<figure><img src="image_120.png" /><figcaption>**FIGURE 38** Cartoon showing the extent of the hydrophobic cavity (colored violet) of Pru p 3, the peach nsLTP (cartoon drawn with the Proteins Plus server (https://proteins-plus/)), and some apolar ligands for the nsLTP</figcaption></figure>

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

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

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

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

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

<figure><img src="image_121.png" /><figcaption>**FIGURE 39** A. Cut section of Pru p 3 in complex with oleic acid (OLA) inserted in the hydrophobic cavity. B. Cartoon showing how oleic acid inserts into the hydrophobic cavity of Pru p 3 in the vicinity of the long C-terminal loop (colored red) of the nsLTP. C. Surface occupied by the C-terminal loop (colored red) of Pru p 3 in contact with OLA. D. Surface occupied by the continuous IgE-binding epitope 3 (colored red) of Pru p 3. The yellow line delineates the region of epitope 3 impacted by binding of oleic acid to the hydrophobic cavity of Pru p 3.</figcaption></figure>

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

## 6 – Other allergens families

For Der p 5, a member of the group 5 mite allergen family, lipid binding has been shown to activate TLR2 signalling in airway epithelial cells.
## 7 – Immunomodulation by lipids independently of allergens

The recognition by dendritic cells (APC) of the nsLTP-lipid (Pru p 3-ligand) complex, is attributed to CD1d molecules, which are receptors structurally similar to MHC-I that process and present lipids and glycolipids to CD1-restricted unconventional T-cells, a particular subset of T-lymphocytes that specifically recognize lipids and phospholipids<sup>485-487</sup> (Figure 40). CD1d on dendritic cells further present the lipid ligand to CD1-restricted invariant NKT cells (iNKT cells). The activation of iNKT cells triggers the release of various cytokines, including IL-4 that promotes the transformation of Tfh0 cells into Tfh2 cells upon the specific recognition of T cell epitopes of the allergens by MHC-II molecules. The Tfh2 cells will trigger the activation of B lymphocytes and their transformation into IgE-producing plasma cells.

Thus, lipid ligands associated to nsLTPs offer a nice example of foreign molecules capable of modulating the allergic response using activation pathways involved in the innate immune response to pathogenic microorganisms and the associated receptors, e.g., the CD1d receptors of dendritic cells.

In addition to phytosphingosine, other lipids and lipid derivatives, either free or in complex with carrier allergens, are capable of activating CD1-restricted T cells. In this respect, also lipids extracted from cypress pollen, olive pollen and Brazil nut seed were capable to activate iNKT cells.<sup>488</sup>

Other receptors specific to the innate immune response such as TLR also recognize the lipid fractions from olive or ryegrass pollens,

dust mites, and cat and dog danders.<sup>479</sup> Additionally, many pollen grains are able to release a number of lipid mediators, the so-called PALMS (pollen-associated lipid mediators), when exposed to water. These PALMS which exhibit pronounced similarities to eicosanoids (leukotrienes, prostaglandins),<sup>489</sup> enhance the inflammatory response and induce a Tfh2 response.<sup>469,490</sup> Finally, nsLTPs appear as lipid carrier allergens that act as adjuvants, and thus trigger and activate pathways occurring in the innate immune response, to reinforce both their allergenic and inflammatory potential.<sup>479</sup>

## 8 – Conclusions

In summary, there is growing interest in the role of small molecules that are present in various tissues either present as ligand of allergenic proteins or as components of the surrounding matrix with a potential effect on the immune response. There is good evidence that ligands bound to a range of allergens provide increased protein stability against degradation, which in turn can have an impact on allergic sensitization. Likewise, IgE-binding activity can increase upon ligand binding as shown for nsLTPs. For some small molecules present in certain plant and animal derived tissues, also their function as immunoactive substances has been confirmed. Nevertheless, the different hypotheses on the role and relevance of additional components from allergenic sources on both, the allergic sensitization and allergic effector phase needs still more studies to provide a better understanding of this immune response.

<figure><img src="image_123.png" /></figure>

**FIGURE 40** Cartoon showing how lipids can contribute to the allergic response. 1. Recognition of the nsLTP-lipid complex by CD1d on dendritic cells (DC). 2. Presentation by CD1d of lipids to iNKT cells promotes the differentiation of 3. Tfh0 (T follicular helper cells type 0) into Tfh2 (T follicular helper cells type 2) cells. 4. Tfh2 cells activate B cells (B) and 5. induce their subsequent differentiation into IgE producing plasma cells (PC).
## 7 – Immunomodulation by lipids independently of allergens

The recognition by dendritic cells (APC) of the nsLTP-lipid (Pru p 3-ligand) complex, is attributed to CD1d molecules, which are receptors structurally similar to MHC-I that process and present lipids and glycolipids to CD1-restricted unconventional T-cells, a particular subset of T-lymphocytes that specifically recognize lipids and phospholipids<sup>485-487</sup> (Figure 40). CD1d on dendritic cells further present the lipid ligand to CD1-restricted invariant NKT cells (iNKT cells). The activation of iNKT cells triggers the release of various cytokines, including IL-4 that promotes the transformation of Tfh0 cells into Tfh2 cells upon the specific recognition of T cell epitopes of the allergens by MHC-II molecules. The Tfh2 cells will trigger the activation of B lymphocytes and their transformation into IgE-producing plasma cells.

Thus, lipid ligands associated to nsLTPs offer a nice example of foreign molecules capable of modulating the allergic response using activation pathways involved in the innate immune response to pathogenic microorganisms and the associated receptors, e.g., the CD1d receptors of dendritic cells.

In addition to phytosphingosine, other lipids and lipid derivatives, either free or in complex with carrier allergens, are capable of activating CD1-restricted T cells. In this respect, also lipids extracted from cypress pollen, olive pollen and Brazil nut seed were capable to activate iNKT cells.<sup>488</sup>

Other receptors specific to the innate immune response such as TLR also recognize the lipid fractions from olive or ryegrass pollens,

dust mites, and cat and dog danders.<sup>479</sup> Additionally, many pollen grains are able to release a number of lipid mediators, the so-called PALMS (pollen-associated lipid mediators), when exposed to water. These PALMS which exhibit pronounced similarities to eicosanoids (leukotrienes, prostaglandins),<sup>489</sup> enhance the inflammatory response and induce a Tfh2 response.<sup>469,490</sup> Finally, nsLTPs appear as lipid carrier allergens that act as adjuvants, and thus trigger and activate pathways occurring in the innate immune response, to reinforce both their allergenic and inflammatory potential.<sup>479</sup>

## 8 – Conclusions

In summary, there is growing interest in the role of small molecules that are present in various tissues either present as ligand of allergenic proteins or as components of the surrounding matrix with a potential effect on the immune response. There is good evidence that ligands bound to a range of allergens provide increased protein stability against degradation, which in turn can have an impact on allergic sensitization. Likewise, IgE-binding activity can increase upon ligand binding as shown for nsLTPs. For some small molecules present in certain plant and animal derived tissues, also their function as immunoactive substances has been confirmed. Nevertheless, the different hypotheses on the role and relevance of additional components from allergenic sources on both, the allergic sensitization and allergic effector phase needs still more studies to provide a better understanding of this immune response.

<figure><img src="image_122.png" /></figure>

**FIGURE 40** Cartoon showing how lipids can contribute to the allergic response. 1. Recognition of the nsLTP-lipid complex by CD1d on dendritic cells (DC). 2. Presentation by CD1d of lipids to iNKT cells promotes the differentiation of 3. Tfh0 (T follicular helper cells type 0) into Tfh2 (T follicular helper cells type 2) cells. 4. Tfh2 cells activate B cells (B) and 5. induce their subsequent differentiation into IgE producing plasma cells (PC).
# A12 - Molecular exposure: Systematic approaches, clinical significance, and harmonization

Martin D Chapman, Adnan Custovic, Clare E N Mills, Thomas Holzhauser

## Highlights

* Molecular allergen exposure assessment is a critical process for the investigation of environmental and food allergens and their relationship to allergic diseases.

* State-of-the-art multiplex technologies, including immunoassays and MS, will facilitate high throughput exposure assessment based on specific allergens that will enable thresholds for risk assessment to be established.

* Molecular exposure assessments, coupled with analyses of other environmental factors and genetic predisposition, will facilitate comprehensive epidemiologic and population studies of the role of the exposome in causing allergic respiratory diseases.

* Harmonization of molecular exposure assessments is urgently needed. This will require mutual collaboration between technology providers, academic and clinical investigators, industry and regulatory authorities to design and execute multi-center studies for validation of sampling plans and analytical detection methods.

Disclaimer: "The views expressed in this review are the personal views of the author Thomas Holzhauser and may not be understood or quoted as being made on behalf of or reflecting the position of the respective national competent authority, the European Medicines Agency, or one of its committees or working parties."

## 1 - Introduction

The use of allergen molecules as markers of environmental exposure was the first practical application of allergens and preceded their use in molecular diagnostics. Instead of counting mites, pets, cockroaches or rodents, specific immunoassays were developed, which measured major allergens (Der p 1, Der f 1, Fel d 1, Can f 1, Bla g 1, Bla g 2, Rat n 1, Mus m 1) in dust, air, and other environmental samples.<sup>491</sup>

Measurements of these allergens provided an objective and quantitative index of exposure that could be directly compared between study populations and cohorts. That the allergens being measured were a primary cause of IgE sensitization underscored this molecular approach to environmental exposure assessment.

Allergen measurements have been widely used for exposure assessments in clinical and epidemiological studies to investigate the relationships between allergen exposure and sensitization; disease associations and risk factors; geographic and climatic differences

in exposure; occupational exposures; and factors influencing the aerodynamic properties of allergens.<sup>492</sup> Other applications include efficacy testing of products, devices, and mitigation processes; assessments of the potency of therapeutic products; and monitoring of allergen exposure in Environmental Exposure Chambers (EEC) as part of clinical trials of allergy therapeutics.<sup>493</sup> Recently, the molecular exposure approach has been extended to include food allergens and pollen allergens. Processing of foods presents challenges for allergen measurements. However, significant progress is being made by monitoring specific allergens in foods and quantifying them in food products, as well as in environmental samples, such as dust. This has become especially important in identifying and quantifying the presence of unintended allergens in foods, which are not added as ingredients but may find their way into foods during food production/preparation and cause allergic reactions.<sup>494</sup> While pollen grain counts remain the standard for assessing pollen exposure, measuring allergen levels in pollen grains has demonstrated differences in geographic variability and allergen potency that are becoming more important with the advent of climate change.<sup>495</sup> Finally, multiplexing of allergen assays has greatly expanded the scope of allergen measurements and for both indoor allergens and foods. The most important allergens can be measured in a single test. The application of mass spectrometry is also providing more information and new ways of quantifying allergens whether in environmental samples or foods and can provide complementary data to immunoassay methods.<sup>496,497</sup>

## 2 - Allergen Exposure: Objectives, Methods, and Applications

The objectives of molecular exposure assessment can be summarized as follows:

(i) To provide consistent and reliable indices of environmental allergen exposure that are directly comparable.

(ii) To use high throughput sampling methods and assay technologies that have adequate sensitivity, specificity and quantification, with validated performance parameters that can be verified through multi-center ring trials.

(iii) To reliably assess the risks and outcomes of allergen exposure in different localities, populations, and circumstances in relation to health effects and as a guide to public policy and improving quality of life.

### Sampling Methods

The standard method for sampling indoor allergens has been to collect reservoir dust samples from bedding, bedrooms and other living spaces using a modified hand-held vacuum cleaner with a dust collection device. Typically, an area of ~0.25 m-2 is sampled for 2 minutes, and results are expressed as ng or µg allergen per gram of dust.<sup>491</sup> This approach accommodates dust mite and cockroach allergens, including Der p 1, Der p 2, Bla g 1, and Bla g 2, which do not readily become
# A12 – Molecular exposure: Systematic approaches, clinical significance, and harmonization

Martin D Chapman, Adnan Custovic, Clare E N Mills, Thomas Holzhauser

## Highlights

* Molecular allergen exposure assessment is a critical process for the investigation of environmental and food allergens and their relationship to allergic diseases.

* State-of-the-art multiplex technologies, including immunoassays and MS, will facilitate high throughput exposure assessment based on specific allergens that will enable thresholds for risk assessment to be established.

* Molecular exposure assessments, coupled with analyses of other environmental factors and genetic predisposition, will facilitate comprehensive epidemiologic and population studies of the role of the exposome in causing allergic respiratory diseases.

* Harmonization of molecular exposure assessments is urgently needed. This will require mutual collaboration between technology providers, academic and clinical investigators, industry and regulatory authorities to design and execute multi-center studies for validation of sampling plans and analytical detection methods.

Disclaimer: "The views expressed in this review are the personal views of the author Thomas Holzhauser and may not be understood or quoted as being made on behalf of or reflecting the position of the respective national competent authority, the European Medicines Agency, or one of its committees or working parties."

## 1 – Introduction

The use of allergen molecules as markers of environmental exposure was the first practical application of allergens and preceded their use in molecular diagnostics. Instead of counting mites, pets, cockroaches or rodents, specific immunoassays were developed, which measured major allergens (Der p 1, Der f 1, Fel d 1, Can f 1, Bla g 1, Bla g 2, Rat n 1, Mus m 1) in dust, air, and other environmental samples.<sup>491</sup>

Measurements of these allergens provided an objective and quantitative index of exposure that could be directly compared between study populations and cohorts. That the allergens being measured were a primary cause of IgE sensitization underscored this molecular approach to environmental exposure assessment.

Allergen measurements have been widely used for exposure assessments in clinical and epidemiological studies to investigate the relationships between allergen exposure and sensitization; disease associations and risk factors; geographic and climatic differences

in exposure; occupational exposures; and factors influencing the aerodynamic properties of allergens.<sup>492</sup> Other applications include efficacy testing of products, devices, and mitigation processes; assessments of the potency of therapeutic products; and monitoring of allergen exposure in Environmental Exposure Chambers (EEC) as part of clinical trials of allergy therapeutics.<sup>493</sup> Recently, the molecular exposure approach has been extended to include food allergens and pollen allergens. Processing of foods presents challenges for allergen measurements. However, significant progress is being made by monitoring specific allergens in foods and quantifying them in food products, as well as in environmental samples, such as dust. This has become especially important in identifying and quantifying the presence of unintended allergens in foods, which are not added as ingredients but may find their way into foods during food production/preparation and cause allergic reactions.<sup>494</sup> While pollen grain counts remain the standard for assessing pollen exposure, measuring allergen levels in pollen grains has demonstrated differences in geographic variability and allergen potency that are becoming more important with the advent of climate change.<sup>495</sup> Finally, multiplexing of allergen assays has greatly expanded the scope of allergen measurements and for both indoor allergens and foods. The most important allergens can be measured in a single test. The application of mass spectrometry is also providing more information and new ways of quantifying allergens whether in environmental samples or foods and can provide complementary data to immunoassay methods.<sup>496,497</sup>

## 2 – Allergen Exposure: Objectives, Methods, and Applications

The objectives of molecular exposure assessment can be summarized as follows:

(i) To provide consistent and reliable indices of environmental allergen exposure that are directly comparable.

(ii) To use high throughput sampling methods and assay technologies that have adequate sensitivity, specificity and quantification, with validated performance parameters that can be verified through multi-center ring trials.

(iii) To reliably assess the risks and outcomes of allergen exposure in different localities, populations, and circumstances in relation to health effects and as a guide to public policy and improving quality of life.

### Sampling Methods

The standard method for sampling indoor allergens has been to collect reservoir dust samples from bedding, bedrooms and other living spaces using a modified hand-held vacuum cleaner with a dust collection device. Typically, an area of ~0.25 m-2 is sampled for 2 minutes, and results are expressed as ng or µg allergen per gram of dust.<sup>491</sup> This approach accommodates dust mite and cockroach allergens, including Der p 1, Der p 2, Bla g 1, and Bla g 2, which do not readily become
airborne. Airborne mite (D. farinae) antigens were measured using a polyclonal ELISA and GSP air samplers.⁴⁹¹ While this is a promising approach, the specific molecular allergen components being measured in the ELISA have yet to be established.⁴⁹⁸ Personal air samplers, such as IOM and GSP samplers, and glass fibre filter cassettes, can be used for allergens of small particle size (2-10 µm diameter) that remain airborne for several hours (e.g., cat, dog and rodent allergens). Measurements of airborne Mus m 1 and Rat n 1 have been used to investigate laboratory animal allergy (LAA).⁴⁹⁹ The pharmaceutical industry has developed programs to monitor rodent allergen exposures as part of its facilities management programs, with the goal of reducing exposures to <5 ng/ m³ to mitigate LAA.⁵⁰⁰ Recently, electrostatic dust collectors have been used to measure settled airborne dust. These collectors are placed at a height of 1.6 m in a room and passively absorb allergens onto 2-4 polyester cloths over a 14-day period.⁵⁰¹ They can then be mailed to a lab for allergen testing. Silent electrokinetic air samplers that plug in to an electrical socket have been developed for high volume sampling and have been used to assess allergen exposure and the microbiomes in US homes.⁵⁰² Sampling of raw ingredients, in-production foods and finished food products presents different issues related to the chance of identifying low level contamination of allergenic ingredients either as a consequence of agricultural comingling or carry-over between manufacturing runs of foods.⁵⁰³ For example, change over between milk and dark chocolate, which can result in levels of milk allergens that pose a risk to allergic consumers.⁵⁰⁴

Immunoassay Methods and Applications

In early studies of environmental exposure, allergen molecules
were measured by two-site enzyme-linked immunosorbent assay

(ELISA) using specific monoclonal antibodies (mAb) for allergen capture and biotinylated mAb (or polyclonal antibodies) for detection. Assays were quantified using purified allergen standards of known protein content that were sub-standardized against international reference preparations, where available. Extracts of household dust samples were compared by ELISA in many epidemiologic studies, including emergency room asthma studies, the US National Institutes of Health Inner-City Asthma Consortium (ICAC) studies, the Manchester Allergy and Asthma Study (MAAS) and the German Multicentre Allergy Study (MAS) prospective birth cohorts.⁵⁰⁵ These studies provided comparative data on multiple allergen exposures across different parts of the world and their relationships to IgE sensitization and allergic disease.⁵⁰⁵ Although ELISA is a high throughput assay, measurement of each allergen in a separate assay was a limitation, especially for large studies involving multiple allergens. Nonetheless, the core components of ELISA, the mAb used for allergen capture and detection could readily be used in other assay systems. The X-ray crystal structure of allergen-mAb complexes has been determined for Der p 1, Der f 1, Der p 2, and Bla g 2 and the amino acid residues that form the allergen epitope are now known.⁵⁰⁶,⁵⁰⁷ This level of molecular analysis is not possible using polyclonal antibodies. The structural data confirm that the mAb epitopes are nonoverlapping and bind to distinct conformational sites on allergen molecules (Figure 41).

The mAb used in ELISA were incorporated into a Multiplex ARray
for Indoor Allergens (MARIA) using Luminex xMAP technology.
Capture mAb were covalently coupled to polystyrene beads with
internal fluorescent dyes. Bound allergen is detected using bioti-
nylated detector mAb and streptavidin-phycoerythrin. The beads
are analysed in a Luminex instrument in which a red laser distin-
guishes the bead set coupled to the capture mAb and a green laser

<figure><img src="image_124.png" /></figure>

**FIGURE 41** X-ray crystal structures of allergen-monoclonal antibody complexes: A, mAb 5H8, 10B9 and 4C1 in complex with Der p 1; B, mAb 7C11 and 4C3 binding to nonoverlapping sites on Bla g 2. Reproduced from Pomés et al, Frontiers in Immunology, with permission⁵⁰⁶
airborne. Airborne mite (D. farinae) antigens were measured using a polyclonal ELISA and GSP air samplers.⁴⁹¹ While this is a promising approach, the specific molecular allergen components being measured in the ELISA have yet to be established.⁴⁹⁸ Personal air samplers, such as IOM and GSP samplers, and glass fibre filter cassettes, can be used for allergens of small particle size (2-10 µm diameter) that remain airborne for several hours (e.g., cat, dog and rodent allergens). Measurements of airborne Mus m 1 and Rat n 1 have been used to investigate laboratory animal allergy (LAA).⁴⁹⁹ The pharmaceutical industry has developed programs to monitor rodent allergen exposures as part of its facilities management programs, with the goal of reducing exposures to <5 ng/ m³ to mitigate LAA.⁵⁰⁰ Recently, electrostatic dust collectors have been used to measure settled airborne dust. These collectors are placed at a height of 1.6 m in a room and passively absorb allergens onto 2-4 polyester cloths over a 14-day period.⁵⁰¹ They can then be mailed to a lab for allergen testing. Silent electrokinetic air samplers that plug in to an electrical socket have been developed for high volume sampling and have been used to assess allergen exposure and the microbiomes in US homes.⁵⁰² Sampling of raw ingredients, in-production foods and finished food products presents different issues related to the chance of identifying low level contamination of allergenic ingredients either as a consequence of agricultural comingling or carry-over between manufacturing runs of foods.⁵⁰³ For example, change over between milk and dark chocolate, which can result in levels of milk allergens that pose a risk to allergic consumers.⁵⁰⁴

Immunoassay Methods and Applications

In early studies of environmental exposure, allergen molecules
were measured by two-site enzyme-linked immunosorbent assay

(ELISA) using specific monoclonal antibodies (mAb) for allergen capture and biotinylated mAb (or polyclonal antibodies) for detection. Assays were quantified using purified allergen standards of known protein content that were sub-standardized against international reference preparations, where available. Extracts of household dust samples were compared by ELISA in many epidemiologic studies, including emergency room asthma studies, the US National Institutes of Health Inner-City Asthma Consortium (ICAC) studies, the Manchester Allergy and Asthma Study (MAAS) and the German Multicentre Allergy Study (MAS) prospective birth cohorts.⁵⁰⁵ These studies provided comparative data on multiple allergen exposures across different parts of the world and their relationships to IgE sensitization and allergic disease.⁵⁰⁵ Although ELISA is a high throughput assay, measurement of each allergen in a separate assay was a limitation, especially for large studies involving multiple allergens. Nonetheless, the core components of ELISA, the mAb used for allergen capture and detection could readily be used in other assay systems. The X-ray crystal structure of allergen-mAb complexes has been determined for Der p 1, Der f 1, Der p 2, and Bla g 2 and the amino acid residues that form the allergen epitope are now known.⁵⁰⁶,⁵⁰⁷ This level of molecular analysis is not possible using polyclonal antibodies. The structural data confirm that the mAb epitopes are nonoverlapping and bind to distinct conformational sites on allergen molecules (Figure 41).

The mAb used in ELISA were incorporated into a Multiplex ARray
for Indoor Allergens (MARIA) using Luminex xMAP technology.
Capture mAb were covalently coupled to polystyrene beads with
internal fluorescent dyes. Bound allergen is detected using bioti-
nylated detector mAb and streptavidin-phycoerythrin. The beads
are analysed in a Luminex instrument in which a red laser distin-
guishes the bead set coupled to the capture mAb and a green laser

<figure><img src="image_123.png" /></figure>

**FIGURE 41** X-ray crystal structures of allergen-monoclonal antibody complexes: A, mAb 5H8, 10B9 and 4C1 in complex with Der p 1; B, mAb 7C11 and 4C3 binding to nonoverlapping sites on Bla g 2. Reproduced from Pomés et al, Frontiers in Immunology, with permission⁵⁰⁶
detects the mean fluorescent intensity and measures the amount of allergen in the sample (Figure 42, Table 10).

