<table><caption>TABLE 5 Basophil activation test to allergen components in food allergy</caption><thead><tr><th>Allergen</th><th>Molecular allergen</th><th>Source</th><th>Concentrations</th><th>Comparison to extract and/or clinical relevance</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="3">Cow's milk</td><td>Casein (nBos d 8)</td><td>Commercially available</td><td>0.1-10-1000 ng/ml</td><td>Lower sensitivity and specificity than cow's milk extract</td><td>[197]</td></tr><tr><td>Casein (Bos d 8)<br>Alpha-Lactalbumin (Bos d 4)<br>Beta-Lactoglobulin (Bos d 5)</td><td>Commercially available</td><td>Casein: 1-400 ng/ml<br>Alpha-Lactalbumin, beta-Lactoglobulin: 10-200 ng/ml</td><td>Not much added value of components compared to cow's milk extract</td><td>[198]</td></tr><tr><td>Ovomucoid (nGal d1)</td><td>Trypsin inhibitor from purified hen egg white ovomucoid type III-0,<br>Commercially available</td><td>0.1-10-1000 ng/ml</td><td>Higher sensitivity and specificity than egg white extract</td><td>[197]</td></tr><tr><td rowspan="2">Egg</td><td>Ovomucoid (Gal d 1)<br>Ovalbumin (Gal d 2)</td><td>0.5 ng/ml - 5 mg/L</td><td></td><td>Difference to egg white not calculated, but in the same range</td><td>[183]</td></tr><tr><td>Omega-5 gliadin (nTri a 19 and rTri a 19)</td><td>Natural protein purified from wheat flour, recombinant protein expressed in E.coli</td><td>10-100-1000-10,000 ng/ml</td><td>Similar sensitivity and specificity as wheat allergen extract</td><td>[199]</td></tr><tr><td rowspan="3">Wheat</td><td>Hydrolyzed wheat protein (HWP), Omega-5 gliadin (nTri a 19)</td><td>Natural protein purified from wheat</td><td>0.0001-1 µg/ml</td><td>Higher activation with HWP in HWP-WDEIA, higher activation with W5-gliadin in CO-WDEIA</td><td>[200]</td></tr><tr><td>ω5-, ω1,2-, α-, γ-gliadins, high- and low molecular-weight wheat flour glutenin subunits (HMW-, LMW-GS, ) gluten</td><td>Natural protein purified from</td><td>0.08-4.0 mg/mL</td><td>Sensitivity of ω5-gliadins: 100% Sensitivity of HMW-GS: 75%</td><td>[201]</td></tr><tr><td>Ara h 1<br>Ara h 2<br>Ara h 6</td><td>Commercially available</td><td>0.1820-4.545 ng/ml</td><td>Ara h 2: Higher sensitivity and specificity than peanut extract, Ara h 1 and Ara h 6</td><td>[178]</td></tr><tr><td rowspan="5">Peanut</td><td>Ara h 2<br>Ara h 6</td><td>Purified peanut proteins</td><td>0.1-1000 µg/mL</td><td>AUC value of Ara h 6 slightly higher than Ara h 2</td><td>[177]</td></tr><tr><td>rAra h 8</td><td>Commercially available</td><td>0.05-500 ng/mL</td><td>More positive CD-sens Ara h 8 compared to peanut in children with IgE-ab to birch and rAra h 8, but not to rAra h 1, Ara h 2 and Ara h 3.</td><td>[202]</td></tr><tr><td>Ara h 1, Ara h 2,<br>Ara h 3, Ara h 6, Ara h 9</td><td>Commercially available</td><td>0.1 µg/mL</td><td>High sensitivity to Ara h 9, but only Ara h 2 was able to discriminate peanut-allergic subjects</td><td>[203]</td></tr><tr><td>3 Ara h 7 isoforms</td><td>Commercially available</td><td>1000 ng/mL</td><td>Sensitisation to Ara h 7.0201 equally potent as Ara h 2.0201 and 6.01</td><td>[204]</td></tr><tr><td>Pru p 3</td><td>Recombinant allergen expressed in P. pastoris</td><td>100-300 ng/ml</td><td>Lower sensitivity and higher specificity compared to peach extract</td><td>[205]</td></tr><tr><td rowspan="3">Peach</td><td>rPru p 3<br>rMal d 3</td><td>Recombinant allergen expressed in P. pastoris</td><td>0.001-0.01-0.1-1 µg/mL</td><td>Symptomatic patients in Barcelona seem to be more sensitive to lower allergen concentrations compared to patients in Antwerpen</td><td>[173]</td></tr><tr><td>Peach-LTPs<br>Pru p 2.0101<br>Pru p 2.0201<br>Pru p 2.0301</td><td>Purified peach proteins (Pru p 2.0101, Pru p 2.0201), recombinant allergen expressed in P. pastoris (Pru p 2.0301)</td><td>25, 10, 1 and 0.1 µg/mL</td><td>Pru p 2.0201 most active of the three isoforms, recognized by 80% of patients (Pru p 2.0101: 60%, Pru p 2.0301: 50%).</td><td>[175]</td></tr><tr><td>Celery/<br>carrot/ apple</td><td>Api g 1<br>Dau c 1<br>Mal d 1</td><td>Commercially available</td><td>10-5-100 µg/mL</td><td>Sensitivity for Mal d 1, Dau c 1, Api g 1: 75%, 65%, 75% specificity: 68%, 100%, 77% in patients with OAS</td><td>[206]</td></tr><tr><td>Strawberry</td><td>8 Fra a 1 proteins</td><td>Recombinant allergens expressed in E.coli</td><td>0.5-500 ng/mL</td><td>Fra a 1.02 and Fra a 1.03 highest activation</td><td>[174]</td></tr><tr><td>Meat/innards</td><td>Alpha-Gal</td><td>Commercially available</td><td>0.022-2272 ng/mL</td><td>Equal sensitivity compared to pork kidney extract</td><td>[180]</td></tr><tr><td>Shrimp</td><td>rPen m 1 (tropomyosin)</td><td>UniProt ID: A1KYZ2; expressed in E. coli</td><td>100-10,000 ng/ml</td><td>Lower sensitivity and higher specificity compared to shrimp extract</td><td>[207]</td></tr><tr><td>Fish</td><td>α- and β-parvalbumins</td><td>Purified fish proteins</td><td>0.1-1000 ng/ml</td><td>Reduced basophil activation by α-parvalbumins compared with β-parvalbumins in individuals allergic to bony fish</td><td>[208]</td></tr></tbody></table>
<table><caption>TABLE 5 Basophil activation test to allergen components in food allergy</caption><thead><tr><th>Allergen</th><th>Molecular allergen</th><th>Source</th><th>Concentrations</th><th>Comparison to extract and/or clinical relevance</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="3">Cow's milk</td><td>Casein (nBos d 8)</td><td>Commercially available</td><td>0.1-10-1000 ng/ml</td><td>Lower sensitivity and specificity than cow's milk extract</td><td>[197]</td></tr><tr><td>Casein (Bos d 8)<br/>Alpha-Lactalbumin (Bos d 4)<br/>Beta-Lactoglobulin (Bos d 5)</td><td>Commercially available</td><td>Casein: 1-400 ng/ml<br/>Alpha-Lactalbumin, beta-Lactoglobulin: 10-200 ng/ml</td><td>Not much added value of components compared to cow's milk extract</td><td>[198]</td></tr><tr><td>Ovomucoid (nGal d1)</td><td>Trypsin inhibitor from purified hen egg white ovomucoid type III-0,<br/>Commercially available</td><td>0.1-10-1000 ng/ml</td><td>Higher sensitivity and specificity than egg white extract</td><td>[197]</td></tr><tr><td rowspan="2">Egg</td><td>Ovomucoid (Gal d 1)<br/>Ovalbumin (Gal d 2)</td><td>0.5 ng/ml - 5 mg/L</td><td></td><td>Difference to egg white not calculated, but in the same range</td><td>[183]</td></tr><tr><td>Omega-5 gliadin (nTri a 19 and rTri a 19)</td><td>Natural protein purified from wheat flour, recombinant protein expressed in E.coli</td><td>10-100-1000-10,000 ng/ml</td><td>Similar sensitivity and specificity as wheat allergen extract</td><td>[199]</td></tr><tr><td rowspan="3">Wheat</td><td>Hydrolyzed wheat protein (HWP), Omega-5 gliadin (nTri a 19)</td><td>Natural protein purified from wheat</td><td>0.0001-1 µg/ml</td><td>Higher activation with HWP in HWP-WDEIA, higher activation with W5-gliadin in CO-WDEIA</td><td>[200]</td></tr><tr><td>ω5-, ω1,2-, α-, γ-gliadins, high- and low molecular-weight wheat flour glutenin subunits (HMW-, LMW-GS, ) gluten</td><td>Natural protein purified from</td><td>0.08-4.0 mg/mL</td><td>Sensitivity of ω5-gliadins: 100% Sensitivity of HMW-GS: 75%</td><td>[201]</td></tr><tr><td>Ara h 1<br/>Ara h 2<br/>Ara h 6<br/>Ara h 2<br/>Ara h 6<br/>rAra h 8</td><td>Commercially available</td><td>0.1820-4.545 ng/ml</td><td>Ara h 2: Higher sensitivity and specificity than peanut extract, Ara h 1 and Ara h 6</td><td>[178]</td></tr><tr><td rowspan="4">Peanut</td><td>Ara h 1, Ara h 2,<br/>Ara h 3, Ara h 6, Ara h 9</td><td>Commercially available</td><td>0.1 µg/mL</td><td>AUC value of Ara h 6 slightly higher than Ara h 2</td><td>[177]</td></tr><tr><td>3 Ara h 7 isoforms</td><td>Commercially available</td><td>1000 ng/mL</td><td>More positive CD-sens Ara h 8 compared to peanut in children with IgE-ab to birch and rAra h 8, but not to rAra h 1, Ara h 2 and Ara h 3.</td><td>[202]</td></tr><tr><td>Peach-LTPs<br/>Pru p 2.0101<br/>Pru p 2.0201<br/>Pru p 2.0301</td><td>Purified peach proteins (Pru p 2.0101, Pru p 2.0201), recombinant allergen expressed in P. pastoris (Pru p 2.0301)</td><td>25, 10, 1 and 0.1 µg/mL</td><td>High sensitivity to Ara h 9, but only Ara h 2 was able to discriminate peanut-allergic subjects</td><td>[203]</td></tr><tr><td>Peach</td><td>Pru p 3<br/>rPru p 3<br/>rMal d 3</td><td>Recombinant allergen expressed in P. pastoris</td><td>100-300 ng/ml<br/>0.001-0.01-0.1-1 µg/mL</td><td>Sensitisation to Ara h 7.0201 equally potent as Ara h 2.0201 and 6.01</td><td>[204]</td></tr><tr><td rowspan="2">Peach</td><td>Peach-LTPs<br/>Pru p 2.0101<br/>Pru p 2.0201<br/>Pru p 2.0301</td><td>Purified peach proteins (Pru p 2.0101, Pru p 2.0201), recombinant allergen expressed in P. pastoris (Pru p 2.0301)</td><td>25, 10, 1 and 0.1 µg/mL</td><td>Symptomatic patients in Barcelona seem to be more sensitive to lower allergen concentrations compared to patients in Antwerpen</td><td>[173]</td></tr><tr><td>Celery/<br/>carrot/ apple</td><td>Api g 1<br/>Dau c 1<br/>Mal d 1</td><td>Commercially available</td><td>10-5-100 µg/mL</td><td>Pru p 2.0201 most active of the three isoforms, recognized by 80% of patients (Pru p 2.0101: 60%, Pru p 2.0301: 50%).</td><td>[175]</td></tr><tr><td>Strawberry</td><td>8 Fra a 1 proteins</td><td>Recombinant allergens expressed in E.coli</td><td>0.5-500 ng/mL</td><td>Sensitivity for Mal d 1, Dau c 1, Api g 1: 75%, 65%, 75% specificity: 68%, 100%, 77% in patients with OAS</td><td>[206]</td></tr><tr><td>Meat/innards</td><td>Alpha-Gal</td><td>Commercially available</td><td>0.022-2272 ng/mL</td><td>Equal sensitivity compared to pork kidney extract</td><td>[180]</td></tr><tr><td>Shrimp</td><td>rPen m 1 (tropomyosin)</td><td>UniProt ID: A1KYZ2; expressed in E. coli</td><td>100-10,000 ng/ml</td><td>Lower sensitivity and higher specificity compared to shrimp extract</td><td>[207]</td></tr><tr><td>Fish</td><td>α- and β-parvalbumins</td><td>Purified fish proteins</td><td>0.1-1000 ng/ml</td><td>Reduced basophil activation by α-parvalbumins compared with β-parvalbumins in individuals allergic to bony fish</td><td>[208]</td></tr></tbody></table>
<figure><img src="image_25.png" /><figcaption>FIGURE 13 Proposed clinical approach to the diagnosis of IgE-mediated food allergy</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>FIGURE 14 Patterns in the BAT over the course of peanut oral immunotherapy can predict the response to treatment and prognosis [186].</figcaption></figure>

basophil parameters can be diagnostically useful.¹⁸⁰ In a different study, BAT to cow's milk was able to predict spontaneous resolution of cow's milk allergy.¹⁸¹

Following a precise diagnosis, BAT can potentially help document the change with specific treatments for food allergy, like allergen-specific immunotherapy or biologicals. Typically, following allergen-specific immunotherapy, a reduction in basophil reactivity and sensitivity is observed with lower proportion of activated basophils for a given allergen concentration, a shift in the dose-response towards higher concentrations of the allergen and lower area under the dose-response curve.¹⁸²,¹⁸³ A reduction in basophil reactivity to bystander allergens and IgE-mediated stimulants during allergen-specific immunotherapy has also been reported.¹⁸⁴ These effects tend to disappear with interruption of treatment, particularly in the patients whose symptoms recur.¹⁸⁵ Importantly, the BAT has shown to be able to predict the response to oral immunotherapy as early as 3 months into treatment with patients with sustained unresponsiveness and patients with transient de sensitization showing different patterns of basophil response.¹⁸⁶ (Figure 14).

Following treatment with omalizumab, two factors have been shown to contribute to the change in basophil response: on one hand, the reduction in IgE on the surface of basophils leads to reduction in basophil reactivity, and on the other hand the reduction in IgE receptor density leads to a greater basophil sensitivity.¹⁸⁷⁻¹⁸⁹ The overall effect results from the combination of these two factors. A better response is expected from patients with a lower IgE specific activity, i.e., a lower proportion of IgE that is specific for that allergen.¹¹⁰

An additional application of the BAT is to determine potency of individual food allergens within a food extract, and to evaluate effects of food processing.⁵¹,¹⁹⁰⁻¹⁹⁵ BAT could potentially be used to

detect the presence of allergens in complex mixtures and to test for possible food allergen contaminations.¹⁹⁶

Further standardization and quality assurance are required for mainstream use of BAT to support food allergy diagnosis and follow up of patients during the course of immunomodulatory treatments. Studies confirming its utility in place of OFC both for diagnosis and follow up in a real-life setting alongside with cost effectiveness and impact studies would be informative to support the incorporation of BAT in clinical guidelines.

## 3 – Insect venom allergy

Hymenoptera venoms are complex mixtures of a variety of substances including numerous potential allergens. The knowledge of the composition of hymenoptera venoms and the use of recombinantly produced CCD (cross-reactive carbohydrate)-free hymenoptera venom allergens has improved diagnostics and led to the field of molecular or component-resolved diagnostics. In recent years, identification and characterization of new allergens of Hymenoptera venoms by biochemical and molecular biological methods have made significant progress, shifting the focus from the whole venom to individual allergenic molecules.²⁰⁹,²¹⁰

The use of recombinant insect venom components in basophil activation testing began with the use of rVes v 1 and rVes v 5 in two yellow-jacket-venom (YJV) sensitized patients showing a CD63 basophil activation of up to 90% and with the recombinant allergens rVes v 3 compared to rVes v 5 and with rApi m 5 compared to rApi m 1 in single patients revealing different individual dose-response curves in insect venom sensitized patients.⁹⁹,²¹¹ Also in 8/13 honey bee venom (HBV) allergic patients rApi m 10 was able to induce
<figure><img src="image_25.png" /><figcaption>FIGURE 13 Proposed clinical approach to the diagnosis of IgE-mediated food allergy</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>FIGURE 14 Patterns in the BAT over the course of peanut oral immunotherapy can predict the response to treatment and prognosis [186].</figcaption></figure>

basophil parameters can be diagnostically useful.¹⁸⁰ In a different study, BAT to cow's milk was able to predict spontaneous resolution of cow's milk allergy.¹⁸¹

Following a precise diagnosis, BAT can potentially help document the change with specific treatments for food allergy, like allergen-specific immunotherapy or biologicals. Typically, following allergen-specific immunotherapy, a reduction in basophil reactivity and sensitivity is observed with lower proportion of activated basophils for a given allergen concentration, a shift in the dose-response towards higher concentrations of the allergen and lower area under the dose-response curve.¹⁸²,¹⁸³ A reduction in basophil reactivity to bystander allergens and IgE-mediated stimulants during allergen-specific immunotherapy has also been reported.¹⁸⁴ These effects tend to disappear with interruption of treatment, particularly in the patients whose symptoms recur.¹⁸⁵ Importantly, the BAT has shown to be able to predict the response to oral immunotherapy as early as 3 months into treatment with patients with sustained unresponsiveness and patients with transient de sensitization showing different patterns of basophil response.¹⁸⁶ (Figure 14).

Following treatment with omalizumab, two factors have been shown to contribute to the change in basophil response: on one hand, the reduction in IgE on the surface of basophils leads to reduction in basophil reactivity, and on the other hand the reduction in IgE receptor density leads to a greater basophil sensitivity.¹⁸⁷⁻¹⁸⁹ The overall effect results from the combination of these two factors. A better response is expected from patients with a lower IgE specific activity, i.e., a lower proportion of IgE that is specific for that allergen.¹¹⁰

An additional application of the BAT is to determine potency of individual food allergens within a food extract, and to evaluate effects of food processing.⁵¹,¹⁹⁰⁻¹⁹⁵ BAT could potentially be used to

detect the presence of allergens in complex mixtures and to test for possible food allergen contaminations.¹⁹⁶

Further standardization and quality assurance are required for mainstream use of BAT to support food allergy diagnosis and follow up of patients during the course of immunomodulatory treatments. Studies confirming its utility in place of OFC both for diagnosis and follow up in a real-life setting alongside with cost effectiveness and impact studies would be informative to support the incorporation of BAT in clinical guidelines.

### 3 – Insect venom allergy

Hymenoptera venoms are complex mixtures of a variety of substances including numerous potential allergens. The knowledge of the composition of hymenoptera venoms and the use of recombinantly produced CCD (cross-reactive carbohydrate)-free hymenoptera venom allergens has improved diagnostics and led to the field of molecular or component-resolved diagnostics. In recent years, identification and characterization of new allergens of Hymenoptera venoms by biochemical and molecular biological methods have made significant progress, shifting the focus from the whole venom to individual allergenic molecules.²⁰⁹,²¹⁰

The use of recombinant insect venom components in basophil activation testing began with the use of rVes v 1 and rVes v 5 in two yellow-jacket-venom (YJV) sensitized patients showing a CD63 basophil activation of up to 90% and with the recombinant allergens rVes v 3 compared to rVes v 5 and with rApi m 5 compared to rApi m 1 in single patients revealing different individual dose-response curves in insect venom sensitized patients.⁹⁹,²¹¹ Also in 8/13 honey bee venom (HBV) allergic patients rApi m 10 was able to induce
basophil activation upon almost 100%.²¹² In 43 patients with YJV allergy the use of the four recombinant allergens rVes v 1, rVes v 2, rVes v 3 and rVes v 5 in the BAT was investigated. BAT with rVes v 5 provided a specificity of 100% and a sensitivity of 81% whereas BAT performed with natural venom showed only a specificity of 94.1 and a sensitivity of 68.3%. Additionally, BAT performed with rVes v 5 followed by rVes v 3 was the most sensitive and specific procedure among all recombinant allergens tested. Furthermore, some patients were detected being negative to rVes v 5 but positive to other recombinant allergens or conventional venom extract in the BAT. Therefore, this test markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to respective IgE detection in serum.²¹³ Antigens 5 are the most potent allergens in vespid venoms and are found in in the venom of nearly all *Vespoidea* species with a varying degree of sequence homology. BATs were performed in 21 YJV-allergic patients with the recombinantly produced antigens 5 of seven allergy-relevant species: Vespula vulgaris (rVes v 5), the hornet Vespa crabro (rVesp c 5), the European paper wasp Polistes dominula (rPol d 5), the American paper wasp Polistes annularis (rPol a 5), the white-faced hornet Dolichovespula maculata (Dol m 5), the fire ant Solenopsis invicta (Sol i 3), and the wasp Polybia scutellaris (rPoly s 5). In the BAT, the YJV-allergic patients showed different activation profiles in response to the different antigens 5. Six of twenty (30%) patients exhibited basophil activation in response to rVes v 5 and/or rVesp c 5 only. The basophils of further 11 patients (55%) were activated by either all or different combinations of antigens 5. However, in most of these patients, the basophil activation was more pronounced in response to rVes v 5 and/or rVesp c 5. Only in two patients the activation pattern was more distinct in response to other allergens than rVes v 5 and/or rVesp c 5. rPoly s 5 was also able to activate patient-derived basophils in this assay. These results demonstrated cross-reactivity of vespid venoms on a molecular basis.²¹⁴

Another allergen of Polistes dominula rPol d 3 showed basophil activation in *Polistes dominula* venom (PDV)—and/or YJV-allergic patients from Spain and HBV- and YJV-allergic patients from Germany and was compared with the other recombinant dipeptidyl peptidase IV allergens rVes v 3 or rApi m 5.²¹⁵

Polistes PLA2 from *Polistes dominula* venom and other HBV components (C1q-like protein (C1q) and PDGF/ VEGF-like (PVF1) were unable to activate basophils of allergic patients despite exhibition of specific IgE reactivity questioning their role in the context of clinically relevant sensitization.²¹⁶,²¹⁷ Similarly, neither the hyaluronidase of Polistes dominula (Pol d 2) nor of Vespula vulgaris (Ves v 2b) showed significant basophil activation in any insect venom allergic patient, whereas the allergen rApi m 2 caused a moderate activation in Api m 2 sensitized HBV allergic patients.²¹⁸

In 9 patients sensitized to Api m 1 and Api m 2, a conventional BAT with HBV extract revealed a higher basophil activation compared with the components nApi m 1 and rApi m 2, but in 8 patients sensitized only to Api m 1 the results were comparable. Nanocrystal-labeled nApi m 1 and rApi m 2 showed a strong positive correlation to nApi m 1 and rApi m 2 and enabled the development of a

multiplex BAT approach incorporating multiple fluorescent-labeled allergen components.²¹⁹ The lack of basophil activation by mimotopes corresponding to Api m 1 IgE epitopes could be important for the development of safer allergen immunotherapy.²²⁰

In summary, rVes v 5 and rVes v 3 appear to increase sensitivity and specificity in BAT compared with wasp venom extract in YJV allergic patients, whereas in bee venom allergic patients nApi m 1, rApi m 5, and rApi m 10 induce higher basophil activation than bee venom extracts only in single patients. Therefore, rVes v 3 and 5, nApi m 1 and 80 rApi m 5 and 10 BAT could reveal the actual and species-specific allergenic activity of those venom components and thus better elucidates the pattern of single /double positivity than components based IgE testing.¹⁷¹ Other components (e.g., antigen 5) showed pronounced cross-reactivity, or no allergenicity in BAT (Table 6).

