grass pollen extract but lacking specific IgE to Phl p 1 should be tested for IgE to other Phl p molecules. The group 1 allergens are the major and clinically most important allergen of subtropical Panicoideae grass pollens. Whilst other allergen components are present in subtropical grass pollens, specific IgE to Pas n 1 of Bahia grass pollens accounts for nearly all of the detectable IgE reactivity to the whole extract.⁶³⁶ Similarly, specific IgE reactivity to Sor h 1 of Johnson grass pollen is highly correlated with IgE reactivity with the whole pollen.⁶³³ For Bermuda grass pollen, Cyn d 1 is the major allergen, but the complexity of described allergen components is broad.⁶²⁴

**IgE to Phl p 5 - Clinical relevance** – Phl p 5 is rarely the only molecule inducing grass pollen sensitization and the presence of specific IgE to Phl p 5—observed in around 50% -95.5% of the European grass pollen allergic patients—confirms that a positive SPT reaction is the expression of true sensitization to grass pollen. However, although IgE to Phl p 5 usually appears later than that to Phl p 1 in the sensitization process, its concentration grows rapidly in many patients and higher and its contribution to patients' symptoms has been demonstrated.⁶³⁷ Testing IgE to Phl p 5 can be useful as a second line test and has been shown to be useful for distinguishing between allergy to grass and olive pollen in Southern Europe. Specific IgE to Phl p 5 may have some prognostic value for indicating disease severity or likely progression from allergic rhinitis to asthma, but this needs to be confirmed with well-designed studies. As group 5 allergens have not been found in subtropical grass pollens, specific IgE to Phl p 5 may particularly indicate sensitization to temperate grass pollens. This needs to be investigated in relevant patient populations.

**IgE to Phl p 12 - Clinical relevance (see also Chapter C01)** – Phl p 12, is the highly cross-reactive profilin of *Phleum pratense*. As a heat-labile, relatively weak allergenic molecule, specific IgE sensitization to profilin comes later in the molecular spreading process, reaches only moderate levels of IgE antibodies and only in a minority of patients. Hence, IgE to Phl p 12 mark in general those patients with a higher atopic background and/or longer disease duration. Patients with a positive skin prick test/serum IgE to grass pollen extract but no detectable IgE to Phl p 1 and/or Phl p 5 must be tested for IgE reactivity to Phl p 12 as these antibodies—that can be induced by other pollens containing profilin—is the first cause of “false” positivity to assays based on grass pollen extract. In the presence of specific IgE to Phl p 12, patients should be asked about Oral Allergic Syndrome triggered by the ingestion of fruits and vegetables containing profilin.

## Tips for the use of molecular diagnostics for grass pollen allergy

* IgE to Phl p 1 is a marker of “true sensitization” to grass pollen

* Exceptions: In a few rare cases with skin test positivity to a grass pollen extract but no detectible IgE to Phl p1, IgE to Phl p5, may confirm the diagnosis of grass pollen allergy.

**IgE to Phl p 7 - Clinical relevance (see also Chapter C06)** – Phl p 7, is the highly cross-reactive polcalcin of *Phleum pratense*. This is a heat-stable, relatively potent allergen that can induce quite high specific IgE antibody levels. An IgE response to Phl p 7 is observed only infrequently among grass pollen allergic patients and usually many years after disease onset. Specific IgE to Phl p 7 indicates a relatively distinct category of grass pollen allergic patients, with more severe symptoms, a higher prevalence of asthma, and a higher frequency of allergic comorbidities.⁶³⁸ Moreover, many other pollens and allergenic sources contain polcalcin so that the original sensitization to polcalcin in a grass-pollen allergic patient must be carefully searched.⁶³⁸,⁶³⁹ These other allergenic sources could be indeed responsible for a more severe disease.

**IgE to Phl p 4 - Clinical relevance (see also Chapter A03)** – Phl p 4 is a major allergenic protein of grass pollen. In its native form, that is still used in most commercial available assays, Phl p 4 contains extremely highly cross-reactive carbohydrate determinants (CCD). This explains why in several epidemiological studies IgE positivity to Phl p 4 scores over 90% of the grass pollen allergic patients. However, when the recombinant form of the molecule is used in assays, about 50% of that positivity is not confirmed any more⁶³⁹,⁶⁴⁰ (Matricardi PM, data on file). As extracts contain the native Phl p 4, a weak positivity to SPT/serum IgE test based on grass pollen extracts can be “false” in some patients and simply explained by IgE recognition of CCD determinants. Phl p 4 may also serve as a marker of sensitization to Bermuda grass pollen due to its similarity with Cyn d 4, a major allergen of Bermuda grass pollen, but this needs to be investigated in relevant patient populations. Recently, sIgE to Phl p 4 as well as Phl p 1 and Phl p 5 have been identified as an early indicator of allergic rhinitis in 763 children from Sweden.⁶⁴⁰

**IgE to Phl p 2, 6, 11** – Phl p 2 and Phl p 11 are both rarely the only molecule inducing grass pollen sensitization and the presence of IgE antibodies to Phl p 2—observed in around 60-80% of the European grass pollen allergic patients—just confirms that a positive SPT reaction is the expression of true sensitization to grass. Phl p 6 is highly cross-reacting with Phl p 5 and does not add a lot of diagnostic information, once IgE to Phl p 5 has been documented.

## 4 – Predictive Value of Specific IgE responses to Allergenic Molecules

**IgE to grass pollen molecules as biomarkers of disease** – The presence of allergen-specific IgE towards airborne allergen molecules has been investigated as a putative predictive biomarker for the development of asthma throughout childhood and adolescence. In the case of seasonal allergic rhinitis to timothy grass pollen, individual risk profiles the predictive power of IgE sensitization to certain marker molecules, such as the profilin grass pollen allergen Phl p 12, which correlates with an increased risk for the development of an Oral Allergy Syndrome (OAS), has been confirmed in a cohort of over 1000 grass pollen allergic Italian children.⁸⁵ Similarly, the same study confirmed a strong association between IgE sensitization to Phl p 7 (polcalcin) and asthma. A molecular combinatorial analysis
grass pollen extract but lacking specific IgE to Phl p 1 should be tested for IgE to other Phl p molecules. The group 1 allergens are the major and clinically most important allergen of subtropical Panicoideae grass pollens. Whilst other allergen components are present in subtropical grass pollens, specific IgE to Pas n 1 of Bahia grass pollens accounts for nearly all of the detectable IgE reactivity to the whole extract.⁶³⁶ Similarly, specific IgE reactivity to Sor h 1 of Johnson grass pollen is highly correlated with IgE reactivity with the whole pollen.⁶³³ For Bermuda grass pollen, Cyn d 1 is the major allergen, but the complexity of described allergen components is broad.⁶²⁴

**IgE to Phl p 5 - Clinical relevance** – Phl p 5 is rarely the only molecule inducing grass pollen sensitization and the presence of specific IgE to Phl p 5—observed in around 50% -95.5% of the European grass pollen allergic patients—confirms that a positive SPT reaction is the expression of true sensitization to grass pollen. However, although IgE to Phl p 5 usually appears later than that to Phl p 1 in the sensitization process, its concentration grows rapidly in many patients and higher and its contribution to patients' symptoms has been demonstrated.⁶³⁷ Testing IgE to Phl p 5 can be useful as a second line test and has been shown to be useful for distinguishing between allergy to grass and olive pollen in Southern Europe. Specific IgE to Phl p 5 may have some prognostic value for indicating disease severity or likely progression from allergic rhinitis to asthma, but this needs to be confirmed with well-designed studies. As group 5 allergens have not been found in subtropical grass pollens, specific IgE to Phl p 5 may particularly indicate sensitization to temperate grass pollens. This needs to be investigated in relevant patient populations.

**IgE to Phl p 12 - Clinical relevance (see also Chapter C01)** – Phl p 12, is the highly cross-reactive profilin of *Phleum pratense*. As a heat-labile, relatively weak allergenic molecule, specific IgE sensitization to profilin comes later in the molecular spreading process, reaches only moderate levels of IgE antibodies and only in a minority of patients. Hence, IgE to Phl p 12 mark in general those patients with a higher atopic background and/or longer disease duration. Patients with a positive skin prick test/serum IgE to grass pollen extract but no detectable IgE to Phl p 1 and/or Phl p 5 must be tested for IgE reactivity to Phl p 12 as these antibodies—that can be induced by other pollens containing profilin—is the first cause of “false” positivity to assays based on grass pollen extract. In the presence of specific IgE to Phl p 12, patients should be asked about Oral Allergic Syndrome triggered by the ingestion of fruits and vegetables containing profilin.

## Tips for the use of molecular diagnostics for grass pollen allergy

* IgE to Phl p 1 is a marker of “true sensitization” to grass pollen

* Exceptions: In a few rare cases with skin test positivity to a grass pollen extract but no detectible IgE to Phl p1, IgE to Phl p5, may confirm the diagnosis of grass pollen allergy.

**IgE to Phl p 7 - Clinical relevance (see also Chapter C06)** – Phl p 7, is the highly cross-reactive polcalcin of *Phleum pratense*. This is a heat-stable, relatively potent allergen that can induce quite high specific IgE antibody levels. An IgE response to Phl p 7 is observed only infrequently among grass pollen allergic patients and usually many years after disease onset. Specific IgE to Phl p 7 indicates a relatively distinct category of grass pollen allergic patients, with more severe symptoms, a higher prevalence of asthma, and a higher frequency of allergic comorbidities.⁶³⁸ Moreover, many other pollens and allergenic sources contain polcalcin so that the original sensitization to polcalcin in a grass-pollen allergic patient must be carefully searched.⁶³⁸,⁶³⁹ These other allergenic sources could be indeed responsible for a more severe disease.

**IgE to Phl p 4 - Clinical relevance (see also Chapter A03)** – Phl p 4 is a major allergenic protein of grass pollen. In its native form, that is still used in most commercial available assays, Phl p 4 contains extremely highly cross-reactive carbohydrate determinants (CCD). This explains why in several epidemiological studies IgE positivity to Phl p 4 scores over 90% of the grass pollen allergic patients. However, when the recombinant form of the molecule is used in assays, about 50% of that positivity is not confirmed any more⁶³⁹,⁶⁴⁰ (Matricardi PM, data on file). As extracts contain the native Phl p 4, a weak positivity to SPT/serum IgE test based on grass pollen extracts can be “false” in some patients and simply explained by IgE recognition of CCD determinants. Phl p 4 may also serve as a marker of sensitization to Bermuda grass pollen due to its similarity with Cyn d 4, a major allergen of Bermuda grass pollen, but this needs to be investigated in relevant patient populations. Recently, sIgE to Phl p 4 as well as Phl p 1 and Phl p 5 have been identified as an early indicator of allergic rhinitis in 763 children from Sweden.⁶⁴⁰

**IgE to Phl p 2, 6, 11** – Phl p 2 and Phl p 11 are both rarely the only molecule inducing grass pollen sensitization and the presence of IgE antibodies to Phl p 2—observed in around 60-80% of the European grass pollen allergic patients—just confirms that a positive SPT reaction is the expression of true sensitization to grass. Phl p 6 is highly cross-reacting with Phl p 5 and does not add a lot of diagnostic information, once IgE to Phl p 5 has been documented.

## 4 - Predictive Value of Specific IgE responses to Allergenic Molecules

**IgE to grass pollen molecules as biomarkers of disease** – The presence of allergen-specific IgE towards airborne allergen molecules has been investigated as a putative predictive biomarker for the development of asthma throughout childhood and adolescence. In the case of seasonal allergic rhinitis to timothy grass pollen, individual risk profiles the predictive power of IgE sensitization to certain marker molecules, such as the profilin grass pollen allergen Phl p 12, which correlates with an increased risk for the development of an Oral Allergy Syndrome (OAS), has been confirmed in a cohort of over 1000 grass pollen allergic Italian children.⁸⁵ Similarly, the same study confirmed a strong association between IgE sensitization to Phl p 7 (polcalcin) and asthma. A molecular combinatorial analysis
confirmed that the qualitative homogeneity of IgE sensitization to the extract of *Phleum pratense* among grass pollen-allergic patients is only apparent. The number of described grass pollen IgE sensitization profiles, originally limited was expanded to 87, suggesting that, theoretically, all the 256 possible combinations could be observed in the general population of patients allergic to timothy grass pollen, limiting potential prognostic value. However, some IgE sensitization profiles were much more frequent than others, so that their hypothetical clinical relevance could be investigated. Thus far, no association between any of those frequent specific profiles of IgE sensitization to the eight most relevant allergenic molecules of *Phleum pratense* pollen, with the clinical phenotype of allergic rhinitis and conjunctivitis could yet be identified.⁶³⁸ In other words, the study excluded that a combinatorial analysis of the spectrum of molecular IgE sensitization to Timothy grass pollen is of any diagnostic relevance, probably for the highly multifactorial origin of allergic rhinitis in this cohort was enriched in highly polysensitized children.⁶³⁸ Whether or not the characterization of the IgE sensitization profile to the full set of eight Phl p molecules could be more relevant in patients sensitized only to grass pollen remains an open research question. Interestingly, a 6-year long prospective study of 401 patients of the same cohort highlighted that the observation of IgE to Phl p 1 is relevant for persistence of seasonal allergic rhinitis; IgE to Phl p 5 is predicting persistence of both rhinitis and asthma,⁶³⁸ which may have clinical value if confirmed in other populations.

**Ryegrass pollen and components as markers for thunderstorm asthma risk** – In other parts of the world, including temperate regions of Australia, where timothy grass is rare and ryegrass (*Lolium perenne*) is common, sensitization to ryegrass pollen, and more specifically Lol p 5 or ryegrass pollen starch granules, containing Lol p 5, have been associated with patients presenting with asthma in the context of thunderstorm asthma epidemics.⁶⁴¹ Southeastern Australia and, in particular, Melbourne, has experienced the highest number of thunderstorm asthma events and the highest number of patients affected globally, making understanding and controlling acute thunderstorm asthma risk an imperative.⁶⁴² Positive skin prick test to ryegrass pollen occurs at increased frequency and magnitude in thunderstorm asthma cases vs. controls presenting with asthma at other times.⁶⁴³,⁶⁴⁴ Circumstantial evidence also indicates that during thunderstorm asthma events, ruptured grass pollen does occur.⁶⁴⁴,⁶⁴⁵ Whilst there is high similarity between the major allergens of timothy grass and ryegrass pollen, the amino acid composition differs between isoforms of Phl p 5 of Lol p 5, and it may be relevant, that slgE to major ryegrass pollen serve as prognostic biomarkers of seasonal allergic asthma risk including thunderstorm asthma risk, in patients with allergic rhinitis in temperate regions of Australia.⁶⁴⁶

## 5 – Specific IgE responses to allergenic molecules and response to allergen immunotherapy

**Nasal IgG4 to Phl p 1 & Phl p 5 and SCIT efficacy** – Encouraging and very interesting information emerged from a trial discovering

that IgG4 to *Phleum pratense* molecules in the nasal secretions are biomarkers of efficacy of allergen immunotherapy in grass pollen allergic patients.⁶⁴⁷ In this trial, the levels of nasal IgG4 to Phl p 1 and to Phl p 5 were increased during the pollen season compared with out of season among the patients treated with SCIT compared to those untreated. IgG-associated inhibitory activity in nasal fluid and serum was significantly increased in the SCIT group compared with the untreated group. Inhibitory activity associated to IgG4 antibodies to Phl p 1 and Phl p 5 correlated therefore closely with the clinical response to allergen immunotherapy in patients with allergic rhinitis with or without asthma. Nasal secretions might become therefore a valuable alternative for *in vitro* tests searching antibodies against allergen molecules in patients with respiratory allergies.⁶⁴⁸ However, recent studies on nasal IgE showed that the concentration of slgE is much lower in nasal secretions than in the serum. Consequently, slgE assays with very high analytical sensitivity and sampling methods with minimal dilution are needed before nasal secretions can be validated as alternative to serum in testing the slgE repertoire in patients with seasonal allergic rhinitis.⁶⁴⁹

**Pre-SCIT I IgE sensitization pattern predicts IgG4 response to SCIT** – In a study of 18 grass pollen allergic patients treated with SCIT, the pretreatment IgE repertoire to the different grass pollen components predicted the repertoire of the induced IgG4 antibodies after completing updosing. This may indicate, that slgE to specific components is a prerequisite for the induction of competing IgG4 antibodies during SCIT⁶⁵⁰ At the same time, this study did not find induction of new sensitizations to grass pollen components, the study subjects had not been sensitized to before starting SCIT.

**BM32 hybrid molecule** – A single recombinant hybrid molecule, consisting of the four major Timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Phl p 6) was suitable for *in vivo* diagnosis of genuine grass pollen allergy in children suffering from pollinosis in Greece²⁸² With this hybrid molecule, genuine grass pollen sensitization was confirmed through SPT in 94% of the children with positive SPT to grass pollen extract by SPT and IgE reactivity to the hybrid. Only 4 hybrid-negative children showed IgE reactivity to SPT with grass pollen extract, but they were confirmed to react against cross-reactive allergens such as Phl p 4, Phl p 11, and Phl p 12 and had also sensitizations to pollen allergens from unrelated plants. This study demonstrated therefore that a recombinant hybrid molecule approach represents a useful tool for *in vivo* diagnosis of genuine grass pollen sensitization and opened a new avenue to the use of bioengineered molecules in *in vivo* diagnostics of allergic diseases in general.

In patients allergic to grass pollens specific IgE testing to allergen molecules should be oriented to answer the following questions:

A. Is the patient really sensitized to grass pollen major allergen molecules? (test Phl p 1, if negative also Phl p 5 and the other molecules);

B. Is the patient sensitized also to highly cross-reacting molecules? (test Phl p 12 and Phl p 7);
confirmed that the qualitative homogeneity of IgE sensitization to the extract of *Phleum pratense* among grass pollen-allergic patients is only apparent. The number of described grass pollen IgE sensitization profiles, originally limited was expanded to 87, suggesting that, theoretically, all the 256 possible combinations could be observed in the general population of patients allergic to timothy grass pollen, limiting potential prognostic value. However, some IgE sensitization profiles were much more frequent than others, so that their hypothetical clinical relevance could be investigated. Thus far, no association between any of those frequent specific profiles of IgE sensitization to the eight most relevant allergenic molecules of *Phleum pratense* pollen, with the clinical phenotype of allergic rhinitis and conjunctivitis could yet be identified.⁶³⁸ In other words, the study excluded that a combinatorial analysis of the spectrum of molecular IgE sensitization to Timothy grass pollen is of any diagnostic relevance, probably for the highly multifactorial origin of allergic rhinitis in this cohort was enriched in highly polysensitized children.⁶³⁸ Whether or not the characterization of the IgE sensitization profile to the full set of eight Phl p molecules could be more relevant in patients sensitized only to grass pollen remains an open research question. Interestingly, a 6-year long prospective study of 401 patients of the same cohort highlighted that the observation of IgE to Phl p 1 is relevant for persistence of seasonal allergic rhinitis; IgE to Phl p 5 is predicting persistence of both rhinitis and asthma,⁶³⁸ which may have clinical value if confirmed in other populations.