Luminex xMAP technology is widely used in allergy/ immunology (e.g., for measuring cytokines). The MARIA assay was validated in an international ring trial and can measure up to 14 aeroallergens simultaneously, under the same assay conditions.⁵⁰⁸ The sensitivity of MARIA is 10-40-fold greater than ELISA, which is especially useful for measuring airborne allergens, e.g., in LAA. The development of MARIA enabled larger population studies and greatly increased the scope of exposome analyses, as illustrated by the U.S. National Health and Nutrition Examination Survey (NHANES, Case Study I).⁵⁰⁹

Molecule-based approaches are increasingly being applied to food allergy. Monoclonal antibody-based ELISA for specific food allergens, including peanut (Ara h 1, Ara h 2, Ara h 3, Ara h 6); soy (Gly m 4, Gly m 8); egg (Gal d 2); milk (Bos d 5, Bos d 11) and carrot (Dau c 1, Dau c 4) have been developed and applied to measure

allergens in foods, processed foods, immunotherapy products and early introduction foods.⁵¹⁰⁻⁵¹⁷ A modified multiplex array, MARIA for Foods, measures up to 17 major food allergens. MARIA for Foods measures all the food allergens that are regulated in the US (peanut, milk, egg, soy, tree nuts, sesame, fish, shellfish, wheat, celery, mustard) and which are also regulated in the European Union as part of a larger list of 14 allergenic foods.⁵¹⁸,⁵¹⁹ The MARIA for Foods has recently been used to measure the specific allergen content of commercial early introduction foods that are marketed to consumers as aids for the prevention of food allergy in infants.⁵²⁰,⁵²¹ MARIA technology has also been applied to measure environmental exposure to indoor allergens and food allergens in schools in the North-eastern US, as part of the Schools Inner-City Asthma Study (SICAS, Case Study II).⁵²²,⁵²³

Alternative methods of molecular exposure assessment have been developed including quantitative PCR (qPCR), DNA-based

<figure><img src="image_125.png" /><figcaption>TABLE 10 Antibodies, Allergen Standards and Bead Sets Used in MARIA for inhaled allergens</figcaption></figure>

<table><thead><tr><th>Source</th><th>Target allergens</th><th>Capture Ab</th><th>Allergen standard</th><th>Magnetic bead set</th><th>Detector Ab</th></tr></thead><tbody><tr><td rowspan="5">Dust Mite</td><td>Der p 1</td><td>10B9</td><td>nDer p 1</td><td>15</td><td>5H8</td></tr><tr><td>Der f 1</td><td>6A8</td><td>nDer f 1</td><td>37</td><td>4C1</td></tr><tr><td>Der p 2</td><td>1D8</td><td>nDer p 2</td><td>45</td><td>7A1</td></tr><tr><td>Der p 23</td><td>7A8</td><td>rDer p 23</td><td>63</td><td>pAb</td></tr><tr><td>Blo t 5</td><td>pAb</td><td>rBlo t 5</td><td>35</td><td>pAb</td></tr><tr><td rowspan="4">Animal</td><td>Fel d 1</td><td>6F9</td><td>nFel d 1</td><td>55</td><td>3E4</td></tr><tr><td>Can f 1</td><td>10D4</td><td>nCan f 1</td><td>12</td><td>6E9</td></tr><tr><td>Mus m 1</td><td>pAb</td><td>nMus m 1</td><td>57</td><td>pAb</td></tr><tr><td>Rat n 1</td><td>RUP-6</td><td>nRat n 1</td><td>64</td><td>RUP-1</td></tr><tr><td>Cockroach</td><td>Bla g 2</td><td>1F3</td><td>nBla g 2</td><td>26</td><td>4C3</td></tr><tr><td rowspan="4">Pollen</td><td>Bet v 1</td><td>6H4</td><td>rBet v 1</td><td>72</td><td>5B4</td></tr><tr><td>Phl p 5</td><td>1D11</td><td>rPhl p 5</td><td>67</td><td>Bo1</td></tr><tr><td>Amb a 1</td><td>2B6</td><td>nAmb a 1</td><td>29</td><td>4H7</td></tr><tr><td>Lol p 1</td><td>5G7</td><td>nLol p 1</td><td>13</td><td>7D8</td></tr></tbody></table>
detects the mean fluorescent intensity and measures the amount of allergen in the sample (Figure 42, Table 10).

Luminex xMAP technology is widely used in allergy/ immunology (e.g., for measuring cytokines). The MARIA assay was validated in an international ring trial and can measure up to 14 aeroallergens simultaneously, under the same assay conditions.⁵⁰⁸ The sensitivity of MARIA is 10-40-fold greater than ELISA, which is especially useful for measuring airborne allergens, e.g., in LAA. The development of MARIA enabled larger population studies and greatly increased the scope of exposome analyses, as illustrated by the U.S. National Health and Nutrition Examination Survey (NHANES, Case Study I).⁵⁰⁹

Molecule-based approaches are increasingly being applied to food allergy. Monoclonal antibody-based ELISA for specific food allergens, including peanut (Ara h 1, Ara h 2, Ara h 3, Ara h 6); soy (Gly m 4, Gly m 8); egg (Gal d 2); milk (Bos d 5, Bos d 11) and carrot (Dau c 1, Dau c 4) have been developed and applied to measure

allergens in foods, processed foods, immunotherapy products and early introduction foods.⁵¹⁰⁻⁵¹⁷ A modified multiplex array, MARIA for Foods, measures up to 17 major food allergens. MARIA for Foods measures all the food allergens that are regulated in the US (peanut, milk, egg, soy, tree nuts, sesame, fish, shellfish, wheat, celery, mustard) and which are also regulated in the European Union as part of a larger list of 14 allergenic foods.⁵¹⁸,⁵¹⁹ The MARIA for Foods has recently been used to measure the specific allergen content of commercial early introduction foods that are marketed to consumers as aids for the prevention of food allergy in infants.⁵²⁰,⁵²¹ MARIA technology has also been applied to measure environmental exposure to indoor allergens and food allergens in schools in the North-eastern US, as part of the Schools Inner-City Asthma Study (SICAS, Case Study II).⁵²²,⁵²³

Alternative methods of molecular exposure assessment have been developed including quantitative PCR (qPCR), DNA-based

<figure><img src="image_124.png" /><figcaption>TABLE 10 Antibodies, Allergen Standards and Bead Sets Used in MARIA for inhaled allergens</figcaption></figure>

<table><thead><tr><th>Source</th><th>Target allergens</th><th>Capture Ab</th><th>Allergen standard</th><th>Magnetic bead set</th><th>Detector Ab</th></tr></thead><tbody><tr><td rowspan="5">Dust Mite</td><td>Der p 1</td><td>10B9</td><td>nDer p 1</td><td>15</td><td>5H8</td></tr><tr><td>Der f 1</td><td>6A8</td><td>nDer f 1</td><td>37</td><td>4C1</td></tr><tr><td>Der p 2</td><td>1D8</td><td>nDer p 2</td><td>45</td><td>7A1</td></tr><tr><td>Der p 23</td><td>7A8</td><td>rDer p 23</td><td>63</td><td>pAb</td></tr><tr><td>Blo t 5</td><td>pAb</td><td>rBlo t 5</td><td>35</td><td>pAb</td></tr><tr><td rowspan="4">Animal</td><td>Fel d 1</td><td>6F9</td><td>nFel d 1</td><td>55</td><td>3E4</td></tr><tr><td>Can f 1</td><td>10D4</td><td>nCan f 1</td><td>12</td><td>6E9</td></tr><tr><td>Mus m 1</td><td>pAb</td><td>nMus m 1</td><td>57</td><td>pAb</td></tr><tr><td>Rat n 1</td><td>RUP-6</td><td>nRat n 1</td><td>64</td><td>RUP-1</td></tr><tr><td>Cockroach</td><td>Bla g 2</td><td>1F3</td><td>nBla g 2</td><td>26</td><td>4C3</td></tr><tr><td rowspan="4">Pollen</td><td>Bet v 1</td><td>6H4</td><td>rBet v 1</td><td>72</td><td>5B4</td></tr><tr><td>Phl p 5</td><td>1D11</td><td>rPhl p 5</td><td>67</td><td>Bol</td></tr><tr><td>Amb a 1</td><td>2B6</td><td>nAmb a 1</td><td>29</td><td>4H7</td></tr><tr><td>Lol p 1</td><td>5G7</td><td>nLol p 1</td><td>13</td><td>7D8</td></tr></tbody></table>
biosensors and mass spectrometry (MS).⁴⁹² These tests show promise but need to be validated for use in environmental studies. Mass spectrometry analysis of dust samples has provided useful qualitative data confirming the presence of peanut allergens in dust.⁵²⁴ Using MS in combination with immunoassay and PCR methods provides a synergistic approach to molecular exposure assessment, which can be modified to suit specific applications and to overcome method-specific limitations.⁵²⁵

### 3 - Special Considerations for Food Exposures

Inadvertent exposure to food allergens can cause serious adverse reactions in food allergic patients, including anaphylaxis. For this reason, the presence of the most common allergens in food is regulated by the US FDA and regulatory authorities in Europe and other countries. The nine allergens regulated in the U.S are milk, egg, peanut, tree nuts, soybean, sesame, wheat, fish and shellfish.⁵¹⁸ In addition to these nine allergens, sesame, lupin, molluscs, celery, and mustard require mandatory labeling as food ingredients in Europe.⁵¹⁹ The first ELISA methods for food allergen detection were published in the mid-1990s, followed by the development of PCR and MS methods. All these methods present specific strengths and limitations for allergen measurements, which have recently been reviewed in detail.⁵²⁶ Immunoassays such as ELISA and lateral flow tests have been most widely used for measuring allergens in foods and in food processing facilities for sensitivity and ease of use. The limitations of these assays often include poorly defined analyte specificities and variability of assay performance, depending on food processing. While PCR methods usually have potential for high specificity for the allergenic food, the DNA-based detection of allergens, which are proteins, remains indirect. Multiplex array technologies, such as xMAP FADA (developed by the US FDA) or MARIA for Foods, are high throughput approaches that enable multiple food allergens to be measured simultaneously.⁵²⁰,⁵²⁷ Immunoassay epitopes may be denatured by food processing procedures such as heat treatment, polymerization or acid precipitation. Under these conditions, targeted allergen specific detection and quantification by LC-MS/MS, using peptides derived from allergen sequences, is a valuable alternative approach.⁵²⁸⁻⁵³⁰ Mass spectrometry is an exciting new tool for molecular exposure assessment of allergens in environmental samples and foods. The common denominator with new immunoassay methods is that both approaches measure specific allergen molecules and should provide greater consistency of allergen measurements when information about molecular allergen components is required. Various MS methods have been developed with allergen-specific peptides of peanut Ara h 1, Ara h 2, Ara h 3, soybean Gly m 5 and Gly m 6, hazelnut Cor a 9, wheat gliadins, cow's milk Bos d 4, Bos d 5, Bos d 9, Bos d 10, and hen's egg Gal d 2, and Gal d 4, allowing a sensitivity of approximately 1-10 mg total protein of the allergenic food per kg of food matrix, or even below, in typical matrices such as cookie, bread, cereals, ice cream and chocolate.⁵²⁶,⁵³⁰,⁵³¹ Multi-analyte methods capable of detecting and quantifying several

allergens have been developed.⁵³² MS methods can also provide absolute quantification, which lends itself to the development of reference methods.⁵³³

### 4 - Clinical Significance of Allergen Exposures

#### Effect on the development of allergen-specific sensitization and allergic diseases

Allergen exposure impacts the risk of sensitization and allergic disease and is influenced by the route of exposure (e.g., inhaled, transcutaneous, oral), its dose, timing, and individual genetic predisposition.⁵³⁴ Allergen exposure is essential for the development of allergen-specific sensitization, but the nature and the direction of this relationship is a matter of a considerable debate. For example, the evidence on the role of house dust mite (HDM) and cat exposure is contradictory. A Swedish birth cohort reported increased risk of cat-specific sensitization at age 4 years with increasing early-life cat allergen exposure.⁵³⁵ A similar dose-response relationship for both HDM and cat exposure was observed in the German Multicenter Allergy Study.⁵³⁶ By contrast, the opposite finding of a protective effect of high cat allergen exposure on cat sensitization (with a bell-shaped dose-response relationship), was reported in several cross-sectional studies in older children and adults (reviewed in⁵³⁴). The reasons for such heterogeneity include the study design (birth cohorts vs. cross-sectional) and the choice of population (high-risk vs. population-based), making direct comparisons difficult.

The limitations of drawing conclusions about the role of early-life exposures from cross-sectional analyses underscore the importance of looking at life-course trajectories. A recent longitudinal analysis showed that sensitization to cat in the first 3 years of life was significantly higher amongst children living in a home with a cat and exposed to high level of Fel d 1, but after this age, the annual increase in sensitization was lower compared to children without a cat. By adolescence the point prevalence of cat sensitization was numerically higher among children without a cat (Figure 43).⁵³⁷ These findings may explain inconsistencies in previous literature and indicate that apparently contradictory findings may be a consequence of different longitudinal trajectories of cat sensitization between those exposed to high and low cat allergen levels.

#### The route of exposure and the role of genetic predisposition

General assumption is that the default route of exposure to inhalant allergens is via inhalation, and to food allergens via the oral route. Sensitization may also be a consequence of allergen presentation through an impaired skin barrier, which is important within the context of filaggrin genotype. In an unselected birth cohort (MAAS), peanut allergen in house dust increased the risk of peanut sensitization and peanut allergy in a dose-response manner in children with filaggrin loss-of-function mutations, but not in those without, providing evidence that (i) transcutaneous exposure is important in food allergy and (ii) that susceptibility to allergen exposure differs between individuals with different genetic predispositions.⁵³⁸ Filaggrin loss-of-function mutations also modify the impact of exposure to HDM
biosensors and mass spectrometry (MS).⁴⁹² These tests show promise but need to be validated for use in environmental studies. Mass spectrometry analysis of dust samples has provided useful qualitative data confirming the presence of peanut allergens in dust.⁵²⁴ Using MS in combination with immunoassay and PCR methods provides a synergistic approach to molecular exposure assessment, which can be modified to suit specific applications and to overcome method-specific limitations.⁵²⁵

### 3 - Special Considerations for Food Exposures

Inadvertent exposure to food allergens can cause serious adverse reactions in food allergic patients, including anaphylaxis. For this reason, the presence of the most common allergens in food is regulated by the US FDA and regulatory authorities in Europe and other countries. The nine allergens regulated in the U.S are milk, egg, peanut, tree nuts, soybean, sesame, wheat, fish and shellfish.⁵¹⁸ In addition to these nine allergens, sesame, lupin, molluscs, celery, and mustard require mandatory labeling as food ingredients in Europe.⁵¹⁹ The first ELISA methods for food allergen detection were published in the mid-1990s, followed by the development of PCR and MS methods. All these methods present specific strengths and limitations for allergen measurements, which have recently been reviewed in detail.⁵²⁶ Immunoassays such as ELISA and lateral flow tests have been most widely used for measuring allergens in foods and in food processing facilities for sensitivity and ease of use. The limitations of these assays often include poorly defined analyte specificities and variability of assay performance, depending on food processing. While PCR methods usually have potential for high specificity for the allergenic food, the DNA-based detection of allergens, which are proteins, remains indirect. Multiplex array technologies, such as xMAP FADA (developed by the US FDA) or MARIA for Foods, are high throughput approaches that enable multiple food allergens to be measured simultaneously.⁵²⁰,⁵²⁷ Immunoassay epitopes may be denatured by food processing procedures such as heat treatment, polymerization or acid precipitation. Under these conditions, targeted allergen specific detection and quantification by LC-MS/MS, using peptides derived from allergen sequences, is a valuable alternative approach.⁵²⁸⁻⁵³⁰ Mass spectrometry is an exciting new tool for molecular exposure assessment of allergens in environmental samples and foods. The common denominator with new immunoassay methods is that both approaches measure specific allergen molecules and should provide greater consistency of allergen measurements when information about molecular allergen components is required. Various MS methods have been developed with allergen-specific peptides of peanut Ara h 1, Ara h 2, Ara h 3, soybean Gly m 5 and Gly m 6, hazelnut Cor a 9, wheat gliadins, cow's milk Bos d 4, Bos d 5, Bos d 9, Bos d 10, and hen's egg Gal d 2, and Gal d 4, allowing a sensitivity of approximately 1-10 mg total protein of the allergenic food per kg of food matrix, or even below, in typical matrices such as cookie, bread, cereals, ice cream and chocolate.⁵²⁶,⁵³⁰,⁵³¹ Multi-analyte methods capable of detecting and quantifying several

allergens have been developed.⁵³² MS methods can also provide absolute quantification, which lends itself to the development of reference methods.⁵³³

### 4 - Clinical Significance of Allergen Exposures

#### Effect on the development of allergen-specific sensitization and allergic diseases

Allergen exposure impacts the risk of sensitization and allergic disease and is influenced by the route of exposure (e.g., inhaled, transcutaneous, oral), its dose, timing, and individual genetic predisposition.⁵³⁴ Allergen exposure is essential for the development of allergen-specific sensitization, but the nature and the direction of this relationship is a matter of a considerable debate. For example, the evidence on the role of house dust mite (HDM) and cat exposure is contradictory. A Swedish birth cohort reported increased risk of cat-specific sensitization at age 4 years with increasing early-life cat allergen exposure.⁵³⁵ A similar dose-response relationship for both HDM and cat exposure was observed in the German Multicenter Allergy Study.⁵³⁶ By contrast, the opposite finding of a protective effect of high cat allergen exposure on cat sensitization (with a bell-shaped dose-response relationship), was reported in several cross-sectional studies in older children and adults (reviewed in⁵³⁴). The reasons for such heterogeneity include the study design (birth cohorts vs. cross-sectional) and the choice of population (high-risk vs. population-based), making direct comparisons difficult.

The limitations of drawing conclusions about the role of early-life exposures from cross-sectional analyses underscore the importance of looking at life-course trajectories. A recent longitudinal analysis showed that sensitization to cat in the first 3 years of life was significantly higher amongst children living in a home with a cat and exposed to high level of Fel d 1, but after this age, the annual increase in sensitization was lower compared to children without a cat. By adolescence the point prevalence of cat sensitization was numerically higher among children without a cat (Figure 43).⁵³⁷ These findings may explain inconsistencies in previous literature and indicate that apparently contradictory findings may be a consequence of different longitudinal trajectories of cat sensitization between those exposed to high and low cat allergen levels.

#### The route of exposure and the role of genetic predisposition

General assumption is that the default route of exposure to inhalant allergens is via inhalation, and to food allergens via the oral route. Sensitization may also be a consequence of allergen presentation through an impaired skin barrier, which is important within the context of filaggrin genotype. In an unselected birth cohort (MAAS), peanut allergen in house dust increased the risk of peanut sensitization and peanut allergy in a dose-response manner in children with filaggrin loss-of-function mutations, but not in those without, providing evidence that (i) transcutaneous exposure is important in food allergy and (ii) that susceptibility to allergen exposure differs between individuals with different genetic predispositions.⁵³⁸ Filaggrin loss-of-function mutations also modify the impact of exposure to HDM
<figure><img src="image_126.png" /><figcaption>FIGURE 43 Longitudinal trajectories of cat sensitization (SPT-skin prick test) among children who lived in a home with a cat in early life cat and those who did not. Predicted value of mean response is shown in graphical format along with 95% confidence intervals (From ⁵³⁷, with permission).</figcaption></figure>

and cat allergens on the development of allergen-specific sensitization. In the MAAS cohort, Der p 1 and Fel d 1 exposure was much greater among children with filaggrin mutations compared to those without.⁵³⁹ This analysis confirmed previous observations that the association between early-life allergen exposures and sensitization changes with time. In real life, individuals are contemporaneously exposed to a range of other environmental exposures. One example is the observation that high allergen exposure combined with an environment rich in specific bacteria may protect against sensitization and atopic wheezing.⁵⁴⁰ Gene-environment interactions add to this complexity.

Increasing early life Der p 1 exposure was associated with increased risk of mite sensitization. The impact of allergen exposure was markedly reduced at high endotoxin exposure, but only among children with specific genotype in CD14.⁵⁴¹ These findings confirmed that sensitization is influenced by allergen exposure, by other environmental exposures, and by genetic predisposition. Consequently, the effects of allergen avoidance may differ between individuals with different genotypes.

### Effect of allergen exposure on asthma severity and exacerbations

Most studies that investigated the impact of exposure on symptoms among sensitized patients with established disease reported increased severity with increasing exposure. Amongst allergic asthmatics, indicators of asthma severity (including increased airway hyper-reactivity and Peak Expiratory Flow Rate (PEFR) variability and diminished lung function) are associated with high exposure to sensitizing allergen, emphasizing the contribution of allergen exposure to the ongoing chronic disease process.⁵⁴² High allergen exposure in sensitized asthmatics interacts with virus infection in increasing the risk of exacerbation in children and adults.⁵⁴³,⁵⁴⁴ Evidence that high exposure to allergens can worsen asthma symptoms indicate that effective allergen avoidance should improve asthma control. However, attempts to replicate clinical benefits observed in occupational asthma, or the studies at high altitude sanatoria, by using

allergen control measures in patients' homes, have provided conflicting results (reviewed in⁵⁴⁵).

**Allergen avoidance in the treatment of asthma** Several systematic reviews and meta-analyses have questioned the role of HDM avoidance in sensitized asthmatics, resulting in a lack of consensus and conflicting recommendations by national and international asthma guidelines. The limitations of such analyses and why one should not disproportionately rely on meta-analyses and systematic reviews to inform clinical practice have been reviewed.⁵⁴⁵,⁵⁴⁶

In adults, two large randomized double-blind, placebo-controlled trials assessing the effectiveness of mite-impermeable bed covers as a single intervention found no benefits on morning PEFR during the first 6 months, or the proportion of patients able to discontinue inhaled corticosteroids during the second six months of the study. The lack of benefits in some domains of the disease (e.g., lung function or symptoms) and some age groups (e.g., adults) does not exclude the possibility of benefits in other domains (such as prevention of exacerbations) and in other age groups. A large randomized double-blind placebo-controlled trial in children at high risk of severe exacerbations (Preventing asthma exacerbations by avoiding mite allergen—PAXAMA) showed that significantly fewer children who received mite-impermeable bed encasings attended hospital with asthma exacerbation compared to the placebo group in the 12-month follow-up period. The risk of hospital presentation was 45% lower in the Active compared with the Placebo group (p=0.006) (Figure 44).⁵⁴⁷ The effect of intervention was highest in children younger than 11 years, mono-sensitized to mite, living in nonsmoking households, and among children requiring more controller medication.