## 4 - Aeroallergens

### Tree pollen allergens

PR-10-like allergens are the major allergens in pollen from trees of the order Fagales. BAT with Bet v 1 is a useful and efficient approach to determine the allergic status in birch sensitized individuals (Table 7).²²¹⁻²³¹ BAT to Mal d 1, Api g 1, and Dau c 1 have been used to characterize PR-10-like allergens in different individuals to better distinguish cross-reactive birch-pollen-associated food allergy from sensitization without food allergy.²⁰⁶,²²⁵,²³² Recently, Que m 1, a major allergen from Mongolian oak, a dominant species in Korea, was cloned, its recombinant protein was produced, and in oak-sensitized subjects, Que m 1 demonstrated a potent basophil activation in comparison to Bet v 1.²²³

Basophil testing can be used to functionally monitor IgG blocking bioactivity and humoral response induced by SCIT or SLIT.²³¹,²³³ Recently, a basophil inhibition assay using stripped basophils sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1, or Que a 1 after incubation with pre-Bet v 1 SLIT or post-Bet v 1 SLIT sample.²²⁴ This study demonstrated highly variable and nonpredictable Bet v 1 SLIT cross-blocking bioactivity to PR-10-like allergens of Fagales tree pollen. Similarly, Bet v 1 SLIT also induced limited cross-blocking bioactivity to Mal d 1.²²⁵

A nonallergenic birch pollen allergy vaccine, consisting of hepatitis PreS-fused Bet v 1 peptides²²⁸ or derivatives of the Bet v 1 obtained by rational sequence reassembly²²⁹ showed reduced allergenic activity when tested in BAT with basophils from patients allergic to birch pollen. Modification of Bet v 1 into trimer showed a more than 10-fold reduced allergenic activity compared with the rBet v 1 wild-type.²³⁰

BAT can also explore the role of organic compounds of airborne particles in the aggravation of IgE-mediated allergic reactions to aeroallergens. Thus, organic extracts of urban aerosol (of PM2.5 or less) or polycyclic aromatic hydrocarbons from diesel emissions
basophil activation upon almost 100%.²¹² In 43 patients with YJV allergy the use of the four recombinant allergens rVes v 1, rVes v 2, rVes v 3 and rVes v 5 in the BAT was investigated. BAT with rVes v 5 provided a specificity of 100% and a sensitivity of 81% whereas BAT performed with natural venom showed only a specificity of 94.1 and a sensitivity of 68.3%. Additionally, BAT performed with rVes v 5 followed by rVes v 3 was the most sensitive and specific procedure among all recombinant allergens tested. Furthermore, some patients were detected being negative to rVes v 5 but positive to other recombinant allergens or conventional venom extract in the BAT. Therefore, this test markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to respective IgE detection in serum.²¹³ Antigens 5 are the most potent allergens in vespid venoms and are found in in the venom of nearly all *Vespoidea* species with a varying degree of sequence homology. BATs were performed in 21 YJV-allergic patients with the recombinantly produced antigens 5 of seven allergy-relevant species: Vespula vulgaris (rVes v 5), the hornet Vespa crabro (rVesp c 5), the European paper wasp Polistes dominula (rPol d 5), the American paper wasp Polistes annularis (rPol a 5), the white-faced hornet Dolichovespula maculata (Dol m 5), the fire ant Solenopsis invicta (Sol i 3), and the wasp Polybia scutellaris (rPoly s 5). In the BAT, the YJV-allergic patients showed different activation profiles in response to the different antigens 5. Six of twenty (30%) patients exhibited basophil activation in response to rVes v 5 and/or rVesp c 5 only. The basophils of further 11 patients (55%) were activated by either all or different combinations of antigens 5. However, in most of these patients, the basophil activation was more pronounced in response to rVes v 5 and/or rVesp c 5. Only in two patients the activation pattern was more distinct in response to other allergens than rVes v 5 and/or rVesp c 5. rPoly s 5 was also able to activate patient-derived basophils in this assay. These results demonstrated cross-reactivity of vespid venoms on a molecular basis.²¹⁴

Another allergen of Polistes dominula rPol d 3 showed basophil activation in *Polistes dominula* venom (PDV)—and/or YJV-allergic patients from Spain and HBV- and YJV-allergic patients from Germany and was compared with the other recombinant dipeptidyl peptidase IV allergens rVes v 3 or rApi m 5.²¹⁵

Polistes PLA2 from *Polistes dominula* venom and other HBV components (C1q-like protein (C1q) and PDGF/ VEGF-like (PVF1) were unable to activate basophils of allergic patients despite exhibition of specific IgE reactivity questioning their role in the context of clinically relevant sensitization.²¹⁶,²¹⁷ Similarly, neither the hyaluronidase of Polistes dominula (Pol d 2) nor of Vespula vulgaris (Ves v 2b) showed significant basophil activation in any insect venom allergic patient, whereas the allergen rApi m 2 caused a moderate activation in Api m 2 sensitized HBV allergic patients.²¹⁸

In 9 patients sensitized to Api m 1 and Api m 2, a conventional BAT with HBV extract revealed a higher basophil activation compared with the components nApi m 1 and rApi m 2, but in 8 patients sensitized only to Api m 1 the results were comparable. Nanocrystal-labeled nApi m 1 and rApi m 2 showed a strong positive correlation to nApi m 1 and rApi m 2 and enabled the development of a

multiplex BAT approach incorporating multiple fluorescent-labeled allergen components.²¹⁹ The lack of basophil activation by mimotopes corresponding to Api m 1 IgE epitopes could be important for the development of safer allergen immunotherapy.²²⁰

In summary, rVes v 5 and rVes v 3 appear to increase sensitivity and specificity in BAT compared with wasp venom extract in YJV allergic patients, whereas in bee venom allergic patients nApi m 1, rApi m 5, and rApi m 10 induce higher basophil activation than bee venom extracts only in single patients. Therefore, rVes v 3 and 5, nApi m 1 and 80 rApi m 5 and 10 BAT could reveal the actual and species-specific allergenic activity of those venom components and thus better elucidates the pattern of single /double positivity than components based IgE testing.¹⁷¹ Other components (e.g., antigen 5) showed pronounced cross-reactivity, or no allergenicity in BAT (Table 6).

## 4 - Aeroallergens

### Tree pollen allergens

PR-10-like allergens are the major allergens in pollen from trees of the order Fagales. BAT with Bet v 1 is a useful and efficient approach to determine the allergic status in birch sensitized individuals (Table 7).²²¹⁻²³¹ BAT to Mal d 1, Api g 1, and Dau c 1 have been used to characterize PR-10-like allergens in different individuals to better distinguish cross-reactive birch-pollen-associated food allergy from sensitization without food allergy.²⁰⁶,²²⁵,²³² Recently, Que m 1, a major allergen from Mongolian oak, a dominant species in Korea, was cloned, its recombinant protein was produced, and in oak-sensitized subjects, Que m 1 demonstrated a potent basophil activation in comparison to Bet v 1.²²³

Basophil testing can be used to functionally monitor IgG blocking bioactivity and humoral response induced by SCIT or SLIT.²³¹,²³³ Recently, a basophil inhibition assay using stripped basophils sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1, or Que a 1 after incubation with pre-Bet v 1 SLIT or post-Bet v 1 SLIT sample.²²⁴ This study demonstrated highly variable and nonpredictable Bet v 1 SLIT cross-blocking bioactivity to PR-10-like allergens of Fagales tree pollen. Similarly, Bet v 1 SLIT also induced limited cross-blocking bioactivity to Mal d 1.²²⁵

A nonallergenic birch pollen allergy vaccine, consisting of hepatitis PreS-fused Bet v 1 peptides²²⁸ or derivatives of the Bet v 1 obtained by rational sequence reassembly²²⁹ showed reduced allergenic activity when tested in BAT with basophils from patients allergic to birch pollen. Modification of Bet v 1 into trimer showed a more than 10-fold reduced allergenic activity compared with the rBet v 1 wild-type.²³⁰

BAT can also explore the role of organic compounds of airborne particles in the aggravation of IgE-mediated allergic reactions to aeroallergens. Thus, organic extracts of urban aerosol (of PM2.5 or less) or polycyclic aromatic hydrocarbons from diesel emissions
<table><caption>TABLE 6 Basophil activation test to allergen components in insect venom allergy. Needs list of Abbreviations</caption><thead><tr><th>Allergen</th><th>Molecular allergen</th><th>Source</th><th>Concentrations</th><th>Comparison to extract and/or clinical relevance</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">Wasp venom</td><td>rVes v 5</td><td>Sf9 insect cells</td><td>0.1, 200 and 2000 ng/mL; 0.08–1000 ng/mL</td><td>Higher sensitivity and specificity than extract</td><td>[211, 99, 213]</td></tr><tr><td>rVes v 3</td><td>Sf9 insect cells</td><td>0.08–1000 ng/mL; 2, 10, 50 and 250 and 1000 ng/mL</td><td>Higher activation compared to extract; lower sensitivity and higher specificity than extract</td><td>[99, 213, 215]</td></tr><tr><td>rVes v 1</td><td>Sf9 insect cells</td><td>0.1, 200 and 2000 ng/mL</td><td>Lower sensitivity and higher specificity than extract</td><td>[211, 213]</td></tr><tr><td>rVes v 2</td><td>Sf9 insect cells</td><td>0.08–1000 ng/mL</td><td>Lower sensitivity and specificity than extract</td><td>[213]</td></tr><tr><td>rVes v 2b</td><td>Sf9 insect cells</td><td>1.6, 8.0, 40, 200, 1000 ng/mL</td><td>No basophil activation</td><td>[218]</td></tr><tr><td rowspan="7">Bee venom</td><td>nApi m 1</td><td>Apis mellifera bee venom</td><td>0.08–1000 ng/mL, 0.001–10 µg/mL</td><td>Higher (64) /slightly lower (71,72) activation compared to extract</td><td>[212, 219, 220]</td></tr><tr><td>Qdot-labeled nApi m 1</td><td>Apis mellifera bee venom; conjugated to Amino (PEG) Quantum Dots or Carboxyl Quantum Dots</td><td>0.012–12.0 nM for NQ705 nApi m 1 0.0185–18.5 nM for CQ705 nApi m 1</td><td>For amino (NQ) Qdot-labeled nApi m 1 similar activation compared to nApi m 1; for carboxyl (CQ) Qdot-labeled nApi m 1 no basophil activation</td><td>[219]</td></tr><tr><td>Mimotopes of Api m 1 epitopes</td><td>E. coli</td><td>0.01–10 µg/mL</td><td>No basophil activation</td><td>[220]</td></tr><tr><td>rApi m 2</td><td>Sf9 insect cells, High Five insect cells</td><td>1.6, 8.0, 40, 200, 1000 ng/mL; 0.1, 1, 10, 100, 1000, 10000 ng/mL</td><td>Lower activation compared to extract</td><td>[218, 219]</td></tr><tr><td>Qdot-labeled rApi m 2</td><td>High Five insect cells; conjugated to Amino (PEG) Quantum Dots or Carboxyl Quantum Dots</td><td>0.01–11.0 nM for NQ800 rApi m 2 0.0031–3.1 nM for CQ800 rApi m 2</td><td>For amino (NQ) Qdot-labeled rApi m 2 similar activation compared to rApi m 2; for carboxyl (CQ) Qdot-labeled rApi m 2 no basophil activation</td><td>[219]</td></tr><tr><td>rApi m 5</td><td>Sf9 insect cells</td><td>0.08–1000 ng/mL; 2, 10, 50 and 250 and 1000 ng/mL</td><td>Higher activation compared to extract</td><td>[99, 215]</td></tr></tbody></table>

enhance Bet v 1-induced activation of basophils from birch pollen-allergic individuals.<sup>234,235</sup>

BP14, the only pollen allergen (cypress) member of the Gibberellin-Regulated Protein (GRP) protein family reported so far, can induce basophil activation in patients with pollen/food-associated syndrome (PFAS) cypress/ peach.<sup>236</sup> BP14 is the cross-reactive allergen of Pru p 7 and Snakin-1.<sup>236</sup> Pru p 7 Vis a predominant cause of severe subtype of Cupressaceae pollinosis underlying cause of severe peach allergy, and Pru p 7 is highly potent in BAT.<sup>237</sup>

## Grass and weed pollen allergens

Both major grass pollen allergens Phl p 1 and Phl p 5 are showing a high allergenicity and basophil activation in a great majority of grass-pollen sensitized subjects.<sup>226,227,238</sup> Similarly, a cross-reacting group 2/3 major grass pollen Phl p 2 allergens induces a positive BAT response in correspondingly sensitized subjects.<sup>221</sup>

BAT is important for the characterization of novel recombinant, hypoallergenic, peptide-based vaccines for grass pollen allergy and has become a major tool for evaluating change in allergenicity when basophils from patients allergic to grass pollen are tested with novel vaccines peptides and/or carriers.<sup>93,239–241</sup> For instance, basophil activation induced by mix of increasing concentrations of the four major timothy grass pollen allergens (rPhl p 1, 2, 5, and 6) was reduced during recombinant B cell epitope-based vaccine (BM32) immunotherapy of patients with grass pollen allergy.<sup>240</sup> Furthermore, rPhl p 5 dependent basophil activation inhibition with SCIT sera demonstrated that immunotherapy-induced allergen-specific IgG antibodies are not long-lasting after treatment discontinuation.<sup>242</sup>

In weed pollen allergy, Parietaria judaica represents one of the main sources of allergens in the Mediterranean area with Par j 1 and Par j 2 as major allergens.<sup>243</sup> Par j 2 demonstrated a positive response in BAT and resembling the allergenic epitopes of Parietaria
<table><caption>TABLE 6 Basophil activation test to allergen components in insect venom allergy. Needs list of Abbreviations</caption><thead><tr><th>Allergen</th><th>Molecular allergen</th><th>Source</th><th>Concentrations</th><th>Comparison to extract and/or clinical relevance</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">Wasp venom</td><td>rVes v 5</td><td>Sf9 insect cells</td><td>0.1, 200 and 2000 ng/mL; 0.08–1000 ng/mL</td><td>Higher sensitivity and specificity than extract</td><td>[211, 99, 213]</td></tr><tr><td>rVes v 3</td><td>Sf9 insect cells</td><td>0.08–1000 ng/mL; 2, 10, 50 and 250 and 1000 ng/mL</td><td>Higher activation compared to extract; lower sensitivity and higher specificity than extract</td><td>[99, 213, 215]</td></tr><tr><td>rVes v 1</td><td>Sf9 insect cells</td><td>0.1, 200 and 2000 ng/mL</td><td>Lower sensitivity and higher specificity than extract</td><td>[211, 213]</td></tr><tr><td>rVes v 2</td><td>Sf9 insect cells</td><td>0.08–1000 ng/mL</td><td>Lower sensitivity and specificity than extract</td><td>[213]</td></tr><tr><td>rVes v 2b</td><td>Sf9 insect cells</td><td>1.6, 8.0, 40, 200, 1000 ng/mL</td><td>No basophil activation</td><td>[218]</td></tr><tr><td rowspan="7">Bee venom</td><td>nApi m 1</td><td>Apis mellifera bee venom</td><td>0.08–1000 ng/mL, 0.001–10 µg/mL</td><td>Higher (64) /slightly lower (71,72) activation compared to extract</td><td>[212, 219, 220]</td></tr><tr><td>Qdot-labeled nApi m 1</td><td>Apis mellifera bee venom; conjugated to Amino (PEG) Quantum Dots or Carboxyl Quantum Dots</td><td>0.012–12.0 nM for NQ705 nApi m 1 0.0185–18.5 nM for CQ705 nApi m 1</td><td>For amino (NQ) Qdot-labeled nApi m 1 similar activation compared to nApi m 1; for carboxyl (CQ) Qdot-labeled nApi m 1 no basophil activation</td><td>[219]</td></tr><tr><td>Mimotopes of Api m 1 epitopes</td><td>E. coli</td><td>0.01–10 µg/mL</td><td>No basophil activation</td><td>[220]</td></tr><tr><td>rApi m 2</td><td>Sf9 insect cells, High Five insect cells</td><td>1.6, 8.0, 40, 200, 1000 ng/mL; 0.1, 1, 10, 100, 1000, 10000 ng/mL</td><td>Lower activation compared to extract</td><td>[218, 219]</td></tr><tr><td>Qdot-labeled rApi m 2</td><td>High Five insect cells; conjugated to Amino (PEG) Quantum Dots or Carboxyl Quantum Dots</td><td>0.01–11.0 nM for NQ800 rApi m 2 0.0031–3.1 nM for CQ800 rApi m 2</td><td>For amino (NQ) Qdot-labeled rApi m 2 similar activation compared to rApi m 2; for carboxyl (CQ) Qdot-labeled rApi m 2 no basophil activation</td><td>[219]</td></tr><tr><td>rApi m 5</td><td>Sf9 insect cells</td><td>0.08–1000 ng/mL; 2, 10, 50 and 250 and 1000 ng/mL</td><td>Higher activation compared to extract</td><td>[99, 215]</td></tr></tbody></table>

enhance Bet v 1-induced activation of basophils from birch pollen-allergic individuals.<sup>234,235</sup>

BP14, the only pollen allergen (cypress) member of the Gibberellin-Regulated Protein (GRP) protein family reported so far, can induce basophil activation in patients with pollen/food-associated syndrome (PFAS) cypress/ peach.<sup>236</sup> BP14 is the cross-reactive allergen of Pru p 7 and Snakin-1.<sup>236</sup> Pru p 7 Vis a predominant cause of severe subtype of Cupressaceae pollinosis underlying cause of severe peach allergy, and Pru p 7 is highly potent in BAT.<sup>237</sup>

Grass and weed pollen allergens

Both major grass pollen allergens Phl p 1 and Phl p 5 are showing a high allergenicity and basophil activation in a great majority of grass-pollen sensitized subjects.<sup>226,227,238</sup> Similarly, a cross-reacting group 2/3 major grass pollen Phl p 2 allergen induces a positive BAT response in correspondingly sensitized subjects.<sup>221</sup>

BAT is important for the characterization of novel recombinant, hypoallergenic, peptide-based vaccines for grass pollen allergy and has become a major tool for evaluating change in allergenicity when basophils from patients allergic to grass pollen are tested with novel vaccines peptides and/or carriers.<sup>93,239–241</sup> For instance, basophil activation induced by mix of increasing concentrations of the four major timothy grass pollen allergens (rPhl p 1, 2, 5, and 6) was reduced during recombinant B cell epitope-based vaccine (BM32) immunotherapy of patients with grass pollen allergy.<sup>240</sup> Furthermore, rPhl p 5 dependent basophil activation inhibition with SCIT sera demonstrated that immunotherapy-induced allergen-specific IgG antibodies are not long-lasting after treatment discontinuation.<sup>242</sup>

In weed pollen allergy, Parietaria judaica represents one of the main sources of allergens in the Mediterranean area with Par j 1 and Par j 2 as major allergens.<sup>243</sup> Par j 2 demonstrated a positive response in BAT and resembling the allergenic epitopes of Parietaria
judaica pollen. Par j 4 a minor Phl p 7 cross-reactive calcium-binding protein was also positive on the BAT.²⁴⁴

## House dust mites and cockroach allergens

BAT was effectively used to characterize the allergenic activity and molecular characteristics of new house dust mite allergens (HDM) Der p 23,²⁴⁵ Der p 18,²⁴⁶ and Der f 24²⁴⁷ and to monitor the change in allergenic activity after genetic engineering and conversion of Der p 1, 2, 5, 7, 21 and/or 23 allergens into hypoallergenic vaccines.²⁴⁸⁻²⁵² Der p 23, which represents a new major HDM allergen, is characterized by high allergenicity comparable with Der p 1, Der p 2, and this was convincingly demonstrated by upregulation of CD203c expression on basophils from HDM allergic patients.²⁴⁸ Basophil activation was also used to assessed inhibition of Der p 1 response through cross-linking of FcεRI with FcγRIIb.²⁵³ In a comprehensive scan of 12 molecular HDM allergens compared with HDM extract, symptomatic patients reacted with more molecules than asymptomatic patients. The number of reactive molecules correlated with the area under the curve of the extract BAT response.²⁵⁴ Recombinant cockroach allergen Per a 5, Per a 9, and Per a 10 expressed in insect cells can activate passively sensitized²⁵⁵ basophils.²⁵⁵,²⁵⁶ Basophil testing is used as a tool to document the biological activity of those recombinant allergens; however, this approach could benefit from standardization. Additionally, as a minimum, the recombinant protein should be compared with extract from the source organism with 5 relevant allergen concentrations.

## Cat, dog, and horse allergens

The response through CD63 and CD203c of 20 cat allergic patients and 19 controls to stimulation with the major allergen Fel d 1 was equivalent and 100% sensitive.²⁵⁶ As CD203c is expressed on resting cells, there is a convention of calculating the stimulation index for this marker rather than using the fraction of activated cells as is done for CD63. BAT was used to characterize recombinant cat albumin Fel d 2, a cross-reactive animal allergen,²⁵⁸ cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1,²⁵⁹ and a novel cat allergen cat-NCP7, with homology to Can f 7.²⁵⁷,²⁶⁰

For the development of hypoallergenic cat vaccine based on Fel d 1-derived peptides fused to hepatitis B PreS allergenic activity of Fel d 1 and the fusion proteins were compared by using basophil activation tests in patients with cat allergy.²⁶¹ The recombinant fusion proteins exhibited more than 1000-fold reduced allergenic activity in BAT in comparison to Fel d 1.²⁶¹ Recombinant mosaic proteins generated by reassembly of non-IgE-reactive peptides of Fel d 1 similarly showed a strong reduction in allergenic activity.²⁶²

The dog allergen Can f 6 is a major allergen in dog-allergic Chinese children and it demonstrated allergenic activity in BAT.²⁶³ Recently, a panel of recombinant dog allergens (Can f 1-6) was quantified in commercial skin prick test (SPT) solutions of dog extracts, and allergenicity to dog extract was assessed by BAT in three patients. Extensive variations in allergen composition were observed in commercial SPT vials resulting in a patient-dependent ability to activate basophils.²⁶⁴

Those observations favoring a recombinant approach in the diagnosis of dog allergy, which is quite common in industrialized countries. Among 58 children sensitized to dog dander, basophil testing with dog allergens was as good at identifying children with clinically relevant sensitization to dogs. All patients with dog allergy-related rhinitis or asthma had relevant basophil activation to Can f 1-6 mix, and the four children that were sensitized to Can f 1 but with a negative BAT response to Can f 1-6 mix seem clinically tolerant to the dog. Those BAT data were clinically more relevant as the measurement of IgEs to Can f 1, Can f 2, or Can f 3.

## 5 – Advantages and limitations

Like any test, the BAT has advantages and limitations, which need to be considered when applying the BAT to clinical practice.²⁶⁵,²⁶⁶ For instance, given the practicalities involved in the performance of the BAT currently, it is not feasible to use in all patients needing allergy testing but rather to use it as a second-line test in all patients for whom the initial set of tests, namely skin and specific IgE testing was not possible or was equivocal, before considering referring the patient for a provocation test, which involves the risk of allergic reaction, or before starting immunotherapy. Should automated methods become available, the use of BAT may become a first-line test. Flow cytometry-based BAT has long replaced the first methods to assess basophil activation, such as histamine and leukotriene release, and has become the method of choice as it is more precise and robust than the former methods.

