergy.

# 5 - Clinical cases

## Case 1 (original)

**History:** Female, 40 years old, stung by unidentified insect in tip of the middle finger of the right hand. Within minutes generalized itching and urticaria, dyspnea and a feeling of tightness in the throat. Care by an emergency physician.

**Skin prick test:** HBV (100 µg/mL) and YJV venom (300 µg/mL) positive.

**In vitro testing (conventional):** tIgE 18.6 kU/L, slgE to HBV 6.9 kU/L, slgE to YJV 1.3 kU/L, baseline tryptase 6.3 µg/L.

**In vitro testing (molecular):** slgE to rApi m 1 5.7 kU/L, slgE to rApi m 2 <0.1 kU/L, slgE to Api m 3 <0.1 kU/L, slgE to Api m 10 1.7 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rVes v 5 <0.1 kU/L, slgE to CCD (MUXF3) 2.4 kU/L.

**Diagnosis:** Sting anaphylaxis with HBV sensitization, cross-reactivity to YJV likely due to CCD-sIgE.

**Recommendation:** VIT with HBV extract, emergency kit with adrenaline autoinjector.

## Case 2 (original)

**History:** Male, 52 years old, history of 3 episodes of severeaphylactic reactions (1 after a honeybee sting and 2 after yellow jacket stings).

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

**In vitro testing (conventional):** tIgE 15 kU/L, slgE to HBV <0.1 kU/L, slgE to YJV 1.1 kU/L, baseline tryptase 18.9 µg/L.

**In vitro testing (molecular):** slgE to rApi m 1 <0.1 kU/L, slgE to rApi m 2 <0.1 kU/L, slgE to rApi m 3 1.28 kU/L, slgE to rApi m 10 0.37 kU/L, slgE to rVes v 1 1.99 kU/L, slgE to rVes v 5 1.53 kU/L, slgE to CCD (MUXF3) <0.1 kU/L.

**Diagnosis:** Sting anaphylaxis with HBV and YJV sensitization, elevated baseline serum tryptase.

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

## Case 3 (original):

**History:** Male, 45 years old, stung by an unidentified insect in the neck, within minutes generalized itching, dyspnea, loss of consciousness. Care by an emergency physician.

**Skin prick test:** HBV (100 µg/mL) and YJV (100 µg/mL) positive.

**In vitro testing (conventional):** tIgE 360 kU/L, slgE to HBV 23.6 kU/L, slgE to YJV 4.3 kU/L, baseline tryptase 3.1 µg/L.

**In vitro testing (molecular):** slgE to rApi m 1 10.9 kU/L, slgE to rApi m 2 2.51 kU/L, slgE to Api m 3 <0.1 kU/L, slgE to Api m 5 2.31 kU/L, slgE to Api m 10 <0.1 kU/L, slgE to rVes v 1 <0.1 kU/L, slgE to rVes v 5 7.4 kU/L, slgE to CCD (MUXF3) <0.1 kU/L.

**Diagnosis:** Sting anaphylaxis with HBV and YJV sensitization.

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

# 6 - Research and future perspectives

The increasing knowledge of the identity of relevant Hymenoptera venom allergens and the availability of their recombinant CCD-free counterparts has led to the development of an advanced CRD in venom allergy. The currently available CRD is a valuable tool to resolve cross-reactivity and primary sensitization; particularly to discriminate between HBV and vespid venom allergy. At this stage, a limitation of CRD in clinical routine is the unavailability of homologous allergen pairs from HBV and vespid venoms that would allow comparative slgE measurements facilitating the evaluation of obtained test results.

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.

Additionally, there is some evidence that some allergens and patients' sensitization profiles may act as biomarkers to identify
particular risk factors in venom allergy. However, further prospective studies are crucial to verify whether allergens such as Api m 4, Api m 10 or others are reliable markers to predict severe side-effects during VIT and/or an elevated risk for treatment failure in bee venom allergy.