For pet-sensitized pet owners with allergic airway disease in whom this allergy is contributing to their symptoms, a double-blind, randomized study of pet removal is not feasible. One small nonrandomized, nonblinded study among pet-allergic patients with asthma indicated that pet removal from home reduced airway responsiveness.⁵⁴⁸ For such patients, the advice based on common sense and
<figure><img src="image_125.png" /><figcaption>FIGURE 43 Longitudinal trajectories of cat sensitization (SPT-skin prick test) among children who lived in a home with a cat in early life cat and those who did not. Predicted value of mean response is shown in graphical format along with 95% confidence intervals (From ⁵³⁷, with permission).</figcaption></figure>

and cat allergens on the development of allergen-specific sensitization. In the MAAS cohort, Der p 1 and Fel d 1 exposure was much greater among children with filaggrin mutations compared to those without.⁵³⁹ This analysis confirmed previous observations that the association between early-life allergen exposures and sensitization changes with time. In real life, individuals are contemporaneously exposed to a range of other environmental exposures. One example is the observation that high allergen exposure combined with an environment rich in specific bacteria may protect against sensitization and atopic wheezing.⁵⁴⁰ Gene-environment interactions add to this complexity.

Increasing early life Der p 1 exposure was associated with increased risk of mite sensitization. The impact of allergen exposure was markedly reduced at high endotoxin exposure, but only among children with specific genotype in CD14.⁵⁴¹ These findings confirmed that sensitization is influenced by allergen exposure, by other environmental exposures, and by genetic predisposition. Consequently, the effects of allergen avoidance may differ between individuals with different genotypes.

### Effect of allergen exposure on asthma severity and exacerbations

Most studies that investigated the impact of exposure on symptoms among sensitized patients with established disease reported increased severity with increasing exposure. Amongst allergic asthmatics, indicators of asthma severity (including increased airway hyper-reactivity and Peak Expiratory Flow Rate (PEFR) variability and diminished lung function) are associated with high exposure to sensitizing allergen, emphasizing the contribution of allergen exposure to the ongoing chronic disease process.⁵⁴² High allergen exposure in sensitized asthmatics interacts with virus infection in increasing the risk of exacerbation in children and adults.⁵⁴³,⁵⁴⁴ Evidence that high exposure to allergens can worsen asthma symptoms indicate that effective allergen avoidance should improve asthma control. However, attempts to replicate clinical benefits observed in occupational asthma, or the studies at high altitude sanatoria, by using

allergen control measures in patients' homes, have provided conflicting results (reviewed in⁵⁴⁵).

**Allergen avoidance in the treatment of asthma** Several systematic reviews and meta-analyses have questioned the role of HDM avoidance in sensitized asthmatics, resulting in a lack of consensus and conflicting recommendations by national and international asthma guidelines. The limitations of such analyses and why one should not disproportionately rely on meta-analyses and systematic reviews to inform clinical practice have been reviewed.⁵⁴⁵,⁵⁴⁶

In adults, two large randomized double-blind, placebo-controlled trials assessing the effectiveness of mite-impermeable bed covers as a single intervention found no benefits on morning PEFR during the first 6 months, or the proportion of patients able to discontinue inhaled corticosteroids during the second six months of the study. The lack of benefits in some domains of the disease (e.g., lung function or symptoms) and some age groups (e.g., adults) does not exclude the possibility of benefits in other domains (such as prevention of exacerbations) and in other age groups. A large randomized double-blind placebo-controlled trial in children at high risk of severe exacerbations (Preventing asthma exacerbations by avoiding mite allergen—PAXAMA) showed that significantly fewer children who received mite-impermeable bed encasings attended hospital with asthma exacerbation compared to the placebo group in the 12-month follow-up period. The risk of hospital presentation was 45% lower in the Active compared with the Placebo group (p=0.006) (Figure 44).⁵⁴⁷ The effect of intervention was highest in children younger than 11 years, mono-sensitized to mite, living in nonsmoking households, and among children requiring more controller medication.

For pet-sensitized pet owners with allergic airway disease in whom this allergy is contributing to their symptoms, a double-blind, randomized study of pet removal is not feasible. One small nonrandomized, nonblinded study among pet-allergic patients with asthma indicated that pet removal from home reduced airway responsiveness.⁵⁴⁸ For such patients, the advice based on common sense and
<figure><img src="image_127.png" /><figcaption>FIGURE 44 Proportion of children who suffered one or more severe exacerbation during the 12 month-follow up period in PAXAMA study (for all children who completed 12 months Follow up, n=241) Results are shown for one or more hospitalizations or emergency department visits requiring systemic corticosteroids because of an asthma exacerbation, and time to first hospitalizations or ED visit because of severe exacerbation of asthma. Active covers (mite-impermeable) (green line) and Placebo covers (blue line). Adapted from ⁵⁴⁷ with permission</figcaption></figure>

clinical experience is to remove the pet from home. Studies of multifaceted interventions tailored towards patients' individual needs reported compelling evidence of improvement in asthma control and are recommended by the U.S. National Asthma Education and Prevention Program expert panel working group.⁵⁴⁹

**Allergen avoidance in the prevention of allergic disease**

Clinical outcomes reported to date by the primary prevention studies are inconsistent. in the Isle of Wight study, allergen avoidance from birth reduced mite sensitization and asthma by age 18 years, while in contrast the Manchester study reported an increase in mite sensitization.⁵⁵⁰,⁵⁵¹ Some intervention studies reported no effect of allergen avoidance. Given such heterogeneity, much longer follow-up and more detailed analyses are required before we can draw conclusions and give meaningful clinical advice.

5 – Harmonization of Allergen Measurement

Harmonization of allergen measurements is required for the comparability of results between analytical laboratories using different methods, often with different analytical responses to the targeted allergens. Reporting units may also differ between laboratories. The goal of allergen standardization and harmonization is to reduce uncertainty and to validate method performance. Several factors may add to uncertainty in the qualitative and/or quantitative determination of allergens in foods, environmental samples, and diagnostic and therapeutic preparations (summarized in Table 11). These factors may be related to the selected method and sample preparation procedure or may be attributable to intrinsic properties of the

target allergens, including their molecular stability and consistency in the investigated sample.⁵²⁶

Antibody-based immunoassays (e.g., ELISA) and physicochemical mass spectrometry are considered as direct methods for measurement of allergenic proteins. By contrast, the detection of nucleic acids as surrogate target molecules, using nucleic acid amplification techniques (NAT), such as PCR are indirect and assume that coexistence of DNA and allergens in the sample is consistent. The NAT methods can be used to verify the biologic identity of source materials for allergen preparations. However, in most cases for the measurement of allergenic proteins, direct methods are preferred. Both ELISA and MS methods usually detect certain epitopes and peptides, respectively, on the allergen molecule.⁵²⁶ These structures must be (made) available and preserved by efficient sample preparation. Specific processes occurring in the environment or that are required to produce the sample may affect allergen integrity. The allergen preparation used for method calibration should be as similar as possible in its composition and presentation compared with the allergen that is measured in a sample. In real-life, this often is hard to achieve. Moreover, depending on the choice and specific type of method and detection modes, differences in the quantitative response to the allergen between the calibrator and sample may result in varying measurement results. Methods often apply different reporting units, further complicating comparability of results between different methods unless appropriate conversion factors are available.⁵²⁶

Compared with environmental samples or food, harmonization/standardization of molecular allergen measurements for pharmaceutical allergen preparations are the most advanced. Harmonization of
<figure><img src="image_126.png" /><figcaption>FIGURE 44 Proportion of children who suffered one or more severe exacerbation during the 12 month-follow up period in PAXAMA study (for all children who completed 12 months Follow up, n=241) Results are shown for one or more hospitalizations or emergency department visits requiring systemic corticosteroids because of an asthma exacerbation, and time to first hospitalizations or ED visit because of severe exacerbation of asthma. Active covers (mite-impermeable) (green line) and Placebo covers (blue line). Adapted from ⁵⁴⁷ with permission</figcaption></figure>

clinical experience is to remove the pet from home. Studies of multifaceted interventions tailored towards patients' individual needs reported compelling evidence of improvement in asthma control and are recommended by the U.S. National Asthma Education and Prevention Program expert panel working group.⁵⁴⁹

**Allergen avoidance in the prevention of allergic disease**

Clinical outcomes reported to date by the primary prevention studies are inconsistent. in the Isle of Wight study, allergen avoidance from birth reduced mite sensitization and asthma by age 18 years, while in contrast the Manchester study reported an increase in mite sensitization.⁵⁵⁰,⁵⁵¹ Some intervention studies reported no effect of allergen avoidance. Given such heterogeneity, much longer follow-up and more detailed analyses are required before we can draw conclusions and give meaningful clinical advice.

## 5 – Harmonization of Allergen Measurement

Harmonization of allergen measurements is required for the comparability of results between analytical laboratories using different methods, often with different analytical responses to the targeted allergens. Reporting units may also differ between laboratories. The goal of allergen standardization and harmonization is to reduce uncertainty and to validate method performance. Several factors may add to uncertainty in the qualitative and/or quantitative determination of allergens in foods, environmental samples, and diagnostic and therapeutic preparations (summarized in Table 11). These factors may be related to the selected method and sample preparation procedure or may be attributable to intrinsic properties of the

target allergens, including their molecular stability and consistency in the investigated sample.⁵²⁶

Antibody-based immunoassays (e.g., ELISA) and physicochemical mass spectrometry are considered as direct methods for measurement of allergenic proteins. By contrast, the detection of nucleic acids as surrogate target molecules, using nucleic acid amplification techniques (NAT), such as PCR are indirect and assume that coexistence of DNA and allergens in the sample is consistent. The NAT methods can be used to verify the biologic identity of source materials for allergen preparations. However, in most cases for the measurement of allergenic proteins, direct methods are preferred. Both ELISA and MS methods usually detect certain epitopes and peptides, respectively, on the allergen molecule.⁵²⁶ These structures must be (made) available and preserved by efficient sample preparation. Specific processes occurring in the environment or that are required to produce the sample may affect allergen integrity. The allergen preparation used for method calibration should be as similar as possible in its composition and presentation compared with the allergen that is measured in a sample. In real-life, this often is hard to achieve. Moreover, depending on the choice and specific type of method and detection modes, differences in the quantitative response to the allergen between the calibrator and sample may result in varying measurement results. Methods often apply different reporting units, further complicating comparability of results between different methods unless appropriate conversion factors are available.⁵²⁶

Compared with environmental samples or food, harmonization/standardization of molecular allergen measurements for pharmaceutical allergen preparations are the most advanced. Harmonization of
<table><caption>TABLE 11 Factors affecting the uncertainty of analytical methods for allergen determination in food, environmental samples, and diagnostic and therapeutic preparations.</caption><thead><tr><td>Factors affecting uncertainty of results</td><td>Examples</td></tr></thead><tbody><tr><td>choice of method</td><td>biochemical (NAT, ELISA), biophysical (MS), qualitative/ quantitative</td></tr><tr><td>type of specific method</td><td>real-time versus digital PCR, sandwich versus inhibition ELISA, MALDI-TOF versus Q-TOF MS, etc.</td></tr><tr><td>detection principle</td><td>amplification of DNA (PCR), non-covalent binding of epitopes by antibodies (ELISA), mass/charge ratio of peptides (MS)</td></tr><tr><td>selection and specificity of detection molecules (target analyte)</td><td>DNA stretch of allergenic source, allergen component, peptides of allergen component</td></tr><tr><td>selection of specific detection reagents or detection modes</td><td>primers in NAT, polyclonal versus monoclonal antibodies and epitope specificity in ELISA, multiple reaction monitoring versus high-resolution in MS; potential signal cross-talk in multi-analyte versus single analyte methods</td></tr><tr><td>reporting unit</td><td>arbitrary units; DNA copies; weight/volume or moles/volume, weight/weight or moles/weight of specific peptides or allergen components or total protein of allergen source per analysed sample</td></tr><tr><td>calibration (selected preparation)</td><td>specific stretch of or total DNA; total protein of allergenic source, selected allergen(s), selected peptide(s) of allergen(s)</td></tr><tr><td>differences in method response, depending on:</td><td>calibrator molecule, target molecule, variations in target molecule (isoform composition, impact of processing or environmental conditions), sample matrix interference, operator, selected detection devices/laboratory</td></tr><tr><td>correlation factors applied (e.g. from DNA to allergen; from epitope or peptide to allergen)</td><td>depending on type of calibration; known if applied for calculation after analysis or unknown if already included in read-out of commercial kit or report of service-lab</td></tr><tr><td>sample preparation</td><td>differences in quality and/or quantity of target analyte depending on extraction efficiency (PCR, ELISA, MS) and impact of additional reagents for sample preparation, e.g. purification (PCR) or enzymatic digestion (MS)</td></tr></tbody></table>

reporting units and the availability and use of certified reference materials and methods is essential to increase comparability of results between commercial allergenic products. Recent work on the

molecular standardization of pollen allergens through the BSP090 project is a good example of this approach. The two major allergens from birch pollen and from timothy grass pollen, recombinant Bet v 1 and Phl p 5, have been made available as European Pharmacopoeia (Ph. Eur.) chemical reference substances through the European Directorate for the Quality of Medicines & Health Care (EDQM).⁵⁵² Their use has so far not become mandatory.⁵⁵³ The current Ph. Eur. Monograph on Allergen Products allows allergen-specific reference standards to be used, when available. In addition to validated reference materials, two allergen-specific ELISA methods for Bet v 1 and for Phl p 5, were evaluated in international ring-trials through EDQM.³⁷²,⁵⁵²,⁵⁵⁴ The implementation of these protocols as general chapters for inclusion in the Ph.Eur. is in progress.⁵⁵³ Standardization and harmonization of test methods in the field of environmental food allergen analysis needs further development. A report on health-based guidance values for allergens in foods by the ad hoc FAO-WHO expert consultation group recommends that test methods report results in mg allergenic ingredient protein/kg of food (https://www.fao.org/3/cb6388en/cb6388en.pdf, accessed 9Feb2022). However, this recommendation may need to be modified to consider allergen detection methods used for verification of food allergen labeling requirements that measure molecular allergen components. Currently, test results are often converted to total protein of the allergenic food by calibration or calculation.⁵²⁶ This allows for comparison of the analytical result with suggested protein reference doses, such as provided by VITAL®, the Australian initiative for voluntary incidental trace allergen labelling and based on clinical reactivity in food challenge studies, at or below which voluntary labelling of noningredient allergen cross-contact is unnecessary for the protection of allergic consumers.⁵⁵⁴

In the field of food allergen risk assessment and labeling, multicenter collaborative studies and ring trials are needed to develop consensus guidelines for use of MS and other allergen specific methods. Harmonized quantitative MS methods are being developed as part of the ThRAII project.⁵⁵⁵ Official Methods Board of the German Government has also launched a new Working Group to standardize MS methods to detect allergens in foods through multi-laboratory validation studies.⁵⁵⁶ There is also a need for more extensive development of certified reference materials for allergen analysis in foods, which will play a key role in allowing harmonization of test methods results both across different platform technologies and between laboratories. Moreover, sample collection can be ad hoc, which makes interpretation of test results complex and uncertain. Consequently, statistically validated sampling plans need to be developed.

In summary, for a few of the many major allergens that are relevant in foods, environmental samples and medicinal allergen products, reference materials and harmonized protocols for methodology are available. Availability of commonly agreed or mandatory reference materials, harmonized protocols for methodology, including commonly agreed reporting units and sampling plans are needed for the inter-laboratory and cross-product comparability of single allergen measurement results.
<table><caption>TABLE 11 Factors affecting the uncertainty of analytical methods for allergen determination in food, environmental samples, and diagnostic and therapeutic preparations.</caption><thead><tr><td>Factors affecting uncertainty of results</td><td>Examples</td></tr></thead><tbody><tr><td>choice of method</td><td>biochemical (NAT, ELISA), biophysical (MS), qualitative/ quantitative</td></tr><tr><td>type of specific method</td><td>real-time versus digital PCR, sandwich versus inhibition ELISA, MALDI-TOF versus Q-TOF MS, etc.</td></tr><tr><td>detection principle</td><td>amplification of DNA (PCR), non-covalent binding of epitopes by antibodies (ELISA), mass/charge ratio of peptides (MS)</td></tr><tr><td>selection and specificity of detection molecules (target analyte)</td><td>DNA stretch of allergenic source, allergen component, peptides of allergen component</td></tr><tr><td>selection of specific detection reagents or detection modes</td><td>primers in NAT, polyclonal versus monoclonal antibodies and epitope specificity in ELISA, multiple reaction monitoring versus high-resolution in MS; potential signal cross-talk in multi-analyte versus single analyte methods</td></tr><tr><td>reporting unit</td><td>arbitrary units; DNA copies; weight/volume or moles/volume, weight/weight or moles/weight of specific peptides or allergen components or total protein of allergen source per analysed sample</td></tr><tr><td>calibration (selected preparation)</td><td>specific stretch of or total DNA; total protein of allergenic source, selected allergen(s), selected peptide(s) of allergen(s)</td></tr><tr><td>differences in method response, depending on:</td><td>calibrator molecule, target molecule, variations in target molecule (isoform composition, impact of processing or environmental conditions), sample matrix interference, operator, selected detection devices/laboratory</td></tr><tr><td>correlation factors applied (e.g. from DNA to allergen; from epitope or peptide to allergen)</td><td>depending on type of calibration; known if applied for calculation after analysis or unknown if already included in read-out of commercial kit or report of service-lab</td></tr><tr><td>sample preparation</td><td>differences in quality and/or quantity of target analyte depending on extraction efficiency (PCR, ELISA, MS) and impact of additional reagents for sample preparation, e.g. purification (PCR) or enzymatic digestion (MS)</td></tr></tbody></table>

reporting units and the availability and use of certified reference materials and methods is essential to increase comparability of results between commercial allergenic products. Recent work on the

molecular standardization of pollen allergens through the BSP090 project is a good example of this approach. The two major allergens from birch pollen and from timothy grass pollen, recombinant Bet v 1 and Phl p 5, have been made available as European Pharmacopoeia (Ph. Eur.) chemical reference substances through the European Directorate for the Quality of Medicines & Health Care (EDQM).⁵⁵² Their use has so far not become mandatory.⁵⁵³ The current Ph. Eur. Monograph on Allergen Products allows allergen-specific reference standards to be used, when available. In addition to validated reference materials, two allergen-specific ELISA methods for Bet v 1 and for Phl p 5, were evaluated in international ring-trials through EDQM.³⁷²,⁵⁵²,⁵⁵⁴ The implementation of these protocols as general chapters for inclusion in the Ph.Eur. is in progress.⁵⁵³ Standardization and harmonization of test methods in the field of environmental food allergen analysis needs further development. A report on health-based guidance values for allergens in foods by the ad hoc FAO-WHO expert consultation group recommends that test methods report results in mg allergenic ingredient protein/kg of food (https://www.fao.org/3/cb6388en/cb6388en.pdf, accessed 9Feb2022). However, this recommendation may need to be modified to consider allergen detection methods used for verification of food allergen labeling requirements that measure molecular allergen components. Currently, test results are often converted to total protein of the allergenic food by calibration or calculation.⁵²⁶ This allows for comparison of the analytical result with suggested protein reference doses, such as provided by VITAL®, the Australian initiative for voluntary incidental trace allergen labelling and based on clinical reactivity in food challenge studies, at or below which voluntary labelling of noningredient allergen cross-contact is unnecessary for the protection of allergic consumers.⁵⁵⁴

In the field of food allergen risk assessment and labeling, multicenter collaborative studies and ring trials are needed to develop consensus guidelines for use of MS and other allergen specific methods. Harmonized quantitative MS methods are being developed as part of the ThRAII project.⁵⁵⁵ Official Methods Board of the German Government has also launched a new Working Group to standardize MS methods to detect allergens in foods through multi-laboratory validation studies.⁵⁵⁶ There is also a need for more extensive development of certified reference materials for allergen analysis in foods, which will play a key role in allowing harmonization of test methods results both across different platform technologies and between laboratories. Moreover, sample collection can be ad hoc, which makes interpretation of test results complex and uncertain. Consequently, statistically validated sampling plans need to be developed.

In summary, for a few of the many major allergens that are relevant in foods, environmental samples and medicinal allergen products, reference materials and harmonized protocols for methodology are available. Availability of commonly agreed or mandatory reference materials, harmonized protocols for methodology, including commonly agreed reporting units and sampling plans are needed for the inter-laboratory and cross-product comparability of single allergen measurement results.
# 6 – Case studies

## Case Study I

The US National Health and Nutrition Examination Survey (NHANES) 2005-6.

* A survey of allergen exposure in US homes that were representative of the general US population⁵⁵⁷.

* A mixed bed and bedroom floor dust sample was obtained from ~7,000 homes.

* Samples were analysed using a MARIA 9-plex array for Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Mus m 1, Rat n 1, Bla g 2, Alt a 1. Over 56,000 data points.

* >90% of homes had detectable levels of 3 allergens, usually Fel d 1, Can f 1, Mus m 1, Der p 1, or Der f 1.

* 15.8% had detectable levels of 7 or more allergens.

* Individual allergen levels in homes varied according to the participants' race/ethnicity, poverty index ratio, age and presence of children in the household. Regional variation, climate factors and level of urbanization also affected dust mite, cockroach and pet allergen levels (see refs for full details).

## Case Study II

The Schools Inner-City Asthma Study (SICAS) 2008-13

* A study of allergen exposure in 37 inner-city elementary schools in the northeastern US.⁵⁵⁸

* ~1,100 dust/air/table wipe samples were collected from the school environment and children's homes.

* Samples were by MARIA 9-plex array for Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Mus m 1, Rat n 1, Bla g 2, Alt a 1 and for endotoxin. Over 11,000 data points.

* Mus m 1 was the most common allergen found in schools and homes, with higher allergen levels found in settled dust from schools (which was highly correlated with airborne Mus m 1 levels)

* In a follow up study (SICAS II), ~450 dust/table wipe samples collected from the school environment and homes were analysed for food allergens:

* Samples were analysed by MARIA for Foods 7-plex array for Ana o 3, Ara h 3, Ara h 6, Bos d 5, Cor a 9, Gal d 1, Gal d 2.

* Milk, peanut and egg allergens were readily detectable in floor samples and table wipes in elementary schools, but not at higher levels than those found in children's homes.⁵²³

# 7 – Salient points

* Molecular allergen exposure assessment is a critical process for the investigation of environmental and food allergens and their relationship to allergic diseases.

* State-of-the-art multiplex technologies, including immunoassays and MS, will facilitate high throughput exposure assessment based on specific allergens that will enable thresholds for risk assessment to be established.

* Molecular exposure assessments, coupled with analyses of other environmental factors and genetic predisposition, will facilitate comprehensive epidemiologic and population studies of the role of the exposome in causing allergic respiratory diseases.

* Harmonization of molecular exposure assessments is urgently needed. This will require mutual collaboration between technology providers, academic and clinical investigators, industry and regulatory authorities to design and execute multi-center studies for validation of sampling plans and analytical detection methods.
# 6 – Case studies

## Case Study I

The US National Health and Nutrition Examination Survey (NHANES) 2005-6.

* A survey of allergen exposure in US homes that were representative of the general US population⁵⁵⁷.

* A mixed bed and bedroom floor dust sample was obtained from ~7,000 homes.

* Samples were analysed using a MARIA 9-plex array for Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Mus m 1, Rat n 1, Bla g 2, Alt a 1. Over 56,000 data points.

* >90% of homes had detectable levels of 3 allergens, usually Fel d 1, Can f 1, Mus m 1, Der p 1, or Der f 1.

* 15.8% had detectable levels of 7 or more allergens.

* Individual allergen levels in homes varied according to the participants' race/ethnicity, poverty index ratio, age and presence of children in the household. Regional variation, climate factors and level of urbanization also affected dust mite, cockroach and pet allergen levels (see refs for full details).