**Ryegrass pollen and components as markers for thunderstorm asthma risk** – In other parts of the world, including temperate regions of Australia, where timothy grass is rare and ryegrass (*Lolium perenne*) is common, sensitization to ryegrass pollen, and more specifically Lol p 5 or ryegrass pollen starch granules, containing Lol p 5, have been associated with patients presenting with asthma in the context of thunderstorm asthma epidemics.⁶⁴¹ Southeastern Australia and, in particular, Melbourne, has experienced the highest number of thunderstorm asthma events and the highest number of patients affected globally, making understanding and controlling acute thunderstorm asthma risk an imperative.⁶⁴² Positive skin prick test to ryegrass pollen occurs at increased frequency and magnitude in thunderstorm asthma cases vs. controls presenting with asthma at other times.⁶⁴³,⁶⁴⁴ Circumstantial evidence also indicates that during thunderstorm asthma events, ruptured grass pollen does occur.⁶⁴⁴,⁶⁴⁵ Whilst there is high similarity between the major allergens of timothy grass and ryegrass pollen, the amino acid composition differs between isoforms of Phl p 5 of Lol p 5, and it may be relevant, that slgE to major ryegrass pollen serve as prognostic biomarkers of seasonal allergic asthma risk including thunderstorm asthma risk, in patients with allergic rhinitis in temperate regions of Australia.⁶⁴⁶

## 5 – Specific IgE responses to allergenic molecules and response to allergen immunotherapy

**Nasal IgG4 to Phl p 1 & Phl p 5 and SCIT efficacy** – Encouraging and very interesting information emerged from a trial discovering

that IgG4 to *Phleum pratense* molecules in the nasal secretions are biomarkers of efficacy of allergen immunotherapy in grass pollen allergic patients.⁶⁴⁷ In this trial, the levels of nasal IgG4 to Phl p 1 and to Phl p 5 were increased during the pollen season compared with out of season among the patients treated with SCIT compared to those untreated. IgG-associated inhibitory activity in nasal fluid and serum was significantly increased in the SCIT group compared with the untreated group. Inhibitory activity associated to IgG4 antibodies to Phl p 1 and Phl p 5 correlated therefore closely with the clinical response to allergen immunotherapy in patients with allergic rhinitis with or without asthma. Nasal secretions might become therefore a valuable alternative for *in vitro* tests searching antibodies against allergen molecules in patients with respiratory allergies.⁶⁴⁸ However, recent studies on nasal IgE showed that the concentration of slgE is much lower in nasal secretions than in the serum. Consequently, slgE assays with very high analytical sensitivity and sampling methods with minimal dilution are needed before nasal secretions can be validated as alternative to serum in testing the slgE repertoire in patients with seasonal allergic rhinitis.⁶⁴⁹

**Pre-SCIT I IgE sensitization pattern predicts IgG4 response to SCIT** – In a study of 18 grass pollen allergic patients treated with SCIT, the pretreatment IgE repertoire to the different grass pollen components predicted the repertoire of the induced IgG4 antibodies after completing updosing. This may indicate, that slgE to specific components is a prerequisite for the induction of competing IgG4 antibodies during SCIT⁶⁵⁰ At the same time, this study did not find induction of new sensitizations to grass pollen components, the study subjects had not been sensitized to before starting SCIT.

**BM32 hybrid molecule** – A single recombinant hybrid molecule, consisting of the four major Timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Phl p 6) was suitable for *in vivo* diagnosis of genuine grass pollen allergy in children suffering from pollinosis in Greece²⁸² With this hybrid molecule, genuine grass pollen sensitization was confirmed through SPT in 94% of the children with positive SPT to grass pollen extract by SPT and IgE reactivity to the hybrid. Only 4 hybrid-negative children showed IgE reactivity to SPT with grass pollen extract, but they were confirmed to react against cross-reactive allergens such as Phl p 4, Phl p 11, and Phl p 12 and had also sensitizations to pollen allergens from unrelated plants. This study demonstrated therefore that a recombinant hybrid molecule approach represents a useful tool for *in vivo* diagnosis of genuine grass pollen sensitization and opened a new avenue to the use of bioengineered molecules in *in vivo* diagnostics of allergic diseases in general.

In patients allergic to grass pollens specific IgE testing to allergen molecules should be oriented to answer the following questions:

A. Is the patient really sensitized to grass pollen major allergen molecules? (test Phl p 1, if negative also Phl p 5 and the other molecules);

B. Is the patient sensitized also to highly cross-reacting molecules? (test Phl p 12 and Phl p 7);
C. In case of negativity to Phl p 1 and the other species-specific allergenic molecules and positivity to Phl p 12 and/or Phl p 7, which pollen is inducing a "false" IgE sensitization to grass pollen extracts?

After having answered these questions the doctor should be able to decide whether the patients' symptoms are consistent or not to IgE sensitization to grass-pollen, and consequently can decide whether to prescribe an AIT based on grass-pollen extract. Whilst orthologues such as Cyn d 7 and Sor h 12 have been reported in subtropical grasses, there is currently limited evidence available of IgE reactivity in relevant patients primarily sensitized to subtropical grass pollens. A diagnostic algorithm for a decision making process, which summarizes the information provided in the previous section is proposed (Figure 51).

### Clinically Relevant

* The major allergen Phl p 1 serves as a specific diagnostic marker for grass pollen allergy in temperate regions. Phl p 5 and Phl p 2 may serve as secondary diagnostic or prognostic markers for some patients. Exceptions: In rare cases a grass pollen allergic patient can lack IgE to Phl p 1.

* Whilst minor pan-allergens Phl p 12 and Phl p 7 may not increase diagnostic specificity, in some geographical regions Phl p 12 IgE appears to be associated with true grass pollen allergy.

## 6 - Clinical cases

### Case 1

**Clinical history:** A 35 year old male from Central Italy patient presented with allergic rhinitis from April to July but not in September or October. The patient had experienced conjunctival and nasal symptoms that did not respond to antihistamines and were only partly controlled with nasal steroids. He reported the condition was steadily increasing each season and that he, occasionally, experienced a tight chest after spending time outside. Since the last year, he had experienced oral symptoms (pruritus, swelling) after eating either melon or watermelon.

**Testing:** SPT positive for birch (5 mm), Timothy grass (8 mm), pellitory (4 mm), olive (3 mm) pollens. Serum IgE antibody levels were 7.1 kU/L to birch, 17.3 kU/L to timothy grass, 6.7 kU/L to pellitory, 3.2 kU/L to olive extracts.

**Treatment:** No AIT was started as the doctor was not sure which pollen(s) was/were responsible of the patient's symptoms.

**Added CRD value:** positive response to Phl p 1 (12.2 kU/l), Phl p 5 (6.5 kU/l), and Phl p 12 (4.3 kU/l) but not to Bet v 1, Ole e 1, and Par j 2. The patient commenced SLIT with grass pollen and responded well to this treatment. OAS was also explained by IgE sensitization to profilin (Phl p 12).

### Case 2

**Clinical history:** A 26 year old Danish woman with a 10 year history of persistent severe seasonal rhinoconjunctivitis during birch and grass pollen season. Symptoms most severe in early summer, with persistent conjunctival (redness, itching, watering, light sensitivity) and nasal (blocked nose, itching and secretion) symptoms. Very poor response to systemic antihistamines and topical antihistamine

<figure><img src="image_137.png" /><figcaption>**FIGURE 51** Diagnostic algorithm for AIT prescription in grass pollen allergic patients—Patients with AR symptoms during the grass pollen-season and a positive SPT/IgE assay to grass pollen extracts are further investigated to detect serum IgE antibodies to Phl p 1, Phl p 2, Phl p 5, Phl p 7, Phl p 11, and Phl p 12. The identification of one or more of IgE antibodies to Phl p 1, Phl p 2, Phl p 5 and/or Phl p 11 is followed by the prescription of grass pollen AIT. The identification of IgE to Phl p 12 (profilin) is followed by further investigation of OAS and influences is relevant to better interpret results of SPT/IgE assays with other pollen extracts or vegetables. The identification of IgE to Phl p 7 alerts the doctor of a worse prognosis and greater severity of the disease.</figcaption></figure>
C. In case of negativity to Phl p 1 and the other species-specific allergenic molecules and positivity to Phl p 12 and/or Phl p 7, which pollen is inducing a "false" IgE sensitization to grass pollen extracts?

After having answered these questions the doctor should be able to decide whether the patients' symptoms are consistent or not to IgE sensitization to grass-pollen, and consequently can decide whether to prescribe an AIT based on grass-pollen extract. Whilst orthologues such as Cyn d 7 and Sor h 12 have been reported in subtropical grasses, there is currently limited evidence available of IgE reactivity in relevant patients primarily sensitized to subtropical grass pollens. A diagnostic algorithm for a decision making process, which summarizes the information provided in the previous section is proposed (Figure 51).

### Clinically Relevant

* The major allergen Phl p 1 serves as a specific diagnostic marker for grass pollen allergy in temperate regions. Phl p 5 and Phl p 2 may serve as secondary diagnostic or prognostic markers for some patients. Exceptions: In rare cases a grass pollen allergic patient can lack IgE to Phl p 1.

* Whilst minor pan-allergens Phl p 12 and Phl p 7 may not increase diagnostic specificity, in some geographical regions Phl p 12 IgE appears to be associated with true grass pollen allergy.

## 6 - Clinical cases

### Case 1

**Clinical history:** A 35 year old male from Central Italy patient presented with allergic rhinitis from April to July but not in September or October. The patient had experienced conjunctival and nasal symptoms that did not respond to antihistamines and were only partly controlled with nasal steroids. He reported the condition was steadily increasing each season and that he, occasionally, experienced a tight chest after spending time outside. Since the last year, he had experienced oral symptoms (pruritus, swelling) after eating either melon or watermelon.

**Testing:** SPT positive for birch (5 mm), Timothy grass (8 mm), pellitory (4 mm), olive (3 mm) pollens. Serum IgE antibody levels were 7.1 kU/L to birch, 17.3 kU/L to timothy grass, 6.7 kU/L to pellitory, 3.2 kU/L to olive extracts.

**Treatment:** No AIT was started as the doctor was not sure which pollen(s) was/were responsible of the patient's symptoms.

**Added CRD value:** positive response to Phl p 1 (12.2 kU/l), Phl p 5 (6.5 kU/l), and Phl p 12 (4.3 kU/l) but not to Bet v 1, Ole e 1, and Par j 2. The patient commenced SLIT with grass pollen and responded well to this treatment. OAS was also explained by IgE sensitization to profilin (Phl p 12).

### Case 2

**Clinical history:** A 26 year old Danish woman with a 10 year history of persistent severe seasonal rhinoconjunctivitis during birch and grass pollen season. Symptoms most severe in early summer, with persistent conjunctival (redness, itching, watering, light sensitivity) and nasal (blocked nose, itching and secretion) symptoms. Very poor response to systemic antihistamines and topical antihistamine

<figure><img src="image_136.png" /></figure>

**FIGURE 51** Diagnostic algorithm for AIT prescription in grass pollen allergic patients—Patients with AR symptoms during the grass pollen-season and a positive SPT/IgE assay to grass pollen extracts are further investigated to detect serum IgE antibodies to Phl p 1, Phl p 2, Phl p 5, Phl p 7, Phl p 11, and Phl p 12. The identification of one or more of IgE antibodies to Phl p 1, Phl p 2, Phl p 5 and/or Phl p 11 is followed by the prescription of grass pollen AIT. The identification of IgE to Phl p 12 (profilin) is followed by further investigation of OAS and influences is relevant to better interpret results of SPT/IgE assays with other pollen extracts or vegetables. The identification of IgE to Phl p 7 alerts the doctor of a worse prognosis and greater severity of the disease.
(eyes and nose) and to intranasal corticosteroid. Had some benefit from systemic corticosteroid. Good symptom control during birch pollen season.

**Testing:** SPT positive for birch and grass pollen 7 mm diameter. IgE to grass pollen 10.9 kU/l, birch pollen 3.9 kU/l

**Treatment:** Starts standard SCIT with natural grass pollen extract¹. Poor clinical effect after 2 years of treatment. Added CRD

value = Missing Step 2b - *In vitro* testing: We found a sensitizations to Phl p 4 in the grass pollen panel, as well as Bet v 1. She was not sensitized to typical other CCD reactive natural molecules. The conclusion was that she truly was grass pollen allergic, and she received anti-IgE treatment during grass pollen season with good clinical outcome.
(eyes and nose) and to intranasal corticosteroid. Had some benefit from systemic corticosteroid. Good symptom control during birch pollen season.

**Testing:** SPT positive for birch and grass pollen 7 mm diameter. IgE to grass pollen 10.9 kU/l, birch pollen 3.9 kU/l

**Treatment:** Starts standard SCIT with natural grass pollen extract¹. Poor clinical effect after 2 years of treatment. Added CRD

value = Missing Step 2b - *In vitro* testing: We found a sensitizations to Phl p 4 in the grass pollen panel, as well as Bet v 1. She was not sensitized to typical other CCD reactive natural molecules. The conclusion was that she truly was grass pollen allergic, and she received anti-IgE treatment during grass pollen season with good clinical outcome.
# B03 - Weed pollen allergy

Michael Hauser, Janet M. Davies, Thomas Hawranek, Gabriele Gadermaier

## Highlights

* Common invasive weeds like ragweed, mugwort and plantain, are important allergen sources typically flowering in summer through to autumn.

* The biogeographical range and pollination periods of allergenic weeds can overlap confounding accurate allergy diagnosis.

* Specific IgE to Amb a 1 can be a useful marker for ragweed sensitization, but it shows cross-reactivity with Art v 6 from mugwort and Hel a 6 from sunflower. Likewise specific IgE to Art v 1 can be a useful marker for mugwort sensitization, but it shows partial cross-reactivity with ragweed Amb a 4.

* Art v 3 reactivity plays a major role in LTP-related allergies in patients from the Mediterranean region and Northern China.

* Par j 2 is a highly specific marker for pellitory sensitization while Pla I 1 is a useful marker for genuine plantain sensitization.

## 1 - The allergen sources

The term "weed" does not constitute a botanical family but rather refers to diverse plants used as culinary herbs, medicinal plants that are ecologically adaptive, as well as invasive segetal plants. Pollen of weeds mediating IgE-related allergic reactions are found in the dicots of the Asteraceae, Urticaceae, Plantaginaceae, Euphorbiaceae, and Amaranthaceae plant families.⁶⁵¹ Pollen of ragweed, mugwort, sunflower, feverfew, pellitory, English plantain, Annual mercury, goosefoot, Russian thistle and amaranth are considered main weed pollen allergy eliciting sources (Figure 52). Allergenic monocot weeds of the family Poaceae (e.g., Johnson grass) can be found in Chapter B02.

The impact of climate change on pollen load, allergenicity, distribution, and flowering season is well acknowledged and is of particular interest in regard to weeds since they can dominate groundcover, adapt to various environmental conditions or reside in ecologic niches.⁶⁵¹,⁶⁵³,⁶⁵⁴,⁸³⁰ Due to globalization, neophytes such as ragweed have been imported to Europe as ballast grain, spreading readily with predictions to reach Northern Europe.⁶⁵¹ Furthermore, significant increases in duration of pollen seasons of ragweed and pellitory were recorded during the last decades.⁶⁵⁵,⁶⁵⁶,¹⁶⁵⁰ Additional influence on the allergenicity might arise from environmental pollution, as was shown for ragweed pollen collected along high-traffic roads presenting elevated IgE reactivity.⁶⁵⁷ Weeds are often considered nondesired invasive species and thus combated using herbicides. On the other hand, there are some species actively cultivated for economic purposes, e.g., sunflower or *Artemisia annua* to obtain the anti-malaria drug artemisinin.

Plants of the genus *Ambrosia* comprise around 50 species native to Northern and Central America. In the past decades, the neophyte

is rapidly spreading in Europe due to the pollen's ability to travel long distances. The genus *Artemisia* comprises around 350 species and representatives can be found throughout the Northern hemisphere and Australia. Mugwort is frequently used as herb in traditional Chinese medicine and *A. annua* is cultivated for harvest of artemisinin. There are parts of Europe and America where the range and pollination of mugwort and ragweed overlap (www.discoverli fe.org; Altas of living Australia and US Department of Agriculture), confounding accurate allergy diagnosis. Common sunflower is primarily grown for commercial use of its oil and birdseed. *Parthenium spp* are found in Southern US, Central and South America and invasive in India, Australia and parts of Africa. Allergenic members of the Parietaria genus are frequently found in Southern and Central Europe showing a long pollination season with recurrent flowering periods. The genus Plantago includes around 250 species and was spreading from Europe throughout the world. *Mercurialis annua* is a highly prevalent weed throughout Europe. *Chenopodium album*, *Salsola kali* and *Amaranthus retroflexus* can be found in arid regions of the Northern hemisphere and Australia. Due to use in greening programs or as ornamental flowers, these weeds are highly abundant in Iran, Kuwait, or Saudi Arabia as well as the South East of Spain.

## 2 - The allergen families

The most relevant weed pollen allergens belong to the families of pectate lyases, defensin-like proteins, nonspecific lipid transfer proteins (nsLTP) and Ole e 1-like proteins (Table 17). Additionally, the pan-allergens profilin and polcalcin have been identified as cross-reactive molecules present in weed pollen (see Chapters C01 and C04). Currently, 70 weed pollen allergens are listed as allergens by the WHO/IUIS Allergen Nomenclature Sub-Committee. Recent additions of relevant allergens include homologues of defensin-like proteins (feverfew and *Artemisia spp*.), nsLTPs (*Artemisia spp*) and a pectate lyase (sunflower). The acidic glycoprotein at 60 kDa in *Artemisia* was identified as galactose oxidase Art an 7.⁶⁵⁵ Furthermore, two new allergens belonging to the cysteine protease and enolase family were identified in ragweed pollen (Table 17).⁶⁵⁸a

## 3 - Allergenic molecules

*Ambrosia spp* are major elicitors of type I pollen allergies in Northern America with a sensitization frequency in the general adult population of 15.3% in Canada and 8.7-26% in U.S.⁶²⁶,⁶⁵⁸b In Europe, SPT positivity among allergic patients was determined to be in average lower (2.6%) but can be very high in distinct regions, e.g., Hungary 53.8%.⁶⁵⁸c In Germany, sensitization to ragweed in the general population is 10%, in allergic patients it ranges between 19.5 and 36.3%.⁶⁵⁹a,⁶⁵⁹b Notably, ragweed sensitization in Europe is estimated to double within the next decades due to progressive spreading of the invasive plant, fuelled by the impact of climatic changes.⁶⁵⁴ Korean children suffering from allergic rhinitis
# B03 - Weed pollen allergy

Michael Hauser, Janet M. Davies, Thomas Hawranek, Gabriele Gadermaier

## Highlights

* Common invasive weeds like ragweed, mugwort and plantain, are important allergen sources typically flowering in summer through to autumn.

* The biogeographical range and pollination periods of allergenic weeds can overlap confounding accurate allergy diagnosis.

* Specific IgE to Amb a 1 can be a useful marker for ragweed sensitization, but it shows cross-reactivity with Art v 6 from mugwort and Hel a 6 from sunflower. Likewise specific IgE to Art v 1 can be a useful marker for mugwort sensitization, but it shows partial cross-reactivity with ragweed Amb a 4.

* Art v 3 reactivity plays a major role in LTP-related allergies in patients from the Mediterranean region and Northern China.

* Par j 2 is a highly specific marker for pellitory sensitization while Pla I 1 is a useful marker for genuine plantain sensitization.

## 1 - The allergen sources

The term "weed" does not constitute a botanical family but rather refers to diverse plants used as culinary herbs, medicinal plants that are ecologically adaptive, as well as invasive segetal plants. Pollen of weeds mediating IgE-related allergic reactions are found in the dicots of the Asteraceae, Urticaceae, Plantaginaceae, Euphorbiaceae, and Amaranthaceae plant families.⁶⁵¹ Pollen of ragweed, mugwort, sunflower, feverfew, pellitory, English plantain, Annual mercury, goosefoot, Russian thistle and amaranth are considered main weed pollen allergy eliciting sources (Figure 52). Allergenic monocot weeds of the family Poaceae (e.g., Johnson grass) can be found in Chapter B02.

The impact of climate change on pollen load, allergenicity, distribution, and flowering season is well acknowledged and is of particular interest in regard to weeds since they can dominate groundcover, adapt to various environmental conditions or reside in ecologic niches.⁶⁵¹,⁶⁵³,⁶⁵⁴,⁸³⁰ Due to globalization, neophytes such as ragweed have been imported to Europe as ballast grain, spreading readily with predictions to reach Northern Europe.⁶⁵¹ Furthermore, significant increases in duration of pollen seasons of ragweed and pellitory were recorded during the last decades.⁶⁵⁵,⁶⁵⁶,¹⁶⁵⁰ Additional influence on the allergenicity might arise from environmental pollution, as was shown for ragweed pollen collected along high-traffic roads presenting elevated IgE reactivity.⁶⁵⁷ Weeds are often considered nondesired invasive species and thus combated using herbicides. On the other hand, there are some species actively cultivated for economic purposes, e.g., sunflower or *Artemisia annua* to obtain the anti-malaria drug artemisinin.

Plants of the genus Ambrosia comprise around 50 species native to Northern and Central America. In the past decades, the neophyte

is rapidly spreading in Europe due to the pollen's ability to travel long distances. The genus Artemisia comprises around 350 species and representatives can be found throughout the Northern hemisphere and Australia. Mugwort is frequently used as herb in traditional Chinese medicine and A. annua is cultivated for harvest of artemisinin. There are parts of Europe and America where the range and pollination of mugwort and ragweed overlap (www.discoverlifefe.org; Altas of living Australia and US Department of Agriculture), confounding accurate allergy diagnosis. Common sunflower is primarily grown for commercial use of its oil and birdseed. *Parthenium spp* are found in Southern US, Central and South America and invasive in India, Australia and parts of Africa. Allergenic members of the Parietaria genus are frequently found in Southern and Central Europe showing a long pollination season with recurrent flowering periods. The genus Plantago includes around 250 species and was spreading from Europe throughout the world. *Mercurialis annua* is a highly prevalent weed throughout Europe. *Chenopodium album, Salsola kali* and *Amaranthus retroflexus* can be found in arid regions of the Northern hemisphere and Australia. Due to use in greening programs or as ornamental flowers, these weeds are highly abundant in Iran, Kuwait, or Saudi Arabia as well as the South East of Spain.