Nevertheless, the ongoing identification and characterization of Hymenoptera venom allergens as well as the growing availability of allergens for CRD will open new perspectives for accurate and personalized patient management and, hence, for precision medicine in Hymenoptera venom allergy.
particular risk factors in venom allergy. However, further prospective studies are crucial to verify whether allergens such as Api m 4, Api m 10 or others are reliable markers to predict severe side-effects during VIT and/or an elevated risk for treatment failure in bee venom allergy.

Nevertheless, the ongoing identification and characterization of Hymenoptera venom allergens as well as the growing availability of allergens for CRD will open new perspectives for accurate and personalized patient management and, hence, for precision medicine in Hymenoptera venom allergy.
# B21 – Wasp Venom Allergy

## Maria Beatrice Bilò, Thilo Jakob, Markus Ollert, Simon Blank

### Highlights

* In yellow jacket venom allergy, CRD is a valuable tool since the diagnostic sensitivity of a combination of the recombinant allergens rVes v 5 and rVes v 1 is very high. In addition, rVes v 5 and rVes v 1 are marker allergens for vespid venom sensitization and allow an excellent discrimination between honeybee and vespid venom sensitization in double-sensitized patients.

* Among paper wasp allergens, Pol d 5 is currently available for routine diagnosis of paper wasp venom allergy on most common slgE singleplex platforms, while Pol d 1 is exclusively available on multiplex platforms.

* CRD currently offers only limited value in case of double/multiple positivity to vespid venoms. Currently, there are no marker allergens available that allow discrimination between yellow jacket and Polistes venom sensitization.

* There are no marker allergens available that allow discrimination between primary yellow jacket and hornet venom sensitization.

* Together with a better knowledge of the molecular composition of different venom extracts and more recombinant vespid allergens available, CRD may contribute to optimize patient-tailored immunotherapy.

## 1 – The allergen sources

The Vespidae family is composed of the Vespinae subfamily, including the genera *Vespula* (V. germanica, V. vulgaris, V. pensylvanica, V. maculifrons, V. flavopilosa, V. squamosa, V. vidua), *Dolichovespula* (D. maculata, D. arenaria, D. saxonica, D. media), and *Vespa* (V. crabro, V. orientalis, V. velutina nigrithorax, V. magnifica, V. mandarinia) and the subfamily *Polistinae*, which includes the genus *Polistes* (P. dominula, P. gallicus, P. exclamans, P. annularis, P. fuscatus), and *Polybia* (P. paulista, P. scutellaris) (Figure 132). The *Formicidae* family contains the allergy-relevant stinging ant species *Solenopsis* spp., *Myrmecia pilosula* and *Pachycondyla chinensis*, which are not covered by this chapter.

*Vespula* (called wasps in Europe, yellow jackets in the USA) are the most important species in Europe (Figure 132). In southern Europe, in addition to *Vespula*,¹²⁸⁶ hornets are a frequent cause of allergic reactions (genus *Vespa*), including the most widespread species *Vespa crabro*. In 2005, *Vespa velutina nigrithorax*, which is from Southeast Asia, was detected in the South of France. *Vespa velutina* is a predator of bees and is rapidly spreading from France to neighboring countries. Anaphylactic reactions have been reported after *Vespa velutina* stings, with a variable degree of cross-reactivity with other vespid.¹²⁸⁷

The species *Polistes dominula* and *Polistes gallicus* are European paper wasps; *P. dominula* has also spread to the northeastern United

States and also been reported in Australia (Figure 133). The species *Polistes exclamans*, *Polistes annularis* and *Polistes fuscatus* are indigenous to North America and not present in Europe. Several of the European species of the vespid family differ from those found in the USA. Furthermore, popular names for vespid in the USA and Europe are different and may lead to confusion (Table 61).

The vespid and apids other than honeybee have stingers, which usually can be extracted from their victims, thus enabling them to sting several times consecutively.