## Case Study II

The Schools Inner-City Asthma Study (SICAS) 2008-13

* A study of allergen exposure in 37 inner-city elementary schools in the northeastern US.⁵⁵⁸

* ~1,100 dust/air/table wipe samples were collected from the school environment and children's homes.

* Samples were by MARIA 9-plex array for Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Mus m 1, Rat n 1, Bla g 2, Alt a 1 and for endotoxin. Over 11,000 data points.

* Mus m 1 was the most common allergen found in schools and homes, with higher allergen levels found in settled dust from schools (which was highly correlated with airborne Mus m 1 levels)

* In a follow up study (SICAS II), ~450 dust/table wipe samples collected from the school environment and homes were analysed for food allergens:

* Samples were analysed by MARIA for Foods 7-plex array for Ana o 3, Ara h 3, Ara h 6, Bos d 5, Cor a 9, Gal d 1, Gal d 2.

* Milk, peanut and egg allergens were readily detectable in floor samples and table wipes in elementary schools, but not at higher levels than those found in children's homes.⁵²³

# 7 – Salient points

* Molecular allergen exposure assessment is a critical process for the investigation of environmental and food allergens and their relationship to allergic diseases.

* State-of-the-art multiplex technologies, including immunoassays and MS, will facilitate high throughput exposure assessment based on specific allergens that will enable thresholds for risk assessment to be established.

* Molecular exposure assessments, coupled with analyses of other environmental factors and genetic predisposition, will facilitate comprehensive epidemiologic and population studies of the role of the exposome in causing allergic respiratory diseases.

* Harmonization of molecular exposure assessments is urgently needed. This will require mutual collaboration between technology providers, academic and clinical investigators, industry and regulatory authorities to design and execute multi-center studies for validation of sampling plans and analytical detection methods.
# B01 - Tree pollen allergy

Enrico Scala, Alessandro Maria Marra, Riccardo Asero

## Highlights

* Cup a 1 reactivity is the specific marker for a sensitization to the Cupressaceae family.

* PR-10 molecules (Bet v 1 like) are the major allergens in Fagales pollen often associated with an oral allergy syndrome.

* Ole e 1 is the most common sensitizing molecule in olive pollen.

* Pla a 1 and Pla a 2 may serve as a marker of primary sensitization to plane tree pollen.

## 1 - The allergen sources

Among over 400,000 plant species,⁵⁶⁰ about 100 flowering (Angiospermae) and nonflowering (Gymnospermae) trees can induce specific sensitization in predisposed individuals. Besides grass pollen and house dust mites, tree pollens belong to the most important respiratory allergen sources. The knowledge of the taxonomical relationship between different tree species allows the prediction of cross-reactivity between closely related plants, which share homologous molecules not found in unrelated plants. The trees most commonly causing allergy belong to the orders Fagales (alder, beech, birch, hazelnut, oak), Lamiales (ash, privet, olive, lilac), Pinales (cypress, Japanese cedar, juniper), and Proteales (plane tree, sycamore).⁵⁵⁹,⁵⁶¹

The geographical distribution of allergenic plants drives patients' sensitization profiles, as a consequence of different local pollen exposure. For instance, in the Mediterranean area, as well as in regions with a Mediterranean climate such as North and South Africa, North and South America, and Australia, trees belonging to the order Lamiales (i.e., olive tree) or Pinales (i.e., cypress tree) are mainly found, whilst Fagales trees play a role as allergen sources mostly in temperate climate regions such as Northern and Central Europe, North America, East Asia and Northwest Africa.⁵⁶²

The order **Fagales** encompasses seven distinct families, but the two most frequently implicated in tree-pollen allergies are (i) *Betulaceae* including the genera Alnus (alder), Betula (birch), Carpinus (hornbeam), Corylus (hazel), and Ostrya (hop hornbeam), and (ii) *Fagaceae*, comprising the genera Castanea (chestnut), Castanopsis (chinkapin), Chrysolepis (chinquapin), Fagus (beech), Lithocarpus (tanoak), and Quercus (oak)⁵⁶³ (Figure 45). A high degree of allergic cross-reactivity among allergens from these plants distributed all over the world has been demonstrated. Birch, followed by alder and hazel, represents the most relevant cause of tree pollen allergy within this order. The flowering period of birch begins at the end of March in Western Europe, from the beginning to mid-April in Central and Eastern Europe and from late April to late May in Northern Europe.⁵⁶⁴ From 1 to 3 weeks after the beginning of the season higher amounts of pollen in the atmosphere are recorded, and the extent of the pollen season is extremely dependent on weather conditions, and thus ranges from 2 to 8 weeks.⁵⁶⁴ An alternation of low and high pollen production per year has been detected. Hazel and alder florescence starts early from December to April, is followed by birch, hornbeam and hop hornbeam and then by oak and beech

<figure><img src="image_128.png" /><figcaption>FIGURE 45 Taxonomy, geographic distribution, and flowering seasons of the most relevant tree pollen allergen sources</figcaption></figure>
# B01 - Tree pollen allergy

Enrico Scala, Alessandro Maria Marra, Riccardo Asero

## Highlights

* Cup a 1 reactivity is the specific marker for a sensitization to the Cupressaceae family.

* PR-10 molecules (Bet v 1 like) are the major allergens in Fagales pollen often associated with an oral allergy syndrome.

* Ole e 1 is the most common sensitizing molecule in olive pollen.

* Pla a 1 and Pla a 2 may serve as a marker of primary sensitization to plane tree pollen.

## 1 - The allergen sources

Among over 400,000 plant species,⁵⁶⁰ about 100 flowering (Angiospermae) and nonflowering (Gymnospermae) trees can induce specific sensitization in predisposed individuals. Besides grass pollen and house dust mites, tree pollens belong to the most important respiratory allergen sources. The knowledge of the taxonomical relationship between different tree species allows the prediction of cross-reactivity between closely related plants, which share homologous molecules not found in unrelated plants. The trees most commonly causing allergy belong to the orders Fagales (alder, beech, birch, hazelnut, oak), Lamiales (ash, privet, olive, lilac), Pinales (cypress, Japanese cedar, juniper), and Proteales (plane tree, sycamore).⁵⁵⁹,⁵⁶¹

The geographical distribution of allergenic plants drives patients' sensitization profiles, as a consequence of different local pollen exposure. For instance, in the Mediterranean area, as well as in regions with a Mediterranean climate such as North and South Africa, North and South America, and Australia, trees belonging to the order Lamiales (i.e., olive tree) or Pinales (i.e., cypress tree) are mainly found, whilst Fagales trees play a role as allergen sources mostly in temperate climate regions such as Northern and Central Europe, North America, East Asia and Northwest Africa.⁵⁶²

The order **Fagales** encompasses seven distinct families, but the two most frequently implicated in tree-pollen allergies are (i) *Betulaceae* including the genera Alnus (alder), Betula (birch), Carpinus (hornbeam), Corylus (hazel), and Ostrya (hop hornbeam), and (ii) *Fagaceae*, comprising the genera Castanea (chestnut), Castanopsis (chinkapin), Chrysolepis (chinquapin), Fagus (beech), Lithocarpus (tanoak), and Quercus (oak)⁵⁶³ (Figure 45). A high degree of allergic cross-reactivity among allergens from these plants distributed all over the world has been demonstrated. Birch, followed by alder and hazel, represents the most relevant cause of tree pollen allergy within this order. The flowering period of birch begins at the end of March in Western Europe, from the beginning to mid-April in Central and Eastern Europe and from late April to late May in Northern Europe.⁵⁶⁴ From 1 to 3 weeks after the beginning of the season higher amounts of pollen in the atmosphere are recorded, and the extent of the pollen season is extremely dependent on weather conditions, and thus ranges from 2 to 8 weeks.⁵⁶⁴ An alternation of low and high pollen production per year has been detected. Hazel and alder florescence starts early from December to April, is followed by birch, hornbeam and hop hornbeam and then by oak and beech

<figure><img src="image_127.png" /><figcaption>FIGURE 45 Taxonomy, geographic distribution, and flowering seasons of the most relevant tree pollen allergen sources</figcaption></figure>
in spring. Chestnuts shed pollen in June and July in Western and Central Europe.

Trees from the family *Oleaceae*, order **Lamiales**, grow on all 5 continents and are among the most important causes of respiratory allergy in the Mediterranean area as well as in some areas of North America, Australia, Japan, and North and South Africa, where these trees are intensively cultivated.⁵⁶⁵ The *Oleaceae* family comprises 4 main genera: olive (*Olea europea*), European ash (*Fraxinus excelsior*), lilac (*Syringa vulgaris*), and common privet (*Ligustrum vulgare*), all able to cause IgE sensitization ⁵⁶⁶ (Figure 45). The pollination period ranges from April to June in warmer regions, and the occurrence of olive tree allergy among patients suffering from tree pollen allergy is about 30-40% in Italy.⁵⁶⁷,⁵⁶⁸ Ash pollen season is during winter-time, rather similar to the birch pollen season, and is very relevant in Central Europe (Austria, North and East of France, Switzerland up to 30% prevalence of pollen allergic patients).

In Mediterranean regions trees of the *Cupressaceae* family (Figure 43) from the order **Pinales** are widely spread. Wind pollination of cypress trees occurs during the winter season, when no other allergenic plants flower, and accounts for up to 40% of the total pollen count in Mediterranean countries.⁵⁶⁹ Cypress tree florescence covers about 30-40 days, from January to April, showing a high variability from year to year, depending on weather conditions, causing difficulty in identifying the beginning and length of pollen season. The high degree of cross-reactivity found among *Cupressaceae* trees (cypress, juniper, and cedar), which have somewhat different but overlapping pollination periods, could extend the cypress pollen season from December to March.⁵⁷⁰

Trees of the Plane-tree family (e.g., *Platanus acerifolia*), from the order **Proteales**, are common species widely spread in Southern Europe, with a short but intense pollen season from March to April, characterized by high pollen counts, reaching one hundred billion pollen grains per tree only a few days after the florescence time. Clinical surveys have acknowledged plane trees as a major cause of pollen with sensitization rates ranging from 8 to 17% in exposed populations. Annual airborne pollen counts differ based on weather conditions but also as a function of human activity, mainly pruning since plane trees or sycamores are widely used for ornamental purposes.⁵⁷¹ Temperature, but not rainfall, is the weather parameter mainly affecting the Platanus pollen season, influencing both start-date and daily pollen counts.

In subtropical regions mesquite (*Prosopsis juliflora*) and Acacia farnesiana (*Vachellia farnesiana*) (Needle bush), trees belonging to the order **Fabales** have been acknowledged as a clinically relevant allergen in North America, India, and the Arabian Peninsula.⁵⁷²,⁵⁷³

## 2 – Allergen families and allergenic molecules

Pollen from **Fagales** trees is one of the most frequent causes of winter/spring respiratory allergy in the temperate areas of the Northern hemisphere. This order includes two main families (*Betulaceae* and *Fagaceae*) comprising different trees characterized

by a rather limited number of homologous, cross-reacting allergens⁵⁷⁴ (Table 12). Moreover, pollen from birch has shown the ability to suppress innate antiviral immunity, independent of allergy.⁵⁷⁵,⁵⁷⁶

Pathogenesis-related-protein group 10 (PR-10) molecules (i.e., Bet v 1 and homologous allergens)⁵⁷⁷ are the major allergens in Fagales pollen and are recognized by virtually all allergic patients, thus representing the major cause of clinical allergy (see also Chapter C02), which includes a large group of aeroallergens and common food allergens. Several PR-10 family members have been described to date within tree pollen belonging to the Fagales order ([i] *Betulaceae*: Aln g 1 from alder, Bet v 1 from birch, Car b 1 from hornbeam, Cas s 1 from chestnut and Cor a 1 from hazel, [ii] *Fagaceae*: Fag s 1 from European beech, Ost c 1 from hop hornbeam, Que a 1 from white oak, Que i 1 from Sawtooth oak, Que i 1 from Holly Oak, and Que m 1 from Mongolian oak).

In addition to PR-10 proteins, several other allergens have been described.

(i) Profilins (e.g., Bet v 2 from birch pollen or Cor a 2 from hazel pollen)⁵⁷⁸ [20] are pan-allergens (see Chapter C01) present in the whole plant kingdom. Profilins are recognized by 10-20% of patients primarily sensitized to birch pollen, but this proportion is higher in areas where grass pollen represents the primary sensitizer. The clinical relevance of profilin as a respiratory allergen is variable.⁵⁷⁹ Profilins may cause secondary plant food allergy to various fruits and vegetables (see Chapter C01).

(ii) Polcalcin-like proteins (calcium-binding proteins; e.g., Bet v 3 and Bet v 4 from birch, and Aln g 4 from alder) are pollen pan-allergens, which generally sensitize less than 10% of pollen-allergic individuals. They cross-react with homologous pollen allergens from botanically unrelated species. The clinical relevance is variable and often limited⁵⁷⁹: in a retrospective study of 854 Italian patients with birch pollen sensitization, Bet v 1 sensitization significantly decreased from the North (95.41%) to the South (58.56%) of the country, whereas both profilin and polcalcin reactivity significantly increased from Northern to Southern Italy⁵⁸⁰;

(iii) phenyl-coumaran benzylic ether reductases or isoflavone reductases (e.g., Bet v 6 from birch, Cor a 6 from hazel, Ole e 12 from olive) are minor allergens that are involved in plant defence reactions, showing a sensitization rate of about 32% among birch allergic people⁵⁸¹;

(iv) cyclophilin (Bet v 7 from birch) is a minor, potentially cross-reactive, allergen;

(v) pectin methylesterase;

(vi) glucanase;

(vii) thaumatin-like protein; and

(viii) Glutathione-S-transferase (GST) (Bet v 8 from birch) are other minor allergens.⁵⁸²

Olive pollen allergy is caused by Ole e 1 allergy in about 70% of cases (Table 13). The Ole e 1-like protein family comprises several
in spring. Chestnuts shed pollen in June and July in Western and Central Europe.

Trees from the family *Oleaceae*, order **Lamiales**, grow on all 5 continents and are among the most important causes of respiratory allergy in the Mediterranean area as well as in some areas of North America, Australia, Japan, and North and South Africa, where these trees are intensively cultivated.⁵⁶⁵ The *Oleaceae* family comprises 4 main genera: olive (*Olea europea*), European ash (*Fraxinus excelsior*), lilac (*Syringa vulgaris*), and common privet (*Ligustrum vulgare*), all able to cause IgE sensitization ⁵⁶⁶ (Figure 45). The pollination period ranges from April to June in warmer regions, and the occurrence of olive tree allergy among patients suffering from tree pollen allergy is about 30-40% in Italy.⁵⁶⁷,⁵⁶⁸ Ash pollen season is during winter-time, rather similar to the birch pollen season, and is very relevant in Central Europe (Austria, North and East of France, Switzerland up to 30% prevalence of pollen allergic patients).

In Mediterranean regions trees of the *Cupressaceae* family (Figure 43) from the order **Pinales** are widely spread. Wind pollination of cypress trees occurs during the winter season, when no other allergenic plants flower, and accounts for up to 40% of the total pollen count in Mediterranean countries.⁵⁶⁹ Cypress tree florescence covers about 30-40 days, from January to April, showing a high variability from year to year, depending on weather conditions, causing difficulty in identifying the beginning and length of pollen season. The high degree of cross-reactivity found among *Cupressaceae* trees (cypress, juniper, and cedar), which have somewhat different but overlapping pollination periods, could extend the cypress pollen season from December to March.⁵⁷⁰

Trees of the Plane-tree family (e.g., *Platanus acerifolia*), from the order **Proteales**, are common species widely spread in Southern Europe, with a short but intense pollen season from March to April, characterized by high pollen counts, reaching one hundred billion pollen grains per tree only a few days after the florescence time. Clinical surveys have acknowledged plane trees as a major cause of pollen with sensitization rates ranging from 8 to 17% in exposed populations. Annual airborne pollen counts differ based on weather conditions but also as a function of human activity, mainly pruning since plane trees or sycamores are widely used for ornamental purposes.⁵⁷¹ Temperature, but not rainfall, is the weather parameter mainly affecting the Platanus pollen season, influencing both start-date and daily pollen counts.

In subtropical regions mesquite (*Prosopsis juliflora*) and Acacia farnesiana (*Vachellia farnesiana*) (Needle bush), trees belonging to the order **Fabales** have been acknowledged as a clinically relevant allergen in North America, India, and the Arabian Peninsula.⁵⁷²,⁵⁷³

## 2 – Allergen families and allergenic molecules

Pollen from **Fagales** trees is one of the most frequent causes of winter/spring respiratory allergy in the temperate areas of the Northern hemisphere. This order includes two main families (*Betulaceae* and *Fagaceae*) comprising different trees characterized

by a rather limited number of homologous, cross-reacting allergens⁵⁷⁴ (Table 12). Moreover, pollen from birch has shown the ability to suppress innate antiviral immunity, independent of allergy.⁵⁷⁵,⁵⁷⁶

Pathogenesis-related-protein group 10 (PR-10) molecules (i.e., Bet v 1 and homologous allergens)⁵⁷⁷ are the major allergens in Fagales pollen and are recognized by virtually all allergic patients, thus representing the major cause of clinical allergy (see also Chapter C02), which includes a large group of aeroallergens and common food allergens. Several PR-10 family members have been described to date within tree pollen belonging to the Fagales order ([i] *Betulaceae*: Aln g 1 from alder, Bet v 1 from birch, Car b 1 from hornbeam, Cas s 1 from chestnut and Cor a 1 from hazel, [ii] *Fagaceae*: Fag s 1 from European beech, Ost c 1 from hop hornbeam, Que a 1 from white oak, Que i 1 from Sawtooth oak, Que i 1 from Holly Oak, and Que m 1 from Mongolian oak).

In addition to PR-10 proteins, several other allergens have been described.

(i) Profilins (e.g., Bet v 2 from birch pollen or Cor a 2 from hazel pollen)⁵⁷⁸ [20] are pan-allergens (see Chapter C01) present in the whole plant kingdom. Profilins are recognized by 10-20% of patients primarily sensitized to birch pollen, but this proportion is higher in areas where grass pollen represents the primary sensitizer. The clinical relevance of profilin as a respiratory allergen is variable.⁵⁷⁹ Profilins may cause secondary plant food allergy to various fruits and vegetables (see Chapter C01).

(ii) Polcalcin-like proteins (calcium-binding proteins; e.g., Bet v 3 and Bet v 4 from birch, and Aln g 4 from alder) are pollen pan-allergens, which generally sensitize less than 10% of pollen-allergic individuals. They cross-react with homologous pollen allergens from botanically unrelated species. The clinical relevance is variable and often limited⁵⁷⁹: in a retrospective study of 854 Italian patients with birch pollen sensitization, Bet v 1 sensitization significantly decreased from the North (95.41%) to the South (58.56%) of the country, whereas both profilin and polcalcin reactivity significantly increased from Northern to Southern Italy⁵⁸⁰;

(iii) phenyl-coumaran benzylic ether reductases or isoflavone reductases (e.g., Bet v 6 from birch, Cor a 6 from hazel, Ole e 12 from olive) are minor allergens that are involved in plant defence reactions, showing a sensitization rate of about 32% among birch allergic people⁵⁸¹;

(iv) cyclophilin (Bet v 7 from birch) is a minor, potentially cross-reactive, allergen;

(v) pectin methylesterase;

(vi) glucanase;

(vii) thaumatin-like protein; and

(viii) Glutathione-S-transferase (GST) (Bet v 8 from birch) are other minor allergens.⁵⁸²

Olive pollen allergy is caused by Ole e 1 allergy in about 70% of cases (Table 13). The Ole e 1-like protein family comprises several
<table><caption>TABLE 12 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead><tbody><tr><td colspan="4"><strong>Alnus glutinosa (Alder)</strong></td></tr><tr><td>Aln g 1</td><td>PR-10, Bet v 1 family member</td><td>100%</td><td>18</td></tr><tr><td>Aln g 4</td><td>Polcalcin</td><td>18%</td><td>6</td></tr><tr><td colspan="4"><strong>Betula verrucosa (Betula pendula) (European white birch)</strong></td></tr><tr><td>Bet v 1</td><td>PR-10, Bet v 1 family member</td><td>95%</td><td>17</td></tr><tr><td>Bet v 2</td><td>Profilin</td><td>22%</td><td>15</td></tr><tr><td>Bet v 3</td><td>Polcalcin-like protein</td><td>10%</td><td>24</td></tr><tr><td>Bet v 4</td><td>Polcalcin</td><td>5%</td><td>7</td></tr><tr><td>Bet v 6</td><td>PhenylCoumaran benzylic ether reductase</td><td>32%</td><td>35</td></tr><tr><td>Bet v 7</td><td>Cyclophilin</td><td>21%</td><td>18</td></tr><tr><td>Bet v 8</td><td>Glutathione-S-transferase</td><td>13%</td><td>27</td></tr><tr><td colspan="4"><strong>Carpinus betulus (Hornbeam)</strong></td></tr><tr><td>Car b 1</td><td>PR-10, Bet v 1 family member</td><td></td><td>17</td></tr><tr><td colspan="4"><strong>Corylus avellana (Hazel)</strong></td></tr><tr><td>Cor a 1</td><td>PR-10, Bet v 1 family member</td><td>100%</td><td>17</td></tr><tr><td>Cor a 2</td><td>Profilin</td><td></td><td>14</td></tr><tr><td>Cor a 6</td><td>Isoflavone reductase homologue</td><td></td><td>35</td></tr><tr><td>Cor a 10</td><td>Luminal binding protein</td><td></td><td>70</td></tr><tr><td colspan="4"><strong>Ostrya carpinifolia (European hophornbeam)</strong></td></tr><tr><td>Ost c 1</td><td>PR-10, Bet v 1 family member</td><td></td><td></td></tr><tr><td colspan="4"><strong>Castanea sativa (Chestnut)</strong></td></tr><tr><td>Cas s 1</td><td>PR-10, Bet v 1 family member</td><td>100%</td><td>22</td></tr><tr><td colspan="4"><strong>Fagus sylvatica (European beech)</strong></td></tr><tr><td>Fag s 1</td><td>PR-10, Bet v 1 family member</td><td></td><td>17</td></tr><tr><td colspan="4"><strong>Quercus Acutissima (Sawtooth oak)</strong></td></tr><tr><td>Que ac 1</td><td>PR-10, Bet v 1 family member</td><td>92%</td><td>17</td></tr><tr><td>Que ac 2</td><td>Profilin</td><td></td><td>16</td></tr><tr><td colspan="4"><strong>Quercus alba (White oak)</strong></td></tr><tr><td>Que a 1</td><td>PR-10, Bet v 1 family member</td><td>64%</td><td>17</td></tr><tr><td colspan="4"><strong>Quercus mongolica (Mongolian oak)</strong></td></tr><tr><td>Que m 1</td><td>Pathogenesis protein 10</td><td>92%</td><td>17</td></tr><tr><td colspan="4"><strong>Quercus ilex (Holly Oak)</strong></td></tr><tr><td>Que i 1</td><td>PR-10, Bet v 1 family member</td><td>55%</td><td>21</td></tr></tbody></table>

other allergenic glycosylated proteins from tree pollen (Fra e 1, Lig v 1, and Syr v 1), whose glycan moieties are involved in their allergenic properties⁵⁸³ Besides Ole e 1, several other molecules