## 2 - The allergen families

The most relevant weed pollen allergens belong to the families of pectate lyases, defensin-like proteins, nonspecific lipid transfer proteins (nsLTP) and Ole e 1-like proteins (Table 17). Additionally, the pan-allergens profilin and polcalcin have been identified as cross-reactive molecules present in weed pollen (see Chapters C01 and C04). Currently, 70 weed pollen allergens are listed as allergens by the WHO/IUIS Allergen Nomenclature Sub-Committee. Recent additions of relevant allergens include homologues of defensin-like proteins (feverfew and *Artemisia spp*.), nsLTPs (*Artemisia spp*) and a pectate lyase (sunflower). The acidic glycoprotein at 60 kDa in *Artemisia* was identified as galactose oxidase Art an 7.⁶⁵⁵ Furthermore, two new allergens belonging to the cysteine protease and enolase family were identified in ragweed pollen (Table 17).⁶⁵⁸a

## 3 - Allergenic molecules

*Ambrosia spp* are major elicitors of type I pollen allergies in Northern America with a sensitization frequency in the general adult population of 15.3% in Canada and 8.7-26% in U.S.⁶²⁶,⁶⁵⁸b In Europe, SPT positivity among allergic patients was determined to be in average lower (2.6%) but can be very high in distinct regions, e.g., Hungary 53.8%.⁶⁵⁸c In Germany, sensitization to ragweed in the general population is 10%, in allergic patients it ranges between 19.5 and 36.3%.⁶⁵⁹a,⁶⁵⁹b Notably, ragweed sensitization in Europe is estimated to double within the next decades due to progressive spreading of the invasive plant, fuelled by the impact of climatic changes.⁶⁵⁴ Korean children suffering from allergic rhinitis
<figure><img src="image_138.png" /><figcaption>FIGURE 52 Important allergenic weeds. Figure adapted from "Marker allergens of weed pollen"<sup>652</sup>. Flowering periods given refer to the Northern Hemisphere.</figcaption></figure>

showed a sensitization prevalence of 0.2-3.6%. Among close to 20,000 requested specific IgE tests in Japan, 17% of adults and 18.1% of children showed positive results for ragweed pollen, and in some regions of Japan 26.1% of adults and 24.9% of children

were sensitized.<sup>660</sup> In eastern parts of Australia, sensitization frequencies ranging from 34-38% were observed among allergic patients.<sup>661</sup> Ragweed allergy is mainly driven by the major allergen Amb a 1, a pectate lyase with high sensitization prevalence and
<figure><img src="image_137.png" /><figcaption>FIGURE 52 Important allergenic weeds. Figure adapted from "Marker allergens of weed pollen"<sup>652</sup>. Flowering periods given refer to the Northern Hemisphere.</figcaption></figure>

showed a sensitization prevalence of 0.2-3.6%. Among close to 20,000 requested specific IgE tests in Japan, 17% of adults and 18.1% of children showed positive results for ragweed pollen, and in some regions of Japan 26.1% of adults and 24.9% of children

were sensitized.<sup>660</sup> In eastern parts of Australia, sensitization frequencies ranging from 34-38% were observed among allergic patients.<sup>661</sup> Ragweed allergy is mainly driven by the major allergen Amb a 1, a pectate lyase with high sensitization prevalence and
<table><caption>TABLE 17 Clinically relevant weeds</caption><thead><tr><th>Allergenic molecule</th><th>Protein family</th><th>Frequency of IgE reactivity</th><th>MW (kDa)</th></tr></thead><tbody><tr><td colspan="4">Ambrosia artemisiifolia (ragweed), Ambrosia spp</td></tr><tr><td>Amb a 1</td><td>Pectate lyase</td><td>>95%</td><td>38</td></tr><tr><td>Amb a 4</td><td>Defensin-like protein with polyproline rich region</td><td>20-40%</td><td>13-15</td></tr><tr><td>Amb a 6</td><td>Non-specific lipid transfer protein</td><td>20%</td><td>10</td></tr><tr><td>Amb a 8</td><td>Profilin</td><td>35-50%</td><td>14</td></tr><tr><td>Amb a 9</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>10-15%</td><td>9</td></tr><tr><td>Amb a 10</td><td>Polcalcin (3 EF-hand calcium binding protein)</td><td>10-15%</td><td>17</td></tr><tr><td>Amb a 11</td><td>Cysteine protease</td><td>66%</td><td>37</td></tr><tr><td>Amb a 12</td><td>Enolase</td><td>66%</td><td>48</td></tr><tr><td colspan="4">Artemisia vulgaris (mugwort), Artemisia spp</td></tr><tr><td>Art v 1*</td><td>Defensin-like protein with polyproline rich region</td><td>95%</td><td>13-15</td></tr><tr><td>Art v 3*</td><td>Non-specific lipid transfer protein</td><td>22-70%</td><td>10</td></tr><tr><td>Art v 4</td><td>Profilin</td><td>35%</td><td>14</td></tr><tr><td>Art v 5</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>10-28%</td><td>10</td></tr><tr><td>Art v 6</td><td>Pectate lyase, Amb a 1-homologue</td><td>26%</td><td>38</td></tr><tr><td>Art an 7</td><td>Galactose oxidase</td><td>16-94%</td><td>62</td></tr><tr><td colspan="4">Helianthus annuus (sunflower)</td></tr><tr><td>Hel a 1</td><td>Potential defensin-like protein with polyproline rich region</td><td>65%</td><td>34</td></tr><tr><td>Hel a 2</td><td>Profilin</td><td>31%</td><td>14</td></tr><tr><td>Hel a 6</td><td>Pectate lyase</td><td>36%</td><td>42</td></tr><tr><td colspan="4">Parthenium hysterophorus (feverfew)</td></tr><tr><td>Par h 1</td><td>Defensin-like protein with polyproline rich region</td><td>40-60%</td><td>12</td></tr><tr><td colspan="4">Parietaria judaica (pellitory)</td></tr><tr><td>Par j 1</td><td>Non-specific lipid transfer protein</td><td>95%</td><td>15</td></tr><tr><td>Par j 2</td><td>Non-specific lipid transfer protein</td><td>80%</td><td>11</td></tr><tr><td>Par j 3</td><td>Profilin</td><td>nd</td><td>14</td></tr><tr><td>Par j 4</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>6%</td><td>9</td></tr><tr><td colspan="4">Plantago lanceolata (English plantain)</td></tr><tr><td>Pla l 1</td><td>Ole e 1-like protein</td><td>86%</td><td>18</td></tr><tr><td>Pla l 2</td><td>Profilin</td><td>86%</td><td>15</td></tr><tr><td colspan="4">Mercurialis annua (pellitory)</td></tr><tr><td>Mer a 1</td><td>Profilin</td><td>50-60%</td><td>14</td></tr><tr><td colspan="4">Chenopodium album (goosefoot)</td></tr><tr><td>Che a 1</td><td>Ole e 1-like protein</td><td>70%</td><td>18</td></tr><tr><td>Che a 2</td><td>Profilin</td><td>55%</td><td>14</td></tr><tr><td>Che a 3</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>46%</td><td>10</td></tr><tr><td colspan="4">Salsola kali (Russian thistle)</td></tr><tr><td>Sal k 1</td><td>Pectin methylesterase family</td><td>65%</td><td>38</td></tr><tr><td>Sal k 3</td><td>Cobalamin independent methionine synthase</td><td>63%</td><td>85 (35+45)</td></tr><tr><td>Sal k 4</td><td>Profilin</td><td>46%</td><td>14</td></tr><tr><td>Sal k 5</td><td>Ole e 1-like protein</td><td>30-60%</td><td>18</td></tr><tr><td>Sal k 6</td><td>Polygalacturonase</td><td>32%</td><td>47</td></tr><tr><td>Sal k 7</td><td>Polcalcin</td><td>40%</td><td>9</td></tr><tr><td colspan="4">Amaranthus retroflexus (amaranth)</td></tr><tr><td>Ama r 1</td><td>Ole e 1-like protein</td><td>38%</td><td>18</td></tr><tr><td>Ama r 2</td><td>Profilin</td><td>25%</td><td>14</td></tr></tbody></table>

Data on sensitization frequency according to Gadermaier et al.⁶⁸⁰ and the WHO/IUIS allergen nomenclature database. nd not determined.

*Besides Art v 1 and Art v 3, several recently identified homologs from Artemisia spp of the defensin-like and nsLTP family are listed in the WHO/IUIS database (https://www.allergen.org).⁶⁸⁰
<table><caption>TABLE 17 Clinically relevant weeds</caption><thead><tr><th>Allergenic molecule</th><th>Protein family</th><th>Frequency of IgE reactivity</th><th>MW (kDa)</th></tr></thead><tbody><tr><td colspan="4">Ambrosia artemisiifolia (ragweed), Ambrosia spp</td></tr><tr><td>Amb a 1</td><td>Pectate lyase</td><td>>95%</td><td>38</td></tr><tr><td>Amb a 4</td><td>Defensin-like protein with polyproline rich region</td><td>20-40%</td><td>13-15</td></tr><tr><td>Amb a 6</td><td>Non-specific lipid transfer protein</td><td>20%</td><td>10</td></tr><tr><td>Amb a 8</td><td>Profilin</td><td>35-50%</td><td>14</td></tr><tr><td>Amb a 9</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>10-15%</td><td>9</td></tr><tr><td>Amb a 10</td><td>Polcalcin (3 EF-hand calcium binding protein)</td><td>10-15%</td><td>17</td></tr><tr><td>Amb a 11</td><td>Cysteine protease</td><td>66%</td><td>37</td></tr><tr><td>Amb a 12</td><td>Enolase</td><td>66%</td><td>48</td></tr><tr><td colspan="4">Artemisia vulgaris (mugwort), Artemisia spp</td></tr><tr><td>Art v 1*</td><td>Defensin-like protein with polyproline rich region</td><td>95%</td><td>13-15</td></tr><tr><td>Art v 3*</td><td>Non-specific lipid transfer protein</td><td>22-70%</td><td>10</td></tr><tr><td>Art v 4</td><td>Profilin</td><td>35%</td><td>14</td></tr><tr><td>Art v 5</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>10-28%</td><td>10</td></tr><tr><td>Art v 6</td><td>Pectate lyase, Amb a 1-homologue</td><td>26%</td><td>38</td></tr><tr><td>Art an 7</td><td>Galactose oxidase</td><td>16-94%</td><td>62</td></tr><tr><td colspan="4">Helianthus annuus (sunflower)</td></tr><tr><td>Hel a 1</td><td>Potential defensin-like protein with polyproline rich region</td><td>65%</td><td>34</td></tr><tr><td>Hel a 2</td><td>Profilin</td><td>31%</td><td>14</td></tr><tr><td>Hel a 6</td><td>Pectate lyase</td><td>36%</td><td>42</td></tr><tr><td colspan="4">Parthenium hysterophorus (feverfew)</td></tr><tr><td>Par h 1</td><td>Defensin-like protein with polyproline rich region</td><td>40-60%</td><td>12</td></tr><tr><td colspan="4">Parietaria judaica (pellitory)</td></tr><tr><td>Par j 1</td><td>Non-specific lipid transfer protein</td><td>95%</td><td>15</td></tr><tr><td>Par j 2</td><td>Non-specific lipid transfer protein</td><td>80%</td><td>11</td></tr><tr><td>Par j 3</td><td>Profilin</td><td>nd</td><td>14</td></tr><tr><td>Par j 4</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>6%</td><td>9</td></tr><tr><td colspan="4">Plantago lanceolata (English plantain)</td></tr><tr><td>Pla l 1</td><td>Ole e 1-like protein</td><td>86%</td><td>18</td></tr><tr><td>Pla l 2</td><td>Profilin</td><td>86%</td><td>15</td></tr><tr><td colspan="4">Mercurialis annua (pellitory)</td></tr><tr><td>Mer a 1</td><td>Profilin</td><td>50-60%</td><td>14</td></tr><tr><td colspan="4">Chenopodium album (goosefoot)</td></tr><tr><td>Che a 1</td><td>Ole e 1-like protein</td><td>70%</td><td>18</td></tr><tr><td>Che a 2</td><td>Profilin</td><td>55%</td><td>14</td></tr><tr><td>Che a 3</td><td>Polcalcin (2 EF-hand calcium binding protein)</td><td>46%</td><td>10</td></tr><tr><td colspan="4">Salsola kali (Russian thistle)</td></tr><tr><td>Sal k 1</td><td>Pectin methylesterase family</td><td>65%</td><td>38</td></tr><tr><td>Sal k 3</td><td>Cobalamin independent methionine synthase</td><td>63%</td><td>85 (35+45)</td></tr><tr><td>Sal k 4</td><td>Profilin</td><td>46%</td><td>14</td></tr><tr><td>Sal k 5</td><td>Ole e 1-like protein</td><td>30-60%</td><td>18</td></tr><tr><td>Sal k 6</td><td>Polygalacturonase</td><td>32%</td><td>47</td></tr><tr><td>Sal k 7</td><td>Polcalcin</td><td>40%</td><td>9</td></tr><tr><td colspan="4">Amaranthus retroflexus (amaranth)</td></tr><tr><td>Ama r 1</td><td>Ole e 1-like protein</td><td>38%</td><td>18</td></tr><tr><td>Ama r 2</td><td>Profilin</td><td>25%</td><td>14</td></tr></tbody></table>

Data on sensitization frequency according to Gadermaier et al.⁶⁸⁰ and the WHO/IUIS allergen nomenclature database. nd not determined.

*Besides Art v 1 and Art v 3, several recently identified homologs from Artemisia spp of the defensin-like and nsLTP family are listed in the WHO/IUIS database (https://www.allergen.org).⁶⁸⁰
allergenic potency. The cysteine protease Amb a 11 is also classified as a major allergen<sup>662</sup>; however, sensitization studies in larger cohorts remain to be conducted.

Artemisia spp contains more than 350 species that seem to be highly similar in their allergen profile, and IgE reactivity. *Artemisia* is considered one of the most relevant allergenic pollen source in Asia with a sensitization prevalence of 11% and 14.5% in allergic adults and children from China, respectively. In the same areas, reactivity to ragweed is only 6.5 – 8.7%.⁶⁶³,¹⁷¹³ Among weed allergics in Northern China, high and correlating IgE reactivity to *Artemisia* (58.3%) and Art v 1 (49%) was found. By contrast, specific IgE to *Ambrosia* (14.7%) and Amb a 1 (11.2%) was lower in frequency and level, not correlated with each other, and uncommon in the absence of specific IgE to Art v 1 suggesting primary sensitization with *Artemisia* species.⁶⁶⁴ Among Korean children with allergic rhinitis, sensitization to mugwort ranges from 2.4-11.7%, depending on the geographic region.⁶⁶⁵ In Japan, 16.1% of adults and 14.1% of children tested showed specific IgE to mugwort pollen; sensitization reached up to 25.4% for adults and 19.9% for children in some areas⁶⁶⁰. Among allergic patients, 26.1 - 27.4% tested positive for mugwort in Germany, while a cross-sectional study among Austrian adolescents revealed a sensitization frequency of 7.2% to the major allergen Art v 1.⁶⁵⁹,⁶⁶⁶ In the Canary islands, *Artemisia*, as determined by Art v 1 sensitization dominates pollen allergy, associated to the endemic species *Artemisia thuscula* and strong trade winds. Up to 40% of pollen allergic patients are mono-sensitized to *Artemisia*.⁶⁶⁷ Both, Art v 1 and the nsLTP Art v 3 present homologous allergens with high similarity and IgE cross-reactivity in the numerous *Artemisia* species, with sensitization frequencies of 84% and 66%, respectively, among Chinese mugwort allergic patients.⁶⁶⁸,⁶⁶⁹ In Northern China, Art v 3 sensitization in mugwort pollen allergic patients is high and in many cases responsible for Pru p 3-related peach allergy.⁶⁶⁹ In addition, the galactose oxidase Art an 7 seems to be a relevant allergen as it was recognized by 87% of *Artemisia*-sensitized patients from China.⁶⁷⁰

Sensitization to *Helianthus* pollen is reported with 23.5% among Turkish sunflower processing workers and 21% among pollen allergic patients from India.⁶⁷¹,⁶⁷² No sequence was so far assigned to the major allergen Hel a 1; however, a highly cross-reactive allergen was detected using Art v 1-specific antibodies.⁶⁷³ Recently, Hel a 6, a pectate lyase from sunflower pollen, was identified showing 57% sensitization prevalence among sunflower pollen allergic patients.³⁵⁶ Positive SPT reactivity to *Parthenium* was noted for 35% of fall pollinosis patients in the U.S. and 35.7% of type IV allergy in atopic dermatitis patients in India.⁶⁷⁴ The recently identified defensin-like allergen Par h 1 was recognized by 60% of Austrian Asteraceae and 40% of Indian feverfew sensitized patients. In addition, other allergenic proteins, e.g., a pectate lyase and pan-allergens were identified in feverfew pollen.⁶⁷⁵

*Parietaria* pollen is one of the most relevant causes of pollen allergy in the Mediterranean region, with an average sensitization prevalence of 46.5% and 58.9% in elderly and adolescent allergics in southern Italy, respectively.⁶⁷⁶,⁶⁷⁷ The major contributors to *Parietaria* allergy are the nsLTPs Par j 1 and Par j 2, which demonstrate no cross-reactivity with nsLTPs from other sources. Although

the weed is highly prevalent throughout Europe, sensitization in the non-Mediterranean population is marginal.⁵⁶⁹

A high sensitization prevalence to *Plantago* was shown in allergics of distinct regions of northern Spain⁵⁶⁹ and in South Australia where 37% SPT positivity is reported (Dr. Frank Kett, personal communication). Recent studies in Central/Northern Europe showed that among German allergic patients sensitization increased from 26.6% to 50.5% within 20 years.⁶⁵⁹ In the general adolescent population in Austria, sensitization to the genuine and major allergen Pla I 1 was as high as 10.4%.⁶⁶⁶ While an association with grass pollen or pan-allergen sensitization is frequently observed, genuine Pla I 1-related plantain allergy represents a true co-sensitization.³⁵⁸,⁶⁷⁸,⁶⁷⁹

High levels of reactivity to *Mercurialis annua* pollen ranging from 28-56% were observed in several areas of Spain.⁶⁸⁰

Due to use of *Chenopodium album* in greening programs, the weed gained relevance in countries with desert and semi-desert areas accounting for up to 70.7% sensitization in asthmatic patients. Clinical incidences have been reported in southern Spain and Saudi Arabia, while they even represent the main sensitizer for allergic rhinitis and asthma in Kuwait and Iran. The ornamental plant Amaranthus retroflexus is also described as a major trigger of allergic reactions in Iran, with a sensitization frequency of 69% among allergic patients.⁶⁸¹ So far, two allergens have been identified and designated Ama r 1 (Ole e 1-like protein) and Ama r 2 (profilin).⁵⁸⁵,¹⁷¹⁴ *Salsola kali* allergy, as determined by specific sensitization to Sal k 1, the major *Salsola* allergen absent in Chenopodiaceae species, is overall the third cause of pollinosis in Spain and a dominant pollen allergen in the

South East region.⁶⁶⁷ In some of the drier areas of the south, up to 80% of the patients suffering from seasonal allergy are sensitized to Sal k 1, and are frequently mono-sensitized. In other areas such as the Ebro river valley it is the second cause of pollinosis after grasses.⁵⁸⁵,⁶⁶⁷ This allergy is also very prevalent in other dry areas like Iran where up to 72.5% of pollen allergic patients are sensitized to *Salsola*.⁶⁸⁰,⁶⁸¹ Additional S. kali allergens have been identified and characterized in detail with IgE frequencies ranging from 30 to 60%.⁶⁸²⁻⁶⁸⁵

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

Exposure to weed pollen and primary sensitization to relevant allergens predominantly leads to rhinoconjunctivitis and/or asthma. Occupational allergies, i.e., to sunflower pollen are observed in workers and citizens in close vicinity. Sensitization to weed pollen allergens can show complex profiles including genuine, specific allergens (e.g., Pla I 1), as well as (partially) cross-reactive allergens from weeds and/or pan-allergens (Table 17). Frequent IgE cross-reactivity is observed within allergenic pollen of the Asteraceae and Amaranthaceae plant family.⁶⁸⁰,⁶⁸⁶ The level and clinical consequence depends on the identity of underlying allergenic molecules while it is to a certain degree patient-specific.