Advantages of the BAT include:

1. High specificity in diagnosis—see previous sections on BAT in food and venom allergy for example.

2. Safety of patients—BAT is safe for the patient, as it does not require *in vivo* exposure to the allergen.

3. Variety of stimulants—almost any material can be tested as long as standardized conditions are used and activity is checked on blood of a nonsensitized control individual if basophils in blood of the patient respond. Many drugs and occupational allergens can be adapted to the test; they are usually added in 10% of the blood volume or in an equal volume.

4. Reproducibility—basophil testing is reproducible for diagnosing allergy²⁶⁷ and assessment of basophil sensitivity as marker of allergy is more reproducible than threshold of allergen provocation.²⁶⁸,²⁶⁹

5. Simple equipment—BAT can be performed on any flow cytometer as limited number of colours is required.

Limitations of the BAT include:

1. Requires fresh blood—BAT needs to be tested preferably within 24 h of blood collection. Blood can be tested at up to 48 hours, but dichotomous negative results must be taken with a grain of salt as they may be false negative. To obviate the
judaica pollen. Par j 4 a minor Phl p 7 cross-reactive calcium-binding protein was also positive on the BAT.²⁴⁴

## House dust mites and cockroach allergens

BAT was effectively used to characterize the allergenic activity and molecular characteristics of new house dust mite allergens (HDM) Der p 23,²⁴⁵ Der p 18,²⁴⁶ and Der f 24²⁴⁷ and to monitor the change in allergenic activity after genetic engineering and conversion of Der p 1, 2, 5, 7, 21 and/or 23 allergens into hypoallergenic vaccines.²⁴⁸⁻²⁵² Der p 23, which represents a new major HDM allergen, is characterized by high allergenicity comparable with Der p 1, Der p 2, and this was convincingly demonstrated by upregulation of CD203c expression on basophils from HDM allergic patients.²⁴⁸ Basophil activation was also used to assessed inhibition of Der p 1 response through cross-linking of FcεRI with FcγRIIb.²⁵³ In a comprehensive scan of 12 molecular HDM allergens compared with HDM extract, symptomatic patients reacted with more molecules than asymptomatic patients. The number of reactive molecules correlated with the area under the curve of the extract BAT response.²⁵⁴ Recombinant cockroach allergen Per a 5, Per a 9, and Per a 10 expressed in insect cells can activate passively sensitized²⁵⁵ basophils.²⁵⁵,²⁵⁶ Basophil testing is used as a tool to document the biological activity of those recombinant allergens; however, this approach could benefit from standardization. Additionally, as a minimum, the recombinant protein should be compared with extract from the source organism with 5 relevant allergen concentrations.

## Cat, dog, and horse allergens

The response through CD63 and CD203c of 20 cat allergic patients and 19 controls to stimulation with the major allergen Fel d 1 was equivalent and 100% sensitive.²⁵⁶ As CD203c is expressed on resting cells, there is a convention of calculating the stimulation index for this marker rather than using the fraction of activated cells as is done for CD63. BAT was used to characterize recombinant cat albumin Fel d 2, a cross-reactive animal allergen,²⁵⁸ cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1,²⁵⁹ and a novel cat allergen cat-NCP7, with homology to Can f 7.²⁵⁷,²⁶⁰

For the development of hypoallergenic cat vaccine based on Fel d 1-derived peptides fused to hepatitis B PreS allergenic activity of Fel d 1 and the fusion proteins were compared by using basophil activation tests in patients with cat allergy.²⁶¹ The recombinant fusion proteins exhibited more than 1000-fold reduced allergenic activity in BAT in comparison to Fel d 1.²⁶¹ Recombinant mosaic proteins generated by reassembly of non-IgE-reactive peptides of Fel d 1 similarly showed a strong reduction in allergenic activity.²⁶²

The dog allergen Can f 6 is a major allergen in dog-allergic Chinese children and it demonstrated allergenic activity in BAT.²⁶³ Recently, a panel of recombinant dog allergens (Can f 1-6) was quantified in commercial skin prick test (SPT) solutions of dog extracts, and allergenicity to dog extract was assessed by BAT in three patients. Extensive variations in allergen composition were observed in commercial SPT vials resulting in a patient-dependent ability to activate basophils.²⁶⁴

Those observations favoring a recombinant approach in the diagnosis of dog allergy, which is quite common in industrialized countries. Among 58 children sensitized to dog dander, basophil testing with dog allergens was as good at identifying children with clinically relevant sensitization to dogs. All patients with dog allergy-related rhinitis or asthma had relevant basophil activation to Can f 1-6 mix, and the four children that were sensitized to Can f 1 but with a negative BAT response to Can f 1-6 mix seem clinically tolerant to the dog. Those BAT data were clinically more relevant as the measurement of IgEs to Can f 1, Can f 2, or Can f 3.

## 5 – Advantages and limitations

Like any test, the BAT has advantages and limitations, which need to be considered when applying the BAT to clinical practice.²⁶⁵,²⁶⁶ For instance, given the practicalities involved in the performance of the BAT currently, it is not feasible to use in all patients needing allergy testing but rather to use it as a second-line test in all patients for whom the initial set of tests, namely skin and specific IgE testing was not possible or was equivocal, before considering referring the patient for a provocation test, which involves the risk of allergic reaction, or before starting immunotherapy. Should automated methods become available, the use of BAT may become a first-line test. Flow cytometry-based BAT has long replaced the first methods to assess basophil activation, such as histamine and leukotriene release, and has become the method of choice as it is more precise and robust than the former methods.

Advantages of the BAT include:

1. High specificity in diagnosis—see previous sections on BAT in food and venom allergy for example.

2. Safety of patients—BAT is safe for the patient, as it does not require *in vivo* exposure to the allergen.

3. Variety of stimulants—almost any material can be tested as long as standardized conditions are used and activity is checked on blood of a nonsensitized control individual if basophils in blood of the patient respond. Many drugs and occupational allergens can be adapted to the test; they are usually added in 10% of the blood volume or in an equal volume.

4. Reproducibility—basophil testing is reproducible for diagnosing allergy²⁶⁷ and assessment of basophil sensitivity as marker of allergy is more reproducible than threshold of allergen provocation.²⁶⁸,²⁶⁹

5. Simple equipment—BAT can be performed on any flow cytometer as limited number of colours is required.

Limitations of the BAT include:

1. Requires fresh blood—BAT needs to be tested preferably within 24 h of blood collection. Blood can be tested at up to 48 hours, but dichotomous negative results must be taken with a grain of salt as they may be false negative. To obviate the
<table><caption>TABLE 7 Basophil activation test to allergen components in respiratory allergy</caption><thead><tr><th>Allergen</th><th>Molecular allergen</th><th>Source</th><th>Concentrations</th><th>Methodological and/or clinical relevance</th><th>Reference</th></tr></thead><tbody><tr><td colspan="6"><strong>Tree pollen</strong></td></tr><tr><td rowspan="10">PR-10-like allergens</td><td>Bet v 1</td><td>E. coli</td><td>10<sup>-7</sup>–10 µg/mL; 1 ng/mL; 1–100 ng/mL; 0.31–20 ng/mL; 0.25–100 ng/mL; 0.002–1000 ng/mL; 10<sup>-5</sup>–10 µg/rnl; 1 µg/mL; 0.1 and 0.3 µg/ml; 0.00001–0.1 µg/ml; 0.005–50 pmol/L; 0.05 pM–0.5 nM</td><td>Evaluation of allergenicity of birch pollen sensitisation</td><td>[234,235,226, 227,238,228, 229,230]</td></tr><tr><td>Mal d 1</td><td></td><td>1 µg/mL; 0.25–100 ng/mL; 0.1 and 0.3 µg/ml</td><td>Identification of cross-reactive non-sensitizers or partial cross-reactive sensitizers; limited Bet v 1 SLIT bioactivity for Mal d 1</td><td>[222, 225, 232]</td></tr><tr><td>Api g 1</td><td></td><td>10 µg/ml</td><td>A major allergen from Mongolian oak pollen.</td><td>[223]</td></tr><tr><td>Dau c 1</td><td></td><td>1 µg/mL</td><td>Limited Bet v 1 SLIT bioactivity for cross-blocking of PR-10-like allergens of Fagales pollen</td><td>[224]</td></tr><tr><td>Que m 1</td><td></td><td>0.08–1000 ng/mL</td><td></td><td></td></tr><tr><td>Aln g 1</td><td></td><td>0.25–100 ng/mL</td><td></td><td></td></tr><tr><td>Car b 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Ost c 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Cor a 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Fag s 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Cas s 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Que a 1</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Bet v 1-derived peptides</td><td></td><td>E. coli; fused to the hepatitis B surface protein, PreS</td><td>0.00001–0.1 µg/ml; 0.005–50 pmol/L</td><td>Highly reduced allergenic activity</td><td>[228, 229]</td></tr><tr><td>rBet v 1 trimer</td><td>E. coli</td><td>0.05 pM–0.5 nM</td><td>10-fold reduced allergenic activity</td><td>[230]</td></tr><tr><td rowspan="2">Cypress pollen</td><td>BP14</td><td>From cypress pollen</td><td>5–5000 ng/mL</td><td>BP14, the only pollen allergen member of the GRP protein, is positive in BAT of patients with pollen-food syndrome (cypress/peach)</td><td>[236]</td></tr><tr><td>Pru p 7 and Snakin-1</td><td>Extract of peaches or Pichia pastoris</td><td>0.25 pg/mL–2.5 µg/mL; 1–1000 ng/mL</td><td>BP14 is cross-reactive with Pru p 7 and Snakin-1; Pru p 7 sensitisation is a predominant cause of severe, cypress pollen-associated peach allergy; Pru p 7 is very potent in BAT</td><td>[236, 237]</td></tr><tr><td colspan="6"><strong>Grass and weed pollen allergens</strong></td></tr><tr><td rowspan="5">Grass pollen</td><td>Phl p 1</td><td rowspan="5">E. coli</td><td>10<sup>-7</sup>–10 µg/mL; 10<sup>-5</sup>–10 µg/mL; 1 µg/ml</td><td rowspan="5">High allergenicity and positive basophil activation in a great majority of grass-pollen sensitised subjects; inhibition with SCIT sera</td><td>[73, 226] [243]</td></tr><tr><td>Phl p 5</td><td></td><td>[227]</td></tr><tr><td>Phl p 2</td><td></td><td></td></tr><tr><td>rPhl p 1 and 5 mix</td><td>0.001–10 µg/ml</td><td>[238]</td></tr><tr><td>rPhl p 1 and 5 mix</td><td></td><td></td></tr></tbody></table>

need for fresh blood, passive sensitization of mast cells lines or basophils from nonallergic donors in place of autologous basophils, i.e., patients' own basophils, can be used.²⁷⁰ An alternative approach is to activate, label, lyse, and fix basophils

at the clinical site, and to analyse them in a centralized flow cytometry service.²⁷¹

2. Nonresponders—10-15% of subjects have nonreleaser basophils (i.e., basophils that do not respond to allergen or the IgE-mediated
<table><caption>TABLE 7 Basophil activation test to allergen components in respiratory allergy</caption><thead><tr><th>Allergen</th><th>Molecular allergen</th><th>Source</th><th>Concentrations</th><th>Methodological and/or clinical relevance</th><th>Reference</th></tr></thead><tbody><tr><td colspan="6"><strong>Tree pollen</strong></td></tr><tr><td rowspan="10">PR-10-like allergens</td><td>Bet v 1</td><td>E. coli</td><td>10<sup>-7</sup>–10 µg/mL; 1 ng/mL; 1–100 ng/mL; 0.31–20 ng/mL; 0.25–100 ng/mL; 0.002–1000 ng/mL; 10<sup>-5</sup>–10 µg/rnl; 1 µg/mL; 0.1 and 0.3 µg/ml; 0.00001–0.1 µg/ml; 0.005–50 pmol/L; 0.05 pM–0.5 nM</td><td>Evaluation of allergenicity of birch pollen sensitisation</td><td>[234,235,226, 227,238,228, 229,230]</td></tr><tr><td>Mal d 1</td><td></td><td>1 µg/mL; 0.25–100 ng/mL; 0.1 and 0.3 µg/ml</td><td>Identification of cross-reactive non-sensitizers or partial cross-reactive sensitizers; limited Bet v 1 SLIT bioactivity for Mal d 1</td><td>[222, 225, 232]</td></tr><tr><td>Api g 1</td><td></td><td>10 µg/ml</td><td>A major allergen from Mongolian oak pollen.</td><td>[223]</td></tr><tr><td>Dau c 1</td><td></td><td>1 µg/mL</td><td>Limited Bet v 1 SLIT bioactivity for cross-blocking of PR-10-like allergens of Fagales pollen</td><td>[224]</td></tr><tr><td>Que m 1</td><td></td><td>0.08–1000 ng/mL</td><td></td><td></td></tr><tr><td>Aln g 1</td><td></td><td>0.25–100 ng/mL</td><td></td><td></td></tr><tr><td>Car b 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Ost c 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Cor a 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Fag s 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Cas s 1</td><td></td><td></td><td></td><td></td></tr><tr><td>Que a 1</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Bet v 1-derived peptides</td><td></td><td>E. coli; fused to the hepatitis B surface protein, PreS</td><td>0.00001–0.1 µg/ml; 0.005–50 pmol/L</td><td>Highly reduced allergenic activity</td><td>[228, 229]</td></tr><tr><td>rBet v 1 trimer</td><td>E. coli</td><td>0.05 pM–0.5 nM</td><td>10-fold reduced allergenic activity</td><td>[230]</td></tr><tr><td rowspan="2">Cypress pollen</td><td>BP14</td><td>From cypress pollen</td><td>5–5000 ng/mL</td><td>BP14, the only pollen allergen member of the GRP protein, is positive in BAT of patients with pollen-food syndrome (cypress/peach)</td><td>[236]</td></tr><tr><td>Pru p 7 and Snakin-1</td><td>Extract of peaches or Pichia pastoris</td><td>0.25 pg/mL–2.5 µg/mL; 1–1000 ng/mL</td><td>BP14 is cross-reactive with Pru p 7 and Snakin-1; Pru p 7 sensitisation is a predominant cause of severe, cypress pollen-associated peach allergy; Pru p 7 is very potent in BAT</td><td>[236, 237]</td></tr><tr><td colspan="6"><strong>Grass and weed pollen allergens</strong></td></tr><tr><td rowspan="5">Grass pollen</td><td>Phl p 1</td><td rowspan="5">E. coli</td><td>10<sup>-7</sup>–10 µg/mL; 10<sup>-5</sup>–10 µg/mL; 1 µg/ml</td><td rowspan="5">High allergenicity and positive basophil activation in a great majority of grass-pollen sensitised subjects; inhibition with SCIT sera</td><td rowspan="5">[73, 226] [243] [227]</td></tr><tr><td>Phl p 5</td><td></td></tr><tr><td>Phl p 2</td><td></td></tr><tr><td>rPhl p 1 and 5 mix</td><td>0.001–10 µg/ml</td></tr><tr><td>rPhl p 1 and 5 mix</td><td></td></tr></tbody></table>

need for fresh blood, passive sensitization of mast cells lines or basophils from nonallergic donors in place of autologous basophils, i.e., patients' own basophils, can be used.²⁷⁰ An alternative approach is to activate, label, lyse, and fix basophils

at the clinical site, and to analyse them in a centralized flow cytometry service.²⁷¹

2. Nonresponders—10-15% of subjects have nonreleaser basophils (i.e., basophils that do not respond to allergen or the IgE-mediated
<table><caption>TABLE 7 (Continued)</caption><tbody><tr><td></td><td>rPhl p 1, 2, 5 and 6 mix</td><td></td><td>1, 5, 25 and 125 pg/ml</td><td>Monitoring of immunotherapy</td><td>[93]</td></tr><tr><td></td><td>Phl p 1-derived peptides</td><td>E. coli; hepatitis B surface</td><td>0.001-1 µg/mL</td><td>Highly reduced allergenic activity</td><td>[239]<br>[93] [239]</td></tr><tr><td></td><td>Phl p 2-derived peptides</td><td>protein, PreS</td><td>0.001-1 µg/mL; 0.05 pM - 20 nM</td><td></td><td>[239] [241]</td></tr><tr><td></td><td>Phl p 5-derived peptides</td><td></td><td>0.001-1 µg/mL; 0.005 pM - 5 nM</td><td></td><td>[239]</td></tr><tr><td></td><td>Phl p 6-derived peptides</td><td></td><td>0.001-1 µg/mL</td><td></td><td>[238]</td></tr><tr><td></td><td>rPhl p 1, 2, 5 and 6 - derived peptides</td><td></td><td>0.001-10 µg/ml</td><td></td><td></td></tr><tr><td>Weed pollen</td><td>Par j 2</td><td>E. coli</td><td>0.01-10 µg/mL</td><td>Positive response in BAT</td><td>[244]</td></tr><tr><td></td><td>Par j 4</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="10">House dust mites</td><td>Der p 1</td><td>E. coli; natural</td><td>0.1-100 ng/ml; 0.04-400 nmol/L;<br>0.00004-10 µg/mL; 0.1-1 µg/ml</td><td rowspan="10">Evaluation of allergenic activity and molecular characteristics of house dust mites allergens</td><td>[245] [250]<br>[252] [253]</td></tr><tr><td>Der p 2</td><td></td><td>0.1-100 ng/ml; 0.066-660 nM; 1.0 µg/ml; 0.0001-10 µg/ml; 0.32-5000 ng/ml</td><td>[246] [252]<br>[248] [226]<br>[249]</td></tr><tr><td>Der p 5</td><td></td><td>0.1-100 ng/ml</td><td>[252]</td></tr><tr><td>Der p 7</td><td></td><td>0.1-100 ng/ml</td><td>[252]</td></tr><tr><td>Der p 18</td><td></td><td>0.0001-10 µg/ml</td><td>[246]</td></tr><tr><td>Der p 21</td><td></td><td>0.1-100 ng/ml</td><td>[252]</td></tr><tr><td>Der p 23</td><td></td><td>0.1-100 ng/ml; 0.012-1200 nM;<br>0.00004-10 µg/mL</td><td>[245][251][205]<br>[247]</td></tr><tr><td>Der f 24</td><td></td><td>1.0 µg/ml</td><td>[252]</td></tr><tr><td>Der p 1, 2, 5, 7, 21, 23 mix</td><td>E. coli; natural; hepatitis B surface protein, PreS</td><td>0.6-600 ng/ml<br>0.04-400 nmol/L</td><td>Highly reduced allergenic activity</td><td>[248]<br>[249] [250]</td></tr><tr><td>Der p 1-derived peptides</td><td></td><td>0.066-660 nM; 0.04-400 nmol/L;<br>0.32 - 5000 ng/ml</td><td></td><td>[248]</td></tr><tr><td>Der p 2-derived peptides</td><td></td><td>0.012-1200 nM</td><td>[251]</td></tr><tr><td>Der p 23-derived peptides</td><td></td><td>0.76-760 ng/ml</td><td>[252]</td></tr><tr><td>American Cockroach</td><td>bPer a 5, iPer a 5<br>Per a 9<br>Per a 10</td><td>E. coli, insect cells</td><td>1 ug/ml</td><td>Comparison of sensitised patients and controls</td><td>[256, 255, 1530,257]</td></tr><tr><td colspan="6">Cat, dog, and horse allergens</td></tr><tr><td rowspan="5">Felix domesticus, cat</td><td>Fel d 1</td><td>E. coli</td><td>0.5 ug/ml; 0.01-0.00001 mg/mL</td><td rowspan="5">Fel d 1 BAT is clinically highly sensitive; characterization of novel cat allergens.</td><td>[257] [261] [262]</td></tr><tr><td>Fel d 2</td><td></td><td>0.1 µg/mL</td><td>[258]</td></tr><tr><td>Fel d 7</td><td></td><td>0.001-10000 ng/ml</td><td>[259]</td></tr><tr><td>CAT-NPC7</td><td></td><td>10 µg/mL</td><td>[260]</td></tr><tr><td>rFel d 1-derived peptides</td><td>E. coli; hepatitis B surface protein, PreS</td><td>0.01-0.00001 mg/mL<br>0.001-10000 ng/ml</td><td>Highly reduced allergenic activity</td><td>[261, 262]<br>[260]</td></tr><tr><td rowspan="3">Canis familiaris, dog</td><td>Can f 1</td><td>E. coli; not specified</td><td>1 ug/ml</td><td rowspan="3">High clinical relevance; Can f 6 is a major allergen in dog-allergic Chinese children</td><td>[264]</td></tr><tr><td>Can f 6</td><td></td><td>0.05 - 500 ng/ml</td><td>[156]</td></tr><tr><td>Can f 1 - Can f 6 mix</td><td></td><td></td><td></td></tr></tbody></table>

positive control but only to the non-IgE-mediated positive control) and their results for BAT cannot be interpreted.