have been identified, and a biological function can be associated with most of these molecules, such as actin-binding protein (the profilin Ole e 2), polcalcin (Ole e 3 and Ole e 8), glucanase (Ole e 9
<table><caption>TABLE 12 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead><tbody><tr><td colspan="4"><strong>Alnus glutinosa (Alder)</strong></td></tr><tr><td>Aln g 1</td><td>PR-10, Bet v 1 family member</td><td>100%</td><td>18</td></tr><tr><td>Aln g 4</td><td>Polcalcin</td><td>18%</td><td>6</td></tr><tr><td colspan="4"><strong>Betula verrucosa (Betula pendula) (European white birch)</strong></td></tr><tr><td>Bet v 1</td><td>PR-10, Bet v 1 family member</td><td>95%</td><td>17</td></tr><tr><td>Bet v 2</td><td>Profilin</td><td>22%</td><td>15</td></tr><tr><td>Bet v 3</td><td>Polcalcin-like protein</td><td>10%</td><td>24</td></tr><tr><td>Bet v 4</td><td>Polcalcin</td><td>5%</td><td>7</td></tr><tr><td>Bet v 6</td><td>PhenylCoumaran benzylic ether reductase</td><td>32%</td><td>35</td></tr><tr><td>Bet v 7</td><td>Cyclophilin</td><td>21%</td><td>18</td></tr><tr><td>Bet v 8</td><td>Glutathione-S-transferase</td><td>13%</td><td>27</td></tr><tr><td colspan="4"><strong>Carpinus betulus (Hornbeam)</strong></td></tr><tr><td>Car b 1</td><td>PR-10, Bet v 1 family member</td><td></td><td>17</td></tr><tr><td colspan="4"><strong>Corylus avellana (Hazel)</strong></td></tr><tr><td>Cor a 1</td><td>PR-10, Bet v 1 family member</td><td>100%</td><td>17</td></tr><tr><td>Cor a 2</td><td>Profilin</td><td></td><td>14</td></tr><tr><td>Cor a 6</td><td>Isoflavone reductase homologue</td><td></td><td>35</td></tr><tr><td>Cor a 10</td><td>Luminal binding protein</td><td></td><td>70</td></tr><tr><td colspan="4"><strong>Ostrya carpinifolia (European hophornbeam)</strong></td></tr><tr><td>Ost c 1</td><td>PR-10, Bet v 1 family member</td><td></td><td></td></tr><tr><td colspan="4"><strong>Castanea sativa (Chestnut)</strong></td></tr><tr><td>Cas s 1</td><td>PR-10, Bet v 1 family member</td><td>100%</td><td>22</td></tr><tr><td colspan="4"><strong>Fagus sylvatica (European beech)</strong></td></tr><tr><td>Fag s 1</td><td>PR-10, Bet v 1 family member</td><td></td><td>17</td></tr><tr><td colspan="4"><strong>Quercus Acutissima (Sawtooth oak)</strong></td></tr><tr><td>Que ac 1</td><td>PR-10, Bet v 1 family member</td><td>92%</td><td>17</td></tr><tr><td>Que ac 2</td><td>Profilin</td><td></td><td>16</td></tr><tr><td colspan="4"><strong>Quercus alba (White oak)</strong></td></tr><tr><td>Que a 1</td><td>PR-10, Bet v 1 family member</td><td>64%</td><td>17</td></tr><tr><td colspan="4"><strong>Quercus mongolica (Mongolian oak)</strong></td></tr><tr><td>Que m 1</td><td>Pathogenesis protein 10</td><td>92%</td><td>17</td></tr><tr><td colspan="4"><strong>Quercus ilex (Holly Oak)</strong></td></tr><tr><td>Que i 1</td><td>PR-10, Bet v 1 family member</td><td>55%</td><td>21</td></tr></tbody></table>

other allergenic glycosylated proteins from tree pollen (Fra e 1, Lig v 1, and Syr v 1), whose glycan moieties are involved in their allergenic properties⁵⁸³ Besides Ole e 1, several other molecules

have been identified, and a biological function can be associated with most of these molecules, such as actin-binding protein (the profilin Ole e 2), polcalcin (Ole e 3 and Ole e 8), glucanase (Ole e 9
<table><caption>TABLE 13 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th colspan="4">Oleaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead></table>

Fraxinus excelsior (Ash)

<table><tr><td>Fra e 1</td><td>Ole e 1-like protein family member</td><td>87%</td><td>20</td></tr></table>

Ligustrum vulgare (Privet)

<table><tr><td>Lig v 1</td><td>Ole e 1-like protein family member</td><td>58%</td><td>20</td></tr></table>

Olea europaea (Olive)

<table><tr><td>Ole e 1</td><td>Common olive group 1</td><td>90%</td><td>16</td></tr><tr><td>Ole e 2</td><td>Profilin</td><td>50%</td><td>15</td></tr><tr><td>Ole e 3</td><td>Polcalcin-like protein (4 EF-hands)</td><td></td><td>9</td></tr><tr><td>Ole e 4</td><td></td><td>80%</td><td>32</td></tr><tr><td>Ole e 5</td><td>Superoxide dismutase [Cu-Zn]</td><td>35%</td><td>16</td></tr><tr><td>Ole e 6</td><td></td><td>15%</td><td>10</td></tr><tr><td>Ole e 7</td><td>non-specific lipid transfer protein</td><td>47%</td><td>9</td></tr><tr><td>Ole e 8</td><td>Polcalcin-like protein</td><td></td><td>21</td></tr><tr><td>Ole e 9</td><td>1,3-β glucanase</td><td>68%</td><td>46</td></tr><tr><td>Ole e 10</td><td>X8 domain containing protein</td><td>90%</td><td>11</td></tr><tr><td>Ole e 11</td><td>Pectin methylesterase</td><td></td><td>39.4</td></tr><tr><td>Ole e 12</td><td>Isoflavone reductase</td><td>4-33%</td><td>37</td></tr><tr><td>Ole e 13</td><td>Thaumatin</td><td>13%</td><td>23</td></tr><tr><td>Ole e 14</td><td>Polygalacturonase</td><td>13%</td><td>46.5</td></tr><tr><td>Ole e 15</td><td>Cyclophilin</td><td>27%</td><td>19</td></tr></table>

Syringa vulgaris (Lilac)

<table><tr><td>Syr v 1</td><td>Ole e 1-like protein family member</td><td>90%</td><td>20</td></tr><tr><td>Syr v 3</td><td>Polcalcin</td><td>90%</td><td>8.9</td></tr></table>

<table><thead><tr><th colspan="4">Fabaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead></table>

Acacia farnesiana (Vachellia farnesiana) (Needle bush)

<table><tr><td>Aca f 1</td><td>Ole e 1-like protein family member</td><td>47%</td><td>17</td></tr><tr><td>Aca f 2</td><td>Profilin</td><td></td><td>14.3</td></tr></table>

Prosopis juliflora (Mesquite)

<table><tr><td>Pro j 1</td><td>Ole e 1-like protein family member</td><td>56%</td><td>18</td></tr><tr><td>Pro j 2</td><td>Profilin</td><td>45%</td><td>14.3</td></tr></table>

and its probable degradation product Ole e 4), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). Olive tree Ole e 7, shares less than 20% of amino acid sequence with Pru p 3.⁵⁶⁷ Even though the homology at the primary sequence level is low, the tertiary structure of nsLTP is rather similar. Immunologically they seem to be distinct, which is also true for Par j 2, the nsLTP from pellitory that does not cross-react with, e.g., Pru p 3 (see Chapter C03).

The glucanase Ole e 9, despite representing less than 0.3% of crude olive pollen content,⁵⁸⁴ induces sensitization in about 50% of patients in some Mediterranean regions with high olive pollen counts during pollen season.⁵⁶⁷,⁵⁸⁵ Patients sensitized to Ole e 9 seem to be at higher risk of suffering adverse side reactions during immunotherapy.⁵⁸⁵ Ole e 7 and Ole e 9 IgE recognition have been recently associated with local or systemic reactions to food,⁵⁸² and
<table><caption>TABLE 13 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th colspan="4">Oleaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead><tbody><tr><td colspan="4"><em>Fraxinus excelsior</em> (Ash)</td></tr><tr><td>Fra e 1</td><td>Ole e 1-like protein family member</td><td>87%</td><td>20</td></tr><tr><td colspan="4"><em>Ligustrum vulgare</em> (Privet)</td></tr><tr><td>Lig v 1</td><td>Ole e 1-like protein family member</td><td>58%</td><td>20</td></tr><tr><td colspan="4"><em>Olea europaea</em> (Olive)</td></tr><tr><td>Ole e 1</td><td>Common olive group 1</td><td>90%</td><td>16</td></tr><tr><td>Ole e 2</td><td>Profilin</td><td>50%</td><td>15</td></tr><tr><td>Ole e 3</td><td>Polcalcin-like protein (4 EF-hands)</td><td></td><td>9</td></tr><tr><td>Ole e 4</td><td></td><td>80%</td><td>32</td></tr><tr><td>Ole e 5</td><td>Superoxide dismutase [Cu-Zn]</td><td>35%</td><td>16</td></tr><tr><td>Ole e 6</td><td></td><td>15%</td><td>10</td></tr><tr><td>Ole e 7</td><td>non-specific lipid transfer protein</td><td>47%</td><td>9</td></tr><tr><td>Ole e 8</td><td>Polcalcin-like protein</td><td></td><td>21</td></tr><tr><td>Ole e 9</td><td>1,3-β glucanase</td><td>68%</td><td>46</td></tr><tr><td>Ole e 10</td><td>X8 domain containing protein</td><td>90%</td><td>11</td></tr><tr><td>Ole e 11</td><td>Pectin methylesterase</td><td></td><td>39.4</td></tr><tr><td>Ole e 12</td><td>Isoflavone reductase</td><td>4-33%</td><td>37</td></tr><tr><td>Ole e 13</td><td>Thaumatin</td><td>13%</td><td>23</td></tr><tr><td>Ole e 14</td><td>Polygalacturonase</td><td>13%</td><td>46.5</td></tr><tr><td>Ole e 15</td><td>Cyclophilin</td><td>27%</td><td>19</td></tr><tr><td colspan="4"><em>Syringa vulgaris</em> (Lilac)</td></tr><tr><td>Syr v 1</td><td>Ole e 1-like protein family member</td><td>90%</td><td>20</td></tr><tr><td>Syr v 3</td><td>Polcalcin</td><td>90%</td><td>8.9</td></tr><tr><th colspan="4">Fabaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr><tr><td colspan="4"><em>Acacia farnesiana</em> (Vachellia farnesiana) (Needle bush)</td></tr><tr><td>Aca f 1</td><td>Ole e 1-like protein family member</td><td>47%</td><td>17</td></tr><tr><td>Aca f 2</td><td>Profilin</td><td></td><td>14.3</td></tr><tr><td colspan="4"><em>Prosopis juliflora</em> (Mesquite)</td></tr><tr><td>Pro j 1</td><td>Ole e 1-like protein family member</td><td>56%</td><td>18</td></tr><tr><td>Pro j 2</td><td>Profilin</td><td>45%</td><td>14.3</td></tr></tbody></table>

and its probable degradation product Ole e 4), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). Olive tree Ole e 7, shares less than 20% of amino acid sequence with Pru p 3.⁵⁶⁷ Even though the homology at the primary sequence level is low, the tertiary structure of nsLTP is rather similar. Immunologically they seem to be distinct, which is also true for Par j 2, the nsLTP from pellitory that does not cross-react with, e.g., Pru p 3 (see Chapter C03).

The glucanase Ole e 9, despite representing less than 0.3% of crude olive pollen content,⁵⁸⁴ induces sensitization in about 50% of patients in some Mediterranean regions with high olive pollen counts during pollen season.⁵⁶⁷,⁵⁸⁵ Patients sensitized to Ole e 9 seem to be at higher risk of suffering adverse side reactions during immunotherapy.⁵⁸⁵ Ole e 7 and Ole e 9 IgE recognition have been recently associated with local or systemic reactions to food,⁵⁸² and
atopic dermatitis,⁵⁸⁶ respectively. Ole e 10 (X8 domain-containing protein) and the pectin methylesterase Ole e 11 are two other major olive pollen allergens.

So far, both Ole e 1-like proteins (Aca f 1 and Pro j 1) and profilins (Aca f 2 and Pro j 2) have been identified and characterized, as relevant allergens in *Acacia farnesiana* and mesquite (*Prosopis juliflora*) tree pollen allergy, respectively.⁵⁸⁷⁻⁵⁸⁹

In the Cypress family, two main groups of proteins have been identified: the pectate lyases and the polygalacturonases⁵⁹⁰ (Table 14). The highly related (95.1% sequence identity) pectate lyases Cup a 1 and Cup s 1 are found in the Mediterranean area, whilst Cry j 1 and Cha o 1 are mainly found in Japan, sharing lower sequence identity (78.6%).

The polygalacturonases, Cha o 2, Cry j 2, and Jun a 2 are also major allergens of Pinales pollen, showing high levels of sequence identities (71%-82%).

Recently cypmaclein, an allergen belonging to the Gibberellin regulated protein (GRP) family, has been isolated from the cypress pollen⁵⁹¹ (Table 14). GRP sensitization is important to define a subset of patients with allergy to cypress pollen and severe peach allergy caused by Pru p 7 (Peamaclein) co-recognition²³¹ (see Chapter C09). Peamaclein sensitization prevalence seems to be quite frequent in France²³⁰ but rare in Italy.⁵⁹²

The most important allergen from London plane tree (*Platanus acerifolia*) pollen is Pla a 1,⁵⁹³ which has an invertase inhibitor function as has the homologous Pla or 1 from Platanus orientalis

<table><caption>TABLE 14 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th colspan="4">Cupressaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead><tbody><tr><td colspan="4"><em>Chamaecyparis obtusa</em> (Japanese cypress)</td></tr><tr><td>Cha o 1</td><td>Pectate lyase</td><td>97.5%</td><td>40.2</td></tr><tr><td>Cha o 2</td><td>Polygalacturonase</td><td>82.5%</td><td>45</td></tr><tr><td>Cha o 3</td><td>Cellulase (glycosyl hydrolase)</td><td>87.5%</td><td>63</td></tr><tr><td colspan="4"><em>Cryptomeria japonica</em> (Sugi)</td></tr><tr><td>Cry j 1</td><td>Pectate lyase</td><td>&gt;90%</td><td>41-45</td></tr><tr><td>Cry j 2</td><td>Polygalacturonase</td><td>&gt;90%</td><td>45</td></tr><tr><td>Cry j 7</td><td>Gibberellin-regulated protein</td><td>100%</td><td>7</td></tr><tr><td colspan="4"><em>Cupressus arizonica</em> (Cypress)</td></tr><tr><td>Cup a 1</td><td>Pectate lyase</td><td>100%</td><td>43</td></tr><tr><td colspan="4"><em>Cupressus sempervirens</em> (Common cypress)</td></tr><tr><td>Cup s 1</td><td>Pectate lyase</td><td></td><td>43</td></tr><tr><td>Cup s 2</td><td>Polygalacturonase</td><td>61.30%</td><td>43</td></tr><tr><td>Cup s 3</td><td>Thaumatin-like protein</td><td></td><td>34</td></tr><tr><td>Cup s 7</td><td>Gibberellin-regulated protein</td><td></td><td>9.5</td></tr><tr><td colspan="4"><em>Juniperus ashei</em> (Mountain cedar)</td></tr><tr><td>Jun a 1</td><td>Pectate lyase</td><td>71.4%</td><td>43</td></tr><tr><td>Jun a 2</td><td>Polygalacturonase</td><td>100%</td><td>43</td></tr><tr><td>Jun a 3</td><td>Thaumatin-like protein</td><td>43%</td><td>30</td></tr><tr><td>Jun a 7</td><td>Gibberellin-regulated protein</td><td>100%</td><td>7</td></tr><tr><td colspan="4"><em>Juniperus oxycedrus</em> (Prickly juniper)</td></tr><tr><td>Jun o 4</td><td>Polcalcin-like protein</td><td>15%</td><td>29</td></tr><tr><td colspan="4"><em>Juniperus virginiana</em> (Eastern red cedar)</td></tr><tr><td>Jun v 1</td><td>Pectate lyase</td><td>46%</td><td>43</td></tr><tr><td>Jun v 3</td><td>Thaumatin-like protein</td><td></td><td>34</td></tr></tbody></table>
atopic dermatitis,⁵⁸⁶ respectively. Ole e 10 (X8 domain-containing protein) and the pectin methylesterase Ole e 11 are two other major olive pollen allergens.

So far, both Ole e 1-like proteins (Aca f 1 and Pro j 1) and profilins (Aca f 2 and Pro j 2) have been identified and characterized, as relevant allergens in *Acacia farnesiana* and mesquite (*Prosopis juliflora*) tree pollen allergy, respectively.⁵⁸⁷⁻⁵⁸⁹

In the Cypress family, two main groups of proteins have been identified: the pectate lyases and the polygalacturonases⁵⁹⁰ (Table 14). The highly related (95.1% sequence identity) pectate lyases Cup a 1 and Cup s 1 are found in the Mediterranean area, whilst Cry j 1 and Cha o 1 are mainly found in Japan, sharing lower sequence identity (78.6%).

The polygalacturonases, Cha o 2, Cry j 2, and Jun a 2 are also major allergens of Pinales pollen, showing high levels of sequence identities (71%-82%).

Recently cypmaclein, an allergen belonging to the Gibberellin regulated protein (GRP) family, has been isolated from the cypress pollen⁵⁹¹ (Table 14). GRP sensitization is important to define a subset of patients with allergy to cypress pollen and severe peach allergy caused by Pru p 7 (Peamaclein) co-recognition²³¹ (see Chapter C09). Peamaclein sensitization prevalence seems to be quite frequent in France²³⁰ but rare in Italy.⁵⁹²

The most important allergen from London plane tree (*Platanus acerifolia*) pollen is Pla a 1,⁵⁹³ which has an invertase inhibitor function as has the homologous Pla or 1 from Platanus orientalis

<table><caption>TABLE 14 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th colspan="4">Cupressaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead><tbody><tr><td colspan="4"><em>Chamaecyparis obtusa</em> (Japanese cypress)</td></tr><tr><td>Cha o 1</td><td>Pectate lyase</td><td>97.5%</td><td>40.2</td></tr><tr><td>Cha o 2</td><td>Polygalacturonase</td><td>82.5%</td><td>45</td></tr><tr><td>Cha o 3</td><td>Cellulase (glycosyl hydrolase)</td><td>87.5%</td><td>63</td></tr><tr><td colspan="4"><em>Cryptomeria japonica</em> (Sugi)</td></tr><tr><td>Cry j 1</td><td>Pectate lyase</td><td>&gt;90%</td><td>41-45</td></tr><tr><td>Cry j 2</td><td>Polygalacturonase</td><td>&gt;90%</td><td>45</td></tr><tr><td>Cry j 7</td><td>Gibberellin-regulated protein</td><td>100%</td><td>7</td></tr><tr><td colspan="4"><em>Cupressus arizonica</em> (Cypress)</td></tr><tr><td>Cup a 1</td><td>Pectate lyase</td><td>100%</td><td>43</td></tr><tr><td colspan="4"><em>Cupressus sempervirens</em> (Common cypress)</td></tr><tr><td>Cup s 1</td><td>Pectate lyase</td><td></td><td>43</td></tr><tr><td>Cup s 2</td><td>Polygalacturonase</td><td>61.30%</td><td>43</td></tr><tr><td>Cup s 3</td><td>Thaumatin-like protein</td><td></td><td>34</td></tr><tr><td>Cup s 7</td><td>Gibberellin-regulated protein</td><td></td><td>9.5</td></tr><tr><td colspan="4"><em>Juniperus ashei</em> (Mountain cedar)</td></tr><tr><td>Jun a 1</td><td>Pectate lyase</td><td>71.4%</td><td>43</td></tr><tr><td>Jun a 2</td><td>Polygalacturonase</td><td>100%</td><td>43</td></tr><tr><td>Jun a 3</td><td>Thaumatin-like protein</td><td>43%</td><td>30</td></tr><tr><td>Jun a 7</td><td>Gibberellin-regulated protein</td><td>100%</td><td>7</td></tr><tr><td colspan="4"><em>Juniperus oxycedrus</em> (Prickly juniper)</td></tr><tr><td>Jun o 4</td><td>Polcalcin-like protein</td><td>15%</td><td>29</td></tr><tr><td colspan="4"><em>Juniperus virginiana</em> (Eastern red cedar)</td></tr><tr><td>Jun v 1</td><td>Pectate lyase</td><td>46%</td><td>43</td></tr><tr><td>Jun v 3</td><td>Thaumatin-like protein</td><td></td><td>34</td></tr></tbody></table>
(Table 15). Pla a 2 and Pla or 2 are major allergens displaying a poly-galacturonase activity. The plane tree Pla a 3 belongs to the family of nonspecific lipid transfer proteins, showing 58.3% sequence identity with the nsLTP Pru p 3 from peach.⁵⁹⁴

Cashew tree pollen appears to be an important cause of rhino-conjunctivitis symptoms in the northeast of Brazil.⁵⁹⁵ Peachtree pollen allergy may be associated with respiratory symptoms in adolescents living in regions where peach trees are grown.⁵⁹⁶

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

In Europe, the prevalence of positive skin prick test to birch pollen allergens ranges from 5% in The Netherlands to 54% in Switzerland, while Scandinavian countries have the highest number of patients with exclusive sensitization to Bet v 1.⁵⁹⁷ Bet v 1-specific IgE levels are not predictive for the development of pollen-related bronchial asthma. PR-10 proteins defend plants against fungi and other micro-organisms. Their homologues are also present in a large number of plant-derived foods, and thus frequently cause IgE cross-sensitization and consequently plant-food allergy (oral allergy syndrome, in most cases). For this reason, up to 70% of patients with sensitization to PR-10 proteins complain about oral symptoms following the ingestion of certain plant foods (e.g., apples, carrots, nuts and stone fruit) (see chapter C02). This indicates from a clinical point of view the need to check at least one representative allergen from both the *Betulaceae* family (i.e., Bet v 1 from birch) and the *Fagaceae* family (i.e., Que a 1 from oak) in all patients in the daily clinical practice. Interestingly, Pru p 9, also belonging to the PR-10 family, is responsible for the respiratory symptoms in patients allergic to peachtree pollen.⁵⁹⁸

The assessment of IgE reactivity to a panel of PR-10 proteins also in birch-free areas may lead to disclosing peculiar relationships between clinical phenotypes and sensitization profiles, such as the association among Bet v 1-, Cor a 1-, and Aln g 1-specific IgE recognition and the occurrence of respiratory symptoms.⁵⁹⁹

Also, *Olea europaea* reactivity seems to be clinically characterized by rhino-conjunctivitis more than bronchial asthma, but a dramatic outbreak of asthma attacks may occur during a thunderstorm in the olive pollen season. Moreover, in olive pollen patients, polysensitization is more common than mono-sensitization. Reactivity to other genera belonging to the Oleaceae family, i.e., *Fraxinus excelsior* or *Ligustrum vulgare*, is relevant in several regions in Central and Southern Europe.⁶⁰⁰ Olive tree Ole e 1 is a 145 amino acid protein sharing both significant sequence identity (82.76% of identity with 120 identical positions and 19 similar positions, as obtained by comparing the single sequences via https://www.uniprot.org/align/) and IgE cross-reactivity with all the other related trees belonging to the Olive family (Fra e 1 from ash, Lig v 1 from privet and Syr v 1 from lilac).⁶⁰¹ Several Ole e 1-like proteins have been described in goose-foot (*Chenopodium album*, Che a 1), timothy (*Phleum pretense*, Phl p 11), rye-grass (Lolium perenne, Lol p 11), English plantain (*Plantago lanceolata*, Pla I 1) and prickly saltwort (*Salsola kali*, Sal k 5), but the real clinical cross-reactivity of these molecules, not belonging to the olive family, with Ole e 1 is somewhat questioned.⁶⁰⁰ As already mentioned, the major allergens of Acacia farnesiana and mesquite (*Prosopis juliflora*) tree are both “Ole e 1 like proteins” (respectively Aca f 1 and Pro j 1): interestingly, Aca f 1 from *Acacia farnesiana* has overall 45.3% of identity with Ole e 1 (68 identical and 41 similar positions).