## 2 – Allergen families and allergenic molecules

Vespid venoms are complex mixtures of powerful allergens and pharmacologically active compounds, primarily made up of proteins. An overview of the Vespoidea venom allergens, which are presently listed in the WHO/ IUIS allergen nomenclature official database, is given in Table 62.

The main marker allergens for yellow jacket (*Vespula vulgaris*–VV) and European paper wasp (*Polistes dominula*) sensitization have been identified in phospholipase A1 (PLA1) (Ves v 1 and Pol d 1) and antigen 5 (Ves v 5 and Pol d 5), respectively (Figures 134 and 135). PLA1 and antigen 5 have been described as relevant venom allergens also in hornets.

The PLA1 allergens of different Vespula species among the *Paravespula* genus share sequence identity of approximately 95% and are thought to be almost completely cross-reactive, while sequence identity is around 70% with the American species V. squamosa and V. vidua belonging to a different subgenus.¹²⁸⁸ Sequence identity between yellow jacket venom (YJV) PLA1 Ves v 1 and hornet venom Vesp c 1 is around 71%. In summary, all PLA1s are structurally similar and cross-reactivity can be observed between PLA1s of most Vespoidea species,¹²⁸⁹,¹⁶⁹¹,¹⁶⁹² making their use difficult for discrimination between allergies to these species. Although catalyzing a related enzymatic reaction, vespid PLA1 allergens share neither sequence identity nor structural similarity with PLA2 allergens from bee venoms. Therefore, PLA1 and PLA2 represent marker allergens that allow discrimination between primary vespid and bee venom sensitization.

As PLA1, antigen 5 (Ag5) allergens are highly abundant proteins in most Vespoida venoms, belonging to the CAP (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins) superfamily, even though their function within the venom remains largely unclear. While Ag5 sensitization represents a clear marker for vespid venom allergy, the Ag5 allergens of various Vespoida species display pronounced structural similarity and cross-reactivity in specific IgE (slgE) measurements as well as using BAT,²¹³ thus preventing them to differentiate between allergies to these species. On the other hand, CAP inhibition and immunoblotting-based studies showed that those techniques remained inconclusive in about 50% of patients with systemic reactions by European hornet stings, suggesting that sensitization against hornet Ag5 is relevant and genuine at least in a subgroup of patients.¹²⁹⁰
# B21 – Wasp Venom Allergy

## Maria Beatrice Bilò, Thilo Jakob, Markus Ollert, Simon Blank

### Highlights

* In yellow jacket venom allergy, CRD is a valuable tool since the diagnostic sensitivity of a combination of the recombinant allergens rVes v 5 and rVes v 1 is very high. In addition, rVes v 5 and rVes v 1 are marker allergens for vespid venom sensitization and allow an excellent discrimination between honeybee and vespid venom sensitization in double-sensitized patients.

* Among paper wasp allergens, Pol d 5 is currently available for routine diagnosis of paper wasp venom allergy on most common slgE singleplex platforms, while Pol d 1 is exclusively available on multiplex platforms.

* CRD currently offers only limited value in case of double/multiple positivity to vespid venoms. Currently, there are no marker allergens available that allow discrimination between yellow jacket and Polistes venom sensitization.

* There are no marker allergens available that allow discrimination between primary yellow jacket and hornet venom sensitization.

* Together with a better knowledge of the molecular composition of different venom extracts and more recombinant vespid allergens available, CRD may contribute to optimize patient-tailored immunotherapy.