3. Manual assay requires significant hands-on time—Automated says are desirable and could circumvent this issue.

4. Subjectivity of data analyses—Interpretation of flow cytometry basophil activation is subjective. Attempts to standardize and automate it using artificial intelligence are underway. There are few

allergens for which there is a data-driven clinical threshold for a positive BAT.¹⁷⁶

As flow cytometers become more ubiquitous and basophil testing by flow cytometry becomes more standardized, basophil testing by flow cytometry will become a more accepted method of supporting a diagnosis and of assessing the allergic status of a patient.
<table><caption>TABLE 7 (Continued)</caption><tbody><tr><td></td><td>rPhl p 1, 2, 5 and 6 mix</td><td></td><td>1, 5, 25 and 125 pg/ml</td><td>Monitoring of immunotherapy</td><td>[93]</td></tr><tr><td></td><td>Phl p 1-derived peptides</td><td>E. coli; hepatitis B surface</td><td>0.001–1 µg/mL</td><td>Highly reduced allergenic activity</td><td>[239]<br>[93] [239]</td></tr><tr><td></td><td>Phl p 2-derived peptides</td><td>protein, PreS</td><td>0.001–1 µg/mL; 0.05 pM – 20 nM</td><td></td><td>[239] [241]</td></tr><tr><td></td><td>Phl p 5-derived peptides</td><td></td><td>0.001–1 µg/mL; 0.005 pM – 5 nM</td><td></td><td>[239]</td></tr><tr><td></td><td>Phl p 6-derived peptides</td><td></td><td>0.001–1 µg/mL</td><td></td><td>[238]</td></tr><tr><td></td><td>rPhl p 1, 2, 5 and 6 – derived peptides</td><td></td><td>0.001–10 µg/ml</td><td></td><td></td></tr><tr><td>Weed pollen</td><td>Par j 2</td><td>E. coli</td><td>0.01–10 µg/mL</td><td>Positive response in BAT</td><td>[244]</td></tr><tr><td></td><td>Par j 4</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="10">House dust mites</td><td>Der p 1</td><td>E. coli; natural</td><td>0.1–100 ng/ml; 0.04–400 nmol/L;<br>0.00004–10 µg/mL; 0.1–1 µg/ml</td><td rowspan="10">Evaluation of allergenic activity and molecular characteristics of house dust mites allergens</td><td>[245] [250]<br>[252] [253]</td></tr><tr><td>Der p 2</td><td></td><td>0.1–100 ng/ml; 0.066–660 nM; 1.0 µg/ml; 0.0001–10 µg/ml; 0.32–5000 ng/ml</td><td>[246] [252]<br>[248] [226]<br>[249]</td></tr><tr><td>Der p 5</td><td></td><td>0.1–100 ng/ml</td><td>[252]</td></tr><tr><td>Der p 7</td><td></td><td>0.1–100 ng/ml</td><td>[252]</td></tr><tr><td>Der p 18</td><td></td><td>0.0001–10 µg/ml</td><td>[246]</td></tr><tr><td>Der p 21</td><td></td><td>0.1–100 ng/ml</td><td>[252]</td></tr><tr><td>Der p 23</td><td></td><td>0.1–100 ng/ml; 0.012–1200 nM;<br>0.00004–10 µg/mL</td><td>[245][251][205]<br>[247]</td></tr><tr><td>Der f 24</td><td></td><td>1.0 µg/ml</td><td>[252]</td></tr><tr><td>Der p 1, 2, 5, 7, 21, 23 mix</td><td>E. coli; natural; hepatitis B surface protein, PreS</td><td>0.6–600 ng/ml<br>0.04–400 nmol/L</td><td>Highly reduced allergenic activity</td><td>[248]<br>[249] [250]</td></tr><tr><td>Der p 1-derived peptides</td><td></td><td>0.066–660 nM; 0.04–400 nmol/L;<br>0.32–5000 ng/ml</td><td></td><td>[248]</td></tr><tr><td>Der p 2-derived peptides</td><td></td><td>0.012–1200 nM</td><td>[251]</td></tr><tr><td>Der p 23-derived peptides</td><td></td><td>0.76–760 ng/ml</td><td>[252]</td></tr><tr><td>American Cockroach</td><td>bPer a 5, iPer a 5<br>Per a 9<br>Per a 10</td><td>E. coli, insect cells</td><td>1 ug/ml</td><td>Comparison of sensitised patients and controls</td><td>[256, 255, 1530,257]</td></tr><tr><td colspan="6" style="text-align: left; font-weight: bold;">Cat, dog, and horse allergens</td></tr><tr><td rowspan="5">Felix domesticus, cat</td><td>Fel d 1</td><td>E. coli</td><td>0.5 ug/ml; 0.01–0.00001 mg/mL</td><td rowspan="5">Fel d 1 BAT is clinically highly sensitive; characterization of novel cat allergens.</td><td>[257] [261] [262]</td></tr><tr><td>Fel d 2</td><td></td><td>0.1 µg/mL</td><td>[258]</td></tr><tr><td>Fel d 7</td><td></td><td>0.001–10000 ng/ml</td><td>[259]</td></tr><tr><td>CAT-NPC7</td><td></td><td>10 µg/mL</td><td>[260]</td></tr><tr><td>rFel d 1-derived peptides</td><td>E. coli; hepatitis B surface protein, PreS</td><td>0.01–0.00001 mg/mL<br>0.001–10000 ng/ml</td><td>Highly reduced allergenic activity</td><td>[261, 262]<br>[260]</td></tr><tr><td rowspan="3">Canis familiaris, dog</td><td>Can f 1</td><td>E. coli; not specified</td><td>1 ug/ml</td><td rowspan="3">High clinical relevance; Can f 6 is a major allergen in dog-allergic Chinese children</td><td>[264]</td></tr><tr><td>Can f 6</td><td></td><td>0.05–500 ng/ml</td><td>[156]</td></tr><tr><td>Can f 1 - Can f 6 mix</td><td></td><td></td><td></td></tr></tbody></table>

positive control but only to the non-IgE-mediated positive control) and their results for BAT cannot be interpreted.

3. Manual assay requires significant hands-on time—Automated says are desirable and could circumvent this issue.

4. Subjectivity of data analyses—Interpretation of flow cytometry basophil activation is subjective. Attempts to standardize and automate it using artificial intelligence are underway. There are few

allergens for which there is a data-driven clinical threshold for a positive BAT.¹⁷⁶

As flow cytometers become more ubiquitous and basophil testing by flow cytometry becomes more standardized, basophil testing by flow cytometry will become a more accepted method of supporting a diagnosis and of assessing the allergic status of a patient.
6 – Future perspectives

Basophil testing is increasingly used as an ex vivo correlate to docu-
ment the clinical relevance of novel allergens and to characterize the
allergenicity of novel recombinant-based immunotherapy vaccines
and carriers; however, there is a need to harmonize requirements
for this documentation. Basophil testing with recombinant allergens

(either in combination with allergen extracts or alone) can also be
used to support clinical diagnosis. Procedures and methodologies
been to be defined and harmonized for BAT to be used more widely.
This is an exciting field, as recombinant allergens used for basophil
testing need less stringent control than allergens used for skin prick
testing and therapy.
6 – Future perspectives

Basophil testing is increasingly used as an ex vivo correlate to docu-
ment the clinical relevance of novel allergens and to characterize the
allergenicity of novel recombinant-based immunotherapy vaccines
and carriers; however, there is a need to harmonize requirements
for this documentation. Basophil testing with recombinant allergens

(either in combination with allergen extracts or alone) can also be
used to support clinical diagnosis. Procedures and methodologies
been to be defined and harmonized for BAT to be used more widely.
This is an exciting field, as recombinant allergens used for basophil
testing need less stringent control than allergens used for skin prick
testing and therapy.
# A06 - In vivo testing

Alexandra F. Santos, Peter Korosec, Leticia de las Vecillas, Nikolaos Douladiris, Barbara Ballmer-Weber

## Highlights

* The clinical history is the cornerstone of allergy diagnosis and should be combined with the results of allergy tests to confirm or exclude the diagnosis.

* Provocation tests are especially helpful when discrepancies exist between the clinical history and other *in vivo* or *in vitro* test results, to phenotype patients and to monitor specific immunotherapy efficacy.

* Only allergen extracts or fresh produce can be used for *in vivo* testing and up to now no molecular allergens based *in vivo* tests are available.

* The use of recombinant allergens in provocations test seems to improve their accuracy; however, it is an unmet need, which requires further investigations.

## 1 - Introduction

The cornerstone for an accurate diagnosis and a targeted appropriate treatment of allergic disease is the clinical history.²⁷² Subsequently, allergy testing should be performed to document the presence of allergen-specific IgE. This can be achieved using skin prick testing and/or specific IgE testing. The combination of a history suggestive of allergy, namely of typical IgE-mediated symptoms following specific allergen exposure, with evidence of IgE sensitization to the allergen allows to confirm the diagnosis of allergy.¹⁶⁹ Conversely, a history suggestive of tolerance or the absence of allergic reactivity to the allergen source combined with undetectable allergen-specific IgE allows excluding the diagnosis of allergy. Unclear history and/or discrepancy between history and IgE sensitization to the suspected allergen requires assessment with a provocation test.⁷²

IgE-mediated allergic reactions can be caused by a wide variety of allergens. Although IgE sensitization to an allergen does not equate to clinical reactivity or allergic disease, IgE based tests can be used to identify the culprit allergen. Persistence, severity and coexistence of allergic reactions are often associated with multisensitization. Concurrent irritant triggers, certain infections, and nonallergic disorders often have a similar presentation to allergy and allergy can drive the underlying inflammatory pathology complicating another disorder. Differential diagnosis is, therefore, an important part of the diagnostic process.

This chapter will cover the importance of and key questions to ask during an allergy-focused clinical history and the performance and diagnostic utility of two forms of *in vivo* testing used to support the diagnosis of IgE-mediated allergy: skin prick testing and provocation tests. Separate chapters will cover *in vitro* testing extensively.

## 2 - Clinical history

A detailed clinical history will provide information on the following important aspects:

A The type of signs and symptoms suggesting the possible underlying immunological mechanism;

B The likelihood of allergy being the main driver of the signs and symptoms;

C The allergen or the allergens as the triggers of the signs and symptoms and drivers of pathology;

D Identification of possible co-factors or facilitators;

E Assessment of the severity of disease and prognosis.

As such, the diagnosis of allergic disease begins as first-line approach, with thorough clinical history and physical examination of the patient. Textbox 9 lists the key questions to ask as part of the clinical history.²⁷²

## Allergy tests

Specific IgE sensitization can be determined using *in vivo* skin prick tests and /or *in vitro* blood tests, as second line. If there is a mismatch between the history and these primary diagnostic tests, third line tests, such as cellular tests, like the basophil activation test, can be used to assess ex vivo the effector cell response to allergen. If, despite the allergy tests, the diagnosis is unclear, provocation tests (e.g., nasal allergen, conjunctival or bronchial challenge, placebo-controlled, or open food challenge) may be needed to clarify the diagnosis.⁷²

An increasing proportion of patients have unclear clinical history and inconclusive allergen extract tests.²⁷³,²⁷⁴ In these circumstances, molecular based diagnostics can be considered particularly in the case of patients with complex symptomatology that mainly originates from:

A Polysensitization to multiple inhalant allergens with overlapping exposure periods to natural and work environment, with graded symptoms. Molecular allergens can be used to efficiently identify genuine sensitization to eliciting allergens, reveal co-sensitization and/or cross-sensitization of closely related or widely different allergens sources and optimize the selection of allergen specific immunotherapy when⁹⁷ needed.

B sensitization to one or more food allergens with graded severity of symptoms that appears on ingestion of graded quantity and/or procession of food. Molecular allergens can be used to characterize the persistence, the severity and assess the future risk of the reaction in relation to stability and any procession of the offending (food) allergen.⁴⁵

C Co-sensitization to inhalant and food allergens present with symptoms of unknown aetiology. Molecular allergens can be used to optimize the decision process of provocation tests,
# A06 - In vivo testing

Alexandra F. Santos, Peter Korosec, Leticia de las Vecillas, Nikolaos Douladiris, Barbara Ballmer-Weber

## Highlights

* The clinical history is the cornerstone of allergy diagnosis and should be combined with the results of allergy tests to confirm or exclude the diagnosis.

* Provocation tests are especially helpful when discrepancies exist between the clinical history and other *in vivo* or *in vitro* test results, to phenotype patients and to monitor specific immunotherapy efficacy.

* Only allergen extracts or fresh produce can be used for *in vivo* testing and up to now no molecular allergens based *in vivo* tests are available.

* The use of recombinant allergens in provocations test seems to improve their accuracy; however, it is an unmet need, which requires further investigations.

## 1 - Introduction

The cornerstone for an accurate diagnosis and a targeted appropriate treatment of allergic disease is the clinical history.²⁷² Subsequently, allergy testing should be performed to document the presence of allergen-specific IgE. This can be achieved using skin prick testing and/or specific IgE testing. The combination of a history suggestive of allergy, namely of typical IgE-mediated symptoms following specific allergen exposure, with evidence of IgE sensitization to the allergen allows to confirm the diagnosis of allergy.¹⁶⁹ Conversely, a history suggestive of tolerance or the absence of allergic reactivity to the allergen source combined with undetectable allergen-specific IgE allows excluding the diagnosis of allergy. Unclear history and/or discrepancy between history and IgE sensitization to the suspected allergen requires assessment with a provocation test.⁷²

IgE-mediated allergic reactions can be caused by a wide variety of allergens. Although IgE sensitization to an allergen does not equate to clinical reactivity or allergic disease, IgE based tests can be used to identify the culprit allergen. Persistence, severity and coexistence of allergic reactions are often associated with multisensitization. Concurrent irritant triggers, certain infections, and nonallergic disorders often have a similar presentation to allergy and allergy can drive the underlying inflammatory pathology complicating another disorder. Differential diagnosis is, therefore, an important part of the diagnostic process.

This chapter will cover the importance of and key questions to ask during an allergy-focused clinical history and the performance and diagnostic utility of two forms of *in vivo* testing used to support the diagnosis of IgE-mediated allergy: skin prick testing and provocation tests. Separate chapters will cover *in vitro* testing extensively.

## 2 - Clinical history

A detailed clinical history will provide information on the following important aspects:

A The type of signs and symptoms suggesting the possible underlying immunological mechanism;

B The likelihood of allergy being the main driver of the signs and symptoms;

C The allergen or the allergens as the triggers of the signs and symptoms and drivers of pathology;

D Identification of possible co-factors or facilitators;

E Assessment of the severity of disease and prognosis.

As such, the diagnosis of allergic disease begins as first-line approach, with thorough clinical history and physical examination of the patient. Textbox 9 lists the key questions to ask as part of the clinical history.²⁷²

## Allergy tests

Specific IgE sensitization can be determined using *in vivo* skin prick tests and /or *in vitro* blood tests, as second line. If there is a mismatch between the history and these primary diagnostic tests, third line tests, such as cellular tests, like the basophil activation test, can be used to assess ex vivo the effector cell response to allergen. If, despite the allergy tests, the diagnosis is unclear, provocation tests (e.g., nasal allergen, conjunctival or bronchial challenge, placebo-controlled, or open food challenge) may be needed to clarify the diagnosis.⁷²

An increasing proportion of patients have unclear clinical history and inconclusive allergen extract tests.²⁷³,²⁷⁴ In these circumstances, molecular based diagnostics can be considered particularly in the case of patients with complex symptomatology that mainly originates from:

A Polysensitization to multiple inhalant allergens with overlapping exposure periods to natural and work environment, with graded symptoms. Molecular allergens can be used to efficiently identify genuine sensitization to eliciting allergens, reveal co-sensitization and/or cross-sensitization of closely related or widely different allergens sources and optimize the selection of allergen specific immunotherapy when⁹⁷ needed.

B sensitization to one or more food allergens with graded severity of symptoms that appears on ingestion of graded quantity and/or procession of food. Molecular allergens can be used to characterize the persistence, the severity and assess the future risk of the reaction in relation to stability and any procession of the offending (food) allergen.⁴⁵

C Co-sensitization to inhalant and food allergens present with symptoms of unknown aetiology. Molecular allergens can be used to optimize the decision process of provocation tests,
### Textbox 9 Aspects to check as part of the clinical history

* Presentation: Which symptoms does the patient present with?

* Timing: How soon after exposure to the allergen do the symptoms develop?

* Likelihood of allergy: To what extent does allergen exposure contribute to the symptoms? Does the patient present with symptoms or conditions other than allergic disease/s?

* Potential mechanism: Does the patient present with characteristic symptoms of allergic disease/s? If so, what is the likely underlying mechanism (IgE, non-IgE-mediated)?

* Consistency: Are the symptoms consistent, i.e., do they develop every time the patient is exposed to the allergen?

* Grading: Are the symptoms of a grading severity, i.e., do they develop graded on exposure to graded quantity of the allergen?

* Seasonality: Do the patient's symptoms worse during any particular time/season of the year? Are they seasonal or perennial?

* Geography: Do the patient's symptoms correlate with a certain place or geographical area?

* Related allergens: Do the patient's symptoms get worse when in contact with known closely related or widely different allergen sources?

* Other triggers: Do other substances, nonallergens or highly suggestive yet undefined allergens, provoke and/or worse these symptoms or add new symptoms and of increased severity?

* Potential co-factors: Do another disease, infection, drug intake (i.e., NSAID) or physical activity provoke and/or worsen these symptoms or increase their severity?

* Family context: Does anybody in the patient's family present the same symptoms or any symptoms characteristic of allergic disease/s currently or in the past?

avoiding costly, time consuming, potentially life-threatening reactions and improve allergen avoidance recommendations.⁴⁵

D Polysensitization to insects' venom allergens present with unclear insect sting history. Molecular allergens can be used to efficiently identify genuine sensitization to eliciting allergens, reveal co-sensitization and/or cross-sensitization to different venom allergens, improve the decision process of insect avoidance recommendations and optimize the selection of venom specific immunotherapy when needed.²⁰⁹,¹⁶²⁹ Molecular Allergology based diagnostics can thus be used as third line investigation in patients with inconclusive history and allergen extract based tests, before considering referral for provocation tests⁴⁵ or initiation of a specific immunotherapy. Currently, molecular allergy tests are only available for *in vitro* testing and not for skin or provocation tests and thus will be covered extensively in other chapters.

### 4 - Skin prick test

Skin prick test (SPT) is a widely available procedure that is usually performed in the physician's office by a qualified healthcare professional.²⁷⁵ SPT consists on the application of allergen extract solution on intact skin followed by puncture using a standardized 1 mm lancet to facilitate the penetration of the allergen through the epidermis into the dermis (Figure 15). If the individual is sensitized to the allergen being tested, this will elicit and wheal and flare reaction at the site. Prior to testing, the skin should be labelled with the different extracts being tested as well as the positive and negative controls, separated by about 1 cm distance to avoid cross-contamination. The positive control is usually a 1 mg/ml histamine solution and the negative control a 0.9% NaCl saline solution. Each control or allergen requires a new lancet and after pricking it is important to clean residual allergen extract/control to avoid contamination. Fifteen minutes later, the results are ready to be read using a ruler and measuring the wheal size diameter. Usually, the wheal diameter is the average of the widest diameter of the wheal and the widest diameter perpendicular to that. The positive control should be at least 3 mm, which is the conventional cut-off for a positive SPT, and the negative control should ideally be 0 mm.⁶⁸

Advantages of SPT compared with serum specific IgE testing are the fact that SPT is inexpensive, provides immediate results, which are evident to patients in clinic, and allows testing with fresh material in addition to allergen extracts. Limitations of SPT are the need for clear skin and to stop anti-histamines a few days prior to testing, being performed by trained skilled healthcare professionals in facilities with equipment and medication required to treat allergic reactions. Systemic allergic reactions of varying degree of severity may result from SPT but are extremely rare.¹⁶⁹ Apart from allergen extracts, fresh produce can also be used for SPT—the so-called “prick-to-prick test” or “modified SPT.” This is particularly important for fruits and vegetables, whose allergens can be labile and get degraded during the extract preparation and therefore can be poorly represented in commercially available allergen extract solutions.²⁷⁶ Using fresh fruits and vegetables allows for more sensitive SPT and for confirmation of allergy in case of sensitization is consistent with the history of allergic reactions to the same food but can induce on the other hand irritation of the skin and false positive results. Using individual allergen molecules could be advantageous regarding specificity and clinical information, but there are limitations in the use of recombinant allergens for SPT.²⁷⁷ Some house dust mites' molecular allergens such as Der f 27 and Der f 29, have been previously used in SPT for research.²⁷⁸, ¹⁶³² Also, Che a 1, Che a 2 and Che a 3, from Chenopodium album and Anis s 1 from Anisakis simplex.²⁷⁹,¹⁶³⁴ Solutions of allergen extracts enriched for certain allergens, such as
### Textbox 9 Aspects to check as part of the clinical history

* Presentation: Which symptoms does the patient present with?

* Timing: How soon after exposure to the allergen do the symptoms develop?

* Likelihood of allergy: To what extent does allergen exposure contribute to the symptoms? Does the patient present with symptoms or conditions other than allergic disease/s?

* Potential mechanism: Does the patient present with characteristic symptoms of allergic disease/s? If so, what is the likely underlying mechanism (IgE, non-IgE-mediated)?

* Consistency: Are the symptoms consistent, i.e., do they develop every time the patient is exposed to the allergen?

* Grading: Are the symptoms of a grading severity, i.e., do they develop graded on exposure to graded quantity of the allergen?

* Seasonality: Do the patient's symptoms worse during any particular time/season of the year? Are they seasonal or perennial?

* Geography: Do the patient's symptoms correlate with a certain place or geographical area?

* Related allergens: Do the patient's symptoms get worse when in contact with known closely related or widely different allergen sources?

* Other triggers: Do other substances, nonallergens or highly suggestive yet undefined allergens, provoke and/or worse these symptoms or add new symptoms and of increased severity?

* Potential co-factors: Do another disease, infection, drug intake (i.e., NSAID) or physical activity provoke and/or worsen these symptoms or increase their severity?

* Family context: Does anybody in the patient's family present the same symptoms or any symptoms characteristic of allergic disease/s currently or in the past?

avoiding costly, time consuming, potentially life-threatening reactions and improve allergen avoidance recommendations.⁴⁵

D Polysensitization to insects' venom allergens present with unclear insect sting history. Molecular allergens can be used to efficiently identify genuine sensitization to eliciting allergens, reveal co-sensitization and/or cross-sensitization to different venom allergens, improve the decision process of insect avoidance recommendations and optimize the selection of venom specific immunotherapy when needed.²⁰⁹,¹⁶²⁹ Molecular Allergology based diagnostics can thus be used as third line investigation in patients with inconclusive history and allergen extract based tests, before considering referral for provocation tests⁴⁵ or initiation of a specific immunotherapy. Currently, molecular allergy tests are only available for *in vitro* testing and not for skin or provocation tests and thus will be covered extensively in other chapters.

### 4 - Skin prick test

Skin prick test (SPT) is a widely available procedure that is usually performed in the physician's office by a qualified healthcare professional.²⁷⁵ SPT consists on the application of allergen extract solution on intact skin followed by puncture using a standardized 1 mm lancet to facilitate the penetration of the allergen through the epidermis into the dermis (Figure 15). If the individual is sensitized to the allergen being tested, this will elicit and wheal and flare reaction at the site. Prior to testing, the skin should be labelled with the different extracts being tested as well as the positive and negative controls, separated by about 1 cm distance to avoid cross-contamination. The positive control is usually a 1 mg/ml histamine solution and the negative control a 0.9% NaCl saline solution. Each control or allergen requires a new lancet and after pricking it is important to clean residual allergen extract/control to avoid contamination. Fifteen minutes later, the results are ready to be read using a ruler and measuring the wheal size diameter. Usually, the wheal diameter is the average of the widest diameter of the wheal and the widest diameter perpendicular to that. The positive control should be at least 3 mm, which is the conventional cut-off for a positive SPT, and the negative control should ideally be 0 mm.⁶⁸

Advantages of SPT compared with serum specific IgE testing are the fact that SPT is inexpensive, provides immediate results, which are evident to patients in clinic, and allows testing with fresh material in addition to allergen extracts. Limitations of SPT are the need for clear skin and to stop anti-histamines a few days prior to testing, being performed by trained skilled healthcare professionals in facilities with equipment and medication required to treat allergic reactions. Systemic allergic reactions of varying degree of severity may result from SPT but are extremely rare.¹⁶⁹ Apart from allergen extracts, fresh produce can also be used for SPT—the so-called “prick-to-prick test” or “modified SPT.” This is particularly important for fruits and vegetables, whose allergens can be labile and get degraded during the extract preparation and therefore can be poorly represented in commercially available allergen extract solutions.²⁷⁶ Using fresh fruits and vegetables allows for more sensitive SPT and for confirmation of allergy in case of sensitization is consistent with the history of allergic reactions to the same food but can induce on the other hand irritation of the skin and false positive results. Using individual allergen molecules could be advantageous regarding specificity and clinical information, but there are limitations in the use of recombinant allergens for SPT.²⁷⁷ Some house dust mites' molecular allergens such as Der f 27 and Der f 29, have been previously used in SPT for research.²⁷⁸, ¹⁶³² Also, Che a 1, Che a 2 and Che a 3, from Chenopodium album and Anis s 1 from Anisakis simplex.²⁷⁹,¹⁶³⁴ Solutions of allergen extracts enriched for certain allergens, such as
<figure><img src="image_27.png" /><figcaption>FIGURE 15 Skin prick test procedure</figcaption></figure>

palm profilin and peach LTP, can be available commercially and may be
helpful in reaching a precise diagnosis in multisensitized patients.²⁸⁰
Molecular allergens from fish have been also tested in a pediatric
population. Wild-type Cyp c 1 (wtCyp c 1) has been shown to be a
useful standardized allergen for skin testing to diagnose patients with
fish allergy.²⁸¹ Recently, a recombinant hybrid molecule including the
major grass pollen allergens detected by a positive result in SPT has
been shown to be a useful and safe tool for in vivo diagnosis of gen-
une sensitization in children, reducing the test to a single extract.²⁸²
Apart from identifying the allergen molecules that are more clinically
relevant, the use of molecular allergens for skin testing may also be
useful to risk-stratify patients (e.g., sensitization to Ara h 2 or Cor a 9
and 14 posing a higher risk of reaction during challenges) and to de-
tect less allergenic molecules to include in immunotherapy vaccines,
after confirming their capacity to induce allergen-specific blocking
IgG antibodies.²⁷⁷,²⁸¹,²⁸² Nevertheless, skin testing with recombinant
or native purified allergens has not entered clinical routine.