Amb a 1 from ragweed pollen represents a dominant, major allergen with moderate IgE cross-reactivity to the minor allergen Art v 6
allergenic potency. The cysteine protease Amb a 11 is also classified as a major allergen<sup>662</sup>; however, sensitization studies in larger cohorts remain to be conducted.

Artemisia spp contains more than 350 species that seem to be highly similar in their allergen profile, and IgE reactivity. *Artemisia* is considered one of the most relevant allergenic pollen source in Asia with a sensitization prevalence of 11% and 14.5% in allergic adults and children from China, respectively. In the same areas, reactivity to ragweed is only 6.5 – 8.7%.⁶⁶³,¹⁷¹³ Among weed allergics in Northern China, high and correlating IgE reactivity to *Artemisia* (58.3%) and Art v 1 (49%) was found. By contrast, specific IgE to *Ambrosia* (14.7%) and Amb a 1 (11.2%) was lower in frequency and level, not correlated with each other, and uncommon in the absence of specific IgE to Art v 1 suggesting primary sensitization with *Artemisia* species.⁶⁶⁴ Among Korean children with allergic rhinitis, sensitization to mugwort ranges from 2.4-11.7%, depending on the geographic region.⁶⁶⁵ In Japan, 16.1% of adults and 14.1% of children tested showed specific IgE to mugwort pollen; sensitization reached up to 25.4% for adults and 19.9% for children in some areas⁶⁶⁰. Among allergic patients, 26.1 - 27.4% tested positive for mugwort in Germany, while a cross-sectional study among Austrian adolescents revealed a sensitization frequency of 7.2% to the major allergen Art v 1.⁶⁵⁹,⁶⁶⁶ In the Canary islands, *Artemisia*, as determined by Art v 1 sensitization dominates pollen allergy, associated to the endemic species *Artemisia thuscula* and strong trade winds. Up to 40% of pollen allergic patients are mono-sensitized to *Artemisia*.⁶⁶⁷ Both, Art v 1 and the nsLTP Art v 3 present homologous allergens with high similarity and IgE cross-reactivity in the numerous *Artemisia* species, with sensitization frequencies of 84% and 66%, respectively, among Chinese mugwort allergic patients.⁶⁶⁸,⁶⁶⁹ In Northern China, Art v 3 sensitization in mugwort pollen allergic patients is high and in many cases responsible for Pru p 3-related peach allergy.⁶⁶⁹ In addition, the galactose oxidase Art an 7 seems to be a relevant allergen as it was recognized by 87% of *Artemisia*-sensitized patients from China.⁶⁷⁰

Sensitization to *Helianthus* pollen is reported with 23.5% among Turkish sunflower processing workers and 21% among pollen allergic patients from India.⁶⁷¹,⁶⁷² No sequence was so far assigned to the major allergen Hel a 1; however, a highly cross-reactive allergen was detected using Art v 1-specific antibodies.⁶⁷³ Recently, Hel a 6, a pectate lyase from sunflower pollen, was identified showing 57% sensitization prevalence among sunflower pollen allergic patients.³⁵⁶ Positive SPT reactivity to *Parthenium* was noted for 35% of fall pollinosis patients in the U.S. and 35.7% of type IV allergy in atopic dermatitis patients in India.⁶⁷⁴ The recently identified defensin-like allergen Par h 1 was recognized by 60% of Austrian Asteraceae and 40% of Indian feverfew sensitized patients. In addition, other allergenic proteins, e.g., a pectate lyase and pan-allergens were identified in feverfew pollen.⁶⁷⁵

*Parietaria* pollen is one of the most relevant causes of pollen allergy in the Mediterranean region, with an average sensitization prevalence of 46.5% and 58.9% in elderly and adolescent allergics in southern Italy, respectively.⁶⁷⁶,⁶⁷⁷ The major contributors to *Parietaria* allergy are the nsLTPs Par j 1 and Par j 2, which demonstrate no cross-reactivity with nsLTPs from other sources. Although

the weed is highly prevalent throughout Europe, sensitization in the non-Mediterranean population is marginal.⁵⁶⁹

A high sensitization prevalence to *Plantago* was shown in allergies of distinct regions of northern Spain⁵⁶⁹ and in South Australia where 37% SPT positivity is reported (Dr. Frank Kett, personal communication). Recent studies in Central/Northern Europe showed that among German allergic patients sensitization increased from 26.6% to 50.5% within 20 years.⁶⁵⁹ In the general adolescent population in Austria, sensitization to the genuine and major allergen Pla I 1 was as high as 10.4%.⁶⁶⁶ While an association with grass pollen or pan-allergen sensitization is frequently observed, genuine Pla I 1-related plantain allergy represents a true co-sensitization.³⁵⁸,⁶⁷⁸,⁶⁷⁹

High levels of reactivity to *Mercurialis annua* pollen ranging from 28-56% were observed in several areas of Spain.⁶⁸⁰

Due to use of *Chenopodium album* in greening programs, the weed gained relevance in countries with desert and semi-desert areas accounting for up to 70.7% sensitization in asthmatic patients. Clinical incidences have been reported in southern Spain and Saudi Arabia, while they even represent the main sensitizer for allergic rhinitis and asthma in Kuwait and Iran. The ornamental plant Amaranthus retroflexus is also described as a major trigger of allergic reactions in Iran, with a sensitization frequency of 69% among allergic patients.⁶⁸¹ So far, two allergens have been identified and designated Ama r 1 (Ole e 1-like protein) and Ama r 2 (profilin).⁵⁸⁵,¹⁷¹⁴ *Salsola kali* allergy, as determined by specific sensitization to Sal k 1, the major *Salsola* allergen absent in Chenopodiaceae species, is overall the third cause of pollinosis in Spain and a dominant pollen allergen in the

South East region.⁶⁶⁷ In some of the drier areas of the south, up to 80% of the patients suffering from seasonal allergy are sensitized to Sal k 1, and are frequently mono-sensitized. In other areas such as the Ebro river valley it is the second cause of pollinosis after grasses.⁵⁸⁵,⁶⁶⁷ This allergy is also very prevalent in other dry areas like Iran where up to 72.5% of pollen allergic patients are sensitized to *Salsola*.⁶⁸⁰,⁶⁸¹ Additional S. kali allergens have been identified and characterized in detail with IgE frequencies ranging from 30 to 60%.⁶⁸²⁻⁶⁸⁵

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

Exposure to weed pollen and primary sensitization to relevant allergens predominantly leads to rhinoconjunctivitis and/or asthma. Occupational allergies, i.e., to sunflower pollen are observed in workers and citizens in close vicinity. Sensitization to weed pollen allergens can show complex profiles including genuine, specific allergens (e.g., Pla I 1), as well as (partially) cross-reactive allergens from weeds and/or pan-allergens (Table 17). Frequent IgE cross-reactivity is observed within allergenic pollen of the Asteraceae and Amaranthaceae plant family.⁶⁸⁰,⁶⁸⁶ The level and clinical consequence depends on the identity of underlying allergenic molecules while it is to a certain degree patient-specific.

Amb a 1 from ragweed pollen represents a dominant, major allergen with moderate IgE cross-reactivity to the minor allergen Art v 6
from mugwort.⁶⁸⁷ Recently, Hel a 6 from sunflower was identified as a major allergen³⁵⁶ and cross-reactivity with ragweed and mugwort pectate lyases was demonstrated (Figure 53A).

The major mugwort pollen allergen Art v 1 shows different degrees of cross-reactivity with Amb a 4 and Par h 1 from ragweed and Parthenium pollen, respectively.⁶⁸⁸ Based on sequence similarity, cross-reactivity with SF18 from sunflower is anticipated. IgE cross-reactivity was demonstrated for the novel defensin-like allergen Api g 7 from celeriac⁶⁸⁹ as well as Aes h 1 from horse chestnut seeds (Gadermaier, unpublished data) (Figure 53B).

Art v 3, the nsLTP from mugwort pollen demonstrates frequent IgE cross-reactivity to homologous molecules in plant food (e.g., Pru p 3) (Figure 53C). By contrast, source constrained sensitizations are observed for the major *Parietaria* allergen Par j 2 and the minor allergen Amb a 6.⁶⁹⁰⁻⁶⁹²

The Ole e 1-like protein Pla I 1 from plantain presents low sequence similarity to other family members and is thus not involved in cross-reactivity. By contrast, substantial cross-reactivity is observed for Che a 1 and Sal k 5 due to 74% sequence identity (Figure 53D).³⁵⁸,⁶⁷⁸,⁶⁸¹

Pollen-food syndromes mediated by weeds are mainly involving mugwort and ragweed allergic patients. In addition to oral

allergy syndromes, more severe clinical pictures as observed, e.g., in the celery-mugwort-spice syndrome are reported. Cross-reactive allergens were identified in the family of nsLTPs, profilins, defensin-like proteins and high-molecular weight allergens including CCDs.⁶⁶⁹,⁶⁸⁹,⁶⁹⁰,⁶⁹³

## 5 – Clinical management

Diagnosis of weed pollen allergy can be difficult due to frequent polysensitization and inconclusive anamnesis owing to overlapping flowering seasons with other pollen. Thus molecule-based allergy diagnosis is particularly advantageous and work-ups facilitating diagnosis of some weed pollen allergies are presented in (Figure 54).

### Case history

Weed pollen allergic patients typically present seasonal respiratory symptoms (rhinitis and/or conjunctivitis and/or asthmatic symptoms, sometimes also itching of the throat and/or contact urticaria). Since clinical symptoms coincide with flowering periods of the respective weeds (Figure 52), principal information can be obtained by narrowing down the eliciting allergen source(s).

<figure><img src="image_139.png" /><figcaption>(B) Defensin-like proteins</figcaption></figure>

<figure><img src="image_140.png" /><figcaption>(C) Lipid transfer proteins</figcaption></figure>

<figure><img src="image_141.png" /><figcaption>(D) Ole e 1 like-proteins</figcaption></figure>

<figure><img src="image_142.png" /></figure>

FIGURE 53 (A) Model of Amb a 1.0101 (template 1PXZ); (B) Structure of Art v 1.0101 defensin-domain (2KPY); (C) Structure of Art v 3.0201 (6FRR); and (D) Structure of Pla I 1.0101 (4Z8W). Models were generated using Swiss-Model (www.swissmodel.expasy.org) and ribbon cartoons are shown using UCSF Chimera (www.cgl.ucsf.edu/chimera). Lines represent documented IgE cross-reactivity, dotted lines represent potential IgE cross-reactivity based on high sequence identity. Lack of lines indicates absent IgE cross-reactivity.
from mugwort.⁶⁸⁷ Recently, Hel a 6 from sunflower was identified as a major allergen³⁵⁶ and cross-reactivity with ragweed and mugwort pectate lyases was demonstrated (Figure 53A).

The major mugwort pollen allergen Art v 1 shows different degrees of cross-reactivity with Amb a 4 and Par h 1 from ragweed and Parthenium pollen, respectively.⁶⁸⁸ Based on sequence similarity, cross-reactivity with SF18 from sunflower is anticipated. IgE cross-reactivity was demonstrated for the novel defensin-like allergen Api g 7 from celeriac⁶⁸⁹ as well as Aes h 1 from horse chestnut seeds (Gadermaier, unpublished data) (Figure 53B).

Art v 3, the nsLTP from mugwort pollen demonstrates frequent IgE cross-reactivity to homologous molecules in plant food (e.g., Pru p 3) (Figure 53C). By contrast, source constrained sensitizations are observed for the major *Parietaria* allergen Par j 2 and the minor allergen Amb a 6.⁶⁹⁰⁻⁶⁹²

The Ole e 1-like protein Pla I 1 from plantain presents low sequence similarity to other family members and is thus not involved in cross-reactivity. By contrast, substantial cross-reactivity is observed for Che a 1 and Sal k 5 due to 74% sequence identity (Figure 53D).³⁵⁸,⁶⁷⁸,⁶⁸¹

Pollen-food syndromes mediated by weeds are mainly involving mugwort and ragweed allergic patients. In addition to oral

allergy syndromes, more severe clinical pictures as observed, e.g., in the celery-mugwort-spice syndrome are reported. Cross-reactive allergens were identified in the family of nsLTPs, profilins, defensin-like proteins and high-molecular weight allergens including CCDs.⁶⁶⁹,⁶⁸⁹,⁶⁹⁰,⁶⁹³

## 5 - Clinical management

Diagnosis of weed pollen allergy can be difficult due to frequent polysensitization and inconclusive anamnesis owing to overlapping flowering seasons with other pollen. Thus molecule-based allergy diagnosis is particularly advantageous and work-ups facilitating diagnosis of some weed pollen allergies are presented in (Figure 54).

### Case history

Weed pollen allergic patients typically present seasonal respiratory symptoms (rhinitis and/or conjunctivitis and/or asthmatic symptoms, sometimes also itching of the throat and/or contact urticaria). Since clinical symptoms coincide with flowering periods of the respective weeds (Figure 52), principal information can be obtained by narrowing down the eliciting allergen source(s).

<figure><img src="image_138.png" /><figcaption>(B) Defensin-like proteins</figcaption></figure>

<figure><img src="image_139.png" /><figcaption>(C) Lipid transfer proteins</figcaption></figure>

<figure><img src="image_140.png" /><figcaption>(D) Ole e 1 like-proteins</figcaption></figure>

<figure><img src="image_141.png" /></figure>

FIGURE 53 (A) Model of Amb a 1.0101 (template 1PXZ); (B) Structure of Art v 1.0101 defensin-domain (2KPY); (C) Structure of Art v 3.0201 (6FRR); and (D) Structure of Pla I 1.0101 (4Z8W). Models were generated using Swiss-Model (www.swissmodel.expasy.org) and ribbon cartoons are shown using UCSF Chimera (www.cgl.ucsf.edu/chimera). Lines represent documented IgE cross-reactivity, dotted lines represent potential IgE cross-reactivity based on high sequence identity. Lack of lines indicates absent IgE cross-reactivity.
## (A) Ragweed and mugwort

**Case history:** pollen-related rhinoconjunctival and/or asthmatic symptoms from late summer to autumn

<figure><img src="image_143.png" /></figure>

## (B) Pellitory

**Case history:** pollen-related rhinitis and/or conjunctivitis in the Mediterranean area during pellitory flowering season

<figure><img src="image_144.png" /><figcaption>FIGURE 54 Diagnostic work-up for (A) ragweed and mugwort, (B) pellitory and (C) English plantain pollen allergy</figcaption></figure>

## (C) English plantain

**Case history:** pollen-related rhinitis and/or conjunctivitis from late summer to autumn

### Skin prick test (SPT)

The choice of commercially available weed pollen extracts for SPT is highly depending on pollen exposure and clinical references. Based on current GA2LEN recommendations for

harmonization of skin prick tests in Europe, mugwort, ragweed and pellitory are included in routine diagnostic panels, while plantain and allergenic pollen of the Amaranthaceae family are not considered. Since the allergological relevance can considerably
## (A) Ragweed and mugwort

**Case history:** pollen-related rhinoconjunctival and/or asthmatic symptoms from late summer to autumn

<figure><img src="image_142.png" /></figure>

## (B) Pellitory

**Case history:** pollen-related rhinitis and/or conjunctivitis in the Mediterranean area during pellitory flowering season

<figure><img src="image_143.png" /><figcaption>FIGURE 54 Diagnostic work-up for (A) ragweed and mugwort, (B) pellitory and (C) English plantain pollen allergy</figcaption></figure>

## (C) English plantain

**Case history:** pollen-related rhinitis and/or conjunctivitis from late summer to autumn

### Skin prick test (SPT)

The choice of commercially available weed pollen extracts for SPT is highly depending on pollen exposure and clinical references. Based on current GA2LEN recommendations for

harmonization of skin prick tests in Europe, mugwort, ragweed and pellitory are included in routine diagnostic panels, while plantain and allergenic pollen of the Amaranthaceae family are not considered. Since the allergological relevance can considerably
vary between regions, local modifications are, however, useful and necessary. Since *Parietaria* extracts are virtually missing profilin, a positive SPT with this weed is generally diagnostic of primary sensitization unless the patient is sensitized to the pollen pan-allergen polcalcin.⁶⁹⁴

### IgE testing

Apart from *in vitro* testing using weed pollen extracts, molecule-based approaches offer a valuable tool for refined diagnosis limiting unspecific results due to polysensitizations. All major allergens of weed pollen are commercially available for diagnosis using the single component ImmunoCAP Specific IgE test, except for Che a 1, which is only present in multiplex arrays (ImmunoCAP ISAC and ALEX²) (Table 18). Components are available as nonglycosylated recombinant molecules (e.g., rPla 1, rPar j 2), CCD (N-glycan)-free, natural molecules like nAmb a 1 (nonglycosylated), nArt v 1 (O-glycosylated), and CCD-containing nSal k 1 (N-glycosylated). Art v 1, Art v 3 and Che a 1 are either provided as purified natural molecules (ImmunoCAP Specific IgE test) or recombinant proteins (ALEX²), respectively. To discriminate ragweed and mugwort primary sensitization, Art v 6 and Amb a 4 would be useful diagnostic markers as they are homologues of the respective major allergens (but these components are limited available for routine diagnosis). In the case of Sal k 1, false-positive results might arise due to N-glycosylation. Mer a 1 is so far the only allergen identified from Annual mercury and owing to expected broad IgE cross-reactivity with other profilins should not be considered a marker allergen for the source (see Chapter C01).

### Therapeutic options

Besides recommendations to generally limit exposure during pollen season, symptomatic treatment is considered a first line of

defence for weed pollen allergic patients. In addition, allergen immunotherapy (AIT) is recommended based on the identification of the primary sensitizer using highly specific marker allergens. Weed allergic patients frequently present multiple sensitizations, and thus typically allergen sources triggering most profound symptoms are selected for therapeutic interventions.

Various subcutaneous extracts of weed pollen (single and combination products) and recently also a tablet for sublingual immunotherapy (ragweed allergen), are available in the U.S. (www.fda.gov), in Canada and in Europe.

In addition, individual recipes may be prescribed for allergens not mentioned in these regulations. However, economic considerations, regulation and standardization requirements are prompting some providers to withdraw their weed pollen products.

## 6 – Clinical Cases

### Case 1 (educational)

**Clinical History:** A 42-year old man from Central Europe with increasing rhinoconjunctival symptoms from February to September for 12 years; additionally asthmatic symptoms for 2 years occurring only between May and September.

**Test with extracts:** Histamine-equivalent sensitization to tree pollen (hazel, alder, birch, ash) and to mugwort; weak skin prick reactivity to ragweed and goosefoot; strong sensitization to grass pollen. Specific IgE to birch (10.7 kU/L), ash (1.31 kU/L), grass (17.7 kU/L), mugwort (10.4 kU/L) and ragweed pollen (1.26 kU/L).

**Test with molecules:** The patient had positive IgE results to Bet v 1 (13.2 kU/L), Ole e 1 (2.56 kU/L), Phl p 1 (9.26 kU/L), Phl p 5 (3.58 kU/L), Art v 1 (12.8 kU/L), Art v 3 (10.8 kU/L) and Amb a 1 (0.68 kU/L).

<table><caption>TABLE 18 Molecular components for diagnosis of weed pollen allergy</caption><thead><tr><td>Allergenic source</td><td>Specific IgE component</td><td>Protein family</td><td>Diagnostic use</td></tr></thead><tbody><tr><td>Ragweed</td><td>nAmb a 1</td><td>Pectate lyase</td><td>Marker for ragweed, IgE cross-reactivity with Art v 6 from mugwort and Hel a 6 from sunflower</td></tr><tr><td>Ragweed</td><td>rAmb a 4<sup>#</sup></td><td>Defensin-like protein</td><td>Minor ragweed allergen with partial IgE cross-reactivity to Art v 1</td></tr><tr><td>Mugwort</td><td>nArt v 1/rArt v 1</td><td>Defensin-like protein</td><td>Marker for mugwort, partial cross-reactivity with Amb a 4 (ragweed), Par h 1 (feverfew) and SF18 (sunflower)</td></tr><tr><td>Mugwort</td><td>nArt v 3/rArt v 3</td><td>nsLTP</td><td>Cross-reactive with nsLTP from food e.g. Pru p 3 from peach or Cor a 8 from hazelnut</td></tr><tr><td>Mugwort</td><td>nArt v 6<sup>*</sup></td><td>Pectate lyase</td><td>Amb a 1 cross-reactive allergen</td></tr><tr><td>Pellitory</td><td>rPar j 2</td><td>nsLTP</td><td>Highly specific marker allergen for pellitory sensitisation</td></tr><tr><td>English plantain</td><td>rPla 1 1</td><td>Ole e 1-like protein</td><td>Highly specific marker allergen for English plantain</td></tr><tr><td>Goosefoot</td><td>rChe a 1<sup>§</sup></td><td>Ole e 1-like protein</td><td>Marker for goosefoot, partial IgE cross-reactivity with minor Salsola allergen Sal k 5</td></tr><tr><td>Russian thistle</td><td>nSal k 1</td><td>Pectin methylesterase</td><td>Marker allergen for Russian thistle allergy, natural allergen contains N-glycans (result might be false positive if patient is CCD positive)</td></tr></tbody></table>

nsLTP, non-specific lipid transfer protein.