Patients allergic to cypress pollen may represent 30% of pollen-sensitized subjects in some areas⁶⁰² and 42% in central and southern Italy.⁵⁶⁸,⁶⁰³ Cypress pectate lyase allergy generally causes seasonal allergic rhinitis, and a very low occurrence of bronchial asthma in sensitized patients has been reported in the literature,⁶⁰⁴ but since asthma is a genetically determined condition, it depends more on the degree of sensitization and exposure rather than on the intrinsic characteristics of the allergens. High sequence identity and IgE cross-reactivity among the pectate lyases belonging to the *Cupressaceae* family (Cha o 1, Cry j 1, Cup a 1, Cup s 1, Jun a 1, Jun c 1, Jun o 1 and Jun v 1) is observed, with an overall 70% of similarity (262 identical and 74 similar amino acid positions, as obtained by comparing the single sequences via https://www.uniprot.org/align).

<table><caption>TABLE 15 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th colspan="4">Platanaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead><tbody><tr><td colspan="4"><em>Platanus acerifolia</em> (London plane tree)</td></tr><tr><td>Pla a 1</td><td>Putative invertase inhibitor</td><td>87.5%</td><td>18</td></tr><tr><td>Pla a 2</td><td>Polygalacturonase</td><td>83%</td><td>43</td></tr><tr><td>Pla a 3</td><td>Non-specific lipid transfer protein type 1</td><td>45%</td><td>10</td></tr><tr><td colspan="4"><em>Platanus orientalis</em> (Oriental plane)</td></tr><tr><td>Pla or 1</td><td>Putative invertase inhibitor</td><td>15.8%</td><td>18</td></tr><tr><td>Pla or 2</td><td>Polygalacturonase</td><td>26.3%</td><td>42</td></tr><tr><td>Pla or 3</td><td>Non-specific lipid transfer protein type 1</td><td>26.3%</td><td>11</td></tr></tbody></table>
(Table 15). Pla a 2 and Pla or 2 are major allergens displaying a poly-galacturonase activity. The plane tree Pla a 3 belongs to the family of nonspecific lipid transfer proteins, showing 58.3% sequence identity with the nsLTP Pru p 3 from peach.⁵⁹⁴

Cashew tree pollen appears to be an important cause of rhino-conjunctivitis symptoms in the northeast of Brazil.⁵⁹⁵ Peachtree pollen allergy may be associated with respiratory symptoms in adolescents living in regions where peach trees are grown.⁵⁹⁶

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

In Europe, the prevalence of positive skin prick test to birch pollen allergens ranges from 5% in The Netherlands to 54% in Switzerland, while Scandinavian countries have the highest number of patients with exclusive sensitization to Bet v 1.⁵⁹⁷ Bet v 1-specific IgE levels are not predictive for the development of pollen-related bronchial asthma. PR-10 proteins defend plants against fungi and other micro-organisms. Their homologues are also present in a large number of plant-derived foods, and thus frequently cause IgE cross-sensitization and consequently plant-food allergy (oral allergy syndrome, in most cases). For this reason, up to 70% of patients with sensitization to PR-10 proteins complain about oral symptoms following the ingestion of certain plant foods (e.g., apples, carrots, nuts and stone fruit) (see chapter C02). This indicates from a clinical point of view the need to check at least one representative allergen from both the *Betulaceae* family (i.e., Bet v 1 from birch) and the *Fagaceae* family (i.e., Que a 1 from oak) in all patients in the daily clinical practice. Interestingly, Pru p 9, also belonging to the PR-10 family, is responsible for the respiratory symptoms in patients allergic to peachtree pollen.⁵⁹⁸

The assessment of IgE reactivity to a panel of PR-10 proteins also in birch-free areas may lead to disclosing peculiar relationships between clinical phenotypes and sensitization profiles, such as the association among Bet v 1-, Cor a 1-, and Aln g 1-specific IgE recognition and the occurrence of respiratory symptoms.⁵⁹⁹

Also, *Olea europaea* reactivity seems to be clinically characterized by rhino-conjunctivitis more than bronchial asthma, but a dramatic outbreak of asthma attacks may occur during a thunderstorm in the olive pollen season. Moreover, in olive pollen patients, polysensitization is more common than mono-sensitization. Reactivity to other genera belonging to the Oleaceae family, i.e., *Fraxinus excelsior* or *Ligustrum vulgare*, is relevant in several regions in Central and Southern Europe.⁶⁰⁰ Olive tree Ole e 1 is a 145 amino acid protein sharing both significant sequence identity (82.76% of identity with 120 identical positions and 19 similar positions, as obtained by comparing the single sequences via https://www.uniprot.org/align/) and IgE cross-reactivity with all the other related trees belonging to the Olive family (Fra e 1 from ash, Lig v 1 from privet and Syr v 1 from lilac).⁶⁰¹ Several Ole e 1-like proteins have been described in goose-foot (*Chenopodium album*, Che a 1), timothy (*Phleum pretense*, Phl p 11), rye-grass (Lolium perenne, Lol p 11), English plantain (*Plantago lanceolata*, Pla I 1) and prickly saltwort (*Salsola kali*, Sal k 5), but the real clinical cross-reactivity of these molecules, not belonging to the olive family, with Ole e 1 is somewhat questioned.⁶⁰⁰ As already mentioned, the major allergens of Acacia farnesiana and mesquite (*Prosopis juliflora*) tree are both “Ole e 1 like proteins” (respectively Aca f 1 and Pro j 1): interestingly, Aca f 1 from *Acacia farnesiana* has overall 45.3% of identity with Ole e 1 (68 identical and 41 similar positions).

Patients allergic to cypress pollen may represent 30% of pollen-sensitized subjects in some areas⁶⁰² and 42% in central and southern Italy.⁵⁶⁸,⁶⁰³ Cypress pectate lyase allergy generally causes seasonal allergic rhinitis, and a very low occurrence of bronchial asthma in sensitized patients has been reported in the literature,⁶⁰⁴ but since asthma is a genetically determined condition, it depends more on the degree of sensitization and exposure rather than on the intrinsic characteristics of the allergens. High sequence identity and IgE cross-reactivity among the pectate lyases belonging to the *Cupressaceae* family (Cha o 1, Cry j 1, Cup a 1, Cup s 1, Jun a 1, Jun c 1, Jun o 1 and Jun v 1) is observed, with an overall 70% of similarity (262 identical and 74 similar amino acid positions, as obtained by comparing the single sequences via https://www.uniprot.org/align).

<table><caption>TABLE 15 The prevalence data shown in the table are derived from www.allergen.org for each allergen indicated.</caption><thead><tr><th colspan="4">Platanaceae</th></tr><tr><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence among patients</th><th>MW (in kDa)</th></tr></thead><tbody><tr><td colspan="4"><em>Platanus acerifolia</em> (London plane tree)</td></tr><tr><td>Pla a 1</td><td>Putative invertase inhibitor</td><td>87.5%</td><td>18</td></tr><tr><td>Pla a 2</td><td>Polygalacturonase</td><td>83%</td><td>43</td></tr><tr><td>Pla a 3</td><td>Non-specific lipid transfer protein type 1</td><td>45%</td><td>10</td></tr><tr><td colspan="4"><em>Platanus orientalis</em> (Oriental plane tree)</td></tr><tr><td>Pla or 1</td><td>Putative invertase inhibitor</td><td>15.8%</td><td>18</td></tr><tr><td>Pla or 2</td><td>Polygalacturonase</td><td>26.3%</td><td>42</td></tr><tr><td>Pla or 3</td><td>Non-specific lipid transfer protein type 1</td><td>26.3%</td><td>11</td></tr></tbody></table>
A 50% sequence identity with the ragweed allergenic pectate lyase Amb a 1 without evidence of cross-reactivity has been described.⁶⁰⁵

In the case of polygalacturonases an IgE cross-reactivity among the homologous allergens belonging to Cupressaceae families (Cri j 2, Cha o 2, Cup a 2, Cup s 2 and Jun a 2) has been observe.⁵⁶¹ Polygalacturonase belonging to timothy grass (Phl p 13) showed also considerable (up to 40%) sequence identity with Cri j 2, without measurable cross-reactivity.⁶⁰⁶

Plane tree Pla a 1 has a 98% identity with Pla or 1. Pla a 2 and Pla or 2 are other major allergens displaying a polygalacturonase activity with only 35% of sequence identity and no cross-reactivity with Cryptomeria japonica Cry j 2.⁵⁹⁴ The plane tree Pla a 3 exhibits approximately 50% sequence identity with peach Pru p 3, and is therefore possibly implicated in plant food-pollen co-sensitization.⁶⁰⁷

## Clinical relevance and patterns

Certain proteins are restricted to a given allergenic biological source, and therefore can be considered as "marker allergens" or a genuine "signature" clinically useful for the identification of patients for whom immunotherapy with a given allergen extract is appropriate. For instance, in the tree pollen model, the major birch pollen, Bet v 1, can identify individuals allergic to the *Betulaceae* family. The olive tree major allergen, Ole e 1, detects sensitization to the *Oleaceae* family, the cypress pollen major allergen, Cup a 1, reveals sensitization to the *Cupressaceae* family, and Pla a 1 detects sensitization to the Platanaceae family. Other allergens exhibit a large cross-reactivity and their distribution is not restricted to a given taxonomical order, but they are rather distributed throughout the entire plant kingdom and are therefore found in all tree pollen families (the so-called pan-allergens). Polcalcin-like proteins and profilins are typical examples of pan-allergens. In the case of polcalcin-like proteins (see Chapter C06), also known as EF-hand calcium-binding allergens (i.e., alder Aln g 4, hornbeam Car b 4, birch Bet v 4, beech Fag s 4, and oak Que a 4), IgE recognition is often associated with multiple pollen (grass, weed and tree) sensitization,⁶⁰⁸ a lower response to immunotherapy, and an association with bronchial asthma.⁶⁰⁹ Patients sensitized to profilins (see Chapter C01) (e.g., Aln g 2 from alder, Bet v 2 from birch, Car b 2 from hornbeam, Cas s 2 from chestnut, Cor a 2 from hazel, Fag s 2 from beech and Que a 2 from oak) are not only reactors to a pan-allergen found in distinct sources but also true plant food multi-sensitized patients.⁶¹⁰ Pan-allergens reactivity could therefore cause a misleading interpretation if allergy testing is carried out only using allergenic extracts. A given patient could have positive extract-based tests to several tree pollen extracts, due to IgE recognition of both genuine and/or pan-allergens, or as a result of an IgE recognition of pan-allergens in the absence of a genuine reactivity to the marker allergens. Despite the high sequence identity observed among constituents of every single group of pan-allergens, testing of several pan-allergens could increase assay reliability and the identification of interesting clinical phenotypes, albeit in daily clinical practice a less expensive approach may often be necessary. Recently, GRPs (see Chapter C09) have turned out to be relevant allergens in multiple fruit allergic reactions, mainly from the *Rosaceae*

family, (i.e., peach, apricot or cherry), due to the high degree of cross-reactivity with the GRPs found in the cypress pollen.²³⁰,⁵⁵⁹

IgE reactivity due to cross-reactive carbohydrate determinants (CCDs) (see Chapter A10) should also be ruled out, since all plant extracts can be weakly recognized by patients' IgE specific for CCDs, with no clinical significance.⁶¹¹ Interestingly, such reactivity does not affect the execution of the skin prick tests, since the IgE binding with the CCDs is not able to induce cutaneous mast cell degranulation. In (Figure 46), genuine markers of sensitization are indicated in green and pan-allergens found in the different pollens are coloured in red.

## Clinical relevance

* Molecular markers of genuine reactivity: Cry j 1 (pectate lyase); Cup a 1 (pectate lyase); Aln g 1 (PR-10 protein); Bet v 1 (PR-10 protein); Cor a 1 (PR-10 protein); Ole e 1 (common olive group 1); Ole e 9 (beta-1,3-glucanase); Pla a 1 (putative invertase inhibitor); Pla a 2 (polygalacturonase).

* Pan-allergens: Profilin (Bet v 2), Polcalcin (Bet v 4), nsLTP (Ole e 7 and Pla a 3), and Gibberellin regulated protein (Cry j 7, Cup s 7, and Jun a 7). Check also the purified N-glycan from bromelain MUXF3 in the case of multiple tree pollen IgE reactivity, to rule out the possibility of CCD reactivity.

## 4 - Clinical Management

Allergen-specific immunotherapy (AIT) should be prescribed only when the clinical relevance of a given allergen source has been reliably demonstrated.⁶¹² In the presence of a multiple IgE sensitization, the first goal is to distinguish patients genuinely reactive from those misrecognizing a given biological source due to reactivity to pan-allergens. Another difficulty in identifying the primary sensitizing source occurs in several countries (i.e., Southern Europe) where overlapping of tree, weed or grass pollination periods takes place. Several molecules have been proposed as markers for the prediction of a better response to AIT: Ole e 1, Cup a 1, Bet v 1, Cor a 1 or Pla a 1 reactivity can be considered as specific signatures for a genuine tree pollen allergy.

Cup a 1 reactivity is the specific marker allergen for sensitization to the pollen of trees of the Cupressaceae family. Also, in this case, the high sequence identity, associated with a high degree of cross-reactivity among Cupressaceae family members, suggest the use of Cup a 1 as a representative marker of the entire family for both diagnostic testing and therapeutic approaches (Figures 46 and 47).

Bet v 1-sensitized individuals often experience an oral allergy syndrome due to the intake of food containing PR-10 proteins.
A 50% sequence identity with the ragweed allergenic pectate lyase Amb a 1 without evidence of cross-reactivity has been described.⁶⁰⁵

In the case of polygalacturonases an IgE cross-reactivity among the homologous allergens belonging to Cupressaceae families (Cri j 2, Cha o 2, Cup a 2, Cup s 2 and Jun a 2) has been observe.⁵⁶¹ Polygalacturonase belonging to timothy grass (Phl p 13) showed also considerable (up to 40%) sequence identity with Cri j 2, without measurable cross-reactivity.⁶⁰⁶

Plane tree Pla a 1 has a 98% identity with Pla or 1. Pla a 2 and Pla or 2 are other major allergens displaying a polygalacturonase activity with only 35% of sequence identity and no cross-reactivity with Cryptomeria japonica Cry j 2.⁵⁹⁴ The plane tree Pla a 3 exhibits approximately 50% sequence identity with peach Pru p 3, and is therefore possibly implicated in plant food-pollen co-sensitization.⁶⁰⁷

## Clinical relevance and patterns

Certain proteins are restricted to a given allergenic biological source, and therefore can be considered as "marker allergens" or a genuine "signature" clinically useful for the identification of patients for whom immunotherapy with a given allergen extract is appropriate. For instance, in the tree pollen model, the major birch pollen, Bet v 1, can identify individuals allergic to the *Betulaceae* family. The olive tree major allergen, Ole e 1, detects sensitization to the *Oleaceae* family, the cypress pollen major allergen, Cup a 1, reveals sensitization to the *Cupressaceae* family, and Pla a 1 detects sensitization to the Platanaceae family. Other allergens exhibit a large cross-reactivity and their distribution is not restricted to a given taxonomical order, but they are rather distributed throughout the entire plant kingdom and are therefore found in all tree pollen families (the so-called pan-allergens). Polcalcin-like proteins and profilins are typical examples of pan-allergens. In the case of polcalcin-like proteins (see Chapter C06), also known as EF-hand calcium-binding allergens (i.e., alder Aln g 4, hornbeam Car b 4, birch Bet v 4, beech Fag s 4, and oak Que a 4), IgE recognition is often associated with multiple pollen (grass, weed and tree) sensitization,⁶⁰⁸ a lower response to immunotherapy, and an association with bronchial asthma.⁶⁰⁹ Patients sensitized to profilins (see Chapter C01) (e.g., Aln g 2 from alder, Bet v 2 from birch, Car b 2 from hornbeam, Cas s 2 from chestnut, Cor a 2 from hazel, Fag s 2 from beech and Que a 2 from oak) are not only reactors to a pan-allergen found in distinct sources but also true plant food multi-sensitized patients.⁶¹⁰ Pan-allergens reactivity could therefore cause a misleading interpretation if allergy testing is carried out only using allergenic extracts. A given patient could have positive extract-based tests to several tree pollen extracts, due to IgE recognition of both genuine and/or pan-allergens, or as a result of an IgE recognition of pan-allergens in the absence of a genuine reactivity to the marker allergens. Despite the high sequence identity observed among constituents of every single group of pan-allergens, testing of several pan-allergens could increase assay reliability and the identification of interesting clinical phenotypes, albeit in daily clinical practice a less expensive approach may often be necessary. Recently, GRPs (see Chapter C09) have turned out to be relevant allergens in multiple fruit allergic reactions, mainly from the *Rosaceae*

family, (i.e., peach, apricot or cherry), due to the high degree of cross-reactivity with the GRPs found in the cypress pollen.²³⁰,⁵⁵⁹

IgE reactivity due to cross-reactive carbohydrate determinants (CCDs) (see Chapter A10) should also be ruled out, since all plant extracts can be weakly recognized by patients' IgE specific for CCDs, with no clinical significance.⁶¹¹ Interestingly, such reactivity does not affect the execution of the skin prick tests, since the IgE binding with the CCDs is not able to induce cutaneous mast cell degranulation. In (Figure 46), genuine markers of sensitization are indicated in green and pan-allergens found in the different pollens are coloured in red.

## Clinical relevance

* Molecular markers of genuine reactivity: Cry j 1 (pectate lyase); Cup a 1 (pectate lyase); Aln g 1 (PR-10 protein); Bet v 1 (PR-10 protein); Cor a 1 (PR-10 protein); Ole e 1 (common olive group 1); Ole e 9 (beta-1,3-glucanase); Pla a 1 (putative invertase inhibitor); Pla a 2 (polygalacturonase).

* Pan-allergens: Profilin (Bet v 2), Polcalcin (Bet v 4), nsLTP (Ole e 7 and Pla a 3), and Gibberellin regulated protein (Cry j 7, Cup s 7, and Jun a 7). Check also the purified N-glycan from bromelain MUXF3 in the case of multiple tree pollen IgE reactivity, to rule out the possibility of CCD reactivity.

## 4 - Clinical Management

Allergen-specific immunotherapy (AIT) should be prescribed only when the clinical relevance of a given allergen source has been reliably demonstrated.⁶¹² In the presence of a multiple IgE sensitization, the first goal is to distinguish patients genuinely reactive from those misrecognizing a given biological source due to reactivity to pan-allergens. Another difficulty in identifying the primary sensitizing source occurs in several countries (i.e., Southern Europe) where overlapping of tree, weed or grass pollination periods takes place. Several molecules have been proposed as markers for the prediction of a better response to AIT: Ole e 1, Cup a 1, Bet v 1, Cor a 1 or Pla a 1 reactivity can be considered as specific signatures for a genuine tree pollen allergy.

Cup a 1 reactivity is the specific marker allergen for sensitization to the pollen of trees of the Cupressaceae family. Also, in this case, the high sequence identity, associated with a high degree of cross-reactivity among Cupressaceae family members, suggest the use of Cup a 1 as a representative marker of the entire family for both diagnostic testing and therapeutic approaches (Figures 46 and 47).

Bet v 1-sensitized individuals often experience an oral allergy syndrome due to the intake of food containing PR-10 proteins.
Cupressaceae

<figure><img src="image_129.png" /></figure>

Betulaceae

<figure><img src="image_130.png" /></figure>

Oleaceae

<figure><img src="image_131.png" /></figure>

Platanaceae

<figure><img src="image_132.png" /><figcaption>FIGURE 46 Overview of the clinically most relevant marker molecules of genuine sensitization (green) and pan-allergens (red)</figcaption></figure>

It has been suggested that birch pollen AIT can improve not only pollen-related respiratory symptoms but also food-related adverse reactions,⁶¹³ but different outcomes without benefit are reported in other studies⁶¹⁴,⁶¹⁵ (see Chapter C02). AIT containing birch extracts can also be used to treat patients allergic to oak, given the cross-reactivity between Bet v 1 and Que a 1.⁶¹⁶

Ole e 1 is the most common sensitizing molecule in olive pollen. It is utilized in both diagnostic and therapeutic extracts for standardization purposes and can determine immunological changes after olive pollen AIT. On the other hand, due to the high degree of cross-reactivity among the Ole e 1-like proteins of the Oleaceae family, in olive-free areas, Ole e 1 reactivity could help to identify individuals reacting with ash or privet pollen as suitable for AIT.⁶¹⁷ In areas with heavy olive pollen exposure, Ole e 7 and Ole e 9 should be tested to identify patients with a more severe allergic phenotype.⁵⁶⁷

Pla a 1 and Pla a 2 may serve as a marker of primary sensitization to plane tree pollen, therefore useful for AIT selection, whilst the nsLTP Pla a 3 has been linked with sensitization to plant-food LTPs.⁵⁹²,⁵⁹⁴

Profilin and polcalcin (see chapters C01 and C06) represent the major causes of cross-reactivity due to their highly conserved structure and ubiquitous distribution. Profilin or polcalcin-reactors

score positive to all tree pollen after extract-based diagnostic testing. Several allergens that are currently available for routine testing (profilins from birch, Bet v 2, and grass, Phl p 12, and polcalcins from birch, rBet v 4, and grass, rPhl p 7), are marker molecules for the entire group of pan-allergens, excluding cypress and Parietaria profilins.⁶¹⁸ As a difference for LTP reactors, GRPs reactors score positive for Cypress on skin testing, but negative for the plane tree, mugwort, or olive tree.⁶¹⁹ IgE sensitization to pan-allergens, despite the ability to induce symptoms in sensitized patients, could affect AIT efficacy in the absence of species-specific molecules reactivity⁶¹² (Figure 47). Interestingly, most pan-allergen reactors are also co-sensitized to species-specific genuine molecules from different pollen sources,⁵⁹⁹ thus potentially require multiple pollen AIT to be successfully treated.