Allergen provocation tests

Provocation tests are often useful to confirm the presence of al-
lergy through the exposure of the patient to a suspected allergen
in a medically supervised environment,⁷² particularly when other
*in vivo* or *in vitro* tests cannot give a conclusive result and to dif-
ferentiate between allergic sensitization not leading to allergic
symptoms (clinically not relevant sensitization) and clinically rel-
evant sensitization leading to reactivity.²⁸³ Provocation tests allow
to identify of phenotypes, culprit relevant allergens and to assess
the evolution/improvement of a patient who underwent specific
immunotherapy in food and airborne allergies including asthma
and rhinoconjunctivitis. These tests also have an important role
in research to identify key allergens to use for allergen-specific
immunotherapy. Before exposing a patient to a possible harmful
substance, a risk stratification must be assessed before the prov-
ocation to ensure the safety and the effectiveness of the proce-
dure.²⁸⁴ Guidelines have recently been published about their use in
different allergic diseases.⁷²,²⁸³,¹⁶³⁰

NASAL ALLERGEN PROVOCATION

Nasal provocation can be potentially useful to identify different
chronic rhinitis phenotypes including the diagnosis and manage-
ment of local allergic rhinitis, which is characterized by the absence

of serum specific sIgE or positive SPT to aeroallergens despite
ongoing symptoms.¹⁶³³ Individual and standardized lyophilized
extracts of Dermatophagoides pteronyssinus, Alternaria alter-
nata, Olea europea and a mix of grass pollen have been used to
assess the nasal reactivity to identify clinically relevant allergens
to tailor a specific treatment such as allergen immunotherapy.²⁸⁵
Recombinant timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl
p 5) and recombinant birch pollen allergens (rBet v 1 and rBet v 2)
used in skin test and nasal provocation have shown a significant
correlation with clinical symptoms, better than serum specific IgE
determination.²⁸⁶ For olive pollen allergy, the challenge with the
major allergen nOle e 1 increases the specificity of the procedure,
compared with a nasal challenge with olive pollen extract.²⁸⁷ Nasal
provocations comparing natural and recombinant Bos d 2 allergen
have also been applied to determine which allergen is more appro-
priate for allergen immunotherapy, based on the reactivity of the
patients.²⁸⁸ Single molecules, however, are not available for routine
nasal provocation testing.

CONJUNCTIVAL ALLERGEN PROVOCATIONS

For conjunctival provocations, the use of different pollen extracts
such as ragweed and grass pollen or recombinant molecular aller-
gens such as rBet v1 in an environmental exposure chamber has
been shown to reproduce a more natural exposure than the tradi-
tional conjunctival allergen challenge in allergic patients.²⁸⁹,²⁹⁰,¹⁶³⁵
Provocation with component Bet v1 and birch extract found
comparable allergenic reactivity of recombinant and natural
products.²⁹¹

BRONCHIAL ALLERGEN PROVOCATION

The first allergen used in bronchial provocation was grass pollen
in 1873.²⁹² Since then, other allergens such as house dust mites
have been used to identify relevant allergens responsible for pa-
tients' symptoms.²⁹³ Although it is not a test to diagnose asthma
itself, bronchial provocation is important in polysensitized patients
to identify the culprit allergen/s, allowing clinicians to use tailored
management and patients to implement environment control ex-
posure or even to choose a healthier lifestyle. A trial comparing
bronchial provocation of asthmatic patients found comparable re-
activity of component Bet v 1 and natural allergen in skin prick test,
nasal and bronchial allergen provocation. However, its utility is lim-
ited by the potential risk of severe asthmatic reactions. Its use in the
diagnosis of occupational allergy has been described with a better
<figure><img src="image_27.png" /><figcaption>FIGURE 15 Skin prick test procedure</figcaption></figure>

palm profilin and peach LTP, can be available commercially and may be
helpful in reaching a precise diagnosis in multisensitized patients.²⁸⁰
Molecular allergens from fish have been also tested in a pediatric
population. Wild-type Cyp c 1 (wtCyp c 1) has been shown to be a
useful standardized allergen for skin testing to diagnose patients with
fish allergy.²⁸¹ Recently, a recombinant hybrid molecule including the
major grass pollen allergens detected by a positive result in SPT has
been shown to be a useful and safe tool for in vivo diagnosis of gen-
une sensitization in children, reducing the test to a single extract.²⁸²
Apart from identifying the allergen molecules that are more clinically
relevant, the use of molecular allergens for skin testing may also be
useful to risk-stratify patients (e.g., sensitization to Ara h 2 or Cor a 9
and 14 posing a higher risk of reaction during challenges) and to de-
tect less allergenic molecules to include in immunotherapy vaccines,
after confirming their capacity to induce allergen-specific blocking
IgG antibodies.²⁷⁷,²⁸¹,²⁸² Nevertheless, skin testing with recombinant
or native purified allergens has not entered clinical routine.

Allergen provocation tests

Provocation tests are often useful to confirm the presence of al-
lergy through the exposure of the patient to a suspected allergen
in a medically supervised environment,⁷² particularly when other
*in vivo* or *in vitro* tests cannot give a conclusive result and to dif-
ferentiate between allergic sensitization not leading to allergic
symptoms (clinically not relevant sensitization) and clinically rel-
evant sensitization leading to reactivity.²⁸³ Provocation tests allow
to identify of phenotypes, culprit relevant allergens and to assess
the evolution/improvement of a patient who underwent specific
immunotherapy in food and airborne allergies including asthma
and rhinoconjunctivitis. These tests also have an important role
in research to identify key allergens to use for allergen-specific
immunotherapy. Before exposing a patient to a possible harmful
substance, a risk stratification must be assessed before the prov-
ocation to ensure the safety and the effectiveness of the proce-
dure.²⁸⁴ Guidelines have recently been published about their use in
different allergic diseases.⁷²,²⁸³,¹⁶³⁰

NASAL ALLERGEN PROVOCATION

Nasal provocation can be potentially useful to identify different
chronic rhinitis phenotypes including the diagnosis and manage-
ment of local allergic rhinitis, which is characterized by the absence

of serum specific sIgE or positive SPT to aeroallergens despite
ongoing symptoms.¹⁶³³ Individual and standardized lyophilized
extracts of Dermatophagoides pteronyssinus, Alternaria alter-
nata, Olea europea and a mix of grass pollen have been used to
assess the nasal reactivity to identify clinically relevant allergens
to tailor a specific treatment such as allergen immunotherapy.²⁸⁵
Recombinant timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl
p 5) and recombinant birch pollen allergens (rBet v 1 and rBet v 2)
used in skin test and nasal provocation have shown a significant
correlation with clinical symptoms, better than serum specific IgE
determination.²⁸⁶ For olive pollen allergy, the challenge with the
major allergen nOle e 1 increases the specificity of the procedure,
compared with a nasal challenge with olive pollen extract.²⁸⁷ Nasal
provocations comparing natural and recombinant Bos d 2 allergen
have also been applied to determine which allergen is more appro-
priate for allergen immunotherapy, based on the reactivity of the
patients.²⁸⁸ Single molecules, however, are not available for routine
nasal provocation testing.

CONJUNCTIVAL ALLERGEN PROVOCATIONS

For conjunctival provocations, the use of different pollen extracts
such as ragweed and grass pollen or recombinant molecular aller-
gens such as rBet v1 in an environmental exposure chamber has
been shown to reproduce a more natural exposure than the tradi-
tional conjunctival allergen challenge in allergic patients.²⁸⁹,²⁹⁰,¹⁶³⁵
Provocation with component Bet v1 and birch extract found
comparable allergenic reactivity of recombinant and natural
products.²⁹¹

BRONCHIAL ALLERGEN PROVOCATION

The first allergen used in bronchial provocation was grass pollen
in 1873.²⁹² Since then, other allergens such as house dust mites
have been used to identify relevant allergens responsible for pa-
tients' symptoms.²⁹³ Although it is not a test to diagnose asthma
itself, bronchial provocation is important in polysensitized patients
to identify the culprit allergen/s, allowing clinicians to use tailored
management and patients to implement environment control ex-
posure or even to choose a healthier lifestyle. A trial comparing
bronchial provocation of asthmatic patients found comparable re-
activity of component Bet v 1 and natural allergen in skin prick test,
nasal and bronchial allergen provocation. However, its utility is lim-
ited by the potential risk of severe asthmatic reactions. Its use in the
diagnosis of occupational allergy has been described with a better
correlation with the hyperreactivity severity than non-specific bronchial challenges.²⁹⁴

## ORAL FOOD CHALLENGE

Oral food challenge is an essential tool in the diagnosis of food allergy. Depending on age, patient characteristics and situation demands it can be performed as an open food challenge or as double-blind placebo-controlled oral food challenge (DBPCFC). It is also relevant to perform risk stratification before starting food allergen-specific immunotherapy, to determine the initial allergen dose based on the threshold of allergen required to trigger a reaction, and to monitor its effectiveness.²⁹⁵ Identifying the molecular

pattern of sensitization of a patient allows to recognize subjects at risk of suffering severe reactions such as IgE sensitization to *Arachis hypogea* 2 (Ara h 2) from peanut.²⁹⁶ However, to improve the safety and reliability of food challenges with allergenic molecules, more clinical evidence is needed.²⁶⁸

Based on previous data proving the usefulness of molecular allergens in *in vitro* and *in vivo* tests to increase the accuracy of the allergy diagnosis, provocation with molecular allergens seems to be more useful than challenges with the whole extracts. However, these molecules are not yet available for routine diagnosis and more research is needed to better standardize these extracts, which differ from those used for *in vitro* testing.²⁹¹
correlation with the hyperreactivity severity than non-specific bronchial challenges.²⁹⁴

## ORAL FOOD CHALLENGE

Oral food challenge is an essential tool in the diagnosis of food allergy. Depending on age, patient characteristics and situation demands it can be performed as an open food challenge or as double-blind placebo-controlled oral food challenge (DBPCFC). It is also relevant to perform risk stratification before starting food allergen-specific immunotherapy, to determine the initial allergen dose based on the threshold of allergen required to trigger a reaction, and to monitor its effectiveness.²⁹⁵ Identifying the molecular

pattern of sensitization of a patient allows to recognize subjects at risk of suffering severe reactions such as IgE sensitization to *Arachis hypogea* 2 (Ara h 2) from peanut.²⁹⁶ However, to improve the safety and reliability of food challenges with allergenic molecules, more clinical evidence is needed.²⁶⁸

Based on previous data proving the usefulness of molecular allergens in *in vitro* and *in vivo* tests to increase the accuracy of the allergy diagnosis, provocation with molecular allergens seems to be more useful than challenges with the whole extracts. However, these molecules are not yet available for routine diagnosis and more research is needed to better standardize these extracts, which differ from those used for *in vitro* testing.²⁹¹
# A07 - Basic and theoretical aspects of allergens

Luis Caraballo, Paolo M Matricardi

## Highlights

* Allergens are defined as molecules inducing and binding specific IgE antibodies

* Various allergens have the capacity to stimulate the innate response before inducing IgE

* Cohort studies show the development of polymolecular sensitization—"molecular spreading"

* Initiator molecules from different sensitization patterns are considered as the optimal targets for allergen immunoprophylaxis

## 1 - Introduction

Like other biomedical sciences, experimental allergology is advancing at great speed. Since the first edition of the EAACI Molecular Allergology User's Guide, important discoveries have been made about the pathophysiological mechanisms of the allergic response and the properties of allergens, which force us to reflect on general theoretical concepts in the field. In this chapter, we will discuss some aspects of allergens on which diverse opinions have been expressed for many years and have to do with ideas and hypotheses that support much of current experimental and clinical work.

## 2 - The allergen concept

Definitions are short expressions of concepts, which in turn are based on current knowledge. Traditionally, allergens have been defined as those molecules inducing and binding specific IgE antibodies; however, one of the most important recent advances in experimental allergology has been the recognition of the inflammatory capacity of various allergens (in addition to proteases) by stimulating the innate response before inducing IgE.⁴⁰,⁵⁵ In fact, this has promoted a great change in our mentality regarding the origin of the allergic responses, currently focused on the epithelium and its pattern recognition receptors (PRR), the rapid production of alarmins, damage-associated molecular patterns (DAMPs) and proinflammatory cytokines that seem to be necessary for the development of a type 2 response. Important questions arise from these findings: Should we broaden the concept of allergen? Are there allergens that act entirely without the involvement of IgE? Will these be able to induce respiratory symptoms on their own? Are there phylogenetic equivalents of this type of immune response? Although there are no conclusive answers to these questions, there are reasons to believe that the allergen concept should include other properties in addition to IgE-binding.⁵⁵

Some evolutionary studies support the idea that prior to the existence of IgE in mammals, some stimuli (for example, bacterial toxins) could elicit an allergic-like inflammatory response, including hypersensitivity and shock reactions in fish.²⁹⁷ Teleost in general, dating back more than 300 million years, have several genes that code for components of the Th2 response,²⁹⁸ as well as effector cells (mast cells, eosinophils, and basophils) that are activated against certain antigenic stimuli in a similar way to those of mammals,²⁹⁹ which suggests, at least since the appearance of these organisms, that an immune response similar to what we now know as allergic has occurred. Th2 lymphocytes, which probably predate IgE,³⁰⁰ are an important source, along with type 2 ILCs, of IL-4 and IL-13. Among alarmins, HMGB-1 is quite conserved and orthologs have been found in C. elegans,³⁰¹ suggesting that this type of almost innate and immediate cellular response could be present before mammals. In addition, though IL-33 has only been found in mammals,³⁰² its specific receptor ST2 appears earlier in the evolutionary ladder and has been detected in birds and fish,³⁰¹ which is similar to what happened with the IgE and its high-affinity receptor, since the latter is in cells of the innate response (such as mast cells) much before than this immunoglobulin.³⁰³

Thus, some mechanisms of innate allergic inflammation that are currently observed recapitulate processes from innate immunity phylogeny. Considering all the above, it seems that there is no theoretical justification for naming as allergens only those molecules that induce and bind IgE. Perhaps if, hypothetically, in the future this were not a criterion, other molecules that trigger an allergic reaction could be discovered and our extent about what is allergy would be broader. For now, considering the recent progresses in molecular allergology and precision medicine, it has been proposed that inducing specific IgE (allergenicity) is not the only property for being an allergen but also the capacity of inducing inflammation (allergenic activity).²⁴,⁵⁵ Therefore, classifying allergens as "major" and "minor," according to the frequency and/or the strength of IgE binding is theoretically incorrect, useless and confusing. Of course, as has been well documented with allergen extracts, not all purified components are expected to have the same clinical impact, and this is an important aspect that has to be tested in different ways.⁵⁵ Whatever it is, all of them are allergens, and are important in terms of personalized allergology and should be named just "allergens".²⁴

## 3 - Why is a molecule an allergen?

This is one of the main interrogates of allergology and the answers have been guided (both theoretically and experimentally) by two general approaches. One is based on the search for molecular intrinsic properties that could make them allergens. The other is centered on the search for genetic variants that determine an allergic response to the molecules. Looking for common patterns among related families of allergens has been taken as evidence of intrinsic allergenic properties.²⁵ On the other hand, common patterns among allergens
# A07 - Basic and theoretical aspects of allergens

Luis Caraballo, Paolo M Matricardi

## Highlights

* Allergens are defined as molecules inducing and binding specific IgE antibodies

* Various allergens have the capacity to stimulate the innate response before inducing IgE

* Cohort studies show the development of polymolecular sensitization—"molecular spreading"

* Initiator molecules from different sensitization patterns are considered as the optimal targets for allergen immunoprophylaxis

## 1 - Introduction

Like other biomedical sciences, experimental allergology is advancing at great speed. Since the first edition of the EAACI Molecular Allergology User's Guide, important discoveries have been made about the pathophysiological mechanisms of the allergic response and the properties of allergens, which force us to reflect on general theoretical concepts in the field. In this chapter, we will discuss some aspects of allergens on which diverse opinions have been expressed for many years and have to do with ideas and hypotheses that support much of current experimental and clinical work.

## 2 - The allergen concept

Definitions are short expressions of concepts, which in turn are based on current knowledge. Traditionally, allergens have been defined as those molecules inducing and binding specific IgE antibodies; however, one of the most important recent advances in experimental allergology has been the recognition of the inflammatory capacity of various allergens (in addition to proteases) by stimulating the innate response before inducing IgE.⁴⁰,⁵⁵ In fact, this has promoted a great change in our mentality regarding the origin of the allergic responses, currently focused on the epithelium and its pattern recognition receptors (PRR), the rapid production of alarmins, damage-associated molecular patterns (DAMPs) and proinflammatory cytokines that seem to be necessary for the development of a type 2 response. Important questions arise from these findings: Should we broaden the concept of allergen? Are there allergens that act entirely without the involvement of IgE? Will these be able to induce respiratory symptoms on their own? Are there phylogenetic equivalents of this type of immune response? Although there are no conclusive answers to these questions, there are reasons to believe that the allergen concept should include other properties in addition to IgE-binding.⁵⁵

Some evolutionary studies support the idea that prior to the existence of IgE in mammals, some stimuli (for example, bacterial toxins) could elicit an allergic-like inflammatory response, including hypersensitivity and shock reactions in fish.²⁹⁷ Teleost in general, dating back more than 300 million years, have several genes that code for components of the Th2 response,²⁹⁸ as well as effector cells (mast cells, eosinophils, and basophils) that are activated against certain antigenic stimuli in a similar way to those of mammals,²⁹⁹ which suggests, at least since the appearance of these organisms, that an immune response similar to what we now know as allergic has occurred. Th2 lymphocytes, which probably predate IgE,³⁰⁰ are an important source, along with type 2 ILCs, of IL-4 and IL-13. Among alarmins, HMGB-1 is quite conserved and orthologs have been found in C. elegans,³⁰¹ suggesting that this type of almost innate and immediate cellular response could be present before mammals. In addition, though IL-33 has only been found in mammals,³⁰² its specific receptor ST2 appears earlier in the evolutionary ladder and has been detected in birds and fish,³⁰¹ which is similar to what happened with the IgE and its high-affinity receptor, since the latter is in cells of the innate response (such as mast cells) much before than this immunoglobulin.³⁰³

Thus, some mechanisms of innate allergic inflammation that are currently observed recapitulate processes from innate immunity phylogeny. Considering all the above, it seems that there is no theoretical justification for naming as allergens only those molecules that induce and bind IgE. Perhaps if, hypothetically, in the future this were not a criterion, other molecules that trigger an allergic reaction could be discovered and our extent about what is allergy would be broader. For now, considering the recent progresses in molecular allergology and precision medicine, it has been proposed that inducing specific IgE (allergenicity) is not the only property for being an allergen but also the capacity of inducing inflammation (allergenic activity).²⁴,⁵⁵ Therefore, classifying allergens as "major" and "minor," according to the frequency and/or the strength of IgE binding is theoretically incorrect, useless and confusing. Of course, as has been well documented with allergen extracts, not all purified components are expected to have the same clinical impact, and this is an important aspect that has to be tested in different ways.⁵⁵ Whatever it is, all of them are allergens, and are important in terms of personalized allergology and should be named just "allergens".²⁴

## 3 - Why is a molecule an allergen?

This is one of the main interrogates of allergology and the answers have been guided (both theoretically and experimentally) by two general approaches. One is based on the search for molecular intrinsic properties that could make them allergens. The other is centered on the search for genetic variants that determine an allergic response to the molecules. Looking for common patterns among related families of allergens has been taken as evidence of intrinsic allergenic properties.²⁵ On the other hand, common patterns among allergens
from distant species have been supporting the hypothesis around the origins of allergens and their IgE-binding.³⁰⁴ Interestingly, so far no "intrinsic property" that makes a molecule an allergen has been discovered; similarly, except for genetic variants determining allergic reactions to some drugs, variants that clearly define the allergic response to common allergens, confirmed with functional studies, have not been found. That a molecule is an allergen is the result of a process influenced by a great number of factors but determined by the genetic background of the immune response. Therefore, the general theoretical background of the "genetic point of view" of the existence of allergens will be examined.

The analysis will be focused not only on allergenicity but also on the allergenic activity. Although it is not always recognized, the existence of antigens in general, including allergens, depends on the existence of the immune response.³⁰⁵ As well as other phenotypes the immune response is under genetic control and is highly polymorphic. Therefore, theoretically, any molecule can be an allergen if it finds the appropriate genotype, that is the set of genetic variants in the genome of a person. For that reason, the same molecule, that is inhaled by everybody in a defined environment, even within a family, is an allergen for some of them and just an antigen for others. Those "susceptible" persons are believed to have a combination of genetic variants (a genotype), which conform a cellular/molecular scenario making the induction of type 2 responses easier or lack variants that exert the opposite effect. The clinical name of this phenotype is atopy. Then it could be said that atopic persons define the existence of allergens. It is known that other factors also influence the intensity of the allergic response, some belonging to the molecule itself and others to the host and the environment,²⁴ but, again, their influence is exerted only on the genetically susceptible individuals. For example, the protease activity of Der p 1 can make it more allergenic, but if this property were defining its allergenicity, the entire exposed population would be allergic to it. In contrast to toxins, to which most people are genetically susceptible, allergens affect only a small percentage of the population. Allergen properties such as abundance and stability (e.g., thermostable food allergens) influence allergenicity because they are associated with greater exposure and more possibilities to get in contact with genetically susceptible individuals. If we speculate about the level of genetic control, we will find that allergenicity is expected to be defined by a less complex genotype than allergenic activity and the latter by a less complex genotype than an allergic disease. The genetic polymorphisms that could define both phenotypes (allergenicity and allergenic activity) range from the first contact with the epithelium, the antigenic recognition and processing to inflammatory cytokine production pathways, including those genes that intervene in the innate response. The expression of these polymorphisms is modified by several mechanisms such as epigenetic pathways and gene-gene interactions. Therefore, discovering the genetic basis of allergenic activity or even allergenicity is not an easy and straightforward task. It needs several approaches and collaborations between groups working on allergy and genetics. This will be very helpful because studying genetic mechanisms not only can explain the origin of allergen activity

but also the pathophysiology of allergy and allergic diseases. Also, defining the mechanisms of how allergens and the environment act in the genetically susceptible population would help to understand the genetic control of the immune response.

## 4 – Initiatory allergen molecules

Atopy is still an enigma, not only because of its complex genetic roots but also because of its poorly defined mechanisms. Consequently, its definition ranges from "the hereditary predisposition to react with IgE" to "the hereditary predisposition to mount a type 2 response" to environmental molecules. Therefore, in clinical and experimental settings, atopy is evaluated by looking at IgE production, usually as a final outcome. However, the process of developing the atopy phenotype involves more than IgE, as has been revealed by the following interesting clinical works.