Marker allergens shown bold, #available only on ALEX2, *test not commercially available, §available only in multiplex analyses.
vary between regions, local modifications are, however, useful and necessary. Since *Parietaria* extracts are virtually missing profilin, a positive SPT with this weed is generally diagnostic of primary sensitization unless the patient is sensitized to the pollen pan-allergen polcalcin.⁶⁹⁴

## IgE testing

Apart from *in vitro* testing using weed pollen extracts, molecule-based approaches offer a valuable tool for refined diagnosis limiting unspecific results due to polysensitizations. All major allergens of weed pollen are commercially available for diagnosis using the single component ImmunoCAP Specific IgE test, except for Che a 1, which is only present in multiplex arrays (ImmunoCAP ISAC and ALEX²) (Table 18). Components are available as nonglycosylated recombinant molecules (e.g., rPla 1, rPar j 2), CCD (N-glycan)-free, natural molecules like nAmb a 1 (nonglycosylated), nArt v 1 (O-glycosylated), and CCD-containing nSal k 1 (N-glycosylated). Art v 1, Art v 3 and Che a 1 are either provided as purified natural molecules (ImmunoCAP Specific IgE test) or recombinant proteins (ALEX²), respectively. To discriminate ragweed and mugwort primary sensitization, Art v 6 and Amb a 4 would be useful diagnostic markers as they are homologues of the respective major allergens (but these components are limited available for routine diagnosis). In the case of Sal k 1, false-positive results might arise due to N-glycosylation. Mer a 1 is so far the only allergen identified from Annual mercury and owing to expected broad IgE cross-reactivity with other profilins should not be considered a marker allergen for the source (see Chapter C01).

## Therapeutic options

Besides recommendations to generally limit exposure during pollen season, symptomatic treatment is considered a first line of

defence for weed pollen allergic patients. In addition, allergen immunotherapy (AIT) is recommended based on the identification of the primary sensitizer using highly specific marker allergens. Weed allergic patients frequently present multiple sensitizations, and thus typically allergen sources triggering most profound symptoms are selected for therapeutic interventions.

Various subcutaneous extracts of weed pollen (single and combination products) and recently also a tablet for sublingual immunotherapy (ragweed allergen), are available in the U.S. (www.fda.gov), in Canada and in Europe.

In addition, individual recipes may be prescribed for allergens not mentioned in these regulations. However, economic considerations, regulation and standardization requirements are prompting some providers to withdraw their weed pollen products.

# 6 – Clinical Cases

## Case 1 (educational)

**Clinical History:** A 42-year old man from Central Europe with increasing rhinoconjunctival symptoms from February to September for 12 years; additionally asthmatic symptoms for 2 years occurring only between May and September.

**Test with extracts:** Histamine-equivalent sensitization to tree pollen (hazel, alder, birch, ash) and to mugwort; weak skin prick reactivity to ragweed and goosefoot; strong sensitization to grass pollen. Specific IgE to birch (10.7 kU/L), ash (1.31 kU/L), grass (17.7 kU/L), mugwort (10.4 kU/L) and ragweed pollen (1.26 kU/L).

**Test with molecules:** The patient had positive IgE results to Bet v 1 (13.2 kU/L), Ole e 1 (2.56 kU/L), Phl p 1 (9.26 kU/L), Phl p 5 (3.58 kU/L), Art v 1 (12.8 kU/L), Art v 3 (10.8 kU/L) and Amb a 1 (0.68 kU/L).

<table><caption>TABLE 18 Molecular components for diagnosis of weed pollen allergy</caption><thead><tr><td>Allergenic source</td><td>Specific IgE component</td><td>Protein family</td><td>Diagnostic use</td></tr></thead><tbody><tr><td>Ragweed</td><td>nAmb a 1</td><td>Pectate lyase</td><td>Marker for ragweed, IgE cross-reactivity with Art v 6 from mugwort and Hel a 6 from sunflower</td></tr><tr><td>Ragweed</td><td>rAmb a 4<sup>#</sup></td><td>Defensin-like protein</td><td>Minor ragweed allergen with partial IgE cross-reactivity to Art v 1</td></tr><tr><td>Mugwort</td><td>nArt v 1/rArt v 1</td><td>Defensin-like protein</td><td>Marker for mugwort, partial cross-reactivity with Amb a 4 (ragweed), Par h 1 (feverfew) and SF18 (sunflower)</td></tr><tr><td>Mugwort</td><td>nArt v 3/rArt v 3</td><td>nsLTP</td><td>Cross-reactive with nsLTP from food e.g. Pru p 3 from peach or Cor a 8 from hazelnut</td></tr><tr><td>Mugwort</td><td>nArt v 6<sup>*</sup></td><td>Pectate lyase</td><td>Amb a 1 cross-reactive allergen</td></tr><tr><td>Pellitory</td><td>rPar j 2</td><td>nsLTP</td><td>Highly specific marker allergen for pellitory sensitisation</td></tr><tr><td>English plantain</td><td>rPla 1 1</td><td>Ole e 1-like protein</td><td>Highly specific marker allergen for English plantain</td></tr><tr><td>Goosefoot</td><td>rChe a 1<sup>§</sup></td><td>Ole e 1-like protein</td><td>Marker for goosefoot, partial IgE cross-reactivity with minor Salsola allergen Sal k 5</td></tr><tr><td>Russian thistle</td><td>nSal k 1</td><td>Pectin methylesterase</td><td>Marker allergen for Russian thistle allergy, natural allergen contains N-glycans (result might be false positive if patient is CCD positive)</td></tr></tbody></table>

nsLTP, non-specific lipid transfer protein.

Marker allergens shown bold, #available only on ALEX2, *test not commercially available, §available only in multiplex analyses.
**Conclusion:** The patient presents a complex pollen sensitization profile involving tree, grass and weed pollen. Symptoms in autumn are mainly caused by mugwort pollen; primary sensitization to ragweed is not indicated as Amb a 1 levels are very low and might arise due to the cross-reactive molecule Art v 6 in mugwort.

### Case 2 (original)

**Clinical History:** A 20-year old woman from Western Austria with severe rhinoconjunctival symptoms from end of April to the middle of September since early childhood.

**Test with extracts:** The patient had a weak SPT positivity with hazel pollen, histamine-equivalent reactivity to grass pollen and strong sensitization to English plantain, all other standard inhalatory allergens negative. Positive *in vitro* IgE test to grass (14.2 kU/L) and plantain pollen (14.6 kU/L).

**Test with molecules:** The patient had positive IgE results to Phl p 1 (9.72 kU/L) and Pla I 1 (15.5 kU/L), while Phl p 7, Phl p 5, and Phl p 12 were negative. Multiplex results showed sensitization to Cyn d 1 (6.20 ISU), Phl p 1 (29.2 ISU), Phl p 2 (10.2 ISU), Phl p 11 (5.25 ISU), and Pla I 1 27.2 ISU.

**Conclusion:** In addition to grass pollen, the patient presents a weed pollen allergy to English plantain, which explains symptoms observed in autumn after the grass pollen season.

### Case 3 (educational)

**Clinical History:** A 36-year-old male living in northern Italy with a long lasting history of mild seasonal rhinitis from the beginning of May to the end of June experiences severe rhino-conjunctivitis associated with asthma as soon as he moves to Sicily for work at the beginning of March 2013. Gradual symptoms worsening force the man to ask for assistance at an Emergency Department where systemic corticosteroids were administered.

**Test with extracts:** Strong skin reactivity to pellitory pollen was observed (12 mm mean wheal diameter) along with a weak sensitivity to grass pollen (3 mm).

**Test with molecules:** The patient had positive IgE results to Par j 2 (37.3 kU/L), Phl p 1 (0.88 kU/L), Phl p 5 (0.41 kU/L).

**Conclusion:** Allergy to pellitory pollen is diagnosed, which was caused by high pollen exposure in Southern Italy.

### Clinical Relevance

* Art v 3 reactivity frequently indicates LTP sensitization and may be associated with oral allergy syndrome.

* Par j 2 is an excellent marker molecule for Parietaria allergy due to its high specificity

* The habitat and flowering period of plantain coincides with grass and should be thus consequently tested especially if symptoms persist for some weeks after the grass pollen season. Pla I 1 is a reliable marker molecule while Ole e 1 should NOT be used as a surrogate diagnostic as it lacks IgE cross-reactivity.

## 7 – Research and future perspectives

Further research on weed pollen allergy includes the following:

* In addition to sunflower, an allergenic cross-reactive pectate lyase was identified in feverfew pollen.⁶⁷⁵

* Recently, two defensin-like proteins from horse chestnut (Aes h 1) und celeriac (Api g 7) were described as novel allergens. Clinical reactivity to horse chestnut is elicited by Aes h 1 and considered a consequence of primary sensitization to Art v 1 from mugwort pollen (Gadermaier et al, unpublished). The involvement of pectate lyases and defensin-like proteins in food allergy remains to be investigated.

* In a murine model, virus-like particles expressing shielded Art v 1 were hypoallergenic, which could be useful for future preventive treatment targeting T cells.⁶⁹⁵

* Structural IgE-binding epitopes of Art v 3 were recently identified by NMR and stable epitope variants revealed strongly hypoallergenic candidate molecules for use in allergen immunotherapy.⁶⁹⁶

In general, effects of climatic changes will lead to a considerable increase in weed pollen load and thus allergic reactions.⁶⁵³,⁶⁵⁴

In light of this, there should be efforts to stop the continuing withdrawal of therapeutic possibilities for weed pollen allergic patients.
**Conclusion:** The patient presents a complex pollen sensitization profile involving tree, grass and weed pollen. Symptoms in autumn are mainly caused by mugwort pollen; primary sensitization to ragweed is not indicated as Amb a 1 levels are very low and might arise due to the cross-reactive molecule Art v 6 in mugwort.

### Case 2 (original)

**Clinical History:** A 20-year old woman from Western Austria with severe rhinoconjunctival symptoms from end of April to the middle of September since early childhood.

**Test with extracts:** The patient had a weak SPT positivity with hazel pollen, histamine-equivalent reactivity to grass pollen and strong sensitization to English plantain, all other standard inhalatory allergens negative. Positive *in vitro* IgE test to grass (14.2 kU/L) and plantain pollen (14.6 kU/L).

**Test with molecules:** The patient had positive IgE results to Phl p 1 (9.72 kU/L) and Pla I 1 (15.5 kU/L), while Phl p 7, Phl p 5, and Phl p 12 were negative. Multiplex results showed sensitization to Cyn d 1 (6.20 ISU), Phl p 1 (29.2 ISU), Phl p 2 (10.2 ISU), Phl p 11 (5.25 ISU), and Pla I 1 27.2 ISU.

**Conclusion:** In addition to grass pollen, the patient presents a weed pollen allergy to English plantain, which explains symptoms observed in autumn after the grass pollen season.

### Case 3 (educational)

**Clinical History:** A 36-year-old male living in northern Italy with a long lasting history of mild seasonal rhinitis from the beginning of May to the end of June experiences severe rhino-conjunctivitis associated with asthma as soon as he moves to Sicily for work at the beginning of March 2013. Gradual symptoms worsening force the man to ask for assistance at an Emergency Department where systemic corticosteroids were administered.

**Test with extracts:** Strong skin reactivity to pellitory pollen was observed (12 mm mean wheal diameter) along with a weak sensitivity to grass pollen (3 mm).

**Test with molecules:** The patient had positive IgE results to Par j 2 (37.3 kU/L), Phl p 1 (0.88 kU/L), Phl p 5 (0.41 kU/L).

**Conclusion:** Allergy to pellitory pollen is diagnosed, which was caused by high pollen exposure in Southern Italy.

### Clinical Relevance

* Art v 3 reactivity frequently indicates LTP sensitization and may be associated with oral allergy syndrome.

* Par j 2 is an excellent marker molecule for Parietaria allergy due to its high specificity

* The habitat and flowering period of plantain coincides with grass and should be thus consequently tested especially if symptoms persist for some weeks after the grass pollen season. Pla I 1 is a reliable marker molecule while Ole e 1 should NOT be used as a surrogate diagnostic as it lacks IgE cross-reactivity.

## 7 – Research and future perspectives

Further research on weed pollen allergy includes the following:

* In addition to sunflower, an allergenic cross-reactive pectate lyase was identified in feverfew pollen.⁶⁷⁵

* Recently, two defensin-like proteins from horse chestnut (Aes h 1) und celeriac (Api g 7) were described as novel allergens. Clinical reactivity to horse chestnut is elicited by Aes h 1 and considered a consequence of primary sensitization to Art v 1 from mugwort pollen (Gadermaier et al, unpublished). The involvement of pectate lyases and defensin-like proteins in food allergy remains to be investigated.

* In a murine model, virus-like particles expressing shielded Art v 1 were hypoallergenic, which could be useful for future preventive treatment targeting T cells.⁶⁹⁵

* Structural IgE-binding epitopes of Art v 3 were recently identified by NMR and stable epitope variants revealed strongly hypoallergenic candidate molecules for use in allergen immunotherapy.⁶⁹⁶

In general, effects of climatic changes will lead to a considerable increase in weed pollen load and thus allergic reactions.⁶⁵³,⁶⁵⁴

In light of this, there should be efforts to stop the continuing withdrawal of therapeutic possibilities for weed pollen allergic patients.
# B04 - Dust mite allergy

Thomas Platts-Mills, Luis Caraballo, Alain Jacquet, Josefina Zakzuk

## Highlights

* House dust mite allergy is an important risk factor for rhinitis and asthma.

* Most recognized house dust mites are *Dermatophagoides pteronyssinus*, *Dermatophagoides farinae* and *Blomia tropicalis*.

* In the majority of cases, a skin test with mite extracts is able to detect sensitization and define the specificity of immunotherapy in asthmatic patients.

* However, the use of component-resolved diagnosis could be useful when genuine sensitization is not clear and has to be defined.

## 1 - The allergen sources

Within the phylum Arthropoda there are three main taxonomic divisions with allergological importance (Figure 55). Among crustaceans, several species are important sources of food allergens. Also, both classes, insects and arachnids, contain species, which are sources of inhalant allergens. Arthropods separated from other animals approximately 600 million years ago (MYA) and the major classes were established within 100 million years. Dust mites and cockroaches, for example, have been separated for at least 400 million years and not surprisingly most of these allergens are so different in their primary sequences that they do not cross-react. This should be compared with the mammals, which separated approximately 65 MYA and still have extensive similarity among proteins. Of the hundreds of thousands of arthropod species only a few have been recognized as significant sources of indoor allergens. Indeed, more than 90%

<figure><img src="image_145.png" /><figcaption>FIGURE 55 A. Phylogenetic tree of the main arthropod taxonomic groups. B. Evolutionary relationship of the main allergenic house dust and storage mite species</figcaption></figure>
# B04 - Dust mite allergy

Thomas Platts-Mills, Luis Caraballo, Alain Jacquet, Josefina Zakzuk

## Highlights

* House dust mite allergy is an important risk factor for rhinitis and asthma.

* Most recognized house dust mites are *Dermatophagoides pteronyssinus*, *Dermatophagoides farinae* and *Blomia tropicalis*.

* In the majority of cases, a skin test with mite extracts is able to detect sensitization and define the specificity of immunotherapy in asthmatic patients.

* However, the use of component-resolved diagnosis could be useful when genuine sensitization is not clear and has to be defined.

## 1 - The allergen sources

Within the phylum Arthropoda there are three main taxonomic divisions with allergological importance (Figure 55). Among crustaceans, several species are important sources of food allergens. Also, both classes, insects and arachnids, contain species, which are sources of inhalant allergens. Arthropods separated from other animals approximately 600 million years ago (MYA) and the major classes were established within 100 million years. Dust mites and cockroaches, for example, have been separated for at least 400 million years and not surprisingly most of these allergens are so different in their primary sequences that they do not cross-react. This should be compared with the mammals, which separated approximately 65 MYA and still have extensive similarity among proteins. Of the hundreds of thousands of arthropod species only a few have been recognized as significant sources of indoor allergens. Indeed, more than 90%

<figure><img src="image_144.png" /><figcaption>FIGURE 55 A. Phylogenetic tree of the main arthropod taxonomic groups. B. Evolutionary relationship of the main allergenic house dust and storage mite species</figcaption></figure>
of the literature is related to five genera: *Dermatophagoides*, *Blomia*, *Euroglyphus*, *Blatella*, and *Periplaneta*. One important aspect of the house dust mite (HDM) allergenic sources is that they can play a very important role in asthma, without their role being obvious to the patients.

While there are many possible explanations for this, the most obvious ones are perennial exposure, that the organisms are small and often not noticed and that particles carrying these allergens are sufficiently large not to remain airborne for more than a few minutes after disturbance.⁶⁹⁷ Because the role of exposure to mite or cockroach allergens had not been obvious to patients, learning about these species has been essential to increase our knowledge on the causal role of allergen exposure in asthma. The relevance of particle size became obvious shortly after the purification of Der p 1 and the development of accurate assays for this allergen.⁸ For *D. pteronyssinus* and *D. farinae*, allergens become airborne via fecal particles and fragmented mite bodies in household dust.⁶⁹⁸

These allergen-carrying particles are particularly relevant for the type of exposure, as allergenic proteins with a size of 15,000 to 50,000 Daltons are not volatile and significant exposure can only occur via particles. Mite fecal pellets have an average diameter of 20–30 µm⁶⁹⁹ and those with a size between 2–6 µm can be transported into small airways.⁷⁰⁰ The nature of these particles is relevant to, both, the induction of an IgE response and the subsequent contribution to inflammation of the nose and lungs. For dust mite particles, they not only carry a high concentration of several allergens but also are an important source of microbial compounds (at least LPS, β-glucans and chitin) capable of eliciting strong innate immune responses.

A significant feature of the epidemiology of HDM allergens is that there are areas of the world where some species are more present, for example, the high prevalence of *B. tropicalis* in the tropics. Both climate and housing conditions play a major role in these differences because mites absorb moisture from their environment and are absolutely dependent on the level of humidity in the air or on either carpets or upholstered material, which will retain humidity for long periods of time. In addition, the immature forms of *D. farinae* can withstand longer periods of dryness than *D. pteronyssinus*.

Before 1960, it was well recognized that house dust contained allergens other than those derived from domestic or pet animals as well as pollens or fungi; indeed, several groups had attempted to identify a house dust atopen by immunochemical analysis of house dust extracts. However, the breakthrough was made by microscopic identification of dust mites in house dust. It was fitting that these observations were made in the Netherlands since the Dutch scientist Antonie van Leeuwenhoek had first described mites in 1693. In addition, to evidence that mites of the genus *Dermatophagoides* were the major source of allergens in house dust, Spieksma and Voorhorst also developed the technique for culturing these organisms.⁷⁰⁰ This in turn made it possible to manufacture dust mite extracts for commercial use and subsequently facilitated the purification of mite allergens.⁸

## 2 – The allergen families

Dust mites belong to the order Astigmata, which is part of the Arachnids (Figure 55). This order includes the well-known Pyroglyphidae family but also the Acaridae and a superfamily (Glycyphagoidea) then divided into three main families: Echimyopodidae (*Blomia tropicalis*), Glycyphagidae (*Glycyphagus domesticus* and *Lepidoglyphus destructor*), and Chortoglyphidae (*Chortoglyphus arcuatus*). *Sarcoptes scabiei* (the scabies mite) is a member of the Sarcoptidae family, which has also been found as allergenic to humans. The mites generally recognized as house dust mites are *D. pteronyssinus*, *D. farinae*, *Euroglyphus maynei*, and *B. tropicalis*. However, four species of storage mites, which are best recognized as pests on farms or in food storage have also been recognized in house dust (Table 19). In addition, mites of the family Tarsonemidae may be found in significant numbers in house dust. The best evidence that a given mite species is relevant to allergic disease comes from studies in an area where a given species dominates all other mites in the house dust. This is true for *D. pteronyssinus* in the UK and New Zealand, for *D. farinae* in some areas of the United States, and for *B. tropicalis* in areas of South America and other tropical regions.⁷⁰¹,⁷⁰²

## 3 – Allergenic molecules. Epidemiology and function

Der p 1 was the first identified dust mite allergen in 1980, purified by conventional chromatographic techniques⁸; the purification of the Group 2 allergens Der p 2 and Der f 2 in 1989 was performed by immunoaffinity chromatography.⁷⁰³ Over the last 20 years, the techniques for cloning and sequencing proteins have become well-defined and much simpler. As a result, many mite-derived proteins have been described with convincing data about their amino acid sequence and the tertiary structure for some of them.⁷⁰⁴⁻⁷⁰⁸ More than 35 HDM allergen groups have been identified so far; this

<table><caption>TABLE 19 Common mite species found in house dust</caption><thead><tr><th>Mite Species</th><th>Number of Identified Allergens*</th></tr></thead><tbody><tr><td><strong>House Dust Mites</strong></td><td></td></tr><tr><td><em>Blomia tropicalis</em></td><td>14</td></tr><tr><td><em>Dermatophagoides pteronyssinus</em></td><td>31</td></tr><tr><td><em>Dermatophagoides farinae</em></td><td>36</td></tr><tr><td><em>Euroglyphus maynei</em></td><td>5</td></tr><tr><td><strong>Storage Mites</strong></td><td></td></tr><tr><td><em>Acarus siro</em></td><td>1</td></tr><tr><td><em>Chortoglyphus arcuatus</em></td><td>1</td></tr><tr><td><em>Glycyphagus domesticus</em></td><td>1</td></tr><tr><td><em>Lepidoglyphus destructor</em></td><td>5</td></tr><tr><td><em>Tyrophagus putrescentiae</em></td><td>15</td></tr></tbody></table>
of the literature is related to five genera: *Dermatophagoides*, *Blomia*, *Euroglyphus*, *Blatella*, and *Periplaneta*. One important aspect of the house dust mite (HDM) allergenic sources is that they can play a very important role in asthma, without their role being obvious to the patients.