## 1 – The allergen sources

The Vespidae family is composed of the Vespinae subfamily, including the genera *Vespula* (V. germanica, V. vulgaris, V. pensylvanica, V. maculifrons, V. flavopilosa, V. squamosa, V. vidua), *Dolichovespula* (D. maculata, D. arenaria, D. saxonica, D. media), and *Vespa* (V. crabro, V. orientalis, V. velutina nigrithorax, V. magnifica, V. mandarinia) and the subfamily *Polistinae*, which includes the genus *Polistes* (P. dominula, P. gallicus, P. exclamans, P. annularis, P. fuscatus), and *Polybia* (P. paulista, P. scutellaris) (Figure 132). The *Formicidae* family contains the allergy-relevant stinging ant species *Solenopsis* spp., *Myrmecia pilosula* and *Pachycondyla chinensis*, which are not covered by this chapter.

*Vespula* (called wasps in Europe, yellow jackets in the USA) are the most important species in Europe (Figure 132). In southern Europe, in addition to *Vespula*,¹²⁸⁶ hornets are a frequent cause of allergic reactions (genus *Vespa*), including the most widespread species *Vespa crabro*. In 2005, *Vespa velutina nigrithorax*, which is from Southeast Asia, was detected in the South of France. *Vespa velutina* is a predator of bees and is rapidly spreading from France to neighboring countries. Anaphylactic reactions have been reported after *Vespa velutina* stings, with a variable degree of cross-reactivity with other vespid.¹²⁸⁷

The species *Polistes dominula* and *Polistes gallicus* are European paper wasps; *P. dominula* has also spread to the northeastern United

States and also been reported in Australia (Figure 133). The species *Polistes exclamans*, *Polistes annularis* and *Polistes fuscatus* are indigenous to North America and not present in Europe. Several of the European species of the vespid family differ from those found in the USA. Furthermore, popular names for vespid in the USA and Europe are different and may lead to confusion (Table 61).

The vespid and apids other than honeybee have stingers, which usually can be extracted from their victims, thus enabling them to sting several times consecutively.

## 2 – Allergen families and allergenic molecules

Vespid venoms are complex mixtures of powerful allergens and pharmacologically active compounds, primarily made up of proteins. An overview of the Vespoidea venom allergens, which are presently listed in the WHO/ IUIS allergen nomenclature official database, is given in Table 62.

The main marker allergens for yellow jacket (*Vespula vulgaris*–VV) and European paper wasp (*Polistes dominula*) sensitization have been identified in phospholipase A1 (PLA1) (Ves v 1 and Pol d 1) and antigen 5 (Ves v 5 and Pol d 5), respectively (Figures 134 and 135). PLA1 and antigen 5 have been described as relevant venom allergens also in hornets.

The PLA1 allergens of different Vespula species among the *Paravespula* genus share sequence identity of approximately 95% and are thought to be almost completely cross-reactive, while sequence identity is around 70% with the American species V. squamosa and V. vidua belonging to a different subgenus.¹²⁸⁸ Sequence identity between yellow jacket venom (YJV) PLA1 Ves v 1 and hornet venom Vesp c 1 is around 71%. In summary, all PLA1s are structurally similar and cross-reactivity can be observed between PLA1s of most Vespoidea species,¹²⁸⁹,¹⁶⁹¹,¹⁶⁹² making their use difficult for discrimination between allergies to these species. Although catalyzing a related enzymatic reaction, vespid PLA1 allergens share neither sequence identity nor structural similarity with PLA2 allergens from bee venoms. Therefore, PLA1 and PLA2 represent marker allergens that allow discrimination between primary vespid and bee venom sensitization.