An age-related increase in the number of allergenic molecules recognized by IgE antibodies has been observed in cross-sectional studies in patient populations, where this number is greater in older patients.³⁰⁶ So far, several birth cohort studies following individual patients since the early years of life have prospectively examined their evolution of IgG and IgE responses to allergen molecules. Specifically, the German Multicenter Allergy Study (MAS), an extensive European birth cohort on atopic diseases, provides insight on the onset and evolution of IgE responses in atopic versus healthy children, as well as on the humoral state prior to the manifestation of IgE or allergic symptoms. Serologic analyses taken from both healthy and atopic children revealed that both groups present a broad repertoire of IgG antibodies to a wide array of allergen molecules already at age 2.³⁰⁷ The intensity and frequency of these "normal" IgG responses differ according to the allergen group, with the highest being animal food allergens, intermediate toward vegetable food allergens, and only minimal to airborne allergens. Of note, a stronger and more frequent IgG response is shown by atopic children, when comparing to their nonatopic peers.³⁰⁷

A second type of "atopic" IgG response was uncovered when studying the IgE response to Bet v 1 and other PR-10 molecules. This response, directed against airborne allergens, was observed to be persistent and accompany IgE production against the same molecule, which led to the assumption that IgE response to Bet v 1 in children with birch allergy represents a broader abnormal humoral response involving IgG antibodies directed to the same antigens. This concept was corroborated in a broader study, which, additionally, showed that the "default" IgG response to major allergenic molecules becomes stronger and persistent in atopic subjects with IgE responses to the same molecules from mites, pollen, or moulds, thus becoming a sort of "atopic" IgG response.³⁰⁸

Various patterns of evolution following the beginning of an IgE response have been described in several cohorts. Within the MAS cohort, subsets of children showed a sequential broadening of the IgE response to *Phleum pratense* molecules. This broadening consisted of a progression from an initial monomolecular through an
from distant species have been supporting the hypothesis around the origins of allergens and their IgE-binding.³⁰⁴ Interestingly, so far no "intrinsic property" that makes a molecule an allergen has been discovered; similarly, except for genetic variants determining allergic reactions to some drugs, variants that clearly define the allergic response to common allergens, confirmed with functional studies, have not been found. That a molecule is an allergen is the result of a process influenced by a great number of factors but determined by the genetic background of the immune response. Therefore, the general theoretical background of the "genetic point of view" of the existence of allergens will be examined.

The analysis will be focused not only on allergenicity but also on the allergenic activity. Although it is not always recognized, the existence of antigens in general, including allergens, depends on the existence of the immune response.³⁰⁵ As well as other phenotypes the immune response is under genetic control and is highly polymorphic. Therefore, theoretically, any molecule can be an allergen if it finds the appropriate genotype, that is the set of genetic variants in the genome of a person. For that reason, the same molecule, that is inhaled by everybody in a defined environment, even within a family, is an allergen for some of them and just an antigen for others. Those "susceptible" persons are believed to have a combination of genetic variants (a genotype), which conform a cellular/molecular scenario making the induction of type 2 responses easier or lack variants that exert the opposite effect. The clinical name of this phenotype is atopy. Then it could be said that atopic persons define the existence of allergens. It is known that other factors also influence the intensity of the allergic response, some belonging to the molecule itself and others to the host and the environment,²⁴ but, again, their influence is exerted only on the genetically susceptible individuals. For example, the protease activity of Der p 1 can make it more allergenic, but if this property were defining its allergenicity, the entire exposed population would be allergic to it. In contrast to toxins, to which most people are genetically susceptible, allergens affect only a small percentage of the population. Allergen properties such as abundance and stability (e.g., thermostable food allergens) influence allergenicity because they are associated with greater exposure and more possibilities to get in contact with genetically susceptible individuals. If we speculate about the level of genetic control, we will find that allergenicity is expected to be defined by a less complex genotype than allergenic activity and the latter by a less complex genotype than an allergic disease. The genetic polymorphisms that could define both phenotypes (allergenicity and allergenic activity) range from the first contact with the epithelium, the antigenic recognition and processing to inflammatory cytokine production pathways, including those genes that intervene in the innate response. The expression of these polymorphisms is modified by several mechanisms such as epigenetic pathways and gene-gene interactions. Therefore, discovering the genetic basis of allergenic activity or even allergenicity is not an easy and straightforward task. It needs several approaches and collaborations between groups working on allergy and genetics. This will be very helpful because studying genetic mechanisms not only can explain the origin of allergen activity

but also the pathophysiology of allergy and allergic diseases. Also, defining the mechanisms of how allergens and the environment act in the genetically susceptible population would help to understand the genetic control of the immune response.

## 4 - Initiatory allergen molecules

Atopy is still an enigma, not only because of its complex genetic roots but also because of its poorly defined mechanisms. Consequently, its definition ranges from "the hereditary predisposition to react with IgE" to "the hereditary predisposition to mount a type 2 response" to environmental molecules. Therefore, in clinical and experimental settings, atopy is evaluated by looking at IgE production, usually as a final outcome. However, the process of developing the atopy phenotype involves more than IgE, as has been revealed by the following interesting clinical works.

An age-related increase in the number of allergenic molecules recognized by IgE antibodies has been observed in cross-sectional studies in patient populations, where this number is greater in older patients.³⁰⁶ So far, several birth cohort studies following individual patients since the early years of life have prospectively examined their evolution of IgG and IgE responses to allergen molecules. Specifically, the German Multicenter Allergy Study (MAS), an extensive European birth cohort on atopic diseases, provides insight on the onset and evolution of IgE responses in atopic versus healthy children, as well as on the humoral state prior to the manifestation of IgE or allergic symptoms. Serologic analyses taken from both healthy and atopic children revealed that both groups present a broad repertoire of IgG antibodies to a wide array of allergen molecules already at age 2.³⁰⁷ The intensity and frequency of these "normal" IgG responses differ according to the allergen group, with the highest being animal food allergens, intermediate toward vegetable food allergens, and only minimal to airborne allergens. Of note, a stronger and more frequent IgG response is shown by atopic children, when comparing to their nonatopic peers.³⁰⁷

A second type of "atopic" IgG response was uncovered when studying the IgE response to Bet v 1 and other PR-10 molecules. This response, directed against airborne allergens, was observed to be persistent and accompany IgE production against the same molecule, which led to the assumption that IgE response to Bet v 1 in children with birch allergy represents a broader abnormal humoral response involving IgG antibodies directed to the same antigens. This concept was corroborated in a broader study, which, additionally, showed that the "default" IgG response to major allergenic molecules becomes stronger and persistent in atopic subjects with IgE responses to the same molecules from mites, pollen, or moulds, thus becoming a sort of "atopic" IgG response.³⁰⁸

Various patterns of evolution following the beginning of an IgE response have been described in several cohorts. Within the MAS cohort, subsets of children showed a sequential broadening of the IgE response to *Phleum pratense* molecules. This broadening consisted of a progression from an initial monomolecular through an
oligomolecular and towards a polymolecular sensitization pattern, so that it could be defined as "molecular spreading" (Figure 16).⁹⁵,³⁰⁹ Interestingly, it was also observed that an initial IgE response is directed against Phl p 1 in almost all grass pollen allergic children, making it a sort of starter or "initiator molecule" of this IgE response. Only a small number of patients developed a full-scale polymolecular response to all 8 allergenic molecules of *P. pratense*. This molecular spreading often begins even in the preclinical stages of sensitization. IgE against Phl p 1 has been observed up to 5 years before the child starts presenting the first allergic symptoms.³¹⁰

A quite similar process has been described in the MAS cohort children sensitized to *D. pteronyssinus*.³¹⁰ Sensitization started against Der p 1 and/or Der p 2 and/or Der p 23 (defined as group A molecules); expanded to Der p 4, Der p 5, Der p 7, and Der p 21 (group B molecules); and eventually progressed to Der p 11, Der p 14, Der p 15, Der p 18, and clone 16 (group C molecules). This evolution has been defined as the "ABC march" of mite allergy (i.e., molecular spreading of the IgE repertoire against *D. pteronyssinus* allergens).²⁸ Moreover, an association was observed between several features and a broader polymolecular IgE sensitization pattern, such as early IgE sensitization onset, parental hay fever, and greater exposure to mites. Another curious conclusion was the correlation between participants reaching the broadest ABC IgE sensitization stage and a significantly greater risk of mite-related allergic rhinitis and asthma, not observed in the monomolecularly sensitized participants. Finally, IgE sensitization to Der p 1 or Der p 23 at 5 years of age or less in healthy children predicted asthma at school age.³¹⁰

The Manchester Asthma and Allergy Study (MAAS) showed a similar trend within 235 children whose sera were tested for IgE

to Timothy grass pollen and mite allergens.³¹¹ Three sensitization slopes towards mite allergy were identified among children aged 5 to 11 years: group 1 sensitization (involving Der f 1 and Der p 1), group 2 (Der f 2, Der p 2), and complete mite sensitization (both). Children with group 1 and complete mite sensitization trajectories had a significantly increased risk for asthma, eczema, and rhinitis, but the highest risk for asthma (odds ratio, 7.15; 95% CI, 3.80–13.44) was within the subjects of the complete mite sensitization group. Regarding the grass sensitization, a molecular progression of the IgE response similar to molecular spreading in MAS participants was observed. A correlation between an early onset of IgE response to grass molecules and asthma along with diminished lung function was observed, as well as between a late onset and allergic rhinitis.³¹¹

In the Swedish Children, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) study, sensitization to Fel d 1 and Can f 1 in childhood, as well as polysensitization to allergen molecules of either cat or dog predicted cat and dog allergy cross-sectionally and longitudinally substantially better than simply IgE to cat or dog extract.³¹² The onset and molecular profile of the IgE responses in food allergic patients during childhood and adolescence appears to be crucial, as shown by the BAMSE population-based birth cohort. Two distinct phenotypes of peanut allergy development before adulthood have been identified. The first is the association between early onset of sensitization to Ara h 2 and an increased risk of systemic reactions after peanut exposure, and the second is, starting later in childhood and being related to Ara h 8, goes along with no systemic reactions after peanut ingestion.³¹³ After expanding the data analysis to the whole set of IgE results against 132 allergen molecules, 4 risk molecules were additionally identified, to which an IgE response was

<figure><img src="image_28.png" /><figcaption>FIGURE 16 Evolution of the prevalence of IgE responses to 16 allergen molecules from 1 to 10 years of age (n = 104). The prevalence of IgE sensitization (≥0.3 ISU) to the 16 allergen molecules by age at follow-up is shown. The number of participants examined at each follow-up is reported under the x-axis. NA, not applicable (no blood drawn at this follow-up).</figcaption></figure>
oligomolecular and towards a polymolecular sensitization pattern, so that it could be defined as "molecular spreading" (Figure 16).⁹⁵,³⁰⁹ Interestingly, it was also observed that an initial IgE response is directed against Phl p 1 in almost all grass pollen allergic children, making it a sort of starter or "initiator molecule" of this IgE response. Only a small number of patients developed a full-scale polymolecular response to all 8 allergenic molecules of *P. pratense*. This molecular spreading often begins even in the preclinical stages of sensitization. IgE against Phl p 1 has been observed up to 5 years before the child starts presenting the first allergic symptoms.³¹⁰

A quite similar process has been described in the MAS cohort children sensitized to *D. pteronyssinus*.³¹⁰ Sensitization started against Der p 1 and/or Der p 2 and/or Der p 23 (defined as group A molecules); expanded to Der p 4, Der p 5, Der p 7, and Der p 21 (group B molecules); and eventually progressed to Der p 11, Der p 14, Der p 15, Der p 18, and clone 16 (group C molecules). This evolution has been defined as the "ABC march" of mite allergy (i.e., molecular spreading of the IgE repertoire against *D. pteronyssinus* allergens).²⁸ Moreover, an association was observed between several features and a broader polymolecular IgE sensitization pattern, such as early IgE sensitization onset, parental hay fever, and greater exposure to mites. Another curious conclusion was the correlation between participants reaching the broadest ABC IgE sensitization stage and a significantly greater risk of mite-related allergic rhinitis and asthma, not observed in the monomolecularly sensitized participants. Finally, IgE sensitization to Der p 1 or Der p 23 at 5 years of age or less in healthy children predicted asthma at school age.³¹⁰

The Manchester Asthma and Allergy Study (MAAS) showed a similar trend within 235 children whose sera were tested for IgE

to Timothy grass pollen and mite allergens.³¹¹ Three sensitization slopes towards mite allergy were identified among children aged 5 to 11 years: group 1 sensitization (involving Der f 1 and Der p 1), group 2 (Der f 2, Der p 2), and complete mite sensitization (both). Children with group 1 and complete mite sensitization trajectories had a significantly increased risk for asthma, eczema, and rhinitis, but the highest risk for asthma (odds ratio, 7.15; 95% CI, 3.80–13.44) was within the subjects of the complete mite sensitization group. Regarding the grass sensitization, a molecular progression of the IgE response similar to molecular spreading in MAS participants was observed. A correlation between an early onset of IgE response to grass molecules and asthma along with diminished lung function was observed, as well as between a late onset and allergic rhinitis.³¹¹

In the Swedish Children, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) study, sensitization to Fel d 1 and Can f 1 in childhood, as well as polysensitization to allergen molecules of either cat or dog predicted cat and dog allergy cross-sectionally and longitudinally substantially better than simply IgE to cat or dog extract.³¹² The onset and molecular profile of the IgE responses in food allergic patients during childhood and adolescence appears to be crucial, as shown by the BAMSE population-based birth cohort. Two distinct phenotypes of peanut allergy development before adulthood have been identified. The first is the association between early onset of sensitization to Ara h 2 and an increased risk of systemic reactions after peanut exposure, and the second is, starting later in childhood and being related to Ara h 8, goes along with no systemic reactions after peanut ingestion.³¹³ After expanding the data analysis to the whole set of IgE results against 132 allergen molecules, 4 risk molecules were additionally identified, to which an IgE response was

<figure><img src="image_28.png" /><figcaption>FIGURE 16 Evolution of the prevalence of IgE responses to 16 allergen molecules from 1 to 10 years of age (n = 104). The prevalence of IgE sensitization (≥0.3 ISU) to the 16 allergen molecules by age at follow-up is shown. The number of participants examined at each follow-up is reported under the x-axis. NA, not applicable (no blood drawn at this follow-up).</figcaption></figure>
associated with an increased risk of asthma, allergic rhinitis, or both, namely Ara h 1 (peanut), Bet v 1 (birch), Fel d 1 (cat), and Phl p 1 (grass). The previously discussed results regarding airborne molecules could be replicated within the MAAS cohort, adding Der f 2 (dust mite) and Phl p 5 (grass) as disease predictors.³¹³

This uncovered understanding of the progressive molecular
spreading of the immune response, especially to airborne allergens,
led to hypothesize that immunologic intervention (AIT) should ide-
ally be anticipated, as it might prevent the development of more

complex and strong IgE responses.⁹⁵ The initiator molecules—Phl p 1 for grass pollen, Bet v 1 for birch pollen, Der p 1, Der p 2, and Der p 23 for mites, and Fel d 1 for cat—are thought to be optimal targets for allergen immunoprophylaxis (AIP) aimed at preventing the onset of allergic rhinitis and asthma in children during a preclinical sensitization stage.⁹⁵,³⁰⁹ An initiating molecule has been defined as “The allergenic molecule within an allergenic source responsible for induction of the first IgE antibody response to that allergenic source”.⁹⁵
associated with an increased risk of asthma, allergic rhinitis, or both, namely Ara h 1 (peanut), Bet v 1 (birch), Fel d 1 (cat), and Phl p 1 (grass). The previously discussed results regarding airborne molecules could be replicated within the MAAS cohort, adding Der f 2 (dust mite) and Phl p 5 (grass) as disease predictors.³¹³

This uncovered understanding of the progressive molecular
spreading of the immune response, especially to airborne allergens,
led to hypothesize that immunologic intervention (AIT) should ide-
ally be anticipated, as it might prevent the development of more

complex and strong IgE responses.⁹⁵ The initiator molecules—Phl p
1 for grass pollen, Bet v 1 for birch pollen, Der p 1, Der p 2, and Der
p 23 for mites, and Fel d 1 for cat—are thought to be optimal tar-
gets for allergen immunoprophylaxis (AIP) aimed at preventing the
onset of allergic rhinitis and asthma in children during a preclinical
sensitization stage.⁹⁵,³⁰⁹ An initiating molecule has been defined
as “The allergenic molecule within an allergenic source responsible
for induction of the first IgE antibody response to that allergenic
source”.⁹⁵
# A08 - Allergen families and databases

## Christian Radauer, Gabriele Gadermaier, Heimo Breiteneder

### Highlights

* Proteins that share a common evolutionary origin—which is reflected primarily by their similar sequences and structures—are grouped into families. Families with a common origin are grouped into superfamilies.

* Very few of the more than 19,000 protein families as described by the protein family database Pfam harbor allergens.

* Most proteins that are described as allergens can be classified roughly into 30 to 40 protein families.

* Allergen databases make curated information available on allergen nomenclature, allergen structures, allergen protein families, and the risk of allergen cross-reactivity, or offer noncurated extensive collections of information on allergens.

## 1 - Introduction

A protein family is a group of proteins that share a common evolutionary origin, which is reflected primarily by their similar sequences and structures but often also by similar biochemical functions and physico-chemical properties (Figure 17). Moreover, in most cases common protein family membership is a prerequisite for immunological cross-reactivity. The current version 34.0 of the Pfam (protein family) database (https://www.ebi.ac.uk/interpro/) describes 19,179

protein families.³¹⁴ Evolutionarily related families are grouped together into superfamilies—called clans in Pfam—(e.g., the prolamin superfamily). As a consequence of diversification during evolution and as the phylogenetic distance increases, the members of different families within a superfamily possess only moderate to low levels of primary sequence identities and little to no immunological cross-reactivity.

Very few protein architectures give rise to allergenic proteins and thus allergens are only found in a rather limited number of protein families. The AllFam allergen family database (http://www.meduniwien.ac.at/allfam/; version of 2017-03-07) assigns all presently known allergens to 216 (1.6%) of the then 16,306 Pfam families. Proteins that are described worldwide as the most important allergens can be classified into roughly 30 to 40 protein families. However, it has to be emphasized that the vast majority of proteins in any given family or superfamily are nonallergenic. The most important families and selected allergenic member proteins are discussed here (Table 8).

Our understanding why exactly these types of proteins are able to trigger Th2-dominated immune responses is incomplete. Allergenic proteins that are able to sensitize predisposed individuals initiate both innate and adaptive immune responses (see chapter A02). Such proteins are recognized by epithelial cells and dendritic cells that produce signals to polarize the immune response towards a Th2 phenotype. It has been shown that the innate immune system plays an important role in the decision whether a protein will be treated as an allergen by the organism.³¹⁵,³¹⁶ It is highly likely that allergenic proteins possess molecular features that enable them to activate the pattern recognition receptors of innate immune cells to induce a Th2 polarization of the ensuing immune response. These

<table><caption>(A)</caption><tr><td>Bet v 1</td><td>1</td><td>MGV F NYETET SVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYN</td><td>83</td></tr><tr><td>Pru av 1</td><td>1</td><td>MGVFTYESEFTSEIPPPRLEKAFVL DADNLVPKIAPOAIIKHSEILEGDGGPGTIIKKITFGEQSQYGYVKHKIDSIDKENYSYS</td><td>83</td></tr><tr><td>Ara h 8</td><td>1</td><td>MGVFTFEDEITSTVPPAKLYNAMK -DADSITPKII -DDVKSVEIVEGNGGPGTIIKKLTIVEDGETKFIILHKVESIDEANYAYN</td><td>81</td></tr><tr><td>Api g 1</td><td>1</td><td>MGVQTHVLELLSSVSAEKIFQGEVIVDVDTVLPAAPGAYKSVEIK -GDGGPGTLKIIITLPDGGPITTMTLRI DGVNKEALTFD</td><td>82</td></tr><tr><td>Bet v 1</td><td>84</td><td>YSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYN</td><td>160</td></tr><tr><td>Pru av 1</td><td>84</td><td>YTLIEGDALGDTLEKISYETKLVASPGGSIIKSTSHYHTKGNVETIKEEHHVKAGKEKASNLFKLIETYLKHPDAYN</td><td>160</td></tr><tr><td>Ara h 8</td><td>82</td><td>YSVVGDAVLPPTAEKITFETKLVEGPNGGSIGKLTILKYHTKGDAKPDEEEELKKGKAKGEGLFRAIEGYVLANPTQY-</td><td>157</td></tr><tr><td>Api g 1</td><td>83</td><td>YSVIDGDILLGFIESIENHVVLVPTADGGSICKTTAIFHTKGDAVVPEENIKYANEQNTALEKALEAYLIAN - - - - -</td><td>154</td></tr></table>

<figure><img src="image_29.png" /><figcaption>FIGURE 17 The Bet v 1 family as an example of a protein family. Sequence alignment (A) and structures (B) of four representative allergenic members. Bet v 1 is from birch pollen, Pru av 1 from cherry, Ara h 8 from peanut, and Api g 1 from celery.</figcaption></figure>
# A08 - Allergen families and databases

## Christian Radauer, Gabriele Gadermaier, Heimo Breiteneder

### Highlights

* Proteins that share a common evolutionary origin—which is reflected primarily by their similar sequences and structures—are grouped into families. Families with a common origin are grouped into superfamilies.

* Very few of the more than 19,000 protein families as described by the protein family database Pfam harbor allergens.

* Most proteins that are described as allergens can be classified roughly into 30 to 40 protein families.

* Allergen databases make curated information available on allergen nomenclature, allergen structures, allergen protein families, and the risk of allergen cross-reactivity, or offer noncurated extensive collections of information on allergens.

## 1 - Introduction

A protein family is a group of proteins that share a common evolutionary origin, which is reflected primarily by their similar sequences and structures but often also by similar biochemical functions and physico-chemical properties (Figure 17). Moreover, in most cases common protein family membership is a prerequisite for immunological cross-reactivity. The current version 34.0 of the Pfam (protein family) database (https://www.ebi.ac.uk/interpro/) describes 19,179

protein families.³¹⁴ Evolutionarily related families are grouped together into superfamilies—called clans in Pfam—(e.g., the prolamin superfamily). As a consequence of diversification during evolution and as the phylogenetic distance increases, the members of different families within a superfamily possess only moderate to low levels of primary sequence identities and little to no immunological cross-reactivity.

Very few protein architectures give rise to allergenic proteins and thus allergens are only found in a rather limited number of protein families. The AllFam allergen family database (http://www.meduniwien.ac.at/allfam/; version of 2017-03-07) assigns all presently known allergens to 216 (1.6%) of the then 16,306 Pfam families. Proteins that are described worldwide as the most important allergens can be classified into roughly 30 to 40 protein families. However, it has to be emphasized that the vast majority of proteins in any given family or superfamily are nonallergenic. The most important families and selected allergenic member proteins are discussed here (Table 8).