While there are many possible explanations for this, the most obvious ones are perennial exposure, that the organisms are small and often not noticed and that particles carrying these allergens are sufficiently large not to remain airborne for more than a few minutes after disturbance.⁶⁹⁷ Because the role of exposure to mite or cockroach allergens had not been obvious to patients, learning about these species has been essential to increase our knowledge on the causal role of allergen exposure in asthma. The relevance of particle size became obvious shortly after the purification of Der p 1 and the development of accurate assays for this allergen.⁸ For *D. pteronyssinus* and *D. farinae*, allergens become airborne via fecal particles and fragmented mite bodies in household dust.⁶⁹⁸

These allergen-carrying particles are particularly relevant for the type of exposure, as allergenic proteins with a size of 15,000 to 50,000 Daltons are not volatile and significant exposure can only occur via particles. Mite fecal pellets have an average diameter of 20–30 µm⁶⁹⁹ and those with a size between 2–6 µm can be transported into small airways.⁷⁰⁰ The nature of these particles is relevant to, both, the induction of an IgE response and the subsequent contribution to inflammation of the nose and lungs. For dust mite particles, they not only carry a high concentration of several allergens but also are an important source of microbial compounds (at least LPS, β-glucans and chitin) capable of eliciting strong innate immune responses.

A significant feature of the epidemiology of HDM allergens is that there are areas of the world where some species are more present, for example, the high prevalence of *B. tropicalis* in the tropics. Both climate and housing conditions play a major role in these differences because mites absorb moisture from their environment and are absolutely dependent on the level of humidity in the air or on either carpets or upholstered material, which will retain humidity for long periods of time. In addition, the immature forms of *D. farinae* can withstand longer periods of dryness than *D. pteronyssinus*.

Before 1960, it was well recognized that house dust contained allergens other than those derived from domestic or pet animals as well as pollens or fungi; indeed, several groups had attempted to identify a house dust atopen by immunochemical analysis of house dust extracts. However, the breakthrough was made by microscopic identification of dust mites in house dust. It was fitting that these observations were made in the Netherlands since the Dutch scientist Antonie van Leeuwenhoek had first described mites in 1693. In addition, to evidence that mites of the genus *Dermatophagoides* were the major source of allergens in house dust, Spieksma and Voorhorst also developed the technique for culturing these organisms.⁷⁰⁰ This in turn made it possible to manufacture dust mite extracts for commercial use and subsequently facilitated the purification of mite allergens.⁸

## 2 – The allergen families

Dust mites belong to the order Astigmata, which is part of the Arachnids (Figure 55). This order includes the well-known Pyroglyphidae family but also the Acaridae and a superfamily (Glycyphagoidea) then divided into three main families: Echimyopodidae (*Blomia tropicalis*), Glycyphagidae (*Glycyphagus domesticus* and *Lepidoglyphus destructor*), and Chortoglyphidae (*Chortoglyphus arcuatus*). *Sarcoptes scabiei* (the scabies mite) is a member of the Sarcoptidae family, which has also been found as allergenic to humans. The mites generally recognized as house dust mites are *D. pteronyssinus*, *D. farinae*, *Euroglyphus maynei*, and *B. tropicalis*. However, four species of storage mites, which are best recognized as pests on farms or in food storage have also been recognized in house dust (Table 19). In addition, mites of the family Tarsonemidae may be found in significant numbers in house dust. The best evidence that a given mite species is relevant to allergic disease comes from studies in an area where a given species dominates all other mites in the house dust. This is true for *D. pteronyssinus* in the UK and New Zealand, for *D. farinae* in some areas of the United States, and for *B. tropicalis* in areas of South America and other tropical regions.⁷⁰¹,⁷⁰²

## 3 – Allergenic molecules. Epidemiology and function

Der p 1 was the first identified dust mite allergen in 1980, purified by conventional chromatographic techniques⁸; the purification of the Group 2 allergens Der p 2 and Der f 2 in 1989 was performed by immunoaffinity chromatography.⁷⁰³ Over the last 20 years, the techniques for cloning and sequencing proteins have become well-defined and much simpler. As a result, many mite-derived proteins have been described with convincing data about their amino acid sequence and the tertiary structure for some of them.⁷⁰⁴⁻⁷⁰⁸ More than 35 HDM allergen groups have been identified so far; this

<table><caption>TABLE 19 Common mite species found in house dust</caption><thead><tr><th>Mite Species</th><th>Number of Identified Allergens*</th></tr></thead><tbody><tr><td><strong>House Dust Mites</strong></td><td></td></tr><tr><td><em>Blomia tropicalis</em></td><td>14</td></tr><tr><td><em>Dermatophagoides pteronyssinus</em></td><td>31</td></tr><tr><td><em>Dermatophagoides farinae</em></td><td>36</td></tr><tr><td><em>Euroglyphus maynei</em></td><td>5</td></tr><tr><td><strong>Storage Mites</strong></td><td></td></tr><tr><td><em>Acarus siro</em></td><td>1</td></tr><tr><td><em>Chortoglyphus arcuatus</em></td><td>1</td></tr><tr><td><em>Glycyphagus domesticus</em></td><td>1</td></tr><tr><td><em>Lepidoglyphus destructor</em></td><td>5</td></tr><tr><td><em>Tyrophagus putrescentiae</em></td><td>15</td></tr></tbody></table>
functional classification was based on amino acid sequence and/or structural homologies. Some of the most studied groups are presented in (Table 20); other groups can be found in the WHO/IUIS Database (www.allergen.org).

Of note, proteomic analyses showed that Der p 1, Der p 2 and Der p 23 together with Der p 3, Der p 6, Der p 9, Der p 15 and Der p 28 represent the most abundant allergens in mite fecal pellets.⁷⁰⁹

In addition, the biological functions of some HDM allergens from groups 1 (Der p 1, Der f 1), 2 (Der p 2, Der f 2), 3 (Der p 3), 5 (Der p 5), 6 (Der p 6), 7 (Blo t 7), 9 (Der p 9), 12 (Blo t 12) and 13 (Der p 13, Blo t 13) were experimentally confirmed.⁴⁰

Component-resolved diagnosis using a collection of purified natural or recombinant HDM allergens greatly improved the characterization of IgE reactivity profiles for each HDM-allergic patient. sensitization patterns in random cohorts and birth cohorts evidenced clear serodominance of Der p 1/Der f 1, Der p 2/Der f 2 and Der p 23 (prevalence above 50%).⁷¹⁰ The IgE-binding frequencies for Der p 4, Der p 5, Der p 7, Der p 21 range from 20 to 40%. Seroprevalence below 20% is commonly measured for the other allergen groups.²⁸,⁷¹¹,⁷¹² Also, there are calculations suggesting that IgE antibodies specific to Der p 1, Der p 2, and Der p 23 account for more than 80% of the IgE to *D. pteronyssinus*. IgE to middle-tier allergens (i.e., 4, 5, 7) and 21 only adds around 10% to the cumulative amount of mite-specific IgE, whereas the contribution of the other identified proteins is even weaker.⁷⁰⁸,⁷¹³

Monosensitizations to groups 1, 2 and 23 can be detected in 3-5% of the HDM allergic patients.⁷¹² In a general way, IgE levels to Der p 1/Der p 2/Der p 23 were higher in HDM allergic patients developing asthma in comparison to those suffering from allergic rhinitis only. Moreover, sensitization profiles measured in HDM allergic asthmatics were broader than those detected in allergic rhinitis cohorts. The percentage of sensitization to mid-tier allergens such as Der p 5, Der p 7 or Der p 21 is also higher in HDM allergic patients developing asthma.⁷¹¹,⁷¹² The pattern of IgE sensitizations and HDM allergen-specific IgE levels displayed clear geographical variability, for example, Blo t 2, Blo t 5 and Blo t 21 are⁷¹⁴ serodominant in allergic patients living in tropical areas and sensitized to *Blomia tropica*lis,⁷¹⁴,⁷¹⁵ strengthening the concept of global personalized medicine to improve allergen immunotherapy (AIT) outcomes.⁷¹²,⁷¹⁵ Another interesting issue is the heterogeneity and molecular spreading of the IgE responses to mite allergens, which is further analyzed in Chapter A07 on “Basic and theoretical aspects of allergens.”

Der p 1 and Der p 2 are present in fecal particles and strongly associated with asthma. In addition, for Der p 1, there is specific evidence about the effects of reducing exposure from 13 to 0.2µg/g on symptoms among mite allergic individuals.⁷¹⁶ Thus, for these allergens there is good evidence of their important clinical role in rhinitis and asthma. Clearly, it will be very difficult to have an accurate assessment of the relative importance of all HDM allergenic components in the development of allergic diseases, but the trends

<table><caption>TABLE 20 Main HDM allergen groups, taken from WHO-IUIS Database (www.allergen.org)</caption><thead><tr><th>Allergen group</th><th>Biological function</th><th><i>D. pteronyssinus</i></th><th><i>D. farinae</i></th><th><i>B. tropicalis</i></th><th><i>E. maynei</i></th></tr></thead><tbody><tr><td>1</td><td>Cysteine protease</td><td>24*</td><td>10</td><td>2</td><td>2</td></tr><tr><td>2</td><td>MD-2-like lipid binding protein</td><td>15</td><td>17</td><td>5</td><td>2</td></tr><tr><td>3</td><td>Trypsin-like serine protease</td><td>1</td><td>1</td><td>2</td><td>1</td></tr><tr><td>4</td><td>Amylase</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>5</td><td>Lipid binding protein</td><td>2</td><td>1</td><td>1</td><td></td></tr><tr><td>6</td><td>Chymotrypsin-like serine protease</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>7</td><td>Lipid binding protein</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>8</td><td>Glutathione-S-Transferase</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>9</td><td>Collagenase-like serine protease</td><td>2</td><td></td><td></td><td></td></tr><tr><td>10</td><td>Tropomyosin</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>11</td><td>Paramyosin</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>12</td><td>Peritrophin</td><td></td><td></td><td>1</td><td></td></tr><tr><td>13</td><td>Fatty acid binding protein</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>15</td><td>Chitinase</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>18</td><td>Chitinase</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>21</td><td>Lipid binding protein</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>23</td><td>Peritrophin-like protein</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>31</td><td>Cofilin</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>32</td><td>Pyrophosphatase</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>33</td><td>α-tubulin</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>35</td><td>MD2-like lipid binding protein</td><td></td><td>1</td><td></td><td></td></tr></tbody></table>

<footnote>*Number of isoforms</footnote>
functional classification was based on amino acid sequence and/or structural homologies. Some of the most studied groups are presented in (Table 20); other groups can be found in the WHO/IUIS Database (www.allergen.org).

Of note, proteomic analyses showed that Der p 1, Der p 2 and Der p 23 together with Der p 3, Der p 6, Der p 9, Der p 15 and Der p 28 represent the most abundant allergens in mite fecal pellets.⁷⁰⁹

In addition, the biological functions of some HDM allergens from groups 1 (Der p 1, Der f 1), 2 (Der p 2, Der f 2), 3 (Der p 3), 5 (Der p 5), 6 (Der p 6), 7 (Blo t 7), 9 (Der p 9), 12 (Blo t 12) and 13 (Der p 13, Blo t 13) were experimentally confirmed.⁴⁰

Component-resolved diagnosis using a collection of purified natural or recombinant HDM allergens greatly improved the characterization of IgE reactivity profiles for each HDM-allergic patient. sensitization patterns in random cohorts and birth cohorts evidenced clear serodominance of Der p 1/Der f 1, Der p 2/Der f 2 and Der p 23 (prevalence above 50%).⁷¹⁰ The IgE-binding frequencies for Der p 4, Der p 5, Der p 7, Der p 21 range from 20 to 40%. Seroprevalence below 20% is commonly measured for the other allergen groups.²⁸,⁷¹¹,⁷¹² Also, there are calculations suggesting that IgE antibodies specific to Der p 1, Der p 2, and Der p 23 account for more than 80% of the IgE to *D. pteronyssinus*. IgE to middle-tier allergens (i.e., 4, 5, 7) and 21 only adds around 10% to the cumulative amount of mite-specific IgE, whereas the contribution of the other identified proteins is even weaker.⁷⁰⁸,⁷¹³

Monosensitizations to groups 1, 2 and 23 can be detected in 3-5% of the HDM allergic patients.⁷¹² In a general way, IgE levels to Der p 1/Der p 2/Der p 23 were higher in HDM allergic patients developing asthma in comparison to those suffering from allergic rhinitis only. Moreover, sensitization profiles measured in HDM allergic asthmatics were broader than those detected in allergic rhinitis cohorts. The percentage of sensitization to mid-tier allergens such as Der p 5, Der p 7 or Der p 21 is also higher in HDM allergic patients developing asthma.⁷¹¹,⁷¹² The pattern of IgE sensitizations and HDM allergen-specific IgE levels displayed clear geographical variability, for example, Blo t 2, Blo t 5 and Blo t 21 are⁷¹⁴ serodominant in allergic patients living in tropical areas and sensitized to *Blomia tropica*lis,⁷¹⁴,⁷¹⁵ strengthening the concept of global personalized medicine to improve allergen immunotherapy (AIT) outcomes.⁷¹²,⁷¹⁵ Another interesting issue is the heterogeneity and molecular spreading of the IgE responses to mite allergens, which is further analyzed in Chapter A07 on “Basic and theoretical aspects of allergens.”

Der p 1 and Der p 2 are present in fecal particles and strongly associated with asthma. In addition, for Der p 1, there is specific evidence about the effects of reducing exposure from 13 to 0.2µg/g on symptoms among mite allergic individuals.⁷¹⁶ Thus, for these allergens there is good evidence of their important clinical role in rhinitis and asthma. Clearly, it will be very difficult to have an accurate assessment of the relative importance of all HDM allergenic components in the development of allergic diseases, but the trends

<table><caption>TABLE 20 Main HDM allergen groups, taken from WHO-IUIS Database (www.allergen.org)</caption><thead><tr><th>Allergen group</th><th>Biological function</th><th><i>D. pteronyssinus</i></th><th><i>D. farinae</i></th><th><i>B. tropicalis</i></th><th><i>E. maynei</i></th></tr></thead><tbody><tr><td>1</td><td>Cysteine protease</td><td>24*</td><td>10</td><td>2</td><td>2</td></tr><tr><td>2</td><td>MD-2-like lipid binding protein</td><td>15</td><td>17</td><td>5</td><td>2</td></tr><tr><td>3</td><td>Trypsin-like serine protease</td><td>1</td><td>1</td><td>2</td><td>1</td></tr><tr><td>4</td><td>Amylase</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>5</td><td>Lipid binding protein</td><td>2</td><td>1</td><td>1</td><td></td></tr><tr><td>6</td><td>Chymotrypsin-like serine protease</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>7</td><td>Lipid binding protein</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>8</td><td>Glutathione-S-Transferase</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>9</td><td>Collagenase-like serine protease</td><td>2</td><td></td><td></td><td></td></tr><tr><td>10</td><td>Tropomyosin</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>11</td><td>Paramyosin</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>12</td><td>Peritrophin</td><td></td><td></td><td>1</td><td></td></tr><tr><td>13</td><td>Fatty acid binding protein</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>15</td><td>Chitinase</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>18</td><td>Chitinase</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>21</td><td>Lipid binding protein</td><td>1</td><td>1</td><td>1</td><td></td></tr><tr><td>23</td><td>Peritrophin-like protein</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>31</td><td>Cofilin</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>32</td><td>Pyrophosphatase</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>33</td><td>α-tubulin</td><td>1</td><td>1</td><td></td><td></td></tr><tr><td>35</td><td>MD2-like lipid binding protein</td><td></td><td>1</td><td></td><td></td></tr></tbody></table>

<footnote>*Number of isoforms</footnote>
of basic and clinical research on HDM allergy indicate that it could be
possible.²⁴ For example, a recent study found associations between
the IgE sensitization to the novel allergen Der p 37 and asthma.⁷¹⁷

4 – Sensitization to individual allergens and their
clinical relevance

Most clinically relevant allergenic molecules have been identified using serum from allergic patients because, by definition, they should bind specific IgE. However, most of them have been characterized only in terms of IgE-binding frequency (allergenicity) (Figure 56) with less robust evidence about allergenic activity. Since allergenic activity (the property of inducing inflammation, not necessarily IgE-mediated) is closer to clinical relevance than IgE-binding alone, during the last years some groups have focused their efforts on determining the allergenic activity of individual allergenic molecules, as was done before for allergenic extracts. Several assays have been useful to explore the allergenic activity of HDM molecules, among them *in vivo* and *in vitro* provocation tests, case-control studies, experimental animal models, mechanisms of action, avoidance studies and, of course, IgE-binding.⁵⁵ Although important advances have been made in this field, only a few HDM allergenic molecules have been evaluated in terms of allergenic activity; (Figure 57) shows examples from *D. pteronyssinus* and *B. tropicalis*.

Non IgE-mediated allergenic activity of HDM allergens

The inflammatory response triggered by HDM allergens is com-
monly mediated by the allergen binding to IgE immobilized to the
cell membrane, inducing cell activation and the release of a plethora
of pro-inflammatory factors; this mechanism seems to be relevant
for all allergens. In addition, some allergens can stimulate innate
immune inflammatory responses acting directly on immune or non-
immune cells, without an IgE-dependent mechanism. The sum of

both reaction types builds the allergenic activity of an IgE-binding
molecule.