Cross-reactive carbohydrate determinants (CCDs) (see Chapter A10) do not behave as allergens *in vivo* and are therefore clinically insignificant,⁵⁰ but the presence of IgE to CCDs could lead to a misleading *in vitro* reactivity also in the case of extract-based testing or when using CCD-containing natural purified glycoproteins such as nCyn d 1, nOle e 1, nCup a 1, nSal k 1, nPla a 2 or nArt v 1.⁶²⁰ Recombinant proteins produced in Escherichia coli bacteria are not affected by CCD recognition, because of the lack of the
Cupressaceae

<figure><img src="image_128.png" /></figure>

Betulaceae

<figure><img src="image_129.png" /></figure>

Oleaceae

<figure><img src="image_130.png" /></figure>

Platanaceae

<figure><img src="image_131.png" /><figcaption>FIGURE 46 Overview of the clinically most relevant marker molecules of genuine sensitization (green) and pan-allergens (red)</figcaption></figure>

It has been suggested that birch pollen AIT can improve not only pollen-related respiratory symptoms but also food-related adverse reactions,⁶¹³ but different outcomes without benefit are reported in other studies⁶¹⁴,⁶¹⁵ (see Chapter C02). AIT containing birch extracts can also be used to treat patients allergic to oak, given the cross-reactivity between Bet v 1 and Que a 1.⁶¹⁶

Ole e 1 is the most common sensitizing molecule in olive pollen. It is utilized in both diagnostic and therapeutic extracts for standardization purposes and can determine immunological changes after olive pollen AIT. On the other hand, due to the high degree of cross-reactivity among the Ole e 1-like proteins of the Oleaceae family, in olive-free areas, Ole e 1 reactivity could help to identify individuals reacting with ash or privet pollen as suitable for AIT.⁶¹⁷ In areas with heavy olive pollen exposure, Ole e 7 and Ole e 9 should be tested to identify patients with a more severe allergic phenotype.⁵⁶⁷

Pla a 1 and Pla a 2 may serve as a marker of primary sensitization to plane tree pollen, therefore useful for AIT selection, whilst the nsLTP Pla a 3 has been linked with sensitization to plant-food LTPs.⁵⁹²,⁵⁹⁴

Profilin and polcalcin (see chapters C01 and C06) represent the major causes of cross-reactivity due to their highly conserved structure and ubiquitous distribution. Profilin or polcalcin-reactors

score positive to all tree pollen after extract-based diagnostic testing. Several allergens that are currently available for routine testing (profilins from birch, Bet v 2, and grass, Phl p 12, and polcalcins from birch, rBet v 4, and grass, rPhl p 7), are marker molecules for the entire group of pan-allergens, excluding cypress and Parietaria profilins.⁶¹⁸ As a difference for LTP reactors, GRPs reactors score positive for Cypress on skin testing, but negative for the plane tree, mugwort, or olive tree.⁶¹⁹ IgE sensitization to pan-allergens, despite the ability to induce symptoms in sensitized patients, could affect AIT efficacy in the absence of species-specific molecules reactivity⁶¹² (Figure 47). Interestingly, most pan-allergen reactors are also co-sensitized to species-specific genuine molecules from different pollen sources,⁵⁹⁹ thus potentially require multiple pollen AIT to be successfully treated.

Cross-reactive carbohydrate determinants (CCDs) (see Chapter A10) do not behave as allergens *in vivo* and are therefore clinically insignificant,⁵⁰ but the presence of IgE to CCDs could lead to a misleading *in vitro* reactivity also in the case of extract-based testing or when using CCD-containing natural purified glycoproteins such as nCyn d 1, nOle e 1, nCup a 1, nSal k 1, nPla a 2 or nArt v 1.⁶²⁰ Recombinant proteins produced in Escherichia coli bacteria are not affected by CCD recognition, because of the lack of the
<figure><img src="image_133.png" /><figcaption>FIGURE 47 Examples of clinical decision algorithms for different setting of reactivity in SPT or IgE testing with allergen extracts.</figcaption></figure>

post-translational glycosylation of proteins⁶²¹ Bromelain (Ana c 2) or the purified N-glycan from bromelain MUXF3 are available to detect CCDs *in vitro* in the vast majority of pollen sources, except for nArt v 1 where CCD reactivity is driven by O-glycans and not N-glycans measurable with bromelain or MUXF3. A positive skin prick test or *in vivo* (i.e., nasal, or conjunctival) challenge with the biological source can prove the genuine IgE recognition.⁵⁰ In (Figure 45) we suggest several algorithms possibly useful to complete the diagnostic work-up of tree pollen allergic patients.

5 - Clinical Cases

Case 1 (original):

**Clinical History:** Male, Italian, 10 years old with seasonal rhinitis between February and June and oral allergy syndrome induced by raw fruits (apple, peach) and vegetables (carrot, celery).

**IgE and skin tests:** in 2016, the boy reacted to Birch, Hazel, Oak and Cypress on skin prick testing, whilst no allergy to food was detected. *In vitro* IgE measurements by extracts showed reactivity to all pollens and plant foods tested, due to strong CCD reactivity.

The component resolved diagnosis by a commercial multiplex platform revealed IgE reactivity to PR10 molecules (rApi g 1: 4,46 ISU-E; rAra h 8: 0,13 ISU-E; rBet v 1: 3,67 ISU-E; rCor a 1.0101 0,21 ISU-E; rCor a 1.0401: 2,32 ISU-E; rGly m 4: 0,52 ISU-E; rMal d 1: 0,19 ISU-E), cypress pollen allergens (nCry j 1: 2,29 ISU-E; nCup a 1: 2,24 ISU-E) and CCD markers of reactivity (MUXF3 (Ana c 2.0101): 4,13 ISU-E; nPla a 2: 3,30 ISU-E; nJug r 2: 0,94 ISU-E).

Subsequently, due to anaphylactic reactions (diffuse angioedema, respiratory distress and hypotension) following the ingestion of peach on one occasion and mandarin in a second case, the young patient was further evaluated by another multiplex platform in 2021 that revealed an IgE sensitization to peamaclein along with the already known reactivity to PR10 (rPru p 7 (Gibberellin-RP): 17,79 kU/L; rAln g 1: 3,4 kU/L; rApi g 1: 9,96 kU/L; rAra h 8: 0,38 kU/L; rBet v 1: 7,08 kU/L; rCor a 1.0103: 15,42 kU/L; rCor a 1.0401: 3,24 kU/L; rDau c 1: 8,51 kU/L; rFag s 1: 23,34 kU/L; rGly m 4: 0,63 kU/L; rMal d 1: 10,79 kU/L).

Interestingly, the latter platform, which includes the inhibition of CCD reactivity, did not show any sensitization to the native cypress or plane tree molecules (nCry j 1, Cup a 1, nPla a 2).

**Conclusion:** In this case, the multiple allergen recognition was associated with CCDs sensitization, and interfered with the correct assessment of the *in vitro* analysis. This patient was probably primarily sensitized to cypress due to Cup s 7 sensitization (which is currently not detectable) but certainly not due to Cup a 1 reactivity that scored positive only due to CCD recognition.

Case 2 (original):

**Clinical History:** Male, Italy, born 1994. Patient suffering from perennial allergic rhinitis. Because of concurrent antihistaminic therapy, the patient underwent a routine specific extract IgE evaluation.

**Test with extracts:** (A) *In-vitro* testing: birch: 25 kU/L; olive tree: 18 kU/L; plane tree; 14 kU/L; Cypress: 30 kU/L;

D. pteronyssinus: 44 kU/L. (B) SPT: After discontinuation of antihistaminic therapy, the patient went through a cutaneous allergic
<figure><img src="image_132.png" /><figcaption>FIGURE 47 Examples of clinical decision algorithms for different setting of reactivity in SPT or IgE testing with allergen extracts.</figcaption></figure>

post-translational glycosylation of proteins⁶²¹ Bromelain (Ana c 2) or the purified N-glycan from bromelain MUXF3 are available to detect CCDs *in vitro* in the vast majority of pollen sources, except for nArt v 1 where CCD reactivity is driven by O-glycans and not N-glycans measurable with bromelain or MUXF3. A positive skin prick test or *in vivo* (i.e., nasal, or conjunctival) challenge with the biological source can prove the genuine IgE recognition.⁵⁰ In (Figure 45) we suggest several algorithms possibly useful to complete the diagnostic work-up of tree pollen allergic patients.

## 5 - Clinical Cases

### Case 1 (original):

**Clinical History:** Male, Italian, 10 years old with seasonal rhinitis between February and June and oral allergy syndrome induced by raw fruits (apple, peach) and vegetables (carrot, celery).

**IgE and skin tests:** in 2016, the boy reacted to Birch, Hazel, Oak and Cypress on skin prick testing, whilst no allergy to food was detected. *In vitro* IgE measurements by extracts showed reactivity to all pollens and plant foods tested, due to strong CCD reactivity.

The component resolved diagnosis by a commercial multiplex platform revealed IgE reactivity to PR10 molecules (rApi g 1: 4,46 ISU-E; rAra h 8: 0,13 ISU-E; rBet v 1: 3,67 ISU-E; rCor a 1.0101 0,21 ISU-E; rCor a 1.0401: 2,32 ISU-E; rGly m 4: 0,52 ISU-E; rMal d 1: 0,19 ISU-E), cypress pollen allergens (nCry j 1: 2,29 ISU-E; nCup a 1: 2,24 ISU-E) and CCD markers of reactivity (MUXF3 (Ana c 2.0101): 4,13 ISU-E; nPla a 2: 3,30 ISU-E; nJug r 2: 0,94 ISU-E).

Subsequently, due to anaphylactic reactions (diffuse angioedema, respiratory distress and hypotension) following the ingestion of peach on one occasion and mandarin in a second case, the young patient was further evaluated by another multiplex platform in 2021 that revealed an IgE sensitization to peamaclein along with the already known reactivity to PR10 (rPru p 7 (Gibberellin-RP): 17,79 kU/L; rAln g 1: 3,4 kU/L; rApi g 1: 9,96 kU/L; rAra h 8: 0,38 kU/L; rBet v 1: 7,08 kU/L; rCor a 1.0103: 15,42 kU/L; rCor a 1.0401: 3,24 kU/L; rDau c 1: 8,51 kU/L; rFag s 1: 23,34 kU/L; rGly m 4: 0,63 kU/L; rMal d 1: 10,79 kU/L).

Interestingly, the latter platform, which includes the inhibition of CCD reactivity, did not show any sensitization to the native cypress or plane tree molecules (nCry j 1, Cup a 1, nPla a 2).

**Conclusion:** In this case, the multiple allergen recognition was associated with CCDs sensitization, and interfered with the correct assessment of the *in vitro* analysis. This patient was probably primarily sensitized to cypress due to Cup s 7 sensitization (which is currently not detectable) but certainly not due to Cup a 1 reactivity that scored positive only due to CCD recognition.

### Case 2 (original):

**Clinical History:** Male, Italy, born 1994. Patient suffering from perennial allergic rhinitis. Because of concurrent antihistaminic therapy, the patient underwent a routine specific extract IgE evaluation.

**Test with extracts:** (A) *In-vitro* testing: birch: 25 kU/L; olive tree: 18 kU/L; plane tree; 14 kU/L; Cypress: 30 kU/L;

D. *pteronyssinus*: 44 kU/L. (B) SPT: After discontinuation of antihistaminic therapy, the patient went through a cutaneous allergic
evaluation that gave negative results for all the four tree pollen tested and mono-reactivity to *D. pteronyssinus* and *D. farinae*.

**Test with molecules:** ImmunoCAP ISAC: Der p 2: 35 ISU-E | Der f 2: 42 ISU-E | Lep d 2: 2.3 ISU-E; rBet v 1: Negative; nCry j 1: 1.2 ISU-E; nCup a 1: 3.3 ISU-E; nOle e 1: 2.8 ISU-E; rPla a 1: Negative; nPla a 2: 4 ISU-E; rPla a 3: Negative; MUXF3: 18 ISU-E.

**Conclusion:** The serology identifies the patient as genuinely sensitized only to house dust mite. The presence of MUXF3 reactivity indicates a CCD recognition further confirmed by the reactivity to native tree pollen molecules (Ole e 1, Pla a 2, Cup a 1 and Cry j 1) in the presence of negative result considering recombinant (not CCD bringing) molecules (rBet v 1, rPla a 1, rPla a 3) and in the absence of skin test reactivity to tree pollen extracts (CCD IgE reactivity is never followed by a positive skin test result).

### Case 3 (original):

**Clinical History:** Female, Italy, born 1973. The patient has been suffering from seasonal allergic rhinitis and asthma since 2000.

After ingestion of 2 walnuts, anaphylactic reaction (abdominal pain, dyspnea, generalized flushing and swelling, low blood pressure), subsequent emergency treatment and hospitalization overnight. No food allergy to peach or other food item is known so far.

**Test with extracts:** (A) SPT: Environmental allergens: Pellitory (Parietaria j): 9 mm × 5 mm; plane tree (Platanus a.): 6 mm × 7 mm; olive tree (Olea e): 7 mm × 10 mm; Mugwort (Artemisia v.): 3 mm × 4 mm. Food allergens: all negative except walnut (Juglans regia nut): 15 mm × 10 mm (peach negative). (B)

**In-vitro testing:** [2014] Total IgE 49.3 kU/l, specific IgE to pellitory (Parietaria j): 2.5 kU/l; plane tree (Platanus a.): 0.56 kU/l; Olive tree (Olea e): 0.78 kU/l; Mugwort (Artemisia v.): 0.42 kU/l; Walnut 3.82 kU/l; rPru p 3: 0.76 kU/l.

**Test with molecules:** (ImmunoCAP ISAC): Par j 2: 1.56 ISU-E; Jug r 3: 1,15 ISU-E; Ole e 7: 9,82 ISU-E; Pla a 3: 1,98 ISU-E, (Pru p 3: negative).

**Conclusion:** Strict avoidance of walnut. AIT prescribed only for Pellitory.
evaluation that gave negative results for all the four tree pollen tested and mono-reactivity to *D. pteronyssinus* and *D. farinae*.

**Test with molecules:** ImmunoCAP ISAC: Der p 2: 35 ISU-E | Der f 2: 42 ISU-E | Lep d 2: 2.3 ISU-E; rBet v 1: Negative; nCry j 1: 1.2 ISU-E; nCup a 1: 3.3 ISU-E; nOle e 1: 2.8 ISU-E; rPla a 1: Negative; nPla a 2: 4 ISU-E; rPla a 3: Negative; MUXF3: 18 ISU-E.

**Conclusion:** The serology identifies the patient as genuinely sensitized only to house dust mite. The presence of MUXF3 reactivity indicates a CCD recognition further confirmed by the reactivity to native tree pollen molecules (Ole e 1, Pla a 2, Cup a 1 and Cry j 1) in the presence of negative result considering recombinant (not CCD bringing) molecules (rBet v 1, rPla a 1, rPla a 3) and in the absence of skin test reactivity to tree pollen extracts (CCD IgE reactivity is never followed by a positive skin test result).

### Case 3 (original):

**Clinical History:** Female, Italy, born 1973. The patient has been suffering from seasonal allergic rhinitis and asthma since 2000.

After ingestion of 2 walnuts, anaphylactic reaction (abdominal pain, dyspnea, generalized flushing and swelling, low blood pressure), subsequent emergency treatment and hospitalization overnight. No food allergy to peach or other food item is known so far.

**Test with extracts:** (A) SPT: Environmental allergens: Pellitory (Parietaria j): 9 mm × 5 mm; plane tree (Platanus a.): 6 mm × 7 mm; olive tree (Olea e): 7 mm × 10 mm; Mugwort (Artemisia v.): 3 mm × 4 mm. Food allergens: all negative except walnut (Juglans regia nut): 15 mm × 10 mm (peach negative). (B)

**In-vitro testing:** [2014] Total IgE 49.3 kU/l, specific IgE to pellitory (Parietaria j): 2.5 kU/l; plane tree (Platanus a.): 0.56 kU/l; Olive tree (Olea e): 0.78 kU/l; Mugwort (Artemisia v.): 0.42 kU/l; Walnut 3.82 kU/l; rPru p 3: 0.76 kU/l.

**Test with molecules:** (ImmunoCAP ISAC): Par j 2: 1.56 ISU-E; Jug r 3: 1,15 ISU-E; Ole e 7: 9,82 ISU-E; Pla a 3: 1,98 ISU-E, (Pru p 3: negative).

**Conclusion:** Strict avoidance of walnut. AIT prescribed only for Pellitory.
# B02 - Grass pollen allergy

Janet M. Davies, Paolo M. Matricardi, Johannes Schmid

## Highlights

* IgE to group 1 allergens (e.g., Phl p 1) is a marker of true grass pollen sensitization.

* Whilst IgE to other major grass pollen allergens are rarely observed in the absence of IgE to Phl p 1, IgE to Phl p 5 or Phl p 2 can serve as a markers of true grass pollen sensitization.

* A number of allergen families exist in grass pollens, but IgE reactivity to Phl p 4, Phl p 7, Phl p 11, or Phl p 12 may be due to cross-reactivity (CCD or pan-allergenicity).

* Early onset of IgE sensitization to grass pollen allergens, particularly Phl p 1, and a high number of sensitizations (Phl p 5, 7 and 12) may be prognostic markers of disease progression, but further studies are needed.

* In temperate climates, patient serum IgE shows broad cross-reactivity between similar allergen components from different temperate grass pollens.

* Group 1 allergens of subtropical grass pollens (Pasn 1, Sorh 1 and Cyn d 1) are more relevant allergens for patients in subtropical regions.

## 1 – The allergen sources

Grasses are found on all continents except Antarctica. In places with a temperate climate, members of the Pooideae subfamily<sup>622</sup> like Timothy grass (*Phleum pratense*), Orchard grass (*Dactylis glomerata*), Perennial ryegrass (*Lolium perenne*) and bluegrass (*Poa pratensis*) are the most common grasses. The pollen of this Pooideae subfamily show extensive IgE cross-reactivity. Grasses have a pollinating season from May to August in Central Europe, peaking in June. In Northern Europe, the grass pollen season starts later, while pollination lasts from March to July for a longer period in Mediterranean Europe (Figure 48). The grass pollen season overlaps with weed pollen (mugwort, ragweed) in most parts of Europe and with tree pollen (olive, plane) in Southern Europe. As in Europe, grass pollen seasons vary in North America, with an earlier onset and longer duration in the warmer parts, leading to different *Phleum pratense* pollen component sensitization patterns in different regions.<sup>623</sup> In subtropical and tropical regions of the world, grass pollen seasons can be perennial, and are still the dominating pollen source flowering during spring, summer and autumn.<sup>624,625</sup> Subtropical sources of allergenic grass pollens include those of the Panicoideae subfamily; Bahia grass (*Paspalum notatum*) and the prolific weed Johnson grass (*Sorghum halepense*), as well as the Chloridoideae subfamily; Bermuda grass (*Cynodon dactylon*).<sup>624</sup> Timothy grass is originally native to Europe and adjacent regions in Africa and Asia. It is widely cultivated throughout

most temperate regions of the world for pasture and hay production. Despite of substantial geographical variations, grass pollen is the most prevalent sensitizing pollen in population surveys of respiratory allergies, with a median prevalence for timothy grass pollen in Europe of 16.5%<sup>626</sup> and for Ryegrass pollen of 19.5% in the United States of America.<sup>627</sup>

## 2 – Major and relevant minor allergenic molecules

At present, ten allergenic molecules from Timothy grass pollen have been officially listed by the IUIS Allergen Nomenclature Subcommittee. Phl p 2, Phl p 3, p 5 and p 6 are specific for grasses from the Pooideae subfamily whereas polcalcin (Phl p 7), profilin (Phl p 12) and oleosin related protein (Phl p 11) are related to allergens of other pollen sources (Figure 49, Table 16).

Orthologues of the group 1 beta-expansin grass pollen allergens represented by Phl p 1 of Timothy grass are ubiquitous and specific for pollen of the *Poaceae* family. Sensitization to Phl p 1 usually precedes other grass pollen sensitizations and is the most prevalent component sensitization in grass pollen allergic patients.<sup>309</sup> It is a useful marker for primary grass pollen sensitization. Phl p 1 is a beta-expansin, bound to the cell wall and important for pollen tube penetration. Phl p 1 is a major grass pollen allergen, with more than 80% homology to group 1 allergens from other members of the Pooideae subfamily.<sup>628</sup> Phl p 1 shares epitopes with group 1 allergens from other grasses and shows IgE cross-reactivity to most other group 1 allergens from grasses, corns and other monocots.<sup>236</sup>

Phl p 5 also is a major pollen allergen specific to temperate grasses with lower sensitization prevalence but often with high specific IgE levels. Phl p 5 is a cytoplasmatic ribonuclease, important in the enzymatic degradation of RNA. It shows broad IgE cross-reactivity with other group 5 allergens from the Pooideae subfamily of temperate grasses, but the isoforms of group 5 allergens can vary within and between species.

Phl p 6 is another major Timothy grass pollen allergen, specific for the Pooideae subfamily. Its function has not yet been described.

Phl p 4 is a tryptase-resistant glycoprotein, berberine bridge enzyme, involved in the synthesis of alkaloids. It can be classified as a major allergen.<sup>629</sup> It shows IgE cross-reactivity with other group 4 grass pollen allergens, including with Cyn 4 to some extent. Moreover, cross-reactivity to the major ragweed allergen Amb a 1 and to Oilseed Rape pollen has been demonstrated. Natural Phl p 4 contains CCD, which may lead to IgE cross-reactivity with a wide range of plants and plant products

The Phl p 2 and Phl p 3 allergens are proteins with homology to the C terminal domain of the beta-expansin protein family.<sup>630</sup> They show substantial similarities and are specific for the Pooideae subfamily. Their biochemical function is not yet known.

Phl p 13 is a polygalactorunase, which is a hydrolytic enzyme, degrading parts of the pectin network in plant cell walls. It is a major allergen, specific for the Pooideae subfamily.
# B02 - Grass pollen allergy

Janet M. Davies, Paolo M. Matricardi, Johannes Schmid

## Highlights

* IgE to group 1 allergens (e.g., Phl p 1) is a marker of true grass pollen sensitization.

* Whilst IgE to other major grass pollen allergens are rarely observed in the absence of IgE to Phl p 1, IgE to Phl p 5 or Phl p 2 can serve as a markers of true grass pollen sensitization.

* A number of allergen families exist in grass pollens, but IgE reactivity to Phl p 4, Phl p 7, Phl p 11, or Phl p 12 may be due to cross-reactivity (CCD or pan-allergenicity).

* Early onset of IgE sensitization to grass pollen allergens, particularly Phl p 1, and a high number of sensitizations (Phl p 5, 7 and 12) may be prognostic markers of disease progression, but further studies are needed.

* In temperate climates, patient serum IgE shows broad cross-reactivity between similar allergen components from different temperate grass pollens.

* Group 1 allergens of subtropical grass pollens (Pasn 1, Sorh 1 and Cyn d 1) are more relevant allergens for patients in subtropical regions.