Our understanding why exactly these types of proteins are able to trigger Th2-dominated immune responses is incomplete. Allergenic proteins that are able to sensitize predisposed individuals initiate both innate and adaptive immune responses (see chapter A02). Such proteins are recognized by epithelial cells and dendritic cells that produce signals to polarize the immune response towards a Th2 phenotype. It has been shown that the innate immune system plays an important role in the decision whether a protein will be treated as an allergen by the organism.³¹⁵,³¹⁶ It is highly likely that allergenic proteins possess molecular features that enable them to activate the pattern recognition receptors of innate immune cells to induce a Th2 polarization of the ensuing immune response. These

<table><caption>(A)</caption><tr><td>Bet v 1</td><td>1</td><td>MGV F NYETET SVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYN</td><td>83</td></tr><tr><td>Pru av 1</td><td>1</td><td>MGV F TYESEFTSEIPPPRLEKAFVL DADNLVPKIAPOAIIKHSEILEGDGGPGTIIKKITFGEQSYGYVKHKIDSIDKENYSYS</td><td>83</td></tr><tr><td>Ara h 8</td><td>1</td><td>MGV F TFEDEITSTVPPAKLYNAMK -DADSITPKII -DDVKSVEIVEGNGGPGTIIKKLTIVEDGETKFLILHKVESIDEANYAYN</td><td>81</td></tr><tr><td>Api g 1</td><td>1</td><td>MGV QTHVLELLSSVSAEKIFQGEVIVDVDTVLPAAPGAYKSVEIK-GDGGPGTLKIIITLPDGGPITTMTLRIDGVNKEALTFD</td><td>82</td></tr><tr><td>Bet v 1</td><td>84</td><td>YSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYN</td><td>160</td></tr><tr><td>Pru av 1</td><td>84</td><td>YTLIEGDALGDTLEKISYETKLVASPGGSIIKSTSHYHTKGNVETIKEEHHVKAGKEKASNLFKLIETYLKHPDAYN</td><td>160</td></tr><tr><td>Ara h 8</td><td>82</td><td>YSVVGDAALPPTAEKITFETKLVEGPNGGSIGKLTILKYHTKGDAKPDEEEELKKGKAKGEGLFRAIEGYVLANPTQY-</td><td>157</td></tr><tr><td>Api g 1</td><td>83</td><td>YSVIDGDILLGFIESIENHVVLVPTADGGSICKTTAIFHTKGDAVVPEENIKYANEQNTALEKALEAYLIAN- - - - - -</td><td>154</td></tr></table>

<figure><img src="image_29.png" /><figcaption>FIGURE 17 The Bet v 1 family as an example of a protein family. Sequence alignment (A) and structures (B) of four representative allergenic members. Bet v 1 is from birch pollen, Pru av 1 from cherry, Ara h 8 from peanut, and Api g 1 from celery.</figcaption></figure>
<table><caption>TABLE 8 Protein superfamilies and families that contain the highest numbers of allergens in descending order. Data were extracted from the AllFam database (http://www.meduniwien.ac.at/allfam/).</caption><thead><tr><th>Superfamily</th><th>Number of allergens</th><th>Family</th><th>Allergen sources</th></tr></thead><tbody><tr><td rowspan="5">Prolamin</td><td rowspan="5">91</td><td>Cereal prolamin</td><td>Grains of cereal grasses</td></tr><tr><td>Bifunctional inhibitor</td><td>Grains of cereal grasses</td></tr><tr><td>2S albumin</td><td>Tree nuts, legumes (e.g. peanut), seeds</td></tr><tr><td>Non-specific lipid-transfer protein type 1</td><td>Fruits, tree nuts, peanut, vegetables, cereal grains, tree and weed pollen, latex</td></tr><tr><td>Non-specific lipid-transfer protein type 2</td><td>Fruits, vegetables, peanut</td></tr><tr><td rowspan="5">EF-hand</td><td rowspan="5">74</td><td>Polcalcin</td><td>Tree, grass, and weed pollen</td></tr><tr><td>Parvalbumin</td><td>Fish</td></tr><tr><td>Sarcoplasmic Ca-binding protein</td><td>Shellfish, insects</td></tr><tr><td>Troponin C</td><td>Mites, cockroaches, shellfish</td></tr><tr><td>Myosin light chain</td><td>Mites, cockroaches, shellfish</td></tr><tr><td>Tropomyosin-like</td><td>64</td><td>Tropomyosin</td><td>Crustaceans, mollusks, the fish parasite <i>Anisakis simplex</i>, mites, cockroaches</td></tr><tr><td>Profilin-like</td><td>53</td><td>Profilin</td><td>Tree, grass, and weed pollen, fruits, vegetables, latex</td></tr><tr><td rowspan="2">Cupin</td><td rowspan="2">37</td><td>Vicilin</td><td>Tree nuts, peanut, legumes, seeds</td></tr><tr><td>Legumin</td><td>Tree nuts, peanut, legumes, seeds</td></tr><tr><td>Bet v 1-like</td><td>29</td><td>Bet v 1</td><td>Fagales tree pollen, fruits, vegetables, legumes, tree nuts</td></tr><tr><td rowspan="2">Calycin</td><td rowspan="2">27</td><td>Secreted lipocalin</td><td>Mammals, milk</td></tr><tr><td>Cytoplasmic fatty acid binding protein</td><td>Mites</td></tr><tr><td rowspan="5">Double-psi beta-barrel</td><td rowspan="5">26</td><td>Triabin</td><td>Insects</td></tr><tr><td>Expansin</td><td>Grass pollen</td></tr><tr><td>Kiwellin</td><td>Kiwifruit</td></tr><tr><td>Barwin-like</td><td><i>Hevea brasiliensis</i> (natural rubber latex), turnip</td></tr><tr><td>Venom antigen 5</td><td>Hymenoptera venoms</td></tr><tr><td rowspan="2">CAP superfamily</td><td rowspan="2">32</td><td>Plant pathogenesis-related protein (PR)-1</td><td>Pollen, fruits</td></tr><tr><td>Pectate lyase</td><td>Pollen from Cupressaceae and Asteraceae</td></tr><tr><td rowspan="2">Pectate lyase-like</td><td rowspan="2">23</td><td>Polygalacturonase</td><td>Pollen</td></tr><tr><td>Pectinesterase</td><td>Pollen, kiwifruit</td></tr><tr><td>Transthyretin</td><td>16</td><td>Ole e 1-like</td><td>Pollen</td></tr><tr><td>Cysteine peptidase clan CA</td><td>13</td><td>Papain-like cysteine protease</td><td>Mites, fruits</td></tr></tbody></table>

features include (i) the ability to bind lipids and to activate Toll-like receptors (TLRs)—shown for Der p 2 from house dust mite³¹⁷ and for Fel d 1 from cat,³¹⁸ (ii) the ability to bind other cell surface or soluble pattern recognition receptors thereby modulating innate immune responses—shown for group 13 mite allergens that interact with serum amyloid A,³⁸ (iii) the presence of glycosylation and thus the ability to bind to C-type lectin receptors—shown for allergens from house dust mites, pollen and peanut,³¹⁹,¹⁶⁴⁷,¹⁶⁴⁸ or (iv) the presence of protease activity, which allows the activation of the protease-activated receptor PAR-2—shown for Der p 3 and Der p 9 from house dust mite.³²⁰

## 2 – The most important allergen containing protein families

### 2.1. Prolamin superfamily

The prolamin superfamily derives its name from the alcohol-soluble proline- and glutamine-rich storage proteins of cereal grains. Members of this superfamily are characterized by the presence of an α-helical globular domain. This domain contains a conserved pattern of cysteine residues that form three to five intra-molecular disulfide bonds. Apart from the conserved cysteine pattern, only little sequence similarities exist between members of different families.
<table><caption>TABLE 8 Protein superfamilies and families that contain the highest numbers of allergens in descending order. Data were extracted from the AllFam database (http://www.meduniwien.ac.at/allfam/).</caption><thead><tr><th>Superfamily</th><th>Number of allergens</th><th>Family</th><th>Allergen sources</th></tr></thead><tbody><tr><td rowspan="5">Prolamin</td><td rowspan="5">91</td><td>Cereal prolamin</td><td>Grains of cereal grasses</td></tr><tr><td>Bifunctional inhibitor</td><td>Grains of cereal grasses</td></tr><tr><td>2S albumin</td><td>Tree nuts, legumes (e.g. peanut), seeds</td></tr><tr><td>Non-specific lipid-transfer protein type 1</td><td>Fruits, tree nuts, peanut, vegetables, cereal grains, tree and weed pollen, latex</td></tr><tr><td>Non-specific lipid-transfer protein type 2</td><td>Fruits, vegetables, peanut</td></tr><tr><td rowspan="5">EF-hand</td><td rowspan="5">74</td><td>Polcalcin</td><td>Tree, grass, and weed pollen</td></tr><tr><td>Parvalbumin</td><td>Fish</td></tr><tr><td>Sarcoplasmic Ca-binding protein</td><td>Shellfish, insects</td></tr><tr><td>Troponin C</td><td>Mites, cockroaches, shellfish</td></tr><tr><td>Myosin light chain</td><td>Mites, cockroaches, shellfish</td></tr><tr><td>Tropomyosin-like</td><td>64</td><td>Tropomyosin</td><td>Crustaceans, mollusks, the fish parasite <i>Anisakis simplex</i>, mites, cockroaches</td></tr><tr><td>Profilin-like</td><td>53</td><td>Profilin</td><td>Tree, grass, and weed pollen, fruits, vegetables, latex</td></tr><tr><td rowspan="2">Cupin</td><td rowspan="2">37</td><td>Vicilin</td><td>Tree nuts, peanut, legumes, seeds</td></tr><tr><td>Legumin</td><td>Tree nuts, peanut, legumes, seeds</td></tr><tr><td>Bet v 1-like</td><td>29</td><td>Bet v 1</td><td>Fagales tree pollen, fruits, vegetables, legumes, tree nuts</td></tr><tr><td rowspan="2">Calycin</td><td rowspan="2">27</td><td>Secreted lipocalin</td><td>Mammals, milk</td></tr><tr><td>Cytoplasmic fatty acid binding protein</td><td>Mites</td></tr><tr><td rowspan="5">Double-psi beta-barrel</td><td rowspan="5">26</td><td>Triabin</td><td>Insects</td></tr><tr><td>Expansin</td><td>Grass pollen</td></tr><tr><td>Kiwellin</td><td>Kiwifruit</td></tr><tr><td>Barwin-like</td><td><i>Hevea brasiliensis</i> (natural rubber latex), turnip</td></tr><tr><td>Venom antigen 5</td><td>Hymenoptera venoms</td></tr><tr><td rowspan="2">CAP superfamily</td><td rowspan="2">32</td><td>Plant pathogenesis-related protein (PR)-1</td><td>Pollen, fruits</td></tr><tr><td>Pectate lyase</td><td>Pollen from Cupressaceae and Asteraceae</td></tr><tr><td rowspan="2">Pectate lyase-like</td><td rowspan="2">23</td><td>Polygalacturonase</td><td>Pollen</td></tr><tr><td>Pectinesterase</td><td>Pollen, kiwifruit</td></tr><tr><td>Transthyretin</td><td>16</td><td>Ole e 1-like</td><td>Pollen</td></tr><tr><td>Cysteine peptidase clan CA</td><td>13</td><td>Papain-like cysteine protease</td><td>Mites, fruits</td></tr></tbody></table>

features include (i) the ability to bind lipids and to activate Toll-like receptors (TLRs)—shown for Der p 2 from house dust mite³¹⁷ and for Fel d 1 from cat,³¹⁸ (ii) the ability to bind other cell surface or soluble pattern recognition receptors thereby modulating innate immune responses—shown for group 13 mite allergens that interact with serum amyloid A,³⁸ (iii) the presence of glycosylation and thus the ability to bind to C-type lectin receptors—shown for allergens from house dust mites, pollen and peanut,³¹⁹,¹⁶⁴⁷,¹⁶⁴⁸ or (iv) the presence of protease activity, which allows the activation of the protease-activated receptor PAR-2—shown for Der p 3 and Der p 9 from house dust mite.³²⁰

## 2 – The most important allergen containing protein families

### 2.1. Prolamin superfamily

The prolamin superfamily derives its name from the alcohol-soluble proline- and glutamine-rich storage proteins of cereal grains. Members of this superfamily are characterized by the presence of an α-helical globular domain. This domain contains a conserved pattern of cysteine residues that form three to five intra-molecular disulfide bonds. Apart from the conserved cysteine pattern, only little sequence similarities exist between members of different families.
Members of the prolamin superfamily include the cereal prolamin seed storage proteins (gliadins and glutenins) and several families of disulfide-rich small proteins including the bifunctional inhibitors, the 2S albumin seed storage proteins, and the non-specific lipid transfer proteins (Figure 18).

Cereal prolamins are seed storage proteins that are exclusively found in the grains of cereal grasses. In contrast to the low molecular weight members of the superfamily, the α-helical domain of the cereal prolamins has been disrupted by an insertion of repetitive sequences.³²¹ Gliadins and glutenins represent the members of the cereal prolamin family. Gliadins are soluble in alcohol and are classified into α/β-, γ-, and ω-gliadins. Glutenins are polymeric proteins that are held together by interchain disulfide bonds. They are divided into high and low molecular weight groups.³²²

Like the cereal prolamins, the bifunctional inhibitors are only present in cereal grains. These allergens sensitize either via the respiratory tract by inhalation of the flour or via the gastro-intestinal tract by eating foods that contain wheat, barley or rice. The bifunctional inhibitors are 12 to 16 kDa proteins that are held together by 4 to 6 disulfide bonds.³²³ Monomeric, dimeric and tetrameric forms are distinguished according to the degree of oligomerization of their subunits. They are the major cause of baker's asthma but also play a role as plant food allergens.³²⁴

The 2S albumins are a major group of plant seed storage proteins. Most 2S albumins are synthesized as single-chain proteins that are subsequently cleaved into a small and a large subunit. Both subunits are held together as compact α-helical molecules by 4 to 5 disulfide bonds.³²⁵ Many of the important seed and tree nut allergens are 2S albumins.

Nonspecific lipid transfer proteins (nsLTPs) have been suggested to mediate the transfer of phospholipids between vesicles and membranes. However, plants use the three-dimensional scaffold of the nsLTPs in various ways and many nsLTPs play a role in plant defense

against fungi and bacteria or in the response to abiotic stress. nsLTPs are typically found in high concentrations in epidermal tissues of plant food. The majority of allergenic representatives belongs to nsLTP type 1 (~10 kDa) while recently some members of the type 2 (~7 kDa) were described. Allergenic nsLTPs are highly resistant to thermal and enzymatic digestion³²⁶. They are major allergens of fruits from the Rosaceae family. In addition, allergenic nsLTPs are present in nuts, seeds, vegetables, and Hevea brasiliensis (natural rubber latex). Besides their presence in plant foods, nsLTPs are also expressed in pollen of weeds, olive, and plane tree.

* Wheat contains several allergenic cereal prolamins. Tri a 19 is an ω-5-gliadin, Tri a 21 an α/β-gliadin, and Tri a 26 a high molecular weight glutenin from wheat (see chapter B16).

* Hor v 15 is a monomeric α-amylase inhibitor from barley. Tri a 28 is a dimeric and Tri a 29 a tetrameric α-amylase inhibitor from wheat (see chapter B16).

* Allergenic 2S albumins include Ara h 2 and Ara h 6 from peanut, Ber e 1 from Brazil nut, Cor a 14 from hazelnut, Jug r 1 from English walnut, Ses i 1 from sesame seeds, and Sin a 1 from yellow mustard (see chapter C08).

* Rosaceae fruit type 1 nsLTPs include Mal d 3 from apple and Pru p 3 from peach. Representative allergenic type 1 nsLTPs from tree nuts are Cor a 8 from hazelnut and Jug r 3 from walnut. Pollen nsLTPs include Pla a 3 from plane tree and Art v 3 from mugwort. Zea m 14 and Tri a 14 are the nsLTPs from maize and wheat. Can s 3 is the nsLTP from Indian hemp (see chapter C03).

* Allergenic type 2 nsLTPs were identified in tomato (Sola l 6), celery tuber (Api g 6) and peanut (Ara h 16).

## 2.2. EF-hand superfamily

A wide range of calcium-binding proteins share a conserved motif consisting of a 12 residue calcium-binding loop flanked on both sides

### Bifunctional inhibitors

<figure><img src="image_30.png" /><figcaption>Tri a 28</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>Cereal grains</figcaption></figure>

### 2S albumins

<figure><img src="image_32.png" /><figcaption>Ara h 6</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>Legumes</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>Tree nuts</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>Other seeds</figcaption></figure>

### Non-specific lipid transfer proteins

<figure><img src="image_36.png" /><figcaption>Pru p 3</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>Fruits</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>Tree nuts</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>Legumes</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>Cereal grains</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>Weed pollen</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>Tree pollen</figcaption></figure>

**FIGURE 18** Distribution of allergenic members of the prolamin superfamily. The representative structures are Tri a 28, the dimeric α-amylase inhibitor from wheat (PDB: 1hss), Ara h 6, a 2S albumin from peanut (PDB: 1w2q), and Pru p 3, the type 1 nsLTP from peach (PDB: 2alg)
Members of the prolamin superfamily include the cereal prolamin seed storage proteins (gliadins and glutenins) and several families of disulfide-rich small proteins including the bifunctional inhibitors, the 2S albumin seed storage proteins, and the non-specific lipid transfer proteins (Figure 18).

Cereal prolamins are seed storage proteins that are exclusively found in the grains of cereal grasses. In contrast to the low molecular weight members of the superfamily, the α-helical domain of the cereal prolamins has been disrupted by an insertion of repetitive sequences.³²¹ Gliadins and glutenins represent the members of the cereal prolamin family. Gliadins are soluble in alcohol and are classified into α/β-, γ-, and ω-gliadins. Glutenins are polymeric proteins that are held together by interchain disulfide bonds. They are divided into high and low molecular weight groups.³²²

Like the cereal prolamins, the bifunctional inhibitors are only present in cereal grains. These allergens sensitize either via the respiratory tract by inhalation of the flour or via the gastro-intestinal tract by eating foods that contain wheat, barley or rice. The bifunctional inhibitors are 12 to 16 kDa proteins that are held together by 4 to 6 disulfide bonds.³²³ Monomeric, dimeric and tetrameric forms are distinguished according to the degree of oligomerization of their subunits. They are the major cause of baker's asthma but also play a role as plant food allergens.³²⁴

The 2S albumins are a major group of plant seed storage proteins. Most 2S albumins are synthesized as single-chain proteins that are subsequently cleaved into a small and a large subunit. Both subunits are held together as compact α-helical molecules by 4 to 5 disulfide bonds.³²⁵ Many of the important seed and tree nut allergens are 2S albumins.

Nonspecific lipid transfer proteins (nsLTPs) have been suggested to mediate the transfer of phospholipids between vesicles and membranes. However, plants use the three-dimensional scaffold of the nsLTPs in various ways and many nsLTPs play a role in plant defense

against fungi and bacteria or in the response to abiotic stress. nsLTPs are typically found in high concentrations in epidermal tissues of plant food. The majority of allergenic representatives belongs to nsLTP type 1 (~10 kDa) while recently some members of the type 2 (~7 kDa) were described. Allergenic nsLTPs are highly resistant to thermal and enzymatic digestion³²⁶. They are major allergens of fruits from the Rosaceae family. In addition, allergenic nsLTPs are present in nuts, seeds, vegetables, and Hevea brasiliensis (natural rubber latex). Besides their presence in plant foods, nsLTPs are also expressed in pollen of weeds, olive, and plane tree.

* Wheat contains several allergenic cereal prolamins. Tri a 19 is an ω-5-gliadin, Tri a 21 an α/β-gliadin, and Tri a 26 a high molecular weight glutenin from wheat (see chapter B16).

* Hor v 15 is a monomeric α-amylase inhibitor from barley. Tri a 28 is a dimeric and Tri a 29 a tetrameric α-amylase inhibitor from wheat (see chapter B16).

* Allergenic 2S albumins include Ara h 2 and Ara h 6 from peanut, Ber e 1 from Brazil nut, Cor a 14 from hazelnut, Jug r 1 from English walnut, Ses i 1 from sesame seeds, and Sin a 1 from yellow mustard (see chapter C08).

* Rosaceae fruit type 1 nsLTPs include Mal d 3 from apple and Pru p 3 from peach. Representative allergenic type 1 nsLTPs from tree nuts are Cor a 8 from hazelnut and Jug r 3 from walnut. Pollen nsLTPs include Pla a 3 from plane tree and Art v 3 from mugwort. Zea m 14 and Tri a 14 are the nsLTPs from maize and wheat. Can s 3 is the nsLTP from Indian hemp (see chapter C03).

* Allergenic type 2 nsLTPs were identified in tomato (Sola l 6), celery tuber (Api g 6) and peanut (Ara h 16).

## 2.2. EF-hand superfamily

A wide range of calcium-binding proteins share a conserved motif consisting of a 12 residue calcium-binding loop flanked on both sides

### Bifunctional inhibitors

<figure><img src="image_30.png" /><figcaption>Tri a 28</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>Cereal grains</figcaption></figure>

### 2S albumins

<figure><img src="image_32.png" /><figcaption>Ara h 6</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>Legumes</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>Tree nuts</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>Other seeds</figcaption></figure>

### Non-specific lipid transfer proteins

<figure><img src="image_36.png" /><figcaption>Pru p 3</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>Fruits</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>Tree nuts</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>Legumes</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>Cereal grains</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>Weed pollen</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>Tree pollen</figcaption></figure>

**FIGURE 18** Distribution of allergenic members of the prolamin superfamily. The representative structures are Tri a 28, the dimeric α-amylase inhibitor from wheat (PDB: 1hss), Ara h 6, a 2S albumin from peanut (PDB: 1w2q), and Pru p 3, the type 1 nsLTP from peach (PDB: 2alg)
by an α-helix of 12 residues in length.³²⁷ The term EF-hand derives from the nomenclature of carp parvalbumin, which possesses 6 α-helices, named A to F. These helices are paired to form the calcium-binding motifs, which are now referred to as EF-hands after the third of those pairs, which folds into a structure prototypical for this motif. The biological functions of EF-hand proteins include signaling and calcium buffering or transport. Allergenic members of the EF-hand superfamily are found mostly in animals with the exception of the plant-specific polcalcins (Figure 19).

Polcalcins are 9 kDa calcium-binding pollen proteins of unknown biological function. While regular polcalcins contain two EF hand domains, several polcalcin-related allergens with three or four EF hand domains have been described.³²⁸ Polcalcins were shown to be minor albeit highly cross-reactive allergens identified in pollen from diverse plant families (see chapter C06).

Parvalbumins are 12 kDa proteins that contain two EF hand domains. They are found in fast-twitch muscle fibers of vertebrates and bind calcium ions during muscle relaxation. Parvalbumins from fish and amphibians are major food allergens eliciting IgE responses in most fish-allergic individuals (see chapter C11).³²⁹

The invertebrate 20-22 kDa sarcoplasmic calcium-binding proteins (SCPs) are cytosolic calcium buffers that are characterized by

four EF-hand signatures of which two or three are functional.³³⁰ SCPs are the functional analogs of parvalbumins in invertebrate fast-twitch muscle. They promote rapid muscle relaxation by facilitating Ca²⁺ translocation from myofibrils to the sarcoplasmic reticulum.³³¹

Troponin Cs, 18-21 kDa proteins with four EF-hand Ca²⁺ binding domains, are part of the macromolecular complex composed of troponins, tropomyosin, actin and myosin, and is specifically involved in the regulation of muscle contraction³³², while parvalbumin promotes rapid relaxation through translocation of Ca²⁺ from troponin C into the sarcoplasmic reticulum via a Ca²⁺ pump.³³¹

Myosin light chains (MLCs) are subunits of myosins, motor proteins that play a role in muscle contraction and other motility processes in eukaryotic cells. The myosin molecule is a hexameric complex made up of two heavy chains and two pairs of calcium-binding light chains.³³³ Myosin light chains contain two Ca²⁺-binding EF-hand motifs.