The intrinsic allergenic activities of HDM allergens stimulate key
innate immune responses in the skin or airway epithelium leading
to the release of epithelial-derived proinflammatory cytokines and
innate alarmins such as IL-1α/β, GM-CSF, IL-25, IL-33 and TSLP.⁷¹⁸

This pro-Th2 environment is central for the development of the HDM allergic response. To date, deciphering the innate sensing of HDM allergens remains extremely challenging by their natural association with microbial/environmental compounds present in mites and house dust, including LPS, β-glucans or chitin, which are potent stimulators of innate immune signaling. The cysteine proteases from group 1 HDM allergens and group 3, 6 and 9 serine proteases are able to disrupt epithelial barrier integrity through cleavages of tight junction proteins.⁷¹⁹ Whereas HDM serine protease allergens can directly activate protease-activated receptor (PAR)-2 and -4, Der p 1 indirectly stimulates PAR-1/PAR-4 signaling pathways through their canonical activator thrombin. This Der p 1-Thrombin-PAR-1/-4 axis generates TLR4-dependent reactive oxygen species (ROS) leading to the release of IL-33.⁷²⁰ Der p 1 disrupts the lung homeostasis through proteolysis of surfactant proteins (SP-A/D) or protease inhibitors (elafin, α-antitrypsin). Furthermore, it cleaves key receptors involved in Th1 responses such as CD40, DC-SIGN, and CD25⁷²¹ or in the control of IgE production (CD23).⁷²²,⁷²³ Finally, nociceptors, transient receptor potential vanilloid 1 (TRPV1)+ sensory neurons, represent the primary sensors of HDM-associated cysteine protease activity and the activation of sensory neurons is necessary for the initiation of the HDM allergic response.⁷²⁴ While the key role of the LPS/TLR4 axis in the HDM airway inflammation has been evidenced,⁷²⁵ HDM allergens with fatty acid/lipid-binding capacity could represent potent activators of TLR2 and/or TLR4 signaling. Group 2 mite allergens, having structural homology with myeloid differentiation factor-2 (MD-2), the TLR4 co-receptor, can present LPS to TLR4.³¹⁷,⁷⁰⁴ However, their large hydrophobic pocket can transport other lipid

<figure><img src="image_146.png" /></figure>

**FIGURE 56** Relevant house dust mite allergens. Those with experimentally detected cross-reactivity with *Ascaris* are shown in orange. In addition, *Blo t 5* and *Der p 5* have moderate cross-reactivity, and there is high cross-reactivity among *Dermatophagoides* species.
of basic and clinical research on HDM allergy indicate that it could be
possible.²⁴ For example, a recent study found associations between
the IgE sensitization to the novel allergen Der p 37 and asthma.⁷¹⁷

4 – Sensitization to individual allergens and their
clinical relevance

Most clinically relevant allergenic molecules have been identified using serum from allergic patients because, by definition, they should bind specific IgE. However, most of them have been characterized only in terms of IgE-binding frequency (allergenicity) (Figure 56) with less robust evidence about allergenic activity. Since allergenic activity (the property of inducing inflammation, not necessarily IgE-mediated) is closer to clinical relevance than IgE-binding alone, during the last years some groups have focused their efforts on determining the allergenic activity of individual allergenic molecules, as was done before for allergenic extracts. Several assays have been useful to explore the allergenic activity of HDM molecules, among them *in vivo* and *in vitro* provocation tests, case-control studies, experimental animal models, mechanisms of action, avoidance studies and, of course, IgE-binding.⁵⁵ Although important advances have been made in this field, only a few HDM allergenic molecules have been evaluated in terms of allergenic activity; (Figure 57) shows examples from *D. pteronyssinus* and *B. tropicalis*.

Non IgE-mediated allergenic activity of HDM allergens

The inflammatory response triggered by HDM allergens is com-
monly mediated by the allergen binding to IgE immobilized to the
cell membrane, inducing cell activation and the release of a plethora
of pro-inflammatory factors; this mechanism seems to be relevant
for all allergens. In addition, some allergens can stimulate innate
immune inflammatory responses acting directly on immune or non-
immune cells, without an IgE-dependent mechanism. The sum of

both reaction types builds the allergenic activity of an IgE-binding
molecule.

The intrinsic allergenic activities of HDM allergens stimulate key
innate immune responses in the skin or airway epithelium leading
to the release of epithelial-derived proinflammatory cytokines and
innate alarmins such as IL-1α/β, GM-CSF, IL-25, IL-33 and TSLP.⁷¹⁸

This pro-Th2 environment is central for the development of the HDM allergic response. To date, deciphering the innate sensing of HDM allergens remains extremely challenging by their natural association with microbial/environmental compounds present in mites and house dust, including LPS, β-glucans or chitin, which are potent stimulators of innate immune signaling. The cysteine proteases from group 1 HDM allergens and group 3, 6 and 9 serine proteases are able to disrupt epithelial barrier integrity through cleavages of tight junction proteins.⁷¹⁹ Whereas HDM serine protease allergens can directly activate protease-activated receptor (PAR)-2 and -4, Der p 1 indirectly stimulates PAR-1/PAR-4 signaling pathways through their canonical activator thrombin. This Der p 1-Thrombin-PAR-1/-4 axis generates TLR4-dependent reactive oxygen species (ROS) leading to the release of IL-33.⁷²⁰ Der p 1 disrupts the lung homeostasis through proteolysis of surfactant proteins (SP-A/D) or protease inhibitors (elafin, α-antitrypsin). Furthermore, it cleaves key receptors involved in Th1 responses such as CD40, DC-SIGN, and CD25⁷²¹ or in the control of IgE production (CD23).⁷²²,⁷²³ Finally, nociceptors, transient receptor potential vanilloid 1 (TRPV1)+ sensory neurons, represent the primary sensors of HDM-associated cysteine protease activity and the activation of sensory neurons is necessary for the initiation of the HDM allergic response.⁷²⁴ While the key role of the LPS/ TLR4 axis in the HDM airway inflammation has been evidenced,⁷²⁵ HDM allergens with fatty acid/lipid-binding capacity could represent potent activators of TLR2 and/or TLR4 signaling. Group 2 mite allergens, having structural homology with myeloid differentiation factor-2 (MD-2), the TLR4 co-receptor, can present LPS to TLR4.³¹⁷,⁷⁰⁴ However, their large hydrophobic pocket can transport other lipid

<figure><img src="image_145.png" /></figure>

**FIGURE 56** Relevant house dust mite allergens. Those with experimentally detected cross-reactivity with *Ascaris* are shown in orange. In addition, *Blo t 5* and *Der p 5* have moderate cross-reactivity, and there is high cross-reactivity among *Dermatophagoides* species.
<figure><img src="image_147.png" /><figcaption>**FIGURE 57** Allergenic activity assays completed for house dust mite IgE-binding molecules. PT: Provocation test. PCA: Passive cutaneous anaphylaxis. AIT: Allergen immunotherapy</figcaption></figure>

cargos than LPS to trigger TLR2/4 signaling pathways. Recombinant forms of Der p 5, Blo t 7, Der p 13 and Der p 21 stimulate TLR2 signaling pathways.<sup>474,726-728</sup> Serum amyloid A1 (SAA1) can sense Der p 13 or Blo t 13 to promote pulmonary type 2 immunity.<sup>38</sup> HDM allergens from groups 12 and 23 or 15 and 18 could stimulate chitin-dependent innate immune mechanisms according to their sequence homologies with chitin-binding peritrophins or glycosyl hydrolase family 18 chitinsases, respectively. However, the chitin-binding capacity of HDM allergens was only evidenced for Blo t 12.<sup>729</sup> Finally, the HDM group tropomyosins can modulate the HDM airway inflammation through interactions with Dectin-1 expressed in airway epithelium.<sup>730</sup> More recently, the pro-inflammatory activity of Der f 38 through TLR4 has been reported.<sup>731</sup> A list of some HDM allergens innate mechanisms of action is presented in (Table 21).

5 - Clinical Management

Diagnosis of sensitization

As stated in the introduction, the role of dust mite allergy in asthma is generally not obvious to all patients. Indeed, histories of specific allergy to mites are not usually clear. Many patients will report sneezing on awakening or sneezing during house cleaning. During vigorous cleaning they may also notice eye irritation or wheezing, but conjunctivitis is not a common symptom of dust mite allergy. Skin prick testing is the primary means of diagnosis, and dust mite extracts are included in all inhalant panels. Most authorities would regard a wheal of 3 mm greater than the negative control as

positive. The common practice is to test with both *D. pteronyssinus* and *D. farinae*. In some areas, one mite or the other is the dominant cause of sensitization, but in general testing for the two provides convincing positive or negative results. However, *D. pteronyssinus* extracts might lack important allergens and often show great variability regarding allergen composition.<sup>732</sup> Notably, intact Der p 23 or Der p 5 are absent in HDM commercial allergen extracts. Moreover, as these allergen extracts are standardized for group 1 and group 2 allergens, the diagnosis of patients sensitized to HDM allergens other than Der p 1/Der p 2 is very challenging. Component-resolved diagnosis based on the use of individual natural/ recombinant HDM allergens could solve these detection issues and could enhance the percentage of successful allergen immunotherapy through the stratification of HDM allergic patients.<sup>733</sup>

Also, testing with *B. tropicalis* has become a very useful routine in tropical regions. *In vitro* assays for IgE to dust mite are well established and the units are given in IU/ml or KA Units/L. *In vitro* testing for specific IgE can be done using extracts of *D. pteronyssinus* (Dp), *D. farinae* (Df) and *B. tropicalis* (Bt). For multiplex testing, assays are available from several manufacturers, for example, rDer p 1, nDer p 1, nDer f 1, rDer f 2, rDer p 2, rDer p 23 and rDer p 10 are available on ImmunoCAP ISAC112 (ThermoFisher Scientific, Uppsala, Sweden). Der p 1, Der p 2, Blo t 5 and Der p 10 are available on the ImmunoCAP Specific IgE test, but Der p 23 is only available on the ImmunoCAP Specific IgE test (d 209) in Europe. Serum assays can provide a wide range of positive results from 0.1 IU/ml to > 300 IU/ml. There is also good evidence that wheal sizes and IgE titers are useful as risk predictors for allergic disease,
<figure><img src="image_146.png" /><figcaption>**FIGURE 57** Allergenic activity assays completed for house dust mite IgE-binding molecules. PT: Provocation test. PCA: Passive cutaneous anaphylaxis. AIT: Allergen immunotherapy</figcaption></figure>

cargos than LPS to trigger TLR2/4 signaling pathways. Recombinant forms of Der p 5, Blo t 7, Der p 13 and Der p 21 stimulate TLR2 signaling pathways.<sup>474,726-728</sup> Serum amyloid A1 (SAA1) can sense Der p 13 or Blo t 13 to promote pulmonary type 2 immunity.<sup>38</sup> HDM allergens from groups 12 and 23 or 15 and 18 could stimulate chitin-dependent innate immune mechanisms according to their sequence homologies with chitin-binding peritrophins or glycosyl hydrolase family 18 chitinsases, respectively. However, the chitin-binding capacity of HDM allergens was only evidenced for Blo t 12.<sup>729</sup> Finally, the HDM group tropomyosins can modulate the HDM airway inflammation through interactions with Dectin-1 expressed in airway epithelium.<sup>730</sup> More recently, the pro-inflammatory activity of Der f 38 through TLR4 has been reported.<sup>731</sup> A list of some HDM allergens innate mechanisms of action is presented in (Table 21).

# 5 - Clinical Management

## Diagnosis of sensitization

As stated in the introduction, the role of dust mite allergy in asthma is generally not obvious to all patients. Indeed, histories of specific allergy to mites are not usually clear. Many patients will report sneezing on awakening or sneezing during house cleaning. During vigorous cleaning they may also notice eye irritation or wheezing, but conjunctivitis is not a common symptom of dust mite allergy. Skin prick testing is the primary means of diagnosis, and dust mite extracts are included in all inhalant panels. Most authorities would regard a wheal of 3 mm greater than the negative control as

positive. The common practice is to test with both *D. pteronyssinus* and *D. farinae*. In some areas, one mite or the other is the dominant cause of sensitization, but in general testing for the two provides convincing positive or negative results. However, *D. pteronyssinus* extracts might lack important allergens and often show great variability regarding allergen composition.<sup>732</sup> Notably, intact Der p 23 or Der p 5 are absent in HDM commercial allergen extracts. Moreover, as these allergen extracts are standardized for group 1 and group 2 allergens, the diagnosis of patients sensitized to HDM allergens other than Der p 1/Der p 2 is very challenging. Component-resolved diagnosis based on the use of individual natural/ recombinant HDM allergens could solve these detection issues and could enhance the percentage of successful allergen immunotherapy through the stratification of HDM allergic patients.<sup>733</sup>

Also, testing with *B. tropicalis* has become a very useful routine in tropical regions. *In vitro* assays for IgE to dust mite are well established and the units are given in IU/ml or KA Units/L. *In vitro* testing for specific IgE can be done using extracts of *D. pteronyssinus* (Dp), *D. farinae* (Df) and *B. tropicalis* (Bt). For multiplex testing, assays are available from several manufacturers, for example, rDer p 1, nDer p 1, nDer f 1, rDer f 2, rDer p 2, rDer p 23 and rDer p 10 are available on ImmunoCAP ISAC112 (ThermoFisher Scientific, Uppsala, Sweden). Der p 1, Der p 2, Blo t 5 and Der p 10 are available on the ImmunoCAP Specific IgE test, but Der p 23 is only available on the ImmunoCAP Specific IgE test (d 209) in Europe. Serum assays can provide a wide range of positive results from 0.1 IU/ml to > 300 IU/ml. There is also good evidence that wheal sizes and IgE titers are useful as risk predictors for allergic disease,
<table><caption>TABLE 21 Main non IgE-mediated allergenic activities of HDM allergens</caption><thead><tr><th>Group</th><th>Allergen</th><th>Non IgE-mediated allergenic activity</th></tr></thead><tbody><tr><td rowspan="3">1</td><td>Der p 1</td><td>Disruption of the epithelial barrier integrity</td></tr><tr><td>Der f 1</td><td>Pro-thrombin, IL-33 maturation</td></tr><tr><td>Blo t 1</td><td>Cleavage of airway antiproteases, SP-A/SP-D, CD23, CD25, CD40, DC-SIGN eliminate :</td></tr><tr><td rowspan="3">2</td><td>Der p 2</td><td>PAR-1/PAR-4; TLR4-ROS-IL-33 axis; TRPV1' sens</td></tr><tr><td>Der f 2</td><td>TLR 4, TLR 2 activation</td></tr><tr><td>Blo t 2</td><td></td></tr><tr><td rowspan="2">3</td><td>Der p 3</td><td>Disruption of the epithelial barrier integrity</td></tr><tr><td>Blo t 3</td><td>PAR-2/PAR-4 activation</td></tr><tr><td>5</td><td>Der p 5</td><td>TLR2 activation</td></tr><tr><td rowspan="2">7</td><td>Blo t 5</td><td></td></tr><tr><td>Der p 7</td><td>TLR2 activation</td></tr><tr><td rowspan="2">9</td><td>Blo t 7</td><td></td></tr><tr><td>Der p 9</td><td>Disruption of the epithelial barrier integrity</td></tr><tr><td rowspan="2">10</td><td>Blo t 9</td><td>TLR2, SAA activation</td></tr><tr><td>Der p 10</td><td>Dectin-1 activation</td></tr><tr><td rowspan="2">13</td><td>Blo t 13</td><td></td></tr><tr><td>Der p 13</td><td>TLR2 activation</td></tr><tr><td>21</td><td>Der p 21</td><td></td></tr><tr><td>31</td><td>Der f 31</td><td>TLR2 activation</td></tr><tr><td>38</td><td>Der f 38</td><td>TLR4 activation</td></tr></tbody></table>

also in combination with a rhinovirus infection in childhood.⁷³⁴ Although the criteria for judging sensitization can be defined convincingly, it is not so easy to define these criteria for the role of dust mites in individual cases. However, several sets of information can help. If the patient is only allergic to mites, or skin tests/IgE says are much stronger for mites than for other allergens, it could be relevant for perennial symptoms. Although not widely accepted as a diagnostic procedure, a nasal provocation test with mite extracts is another important tool for defining the clinical relevance of sensitization and detecting cases of local rhinitis associated to negative skin tests. Also, conjunctival and nasal provocation tests with *B. tropicalis* have been suggested as a diagnostic procedure in the clinic.⁷³⁵ Measurement of mite allergens in dust from the house can be very helpful. However, the criteria of >2 µg Der p 1 per gram of dust for sensitization and >10 µg Der p 1 per gram of dust for severe symptoms should not be regarded as more than an orientation guide. Still, it is a major advantage to know the average levels of mite allergens in homes or apartments in the area where the patient lives. Cross-reactivity between *D. pteronyssinus* and *D. farinae* extracts is high but between *Dermatophagoides* and *B. tropicalis* is low. The use of species-specific components might be necessary in places like the tropics where co-exposure to both genera is common. Tropomyosin is the main cause of cross-reactivity among mites, cockroaches, shellfish and helminths (e.g., Ascaris lumbricoides), but Glutathione-S-Transferase may also be involved⁷³⁶ (Figure 58).

In the majority of cases skin test with mite extracts can detect sensitization and define the specificity of immunotherapy in asthmatic patients. However, the use of component-resolved diagnosis could be useful in special circumstances where genuine sensitization is not clear and has to be defined. Figures 59 and 60 represent algorithms that could be applied for diagnosing mite allergy in temperate and tropical countries. Not all suggested components are commercially available.

## Management

The management of allergic disease in patients who are allergic to dust mites consists of several different phases, most of which are like those for many other inhalant allergens (Table 22). However, education and allergen avoidance require extra care because of the complex biology of dust mites, and the fact that their presence in the home is not visible. Therefore, significant education in relation to avoidance is needed.

There is a wide range of evidence that dramatically decreasing exposure to dust mite allergens can help both asthma and rhinitis related to dust mites. This comes both from controlled trials of avoidance and from moving patients to a sanatorium or a hospital-based allergen "free" unit.²⁴,⁴⁹¹,⁷³⁷,⁷³⁸ Some of the most dramatic results have come from sanatoria in the Alps, but these are complicated to interpret because exposure to animal dander and fungi, as well as mites will be reduced.⁷³⁸⁻⁷⁴⁰ In addition, most of these sanatoria have regular exercise regimes, which may also contribute to the improved lung function and decrease in nonspecific bronchial hyper-reactivity
<table><caption>TABLE 21 Main non IgE-mediated allergenic activities of HDM allergens</caption><thead><tr><th>Group</th><th>Allergen</th><th>Non IgE-mediated allergenic activity</th></tr></thead><tbody><tr><td rowspan="3">1</td><td>Der p 1</td><td>Disruption of the epithelial barrier integrity</td></tr><tr><td>Der f 1</td><td>Pro-thrombin, IL-33 maturation</td></tr><tr><td>Blo t 1</td><td>Cleavage of airway antiproteases, SP-A/SP-D, CD23, CD25, CD40, DC-SIGN eliminate :</td></tr><tr><td rowspan="3">2</td><td>Der p 2</td><td>PAR-1/PAR-4; TLR4-ROS-IL-33 axis; TRPV1' sens</td></tr><tr><td>Der f 2</td><td>TLR 4, TLR 2 activation</td></tr><tr><td>Blo t 2</td><td></td></tr><tr><td rowspan="2">3</td><td>Der p 3</td><td>Disruption of the epithelial barrier integrity</td></tr><tr><td>Blo t 3</td><td>PAR-2/PAR-4 activation</td></tr><tr><td>5</td><td>Der p 5</td><td>TLR2 activation</td></tr><tr><td rowspan="2">7</td><td>Blo t 5</td><td></td></tr><tr><td>Der p 7</td><td>TLR2 activation</td></tr><tr><td rowspan="2">9</td><td>Blo t 7</td><td></td></tr><tr><td>Der p 9</td><td>Disruption of the epithelial barrier integrity</td></tr><tr><td rowspan="2">10</td><td>Blo t 9</td><td>TLR2, SAA activation</td></tr><tr><td>Der p 10</td><td>Dectin-1 activation</td></tr><tr><td rowspan="2">13</td><td>Blo t 13</td><td></td></tr><tr><td>Der p 13</td><td>TLR2 activation</td></tr><tr><td>21</td><td>Der p 21</td><td></td></tr><tr><td>31</td><td>Der f 31</td><td>TLR2 activation</td></tr><tr><td>38</td><td>Der f 38</td><td>TLR4 activation</td></tr></tbody></table>

also in combination with a rhinovirus infection in childhood.⁷³⁴ Although the criteria for judging sensitization can be defined convincingly, it is not so easy to define these criteria for the role of dust mites in individual cases. However, several sets of information can help. If the patient is only allergic to mites, or skin tests/IgE says are much stronger for mites than for other allergens, it could be relevant for perennial symptoms. Although not widely accepted as a diagnostic procedure, a nasal provocation test with mite extracts is another important tool for defining the clinical relevance of sensitization and detecting cases of local rhinitis associated to negative skin tests. Also, conjunctival and nasal provocation tests with *B. tropicalis* have been suggested as a diagnostic procedure in the clinic.⁷³⁵ Measurement of mite allergens in dust from the house can be very helpful. However, the criteria of >2 µg Der p 1 per gram of dust for sensitization and >10 µg Der p 1 per gram of dust for severe symptoms should not be regarded as more than an orientation guide. Still, it is a major advantage to know the average levels of mite allergens in homes or apartments in the area where the patient lives. Cross-reactivity between *D. pteronyssinus* and *D. farinae* extracts is high but between *Dermatophagoides* and *B. tropicalis* is low. The use of species-specific components might be necessary in places like the tropics where co-exposure to both genera is common. Tropomyosin is the main cause of cross-reactivity among mites, cockroaches, shellfish and helminths (e.g., Ascaris lumbricoides), but Glutathione-S-Transferase may also be involved⁷³⁶ (Figure 58).

In the majority of cases skin test with mite extracts can detect sensitization and define the specificity of immunotherapy in asthmatic patients. However, the use of component-resolved diagnosis could be useful in special circumstances where genuine sensitization is not clear and has to be defined. Figures 59 and 60 represent algorithms that could be applied for diagnosing mite allergy in temperate and tropical countries. Not all suggested components are commercially available.