## 1 – The allergen sources

Grasses are found on all continents except Antarctica. In places with a temperate climate, members of the Pooideae subfamily<sup>622</sup> like Timothy grass (*Phleum pratense*), Orchard grass (*Dactylis glomerata*), Perennial ryegrass (*Lolium perenne*) and bluegrass (*Poa pratensis*) are the most common grasses. The pollen of this Pooideae subfamily show extensive IgE cross-reactivity. Grasses have a pollinating season from May to August in Central Europe, peaking in June. In Northern Europe, the grass pollen season starts later, while pollination lasts from March to July for a longer period in Mediterranean Europe (Figure 48). The grass pollen season overlaps with weed pollen (mugwort, ragweed) in most parts of Europe and with tree pollen (olive, plane) in Southern Europe. As in Europe, grass pollen seasons vary in North America, with an earlier onset and longer duration in the warmer parts, leading to different *Phleum pratense* pollen component sensitization patterns in different regions.<sup>623</sup> In subtropical and tropical regions of the world, grass pollen seasons can be perennial, and are still the dominating pollen source flowering during spring, summer and autumn.<sup>624,625</sup> Subtropical sources of allergenic grass pollens include those of the Panicoideae subfamily; Bahia grass (*Paspalum notatum*) and the prolific weed Johnson grass (*Sorghum halepense*), as well as the Chloridoideae subfamily; Bermuda grass (*Cynodon dactylon*).<sup>624</sup> Timothy grass is originally native to Europe and adjacent regions in Africa and Asia. It is widely cultivated throughout

most temperate regions of the world for pasture and hay production. Despite of substantial geographical variations, grass pollen is the most prevalent sensitizing pollen in population surveys of respiratory allergies, with a median prevalence for timothy grass pollen in Europe of 16.5%<sup>626</sup> and for Ryegrass pollen of 19.5% in the United States of America.<sup>627</sup>

## 2 – Major and relevant minor allergenic molecules

At present, ten allergenic molecules from Timothy grass pollen have been officially listed by the IUIS Allergen Nomenclature Subcommittee. Phl p 2, Phl p 3, p 5 and p 6 are specific for grasses from the Pooideae subfamily whereas polcalcin (Phl p 7), profilin (Phl p 12) and oleosin related protein (Phl p 11) are related to allergens of other pollen sources (Figure 49, Table 16).

Orthologues of the group 1 beta-expansin grass pollen allergens represented by Phl p 1 of Timothy grass are ubiquitous and specific for pollen of the *Poaceae* family. Sensitization to Phl p 1 usually precedes other grass pollen sensitizations and is the most prevalent component sensitization in grass pollen allergic patients.<sup>309</sup> It is a useful marker for primary grass pollen sensitization. Phl p 1 is a beta-expansin, bound to the cell wall and important for pollen tube penetration. Phl p 1 is a major grass pollen allergen, with more than 80% homology to group 1 allergens from other members of the Pooideae subfamily.<sup>628</sup> Phl p 1 shares epitopes with group 1 allergens from other grasses and shows IgE cross-reactivity to most other group 1 allergens from grasses, corns and other monocots.<sup>236</sup>

Phl p 5 also is a major pollen allergen specific to temperate grasses with lower sensitization prevalence but often with high specific IgE levels. Phl p 5 is a cytoplasmatic ribonuclease, important in the enzymatic degradation of RNA. It shows broad IgE cross-reactivity with other group 5 allergens from the Pooideae subfamily of temperate grasses, but the isoforms of group 5 allergens can vary within and between species.

Phl p 6 is another major Timothy grass pollen allergen, specific for the Pooideae subfamily. Its function has not yet been described.

Phl p 4 is a tryptase-resistant glycoprotein, berberine bridge enzyme, involved in the synthesis of alkaloids. It can be classified as a major allergen.<sup>629</sup> It shows IgE cross-reactivity with other group 4 grass pollen allergens, including with Cyn 4 to some extent. Moreover, cross-reactivity to the major ragweed allergen Amb a 1 and to Oilseed Rape pollen has been demonstrated. Natural Phl p 4 contains CCD, which may lead to IgE cross-reactivity with a wide range of plants and plant products

The Phl p 2 and Phl p 3 allergens are proteins with homology to the C terminal domain of the beta-expansin protein family.<sup>630</sup> They show substantial similarities and are specific for the Pooideae subfamily. Their biochemical function is not yet known.

Phl p 13 is a polygalactorunase, which is a hydrolytic enzyme, degrading parts of the pectin network in plant cell walls. It is a major allergen, specific for the Pooideae subfamily.
<figure><img src="image_134.png" /><figcaption>FIGURE 48 Clinically important examples of common temperate (Pooideae) and subtropical (Panicoidae and Chloridoideae) grass pollen allergen sources. Species origin, geographical distribution and peak pollinating periods in common regions are included. Timing of pollination of Pooideae species refers to the Northern Hemisphere.</figcaption></figure>

Phl p 11 belongs to the Ole e 1 related proteins and hence exhibits a broad range of cross-reactivity to pollen from different plants as olive, ash, privet, saffron crocus, thistle, plantain and corn. It is an acidic polypeptide with homology to the tryptase inhibitor of soybean.

Phl p 7 and Phl p 12 are minor allergens, representing pan-allergens from the plant world. Phl p 7, polcalcin, is a calcium-binding protein present in many different types of pollen, hence representing a broad cross-reacting allergen: birch, alder, juniper, ragweed, mugwort, olive, goosefoot etc. sensitization to Phl p 7 can be used as a marker of a wide pollen sensitization.

Phl p 12 is a member of the profilin family, an actin-binding protein that is present throughout the whole plant world. As profilins are ubiquitous in plant cells, profilin sensitization gives rise to a long range of cross-reacting plants and plant derivatives such as birch, soybean, corn, latex and plant foods.

Subtropical grass pollens of Bahia grass (Paspalum notatum) and Bermuda grass (Cynodon dactylon) show only limited IgE cross-reactivity with the pollens from the Pooideae subfamily.²³⁶ Patterns of IgE cross-reactivity between subtropical and temperate grass pollen appear to depend of the geographical region of the patient population being investigated.

Subtropical grass pollens contain the major beta expansin group 1 allergen family; Pas n 1 of Bahia grass, multiple isoforms of Sor h 1 of Johnson grass and Cyn d 1 of Bermuda grass. A number of group 1 pollen allergens have recently been described from tropical regions of Asia from other Panicoidae subtropical grasses; Para (Urochloa mutica)⁶³¹ and Manila grass (Zoysia matrella).⁶³² The polygalacturonase components Pas n 13 of Bahia and Sor h 13 of Johnson grasses, are the second most abundant protein and frequently recognized allergens from pollen of Panicoidae family of subtropical regions.
<figure><img src="image_133.png" /><figcaption>FIGURE 48 Clinically important examples of common temperate (Pooideae) and subtropical (Panicoidae and Chloridoideae) grass pollen allergen sources. Species origin, geographical distribution and peak pollinating periods in common regions are included. Timing of pollination of Pooideae species refers to the Northern Hemisphere.</figcaption></figure>

Phl p 11 belongs to the Ole e 1 related proteins and hence exhibits a broad range of cross-reactivity to pollen from different plants as olive, ash, privet, saffron crocus, thistle, plantain and corn. It is an acidic polypeptide with homology to the tryptase inhibitor of soybean.

Phl p 7 and Phl p 12 are minor allergens, representing pan-allergens from the plant world. Phl p 7, polcalcin, is a calcium-binding protein present in many different types of pollen, hence representing a broad cross-reacting allergen: birch, alder, juniper, ragweed, mugwort, olive, goosefoot etc. sensitization to Phl p 7 can be used as a marker of a wide pollen sensitization.

Phl p 12 is a member of the profilin family, an actin-binding protein that is present throughout the whole plant world. As profilins are ubiquitous in plant cells, profilin sensitization gives rise to a long range of cross-reacting plants and plant derivatives such as birch, soybean, corn, latex and plant foods.

Subtropical grass pollens of Bahia grass (Paspalum notatum) and Bermuda grass (Cynodon dactylon) show only limited IgE cross-reactivity with the pollens from the Pooideae subfamily.²³⁶ Patterns of IgE cross-reactivity between subtropical and temperate grass pollen appear to depend of the geographical region of the patient population being investigated.

Subtropical grass pollens contain the major beta expansin group 1 allergen family; Pas n 1 of Bahia grass, multiple isoforms of Sor h 1 of Johnson grass and Cyn d 1 of Bermuda grass. A number of group 1 pollen allergens have recently been described from tropical regions of Asia from other Panicoideae subtropical grasses; Para (Urochloa mutica)⁶³¹ and Manila grass (Zoysia matrella).⁶³² The polygalacturonase components Pas n 13 of Bahia and Sor h 13 of Johnson grasses, are the second most abundant protein and frequently recognized allergens from pollen of Panicoidae family of subtropical regions.
<figure><img src="image_135.png" /><figcaption>FIGURE 49 A Venn diagram showing allergens of different sources that are similar to temperate and/or subtropical grass pollen allergens. Major allergens are in bold. Allergens of the same biochemical family are shown in boxes of the same colour. Pan-allergens including profilins (dark green boxes, e.g., Phl p12) and polcalcin (pink boxes, e.g., Phl p7) are in small font.</figcaption></figure>

<table><caption>TABLE 16 Overview of the most relevant allergenic molecules from timothy grass and clinically relevant subtropical grasses.</caption><thead><tr><th>Allergen group</th><th>allergenicity</th><th>Allergome code</th><th>Biochemical name</th><th>Function</th><th>MW (kDa)</th><th>Isoform</th></tr></thead><tbody><tr><td colspan="7"><strong>Timothy grass [1696][2][1697]</strong></td></tr><tr><td>Phl p 1</td><td>83-95 %</td><td>549-551</td><td>CCD-bearing protein</td><td>Beta-expansin</td><td>27</td><td>2</td></tr><tr><td>Phl p 2</td><td>55-65 %</td><td>555</td><td>Grass group 2</td><td>Unknown</td><td>10-12</td><td>1</td></tr><tr><td>Phl p 3</td><td>60 %</td><td>556</td><td>Grass group 3</td><td>Unknown</td><td>10-11</td><td>2</td></tr><tr><td>Phl p 4</td><td>70-75 %</td><td>557</td><td>CCD-bearing protein</td><td>Berberine-bridge enzyme</td><td>55</td><td>6</td></tr><tr><td>Phl p 5</td><td>50-95 %</td><td>558</td><td>Grass group 5</td><td>Ribonuclease</td><td>29-32</td><td>16</td></tr><tr><td>Phl p 6</td><td>44-75 %</td><td>569</td><td>Grass group 6</td><td>unknown</td><td>11</td><td>2</td></tr><tr><td>Phl p 7</td><td>7-10 %</td><td>570</td><td>Polcalcin</td><td>Calcium-binding protein</td><td>6</td><td>1</td></tr><tr><td>Phl p 11</td><td>32-43 %</td><td>552</td><td>Ole e 1-related protein</td><td>Trypsin inhibitor</td><td>20</td><td>1</td></tr><tr><td>Phl p 12</td><td>15 %</td><td>553</td><td>Profilin</td><td>Actin-binding protein</td><td>14</td><td>3</td></tr><tr><td>Phl p 13</td><td>50 %</td><td>554</td><td>Grass group 13</td><td>Polygalacturonase</td><td>55</td><td>1</td></tr><tr><td colspan="7"><strong>Subtropical grasses</strong></td></tr><tr><td>Cyn d 1</td><td>76-100%</td><td>266</td><td>Glycoprotein</td><td>Beta-expansin</td><td>32</td><td>12</td></tr><tr><td>Cyn d 4</td><td>100%</td><td>819</td><td>Glycoprotein</td><td>Berberine bridge enzyme</td><td>60</td><td>1</td></tr><tr><td>Pas n 1</td><td>85-92%</td><td>874</td><td>Glycoprotein</td><td>Beta-expansin</td><td>29-30</td><td>2</td></tr><tr><td>Pas n 13</td><td>48%</td><td>9456</td><td>Glycoprotein</td><td>Polygalacturonase</td><td>50</td><td>4</td></tr><tr><td>Sor h 1</td><td>76%</td><td>643</td><td>Glycoprotein</td><td>Beta-expansin</td><td>28.4</td><td>2</td></tr><tr><td>Uro m 1</td><td>100% of 6 patients</td><td>12168</td><td>Glycoprotein</td><td>Beta-expansin</td><td>30</td><td>2</td></tr><tr><td>Zoy m 1</td><td>100% of 7 patients</td><td>No</td><td>Glycoprotein</td><td>Beta-expansin</td><td>30</td><td>1</td></tr></tbody></table>
<figure><img src="image_134.png" /><figcaption>FIGURE 49 A Venn diagram showing allergens of different sources that are similar to temperate and/or subtropical grass pollen allergens. Major allergens are in bold. Allergens of the same biochemical family are shown in boxes of the same colour. Pan-allergens including profilins (dark green boxes, e.g., Phl p12) and polcalcin (pink boxes, e.g., Phl p7) are in small font.</figcaption></figure>

<table><caption>TABLE 16 Overview of the most relevant allergenic molecules from timothy grass and clinically relevant subtropical grasses.</caption><thead><tr><th>Allergen group</th><th>allergenicity</th><th>Allergome code</th><th>Biochemical name</th><th>Function</th><th>MW (kDa)</th><th>Isoform</th></tr></thead><tbody><tr><td colspan="7"><strong>Timothy grass [1696][2][1697]</strong></td></tr><tr><td>Phl p 1</td><td>83-95 %</td><td>549-551</td><td>CCD-bearing protein</td><td>Beta-expansin</td><td>27</td><td>2</td></tr><tr><td>Phl p 2</td><td>55-65 %</td><td>555</td><td>Grass group 2</td><td>Unknown</td><td>10-12</td><td>1</td></tr><tr><td>Phl p 3</td><td>60 %</td><td>556</td><td>Grass group 3</td><td>Unknown</td><td>10-11</td><td>2</td></tr><tr><td>Phl p 4</td><td>70-75 %</td><td>557</td><td>CCD-bearing protein</td><td>Berberine-bridge enzyme</td><td>55</td><td>6</td></tr><tr><td>Phl p 5</td><td>50-95 %</td><td>558</td><td>Grass group 5</td><td>Ribonuclease</td><td>29-32</td><td>16</td></tr><tr><td>Phl p 6</td><td>44-75 %</td><td>569</td><td>Grass group 6</td><td>unknown</td><td>11</td><td>2</td></tr><tr><td>Phl p 7</td><td>7-10 %</td><td>570</td><td>Polcalcin</td><td>Calcium-binding protein</td><td>6</td><td>1</td></tr><tr><td>Phl p 11</td><td>32-43 %</td><td>552</td><td>Ole e 1-related protein</td><td>Trypsin inhibitor</td><td>20</td><td>1</td></tr><tr><td>Phl p 12</td><td>15 %</td><td>553</td><td>Profilin</td><td>Actin-binding protein</td><td>14</td><td>3</td></tr><tr><td>Phl p 13</td><td>50 %</td><td>554</td><td>Grass group 13</td><td>Polygalacturonase</td><td>55</td><td>1</td></tr><tr><td colspan="7"><strong>Subtropical grasses</strong></td></tr><tr><td>Cyn d 1</td><td>76-100%</td><td>266</td><td>Glycoprotein</td><td>Beta-expansin</td><td>32</td><td>12</td></tr><tr><td>Cyn d 4</td><td>100%</td><td>819</td><td>Glycoprotein</td><td>Berberine bridge enzyme</td><td>60</td><td>1</td></tr><tr><td>Pas n 1</td><td>85-92%</td><td>874</td><td>Glycoprotein</td><td>Beta-expansin</td><td>29-30</td><td>2</td></tr><tr><td>Pas n 13</td><td>48%</td><td>9456</td><td>Glycoprotein</td><td>Polygalacturonase</td><td>50</td><td>4</td></tr><tr><td>Sor h 1</td><td>76%</td><td>643</td><td>Glycoprotein</td><td>Beta-expansin</td><td>28.4</td><td>2</td></tr><tr><td>Uro m 1</td><td>100% of 6 patients</td><td>12168</td><td>Glycoprotein</td><td>Beta-expansin</td><td>30</td><td>2</td></tr><tr><td>Zoy m 1</td><td>100% of 7 patients</td><td>No</td><td>Glycoprotein</td><td>Beta-expansin</td><td>30</td><td>1</td></tr></tbody></table>
Several allergens have been described from Bermuda grass pollen including the berberine bridge enzyme orthologue Cyn d 4 that is a major allergen. IgE reactivity with a group 2 allergen Sor h 2 of Johnson grass pollen has recently been discovered.⁶³³ Notably, however, to date no allergen with significant homology with the *Pooideae* group 5 allergen has been discovered by proteomic or transcriptomic analysis of subtropical grass pollens.

As mentioned above allergen molecules from different members of the Pooideae subfamily are highly IgE cross-reactive.⁶²⁹ As both wild and cultivated grasses in the temperate climate zones belong to the Pooideae subfamily, *Phleum pratense* allergens can be used for diagnostic and therapeutic purposes in grass pollen allergic patients living in the temperate parts of the world. However, depending on the biogeographical region and presence of different types of grasses, sensitization to pollen of different subfamilies differs across Australia which climate zones range from tropical to temperate,⁶³⁴ and there were significant differences in inhibition of slgE to Lol p 1 (*Pooideae*), Cyn d 1 (*Chloridoideae*), and Pas n 1 (*Panicoidae*) by pollen extracts from these different subfamilies for patient originating from temperate compared with subtropical regions.

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

The specific IgE response against grass pollen (e.g., *Phleum pratense*) usually evolves from an initial, monomolecular stage to an oligomolecular stage and eventually to a polymolecular sensitization stage.³⁰⁹ This phenomenon has been described during childhood and is defined as “molecular spreading,” that is, “The sequential development of antibody (IgE) response to distinct non-cross-reacting molecules from the same antigenic (allergenic) source, starting with an “initiator” (allergenic) molecule.”⁹⁵ (Figure 50). Phl p 1 is the probable “initiator” molecule in most patients with (temperate) grass pollen allergy, and the response involves then Phl p 4 or Phl p 5,

thereafter also Phl p 2 and Phl p 11 and at a later stage Phl p 12 or Phl p 7. This has been confirmed in other birth cohort studies.³¹¹ The pathophysiological consequence of this phenomenon is that the longer is the duration of disease, the broader is the repertoire of IgE sensitization against the different molecules of the pollen source.⁶³⁵

This has led to the consideration that AIT could be started earlier in a patient's clinical care, possibly even immediately after the first season in which the allergic respiratory symptoms are initiated (“early-AIT”).⁹⁵ However, the molecular spreading process follows different pathways in different children: some patients remain sensitized only to the “initiator” molecule while a few patients become sensitized to most or all allergenic molecules. Consequently, a population of grass-pollen allergic patients “apparently” homogeneous if tested with an allergen extract reveals remarkably heterogeneous when examined with the corresponding molecules.⁸²,³¹¹ clinical relevance of the individual profile of sensitization is being tested in large populations both in cross-sectional, observational studies (Dramburg S. et al., in preparation) and in longitudinal intervention studies (Potapova et al. submitted). However, only few data have yet been published; in a recent study a higher risk of asthma at 11 years was observed in children being sensitized at 5 years to almost all grass pollen allergen molecules in comparison with those who had a late onset of sensitization.³¹¹,⁶³⁵

**IgE to Phl p 1 - Clinical relevance** – Phl p 1 (or another one of the “group 1” antigens of grass pollen, such as Lol p 1, from *Lolium perenne*) is the “initiator” molecule in most patients. Moreover, even in the few grass-pollen allergic patients who start their sensitization process with other molecules, IgE against Phl p 1 is produced soon thereafter. Consequently, IgE sensitization to Phl p 1 is an essential marker to establish “true sensitization” in grass pollen allergic patients. The presence of IgE to Phl p 1 confirms that the patient with a positive Skin Prick Test or serum IgE assay to extract is truly sensitized to grass pollen. The absence of IgE to Phl p 1 does not exclude “true” sensitization to grass pollen, which might be due (in a few cases) to isolated IgE sensitization to other major allergenic proteins (e.g., Phl p 5) but makes it rather unlikely. Then patients with skin prick test-IgE positivity to a

<figure><img src="image_136.png" /><figcaption>FIGURE 50 The molecular spreading of the IgE response to Timothy grass and possible implications for AIT intervention.</figcaption></figure>
Several allergens have been described from Bermuda grass pollen including the berberine bridge enzyme orthologue Cyn d 4 that is a major allergen. IgE reactivity with a group 2 allergen Sor h 2 of Johnson grass pollen has recently been discovered.⁶³³ Notably, however, to date no allergen with significant homology with the *Pooideae* group 5 allergen has been discovered by proteomic or transcriptomic analysis of subtropical grass pollens.

As mentioned above allergen molecules from different members of the Pooideae subfamily are highly IgE cross-reactive.⁶²⁹ As both wild and cultivated grasses in the temperate climate zones belong to the Pooideae subfamily, *Phleum pratense* allergens can be used for diagnostic and therapeutic purposes in grass pollen allergic patients living in the temperate parts of the world. However, depending on the biogeographical region and presence of different types of grasses, sensitization to pollen of different subfamilies differs across Australia which climate zones range from tropical to temperate,⁶³⁴ and there were significant differences in inhibition of slgE to Lol p 1 (*Pooideae*), Cyn d 1 (*Chloridoideae*), and Pas n 1 (*Panicoidae*) by pollen extracts from these different subfamilies for patient originating from temperate compared with subtropical regions.

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

The specific IgE response against grass pollen (e.g., *Phleum pratense*) usually evolves from an initial, monomolecular stage to an oligomolecular stage and eventually to a polymolecular sensitization stage.³⁰⁹ This phenomenon has been described during childhood and is defined as “molecular spreading,” that is, “The sequential development of antibody (IgE) response to distinct non-cross-reacting molecules from the same antigenic (allergenic) source, starting with an “initiator” (allergenic) molecule.”⁹⁵ (Figure 50). Phl p 1 is the probable “initiator” molecule in most patients with (temperate) grass pollen allergy, and the response involves then Phl p 4 or Phl p 5,

thereafter also Phl p 2 and Phl p 11 and at a later stage Phl p 12 or Phl p 7. This has been confirmed in other birth cohort studies.³¹¹ The pathophysiological consequence of this phenomenon is that the longer is the duration of disease, the broader is the repertoire of IgE sensitization against the different molecules of the pollen source.⁶³⁵

This has led to the consideration that AIT could be started earlier in a patient's clinical care, possibly even immediately after the first season in which the allergic respiratory symptoms are initiated (“early-AIT”).⁹⁵ However, the molecular spreading process follows different pathways in different children: some patients remain sensitized only to the “initiator” molecule while a few patients become sensitized to most or all allergenic molecules. Consequently, a population of grass-pollen allergic patients “apparently” homogeneous if tested with an allergen extract reveals remarkably heterogeneous when examined with the corresponding molecules.⁸²,³¹¹ clinical relevance of the individual profile of sensitization is being tested in large populations both in cross-sectional, observational studies (Dramburg S. et al., in preparation) and in longitudinal intervention studies (Potapova et al. submitted). However, only few data have yet been published; in a recent study a higher risk of asthma at 11 years was observed in children being sensitized at 5 years to almost all grass pollen allergen molecules in comparison with those who had a late onset of sensitization.³¹¹,⁶³⁵

**IgE to Phl p 1 - Clinical relevance** – Phl p 1 (or another one of the “group 1” antigens of grass pollen, such as Lol p 1, from *Lolium perenne*) is the “initiator” molecule in most patients. Moreover, even in the few grass-pollen allergic patients who start their sensitization process with other molecules, IgE against Phl p 1 is produced soon thereafter. Consequently, IgE sensitization to Phl p 1 is an essential marker to establish “true sensitization” in grass pollen allergic patients. The presence of IgE to Phl p 1 confirms that the patient with a positive Skin Prick Test or serum IgE assay to extract is truly sensitized to grass pollen. The absence of IgE to Phl p 1 does not exclude “true” sensitization to grass pollen, which might be due (in a few cases) to isolated IgE sensitization to other major allergenic proteins (e.g., Phl p 5) but makes it rather unlikely. Then patients with skin prick test-IgE positivity to a

<figure><img src="image_135.png" /><figcaption>FIGURE 50 The molecular spreading of the IgE response to Timothy grass and possible implications for AIT intervention.</figcaption></figure>