* Allergenic 2 EF-hand polcalcins include the monomeric Bet v 4 from birch and the dimeric Phl p 7 from timothy grass and Che a 3 from white goosefoot.

* Allergenic 4 EF-hand polcalcin-like proteins are Bet v 3 from birch, Amb a 10 from ragweed and Ole e 8 from olive pollen.

## Polcalcins

<figure><img src="image_43.png" /></figure>

Bet v 4

<figure><img src="image_44.png" /></figure>

Grass pollen

<figure><img src="image_45.png" /></figure>

Tree pollen

<figure><img src="image_46.png" /></figure>

Weed pollen

## Parvalbumins

<figure><img src="image_47.png" /></figure>

Cyp c 1

<figure><img src="image_48.png" /></figure>

Carp

Cod

Salmon

## Sarcoplasmic Ca-binding proteins

<figure><img src="image_49.png" /></figure>

Bra 1 SCP

<figure><img src="image_50.png" /></figure>

Crustaceans

<figure><img src="image_51.png" /></figure>

Insects

<figure><img src="image_52.png" /></figure>

Molluscs

## Troponin C

## Myosin light chain

<figure><img src="image_53.png" /></figure>

Let i TnC

<figure><img src="image_54.png" /></figure>

Sch m MLC

<figure><img src="image_55.png" /></figure>

Crustaceans

<figure><img src="image_56.png" /></figure>

Mites

<figure><img src="image_57.png" /></figure>

Cockroaches

**FIGURE 19** Distribution of allergenic members of the EF-hand superfamily. The representative structures of allergens are Bet v 4, the polcalcin from birch pollen (PDB: 1h4b) and Cyp c 1, the parvalbumin from carp (PDB: 4cpv). For the other families, representative structures of nonallergenic proteins are shown: Bra I SCP, a sarcoplasmic Ca-binding protein from amphioxus (*Branchiostoma lanceolatum*; PDB: 2sas), Let i TnC, a troponin C from water bug (*Lethocerus indicus*; PDB: 2jnf), and Sch m MLC, the myosin light chain from *Schistosoma mansoni* (PDB: 3jax). Bound calcium ions are represented by pink spheres. No structures of allergenic EF-hand superfamily members other than polcalcins and parvalbumins have been described.
by an α-helix of 12 residues in length.³²⁷ The term EF-hand derives from the nomenclature of carp parvalbumin, which possesses 6 α-helices, named A to F. These helices are paired to form the calcium-binding motifs, which are now referred to as EF-hands after the third of those pairs, which folds into a structure prototypical for this motif. The biological functions of EF-hand proteins include signaling and calcium buffering or transport. Allergenic members of the EF-hand superfamily are found mostly in animals with the exception of the plant-specific polcalcins (Figure 19).

Polcalcins are 9 kDa calcium-binding pollen proteins of unknown biological function. While regular polcalcins contain two EF hand domains, several polcalcin-related allergens with three or four EF hand domains have been described.³²⁸ Polcalcins were shown to be minor albeit highly cross-reactive allergens identified in pollen from diverse plant families (see chapter C06).

Parvalbumins are 12 kDa proteins that contain two EF hand domains. They are found in fast-twitch muscle fibers of vertebrates and bind calcium ions during muscle relaxation. Parvalbumins from fish and amphibians are major food allergens eliciting IgE responses in most fish-allergic individuals (see chapter C11).³²⁹

The invertebrate 20-22 kDa sarcoplasmic calcium-binding proteins (SCPs) are cytosolic calcium buffers that are characterized by

four EF-hand signatures of which two or three are functional.³³⁰ SCPs are the functional analogs of parvalbumins in invertebrate fast-twitch muscle. They promote rapid muscle relaxation by facilitating Ca²⁺ translocation from myofibrils to the sarcoplasmic reticulum.³³¹

Troponin Cs, 18-21 kDa proteins with four EF-hand Ca²⁺ binding domains, are part of the macromolecular complex composed of troponins, tropomyosin, actin and myosin, and is specifically involved in the regulation of muscle contraction³³², while parvalbumin promotes rapid relaxation through translocation of Ca²⁺ from troponin C into the sarcoplasmic reticulum via a Ca²⁺ pump.³³¹

Myosin light chains (MLCs) are subunits of myosins, motor proteins that play a role in muscle contraction and other motility processes in eukaryotic cells. The myosin molecule is a hexameric complex made up of two heavy chains and two pairs of calcium-binding light chains.³³³ Myosin light chains contain two Ca²⁺-binding EF-hand motifs.

* Allergenic 2 EF-hand polcalcins include the monomeric Bet v 4 from birch and the dimeric Phl p 7 from timothy grass and Che a 3 from white goosefoot.

* Allergenic 4 EF-hand polcalcin-like proteins are Bet v 3 from birch, Amb a 10 from ragweed and Ole e 8 from olive pollen.

## Polcalcins

<figure><img src="image_43.png" /></figure>

Bet v 4

<figure><img src="image_44.png" /></figure>

Grass pollen

<figure><img src="image_45.png" /></figure>

Tree pollen

<figure><img src="image_46.png" /></figure>

Weed pollen

## Parvalbumins

<figure><img src="image_47.png" /></figure>

Cyp c 1

<figure><img src="image_48.png" /></figure>

Carp

Cod

Salmon

## Sarcoplasmic Ca-binding proteins

<figure><img src="image_49.png" /></figure>

Bra 1 SCP

<figure><img src="image_50.png" /></figure>

Crustaceans

<figure><img src="image_51.png" /></figure>

Insects

<figure><img src="image_52.png" /></figure>

Molluscs

## Troponin C

## Myosin light chain

<figure><img src="image_53.png" /></figure>

Let i TnC

<figure><img src="image_54.png" /></figure>

Sch m MLC

<figure><img src="image_55.png" /></figure>

Crustaceans

<figure><img src="image_56.png" /></figure>

Mites

<figure><img src="image_57.png" /></figure>

Cockroaches

**FIGURE 19** Distribution of allergenic members of the EF-hand superfamily. The representative structures of allergens are Bet v 4, the polcalcin from birch pollen (PDB: 1h4b) and Cyp c 1, the parvalbumin from carp (PDB: 4cpv). For the other families, representative structures of nonallergenic proteins are shown: Bra I SCP, a sarcoplasmic Ca-binding protein from amphioxus (*Branchiostoma lanceolatum*; PDB: 2sas), Let i TnC, a troponin C from water bug (*Lethocerus indicus*; PDB: 2jnf), and Sch m MLC, the myosin light chain from *Schistosoma mansoni* (PDB: 3jax). Bound calcium ions are represented by pink spheres. No structures of allergenic EF-hand superfamily members other than polcalcins and parvalbumins have been described.
* Allergenic parvalbumins include Gad m 1 from Atlantic cod, Sal s 1 from Atlantic salmon, and Cyp c 1 from carp.

* Allergenic SCPs have been described in shrimps (e.g., Lit v 4–white shrimp), crabs (e.g., Scy p 4–mud crab), insects (e.g., Aed a 5–yellow fever mosquito), and mollusks (e.g., Cra a 4–Pacific oyster).

* Allergenic troponin C proteins are found in arthropods including crustaceans (e.g., Hom a 6–American lobster), mites (e.g., Der p 39–house dust mite) and cockroaches (e.g., Bla g 6–German cockroach). Allergenic myosin light chains are found in arthropods such as crustaceans (e.g., Scy p 3–mud crab), mites (e.g., Der p 26–house dust mite) and cockroaches (e.g., Bla g 8–German cockroach).

## 2.3. Tropomyosin-like superfamily

Tropomyosins are present in muscle and nonmuscle cells. In striated muscle, they mediate the interactions between the troponin complex and actin to regulate muscle contraction. Tropomyosin is an α-helical protein that forms a coiled-coil structure of two parallel helices containing two sets of seven alternating actin-binding sites. Tropomyosins were identified as animal food allergens in crustaceans, mollusks, and the fish parasite *Anisakis simplex*.334 Tropomyosins were also identified as respiratory allergens in arthropods (mites, cockroaches). Tropomyosin sequences are highly conserved, which explains the frequent cross-sensitization among tropomyosin-containing allergen sources (see chapter C05).335 Pen i 1 from Indian prawn, Bla g 7 from German cockroach, and Der p 10 from house dust mite are well-known allergenic tropomyosins (Figure 20).

## 2.4. Profilin-like superfamily

Profilins are small cytosolic proteins that are found in all eukaryotic cells. They bind to monomeric actin and various other proteins, thus regulating the dynamics of actin polymerization during processes

such as cell movement, cytokinesis, and signaling. Profilins from higher plants are highly conserved showing amino acid sequence identities of > 75% even between members from distantly related organisms.336 Due to extensive IgE cross-reactivity, extract-based diagnosis can be hampered by clinically irrelevant profilin reactivity. However, profilin sensitization is considered a risk factor for pollen-associated food allergy.337 Profilins are heat and digestion labile, but co-factors like antacids and fasting as well as damage of the oral mucosae may facilitate allergic food reactions.338,339 No substantial cross-reactivity between plant and human profilins has been shown so far (see chapter C01).

Allergenic profilins include Phl p 12 from grass pollen, Art v 4 from mugwort pollen, Bet v 2 from birch pollen, Ole e 2 from olive pollen, Cit s 2 from orange, Cuc m 2 from melon, and Mus a 1 from banana [Figure 21].

## 2.5. Cupin superfamily

The cupins are a large and functionally immensely diverse superfamily of proteins whose evolution can be followed from bacteria to eukaryotes including animals and higher plants. Cupin proteins are currently classified into 66 protein families. The largest families of bicupins (i.e., proteins that contain two cupin domains) are the 7/8S and 11S seed storage globulins that are the major components of plant seeds. They are important sources of proteins for the human diet but are also major allergens (see chapter C08).341,1649

7S globulins or vicilins are homotrimeric proteins of about 150 to 190 kDa. Their detailed subunit compositions vary considerably due to differences in proteolytic processing and glycosylation of the monomers. In mature 11S globulins (legumins), two trimers associate to form hexameric proteins (Figure 22).

* Allergenic vicilins include Ara h 1 from peanut, Gly m 5 from soybean, Jug r 2 from walnut, and Ses i 3 from sesame.

<figure><img src="image_58.png" /><figcaption>Rat Tpm</figcaption></figure>

<figure><img src="image_59.png" /><figcaption>Crustaceans</figcaption></figure>

<figure><img src="image_60.png" /><figcaption>Molluscs</figcaption></figure>

<figure><img src="image_61.png" /><figcaption>Mites</figcaption></figure>

<figure><img src="image_62.png" /><figcaption>Cockroaches</figcaption></figure>

**FIGURE 20** Distribution of allergenic tropomyosins. The structure shown is from the nonallergenic rat tropomyosin (PDB: 2b9c). No structure of an allergenic tropomyosin is available.

<figure><img src="image_63.png" /><figcaption>Bet v 2</figcaption></figure>

<figure><img src="image_64.png" /><figcaption>Fruits</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>Vegetables</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>Legumes</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>Tree nuts</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>Latex</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>Grass pollen</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>Weed pollen</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>Tree pollen</figcaption></figure>

**FIGURE 21** Distribution of allergenic profilins. The representative structure is from Bet v 2, the profilin from birch pollen (PDB: 1cqa).
* Allergenic parvalbumins include Gad m 1 from Atlantic cod, Sal s 1 from Atlantic salmon, and Cyp c 1 from carp.

* Allergenic SCPs have been described in shrimps (e.g., Lit v 4–white shrimp), crabs (e.g., Scy p 4–mud crab), insects (e.g., Aed a 5–yellow fever mosquito), and mollusks (e.g., Cra a 4–Pacific oyster).

* Allergenic troponin C proteins are found in arthropods including crustaceans (e.g., Hom a 6–American lobster), mites (e.g., Der p 39–house dust mite) and cockroaches (e.g., Bla g 6–German cockroach). Allergenic myosin light chains are found in arthropods such as crustaceans (e.g., Scy p 3–mud crab), mites (e.g., Der p 26–house dust mite) and cockroaches (e.g., Bla g 8–German cockroach).

## 2.3. Tropomyosin-like superfamily

Tropomyosins are present in muscle and nonmuscle cells. In striated muscle, they mediate the interactions between the troponin complex and actin to regulate muscle contraction. Tropomyosin is an α-helical protein that forms a coiled-coil structure of two parallel helices containing two sets of seven alternating actin-binding sites. Tropomyosins were identified as animal food allergens in crustaceans, mollusks, and the fish parasite *Anisakis simplex*.334 Tropomyosins were also identified as respiratory allergens in arthropods (mites, cockroaches). Tropomyosin sequences are highly conserved, which explains the frequent cross-sensitization among tropomyosin-containing allergen sources (see chapter C05).335 Pen i 1 from Indian prawn, Bla g 7 from German cockroach, and Der p 10 from house dust mite are well-known allergenic tropomyosins (Figure 20).

## 2.4. Profilin-like superfamily

Profilins are small cytosolic proteins that are found in all eukaryotic cells. They bind to monomeric actin and various other proteins, thus regulating the dynamics of actin polymerization during processes

such as cell movement, cytokinesis, and signaling. Profilins from higher plants are highly conserved showing amino acid sequence identities of > 75% even between members from distantly related organisms.336 Due to extensive IgE cross-reactivity, extract-based diagnosis can be hampered by clinically irrelevant profilin reactivity. However, profilin sensitization is considered a risk factor for pollen-associated food allergy.337 Profilins are heat and digestion labile, but co-factors like antacids and fasting as well as damage of the oral mucosae may facilitate allergic food reactions.338,339 No substantial cross-reactivity between plant and human profilins has been shown so far (see chapter C01).

Allergenic profilins include Phl p 12 from grass pollen, Art v 4 from mugwort pollen, Bet v 2 from birch pollen, Ole e 2 from olive pollen, Cit s 2 from orange, Cuc m 2 from melon, and Mus a 1 from banana [Figure 21].

## 2.5. Cupin superfamily

The cupins are a large and functionally immensely diverse superfamily of proteins whose evolution can be followed from bacteria to eukaryotes including animals and higher plants. Cupin proteins are currently classified into 66 protein families. The largest families of bicupins (i.e., proteins that contain two cupin domains) are the 7/8S and 11S seed storage globulins that are the major components of plant seeds. They are important sources of proteins for the human diet but are also major allergens (see chapter C08).341,1649

7S globulins or vicilins are homotrimeric proteins of about 150 to 190 kDa. Their detailed subunit compositions vary considerably due to differences in proteolytic processing and glycosylation of the monomers. In mature 11S globulins (legumins), two trimers associate to form hexameric proteins (Figure 22).

* Allergenic vicilins include Ara h 1 from peanut, Gly m 5 from soybean, Jug r 2 from walnut, and Ses i 3 from sesame.

<figure><img src="image_58.png" /><figcaption>Rat Tpm</figcaption></figure>

<figure><img src="image_59.png" /><figcaption>Crustaceans</figcaption></figure>

<figure><img src="image_60.png" /><figcaption>Molluscs</figcaption></figure>

<figure><img src="image_61.png" /><figcaption>Mites</figcaption></figure>

<figure><img src="image_62.png" /><figcaption>Cockroaches</figcaption></figure>

**FIGURE 20** Distribution of allergenic tropomyosins. The structure shown is from the nonallergenic rat tropomyosin (PDB: 2b9c). No structure of an allergenic tropomyosin is available.

<figure><img src="image_63.png" /><figcaption>Bet v 2</figcaption></figure>

<figure><img src="image_64.png" /><figcaption>Fruits</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>Vegetables</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>Legumes</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>Tree nuts</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>Latex</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>Grass pollen</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>Weed pollen</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>Tree pollen</figcaption></figure>

**FIGURE 21** Distribution of allergenic profilins. The representative structure is from Bet v 2, the profilin from birch pollen (PDB: 1cqa).
* Allergenic legumins include the peanut allergen Ara h 3, Gly m 6 from soybean, Ber e 2 from Brazil nut, and Fag e 1 from buckwheat.

## 2.6. Bet v 1-like superfamily

The version 34.0 of the Pfam database attributes 104,941 proteins from 7,238 species with structures related to the major birch pollen allergen Bet v 1 to the Bet v 1-like superfamily (https://www.ebi.ac.uk/interpro/set/pfam/CL0209/, accessed 08/2021). These proteins are found in all domains of life and are distributed between 24 families. The members of this superfamily share the same structure, which is composed of a 7-stranded antiparallel β-sheet and 3 α-helices.³⁴⁰ There is a cavity between the β-sheet and the long C terminal α-helix, which is able to bind a variety of lipid and flavonoid molecules. So far, allergens were identified only in the Bet v 1 family, whose members are found exclusively in plants (Figure 23).

<figure><img src="image_72.png" /><figcaption>**FIGURE 22** Distribution of allergenic members of the cupin superfamily. The depicted structures are Ara h 1, the 7S globulin from peanut (PDB: 3smh) and Ara h 3, the 11S globulin from peanut (PDB: 3c3v).</figcaption></figure>

The PR-10 proteins are the largest of the 11 subfamilies within the Bet v 1 family. The expression of these proteins is either induced by pathogen attack or abiotic stress, or it is developmentally regulated. PR-10 proteins are expressed in high concentrations in reproductive tissues such as pollen, seeds and fruits. Allergenic PR-10 proteins from pollen are found exclusively in pollen from members of the order Fagales (birch-related and beech-related trees, e.g., Cor a 1 from hazel, Aln g 1 from alder and Que a 1 from oak). Many birch pollen-allergic patients show allergic reactions to various fruits and vegetables, which are caused by IgE cross-reactivity between Bet v 1 and homologous allergens from plant foods. Most Bet v 1-related food allergens have been found in members of certain plant families: Rosaceae (e.g., Mal d 1 from apple, Pyr c 1 from pear and Pru p 1 from peach), Apiaceae (Api g 1 from celery and Dau c 1 from carrot), and Fabaceae (Gly m 4 from soybean and Ara h 8 from peanut). In addition, not all close homologues of Bet v 1 are allergens (see chapter CO2).

Two other subfamilies, whose members show only low sequence similarities and IgE-cross-reactivities with PR-10 subfamily members, contain allergenic members: Act d 11, a minor allergen from kiwifruit, is the first described allergen from the RRP/MLP (ripening-related proteins/major latex proteins) subfamily.³⁴² Vig r 6 from mung bean is the first described allergen from the CSBP (cytokinin-specific binding proteins) subfamily of the Bet v 1 family.³⁴³

## 2.7. Calicyn superfamily

The calycin superfamily comprises 16 families. Although structurally similar, calycins have rather low sequence similarities. The calycin architecture is based on an eight- or nine-stranded β-barrel, which can bind a variety of different ligands.³⁴⁴

Lipocalins are transporters for small hydrophobic molecules, such as lipids, steroid hormones, bilins, and retinoids. Allergens from this protein family include β-lactoglobulins, mammalian dander allergens, and cytoplasmic fatty acid-binding proteins.²⁶,³⁴⁵ Beta-lactoglobulins are the major whey protein of ruminant species. Bos d 5, a β-lactoglobulin, is a major cow's milk allergen. Cross-reactions to milk proteins from other species have also been described.

<figure><img src="image_73.png" /><figcaption>**FIGURE 23** Distribution of allergenic members of the Bet v 1 family. The representative structures shown are Bet v 1, the PR-10 from birch pollen (PDB: 1bv1), Act d 11, the MLP/RRP from kiwifruit (PDB: 4igv) and Vig r 6, the CSBP from mung bean (PDB: 2flh).</figcaption></figure>
* Allergenic legumins include the peanut allergen Ara h 3, Gly m 6 from soybean, Ber e 2 from Brazil nut, and Fag e 1 from buckwheat.

## 2.6. Bet v 1-like superfamily

The version 34.0 of the Pfam database attributes 104,941 proteins from 7,238 species with structures related to the major birch pollen allergen Bet v 1 to the Bet v 1-like superfamily (https://www.ebi.ac.uk/interpro/set/pfam/CL0209/, accessed 08/2021). These proteins are found in all domains of life and are distributed between 24 families. The members of this superfamily share the same structure, which is composed of a 7-stranded antiparallel β-sheet and 3 α-helices.³⁴⁰ There is a cavity between the β-sheet and the long C terminal α-helix, which is able to bind a variety of lipid and flavonoid molecules. So far, allergens were identified only in the Bet v 1 family, whose members are found exclusively in plants (Figure 23).

<figure><img src="image_72.png" /><figcaption>**FIGURE 22** Distribution of allergenic members of the cupin superfamily. The depicted structures are Ara h 1, the 7S globulin from peanut (PDB: 3smh) and Ara h 3, the 11S globulin from peanut (PDB: 3c3v).</figcaption></figure>

The PR-10 proteins are the largest of the 11 subfamilies within the Bet v 1 family. The expression of these proteins is either induced by pathogen attack or abiotic stress, or it is developmentally regulated. PR-10 proteins are expressed in high concentrations in reproductive tissues such as pollen, seeds and fruits. Allergenic PR-10 proteins from pollen are found exclusively in pollen from members of the order Fagales (birch-related and beech-related trees, e.g., Cor a 1 from hazel, Aln g 1 from alder and Que a 1 from oak). Many birch pollen-allergic patients show allergic reactions to various fruits and vegetables, which are caused by IgE cross-reactivity between Bet v 1 and homologous allergens from plant foods. Most Bet v 1-related food allergens have been found in members of certain plant families: Rosaceae (e.g., Mal d 1 from apple, Pyr c 1 from pear and Pru p 1 from peach), Apiaceae (Api g 1 from celery and Dau c 1 from carrot), and Fabaceae (Gly m 4 from soybean and Ara h 8 from peanut). In addition, not all close homologues of Bet v 1 are allergens (see chapter CO2).

Two other subfamilies, whose members show only low sequence similarities and IgE-cross-reactivities with PR-10 subfamily members, contain allergenic members: Act d 11, a minor allergen from kiwifruit, is the first described allergen from the RRP/MLP (ripening-related proteins/major latex proteins) subfamily.³⁴² Vig r 6 from mung bean is the first described allergen from the CSBP (cytokinin-specific binding proteins) subfamily of the Bet v 1 family.³⁴³

## 2.7. Calicyn superfamily

The calycin superfamily comprises 16 families. Although structurally similar, calycins have rather low sequence similarities. The calycin architecture is based on an eight- or nine-stranded β-barrel, which can bind a variety of different ligands.³⁴⁴

Lipocalins are transporters for small hydrophobic molecules, such as lipids, steroid hormones, bilins, and retinoids. Allergens from this protein family include β-lactoglobulins, mammalian dander allergens, and cytoplasmic fatty acid-binding proteins.²⁶,³⁴⁵ Beta-lactoglobulins are the major whey protein of ruminant species. Bos d 5, a β-lactoglobulin, is a major cow's milk allergen. Cross-reactions to milk proteins from other species have also been described.

<figure><img src="image_73.png" /><figcaption>**FIGURE 23** Distribution of allergenic members of the Bet v 1 family. The representative structures shown are Bet v 1, the PR-10 from birch pollen (PDB: 1bv1), Act d 11, the MLP/RRP from kiwifruit (PDB: 4igv) and Vig r 6, the CSBP from mung bean (PDB: 2flh).</figcaption></figure>