As PLA1, antigen 5 (Ag5) allergens are highly abundant proteins in most Vespoida venoms, belonging to the CAP (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins) superfamily, even though their function within the venom remains largely unclear. While Ag5 sensitization represents a clear marker for vespid venom allergy, the Ag5 allergens of various Vespoida species display pronounced structural similarity and cross-reactivity in specific IgE (slgE) measurements as well as using BAT,²¹³ thus preventing them to differentiate between allergies to these species. On the other hand, CAP inhibition and immunoblotting-based studies showed that those techniques remained inconclusive in about 50% of patients with systemic reactions by European hornet stings, suggesting that sensitization against hornet Ag5 is relevant and genuine at least in a subgroup of patients.¹²⁹⁰
<figure><img src="image_245.png" /><figcaption>FIGURE 132 Taxonomy of allergy-relevant vespid species. As the taxonomy of the order Hymenoptera is highly complex, only a selection of allergy-relevant taxa is shown. Only selected species with particular relevance for allergy are included. For taxonomic overview of allergy-relevant bees (Apidae) refer to the bee venom chapter. The family Formicidae (ants) also contains species with relevance for Hymenoptera venom allergy.</figcaption></figure>

Hyaluronidases are common components of vespid venoms (e.g., Ves v 2 and Pol d 2). In YJV an enzymatically active (Ves v 2.0101) and an inactive (Ves v 2.0201) hyaluronidase were identified that share sequence identity of 59%, whereby the latter seems to be the predominant isoform.¹²⁹¹ The extent of cross-reactivity between YJV hyaluronidases and their homologue of P. dominula venom (Pol d 2) so far remains elusive. By contrast, cross-reactivity between vespid hyaluronidases (Ves v 2, Pol d 2) and honeybee venom (HBV) hyaluronidase (Api m 2) beyond cross-reactive carbohydrate (CCD) reactivity seems to be limited (Figure 136) (see chapter B20).

Significant progress has been achieved mainly by proteomic approaches in identifying important allergens of low abundance. The genes of the 100 kDa dipeptidyl peptidases IV (DPPIV) from YJV (Ves v 3) and Polistes dominula venom (Pol d 3), a new class of homologous and cross-reactive Hymenoptera venom enzymes, were identified.²¹⁴,¹²⁹³ While in YJV DPPIV catalyzes the reaction from promastoparan to mastoparan,¹²⁹² the substrate of Polistes dominula venom (PDV) DPPIV remains unclear (the insects probably protect themselves against toxic effects of the peptide substrates). Ves v 3 and Pol d 3 share sequence identity of 76%, resulting in
<figure><img src="image_251.png" /><figcaption>FIGURE 132 Taxonomy of allergy-relevant vespid species. As the taxonomy of the order Hymenoptera is highly complex, only a selection of allergy-relevant taxa is shown. Only selected species with particular relevance for allergy are included. For taxonomic overview of allergy-relevant bees (Apidae) refer to the bee venom chapter. The family Formicidae (ants) also contains species with relevance for Hymenoptera venom allergy.</figcaption></figure>

Hyaluronidases are common components of vespid venoms (e.g., Ves v 2 and Pol d 2). In YJV an enzymatically active (Ves v 2.0101) and an inactive (Ves v 2.0201) hyaluronidase were identified that share sequence identity of 59%, whereby the latter seems to be the predominant isoform.¹²⁹¹ The extent of cross-reactivity between YJV hyaluronidases and their homologue of P. dominula venom (Pol d 2) so far remains elusive. By contrast, cross-reactivity between vespid hyaluronidases (Ves v 2, Pol d 2) and honeybee venom (HBV) hyaluronidase (Api m 2) beyond cross-reactive carbohydrate (CCD) reactivity seems to be limited (Figure 136) (see chapter B20).

Significant progress has been achieved mainly by proteomic approaches in identifying important allergens of low abundance. The genes of the 100 kDa dipeptidyl peptidases IV (DPPIV) from YJV (Ves v 3) and Polistes dominula venom (Pol d 3), a new class of homologous and cross-reactive Hymenoptera venom enzymes, were identified.²¹⁴,¹²⁹³ While in YJV DPPIV catalyzes the reaction from promastoparan to mastoparan,¹²⁹² the substrate of Polistes dominula venom (PDV) DPPIV remains unclear (the insects probably protect themselves against toxic effects of the peptide substrates). Ves v 3 and Pol d 3 share sequence identity of 76%, resulting in