## Management

The management of allergic disease in patients who are allergic to dust mites consists of several different phases, most of which are like those for many other inhalant allergens (Table 22). However, education and allergen avoidance require extra care because of the complex biology of dust mites, and the fact that their presence in the home is not visible. Therefore, significant education in relation to avoidance is needed.

There is a wide range of evidence that dramatically decreasing exposure to dust mite allergens can help both asthma and rhinitis related to dust mites. This comes both from controlled trials of avoidance and from moving patients to a sanatorium or a hospital-based allergen “free” unit.²⁴,⁴⁹¹,⁷³⁷,⁷³⁸ Some of the most dramatic results have come from sanatoria in the Alps, but these are complicated to interpret because exposure to animal dander and fungi, as well as mites will be reduced.⁷³⁸⁻⁷⁴⁰ In addition, most of these sanatoria have regular exercise regimes, which may also contribute to the improved lung function and decrease in nonspecific bronchial hyper-reactivity
<figure><img src="image_148.png" /><figcaption>FIGURE 58 Clinically relevant cross-reactivity of mite allergens. Species-specific components are shown in green.</figcaption></figure>

<figure><img src="image_149.png" /><figcaption>FIGURE 59 Possible decision algorithm for mite allergy in temperate countries. SPT: Skin Prick Test. CRD: Component Resolved Diagnosis. CR: Cross-reactivity. EC: Environmental Control. AIT: Allergen Immunotherapy. Dp: Dermatophagoides pteronyssinus extract. Df: Dermatophagoides farinae extract</figcaption></figure>

(BHR). Following the initial study in Davos (Switzerland), further studies were carried out in Briancon (France) and Misurina (Italy). The studies in Misurina provided compelling evidence that there was a progressive decrease in inflammatory markers in parallel with decreases in BHR among mite allergic children who spent 3 months in the sanatorium. To study the role of mite allergens, mite allergic

asthmatics in London spent 3 or more months living in a hospital room, which had filtered air and was designed to have no sites where mites could live. The level of Der p 1 in dust from their homes was 13.6 µg/g, while dust from the hospital room had less than 0.2 µg/g. The patients not only improved their symptoms but also experienced a major decrease in BHR.⁷¹⁶
<figure><img src="image_147.png" /><figcaption>FIGURE 58 Clinically relevant cross-reactivity of mite allergens. Species-specific components are shown in green.</figcaption></figure>

<figure><img src="image_148.png" /><figcaption>FIGURE 59 Possible decision algorithm for mite allergy in temperate countries. SPT: Skin Prick Test. CRD: Component Resolved Diagnosis. CR: Cross-reactivity. EC: Environmental Control. AIT: Allergen Immunotherapy. Dp: Dermatophagoides pteronyssinus extract. Df: Dermatophagoides farinae extract</figcaption></figure>

(BHR). Following the initial study in Davos (Switzerland), further studies were carried out in Briancon (France) and Misurina (Italy). The studies in Misurina provided compelling evidence that there was a progressive decrease in inflammatory markers in parallel with decreases in BHR among mite allergic children who spent 3 months in the sanatorium. To study the role of mite allergens, mite allergic

asthmatics in London spent 3 or more months living in a hospital room, which had filtered air and was designed to have no sites where mites could live. The level of Der p 1 in dust from their homes was 13.6 µg/g, while dust from the hospital room had less than 0.2 µg/g. The patients not only improved their symptoms but also experienced a major decrease in BHR.⁷¹⁶
<figure><img src="image_150.png" /></figure>

**FIGURE 60** Possible decision algorithm for mite allergy in the tropics. SPT: Skin Prick Test. CRD: Component Resolved Diagnosis. CR: Cross-Reactivity. EC: Environmental Control. AIT: Allergen Immunotherapy. Dp: *Dermatophagoides pteronyssinus* extract. Df: Dermatophagoides farinae extract. Bt: *Blomia tropicalis*.

<table><caption>**TABLE 22** Management of Allergic Disease Related to Dust Mite</caption><tr><td>A</td><td>Skin tests or serum assays for IgE antibodies using dust mite extract. CRD could be helpful to define allergen immunotherapy</td></tr><tr><td>B</td><td>Education about the relevance of these allergens to both acute and chronic symptoms</td></tr><tr><td>C</td><td>Advice about avoidance including a written plan and in some cases measurement of mite allergen in houses</td></tr><tr><td>D</td><td>A plan for pharmaceutical management for both the nose and the lungs</td></tr><tr><td>E</td><td>Subcutaneous or sublingual immunotherapy using dust mite allergen</td></tr><tr><td>F</td><td>In cases of asthma that are poorly controlled, treatment with a variety of monoclonal antibodies is now available and may also be recommended</td></tr></table>

## 6 - Clinical Case

**Clinical history:** A 32-year-old male faculty member in cardiology presented to clinic because of increasing episodes of shortness of breath during exercise. He was an enthusiastic runner (up to 10 miles) and had only developed symptoms since moving into a basement apartment one year earlier. His history did not include seasonal nasal symptoms or reactions on exposure to animals. When seen in clinic, his examination and spirometry were normal (Table 22).

**Tests with extracts:** Skin prick tests were strongly positive for *D. pteronyssinus* and *D. farinae* with 8×8 and 7×6 mm wheals. Blood count was unremarkable with absolute eosinophil count of 350. Total IgE 230 KU/L; IgE to *D. pteronyssinus* extract was 32 UA/ml. He was given a peak flow meter (Mini Wright) and instructed to record values before and after running, in addition, we arranged to collect samples from his apartment.

**Test with molecules:** Serum assays for components using ImmunoCAP ISAC (TFS, Uppsala, Sweden) showed Der p 1 IgE 28 ISU/ml; Der p 2 33 IgE ISU/ml; Der p 10 IgE (tropomyosin) <0.5 ISU/ml. Peak flow value (mean of 3 values) before running was 510 +20 liters/min and fell to 400 +40 liters/min and 320 +20 liters/min 2 and 4 minutes after running for 6 minutes. Dust samples from his apartment: 8.4 µg Der p 1/g carpet dust, 10.6 µg Der p 1/g sofa dust and 4.6 µg Der p 1/g bedding dust.

**Treatment Advice and Outcome:** Initially he was treated with albuterol inhaler, two puffs 10 min. prior to exercise and inhaled fluticasone 100 µg twice a day. In addition, he was advised to move to a second-floor apartment without carpeting and with minimal upholstered furniture. He was given routine advice about controlling mites in his bedding. He moved one month later and within 3 months his exercise breathing returned to normal. When seen 1 year later, he was no longer using inhalers and was without significant symptoms.

## 7 - Research and future perspectives

Given that HDM sensitivity is a major risk factor for asthma worldwide, the definition of the clinical impact of each of the
<figure><img src="image_149.png" /></figure>

**FIGURE 60** Possible decision algorithm for mite allergy in the tropics. SPT: Skin Prick Test. CRD: Component Resolved Diagnosis. CR: Cross-Reactivity. EC: Environmental Control. AIT: Allergen Immunotherapy. Dp: *Dermatophagoides pteronyssinus* extract. Df: Dermatophagoides farinae extract. Bt: *Blomia tropicalis*.

<table><caption>**TABLE 22** Management of Allergic Disease Related to Dust Mite</caption><tr><td>A</td><td>Skin tests or serum assays for IgE antibodies using dust mite extract. CRD could be helpful to define allergen immunotherapy</td></tr><tr><td>B</td><td>Education about the relevance of these allergens to both acute and chronic symptoms</td></tr><tr><td>C</td><td>Advice about avoidance including a written plan and in some cases measurement of mite allergen in houses</td></tr><tr><td>D</td><td>A plan for pharmaceutical management for both the nose and the lungs</td></tr><tr><td>E</td><td>Subcutaneous or sublingual immunotherapy using dust mite allergen</td></tr><tr><td>F</td><td>In cases of asthma that are poorly controlled, treatment with a variety of monoclonal antibodies is now available and may also be recommended</td></tr></table>

## 6 - Clinical Case

**Clinical history:** A 32-year-old male faculty member in cardiology presented to clinic because of increasing episodes of shortness of breath during exercise. He was an enthusiastic runner (up to 10 miles) and had only developed symptoms since moving into a basement apartment one year earlier. His history did not include seasonal nasal symptoms or reactions on exposure to animals. When seen in clinic, his examination and spirometry were normal (Table 22).

**Tests with extracts:** Skin prick tests were strongly positive for *D. pteronyssinus* and *D. farinae* with 8×8 and 7×6 mm wheals. Blood count was unremarkable with absolute eosinophil count of 350. Total IgE 230 KU/L; IgE to *D. pteronyssinus* extract was 32 UA/ml. He was given a peak flow meter (Mini Wright) and instructed to record values before and after running, in addition, we arranged to collect samples from his apartment.

**Test with molecules:** Serum assays for components using ImmunoCAP ISAC (TFS, Uppsala, Sweden) showed Der p 1 IgE 28 ISU/ml; Der p 2 33 IgE ISU/ml; Der p 10 IgE (tropomyosin) <0.5 ISU/ml. Peak flow value (mean of 3 values) before running was 510 +20 liters/min and fell to 400 +40 liters/min and 320 +20 liters/min 2 and 4 minutes after running for 6 minutes. Dust samples from his apartment: 8.4 µg Der p 1/g carpet dust, 10.6 µg Der p 1/g sofa dust and 4.6 µg Der p 1/g bedding dust.

**Treatment Advice and Outcome:** Initially he was treated with albuterol inhaler, two puffs 10 min. prior to exercise and inhaled fluticasone 100 µg twice a day. In addition, he was advised to move to a second-floor apartment without carpeting and with minimal upholstered furniture. He was given routine advice about controlling mites in his bedding. He moved one month later and within 3 months his exercise breathing returned to normal. When seen 1 year later, he was no longer using inhalers and was without significant symptoms.

## 7 - Research and future perspectives

Given that HDM sensitivity is a major risk factor for asthma worldwide, the definition of the clinical impact of each of the
serodominant and mid-tier IgE-binding molecules is essential. Although there are no important difficulties for diagnosing HDM allergy using the whole extracts and some molecules, the complete profile of cross-reactivity among individual components remains to be experimentally analyzed. For example, groups 1 and 2 cross-reactivities are very high as these allergens share most of their conformational IgE-binding epitopes.⁷⁴¹ The complete mapping of these antigenic determinants would allow the identification of unique IgE-binding sites in Der p 1 or Der f 1 as well as Der p 2 or Der f 2.⁵⁰⁷,⁷⁴² This research would improve the elucidation of HDM sensitization profiles.

Studies showing geographical variabilities in the patterns of HDM sensitization have been mainly focused on cohorts of Caucasian HDM allergic patients.⁷¹²,⁷¹³ The elucidation of the IgE reactivity profiles must be further broadened to populations from other ethnicities. This aim, together with the definition of the allergenic activity of additional IgE-binding molecules, could show the need for regional arrays for component-resolved diagnosis. In addition, since

the HDM allergen repertoire has been recently extended through “omic” analysis, recombinant forms of these new HDM allergens could complete the panels of molecules to improve the elucidation of the sensitization profile.

Future research on HDM allergy will keep the interest on the definition of the clinical impact of individual IgE-binding molecules,⁵⁵ and to investigate the mechanisms by which they induce symptoms. To achieve this, other technologic resources will be very helpful, but it is also necessary to better understand the relative role of IgE in the allergic responses, considering the increasing evidence of non-IgE-mediated inflammatory mechanisms associated to the allergenic activity of IgE-binding molecules. Therefore, a wider idea of the potential mechanisms of action of allergens will improve the research proposals in this field. In connection with this, it will be necessary to search for new ways to define the clinical impact of allergens, leaving the constraints of the “major and minor” allergen classification.
serodominant and mid-tier IgE-binding molecules is essential. Although there are no important difficulties for diagnosing HDM allergy using the whole extracts and some molecules, the complete profile of cross-reactivity among individual components remains to be experimentally analyzed. For example, groups 1 and 2 cross-reactivities are very high as these allergens share most of their conformational IgE-binding epitopes.⁷⁴¹ The complete mapping of these antigenic determinants would allow the identification of unique IgE-binding sites in Der p 1 or Der f 1 as well as Der p 2 or Der f 2.⁵⁰⁷,⁷⁴² This research would improve the elucidation of HDM sensitization profiles.

Studies showing geographical variabilities in the patterns of HDM sensitization have been mainly focused on cohorts of Caucasian HDM allergic patients.⁷¹²,⁷¹³ The elucidation of the IgE reactivity profiles must be further broadened to populations from other ethnicities. This aim, together with the definition of the allergenic activity of additional IgE-binding molecules, could show the need for regional arrays for component-resolved diagnosis. In addition, since

the HDM allergen repertoire has been recently extended through “omic” analysis, recombinant forms of these new HDM allergens could complete the panels of molecules to improve the elucidation of the sensitization profile.

Future research on HDM allergy will keep the interest on the definition of the clinical impact of individual IgE-binding molecules,⁵⁵ and to investigate the mechanisms by which they induce symptoms. To achieve this, other technologic resources will be very helpful, but it is also necessary to better understand the relative role of IgE in the allergic responses, considering the increasing evidence of non-IgE-mediated inflammatory mechanisms associated to the allergenic activity of IgE-binding molecules. Therefore, a wider idea of the potential mechanisms of action of allergens will improve the research proposals in this field. In connection with this, it will be necessary to search for new ways to define the clinical impact of allergens, leaving the constraints of the “major and minor” allergen classification.
# B05 - Cockroach allergy

Luis Caraballo, Thomas Platts-Mills, Anna Pomés, L. Karla Arruda

## Highlights

* Cockroach allergens are strong inducers of sensitization and asthma.

* Clinically important species include American, German, Oriental, Asian, brown-banded and smoky-brown cockroaches.

* Bla g 2 and Bla g 5 have the higher frequency of IgE positivity among cockroach allergens, but there are important differences among individual patients and populations.

* Currently, diagnosis is performed by skin testing and/or measurement of specific IgE to cockroach, using crude extracts.

## 1 - The allergen sources

Cockroaches belong to the phylum Arthropoda; class Insecta, Order Blattaria or Blattodea. Species causing allergy symptoms such as asthma are listed in (Table 23). These ubiquitous scavenger organisms have inhabited the planet long ago and domiciliary species are currently a serious problem for humans. Those that live in human dwellings (around 25 species) include American, German, Oriental, and Asian, which, together with the brown-banded and the smoky-brown cockroaches are sources of important allergens, inducers of allergic asthma.⁷⁴³

sensitization to cockroach usually occurs by inhalation. Potential sources of relevant allergens in the environment include whole bodies, cast skins, secretions, egg casings, and fecal material. Level of exposure for increased risk of asthma symptoms is 8 U/g of dust (104 ng/unit for Bla g 1 and 40 ng/unit for Bla g 2⁷⁴⁴) and a US national study found that 10% of living rooms were above this point. There is inter-species cross-reactivity (e.g., American, German, Asian and Oriental) and extra-species cross-reactivity ("pan-allergy") with several other arthropods such as crustaceans (shrimp, crab, and lobster), insects (silverfish, butterflies), arachnids (dust mites) and mollusks

(oysters, mussels, scallops, clams). Since both exposure and allergy to cockroach are very common, patients with asthma or rhinitis should be routinely evaluated for this type of allergy. Based on their molecular and biological properties, cockroach allergens have been distributed in several groups, most of them are shown in (Figure 61) and (Table 24).

## 2 - The allergen families

**Group 1** allergens are composed by multiple consecutive amino acid repeats originated by gene duplication of an original 100 amino acid domain.⁷⁴⁵ Two repeats (each with 6 alpha-helices) define a basic structural unit and encapsulate a large and nearly spherical hydrophobic cavity that binds lipids such as palmitic, oleic, and stearic acids (Figure 62). There is cross-reactivity between Bla g 1, Per a 1 and homologous proteins from other cockroach species such as *P. fuliginosa* and *Blatta orientalis* and from other insects. The protein is most prevalent in the midgut, probably because the Bla g 1 gene is exclusively expressed by midgut cells. The presence of Bla g 1 in fecal particles makes this molecule, together with Bla g 2, a good marker of cockroach allergen exposure.

**Group 2** Bla g 2 is an unusual (inactive) aspartic protease with strong allergenic properties. There is three times more Bla g 2 in cockroach feces compared with the whole extract. It was originally found to be the allergen with the highest IgE antibody prevalence among 5 cockroach allergens in a US population.⁷⁴⁶ The crystal structure shows that Bla g 2 has a bilobal shape.⁷⁴⁷ The antigenic structure of this allergen was analyzed by X-ray crystallography and site-directed mutagenesis, providing important information about function, key amino acids and carbohydrates determining epitopes and antigen-antibody interactions⁷⁴⁸,⁷⁴⁹ (Figure 62). Even a role for Bla g 2-associated glycans in allergen-induced immune reactions through basophil activation has been reported.⁷⁵⁰

**Group 3** Allergens of this group show high homology to insect hemolymph proteins. Per a 3 induces IL-4 expression in PBMC from allergic patients and this correlates with skin reactivity and clinical symptoms.⁷⁵¹

<table><caption>TABLE 23 Clinically relevant cockroaches species</caption><thead><tr><th>Family</th><th>Genus/species</th><th>Common name</th><th>Geographic distribution</th></tr></thead><tbody><tr><td>Blaberidae</td><td>Leucophaea maderae</td><td>Madeira</td><td>Asia, Africa, America, Oceania</td></tr><tr><td>Ectobiidae</td><td>Blattella germanica</td><td>German</td><td>Mainly temperate dry zones</td></tr><tr><td>(formerly Blattellidae)</td><td>Blattella asahinai</td><td>Asian</td><td>Japan, tropical and subtropical</td></tr><tr><td></td><td>Supella longipalpa</td><td>Brown-banded</td><td>Tropical</td></tr><tr><td rowspan="6">Blattidae</td><td>Periplaneta americana</td><td>American</td><td>Mainly tropical and subtropical</td></tr><tr><td>Periplaneta australasiae</td><td>Australian</td><td>Cosmopolitan</td></tr><tr><td>Periplaneta brunnea</td><td>Brown</td><td>Mainly tropical</td></tr><tr><td>Periplaneta fuliginosa</td><td>Smoky brown</td><td>China, Russia, Korea, Japan, Australia and USA</td></tr><tr><td>Blatta orientalis</td><td>Oriental</td><td>America, United Kingdom, Germany</td></tr></tbody></table>
# B05 - Cockroach allergy

Luis Caraballo, Thomas Platts-Mills, Anna Pomés, L. Karla Arruda

## Highlights

* Cockroach allergens are strong inducers of sensitization and asthma.

* Clinically important species include American, German, Oriental, Asian, brown-banded and smoky-brown cockroaches.

* Bla g 2 and Bla g 5 have the higher frequency of IgE positivity among cockroach allergens, but there are important differences among individual patients and populations.

* Currently, diagnosis is performed by skin testing and/or measurement of specific IgE to cockroach, using crude extracts.

## 1 - The allergen sources

Cockroaches belong to the phylum Arthropoda; class Insecta, Order Blattaria or Blattodea. Species causing allergy symptoms such as asthma are listed in (Table 23). These ubiquitous scavenger organisms have inhabited the planet long ago and domiciliary species are currently a serious problem for humans. Those that live in human dwellings (around 25 species) include American, German, Oriental, and Asian, which, together with the brown-banded and the smoky-brown cockroaches are sources of important allergens, inducers of allergic asthma.⁷⁴³

sensitization to cockroach usually occurs by inhalation. Potential sources of relevant allergens in the environment include whole bodies, cast skins, secretions, egg casings, and fecal material. Level of exposure for increased risk of asthma symptoms is 8 U/g of dust (104 ng/unit for Bla g 1 and 40 ng/unit for Bla g 2⁷⁴⁴) and a US national study found that 10% of living rooms were above this point. There is inter-species cross-reactivity (e.g., American, German, Asian and Oriental) and extra-species cross-reactivity ("pan-allergy") with several other arthropods such as crustaceans (shrimp, crab, and lobster), insects (silverfish, butterflies), arachnids (dust mites) and mollusks

(oysters, mussels, scallops, clams). Since both exposure and allergy to cockroach are very common, patients with asthma or rhinitis should be routinely evaluated for this type of allergy. Based on their molecular and biological properties, cockroach allergens have been distributed in several groups, most of them are shown in (Figure 61) and (Table 24).

## 2 - The allergen families

Group 1 allergens are composed by multiple consecutive amino acid repeats originated by gene duplication of an original 100 amino acid domain.⁷⁴⁵ Two repeats (each with 6 alpha-helices) define a basic structural unit and encapsulate a large and nearly spherical hydrophobic cavity that binds lipids such as palmitic, oleic, and stearic acids (Figure 62). There is cross-reactivity between Bla g 1, Per a 1 and homologous proteins from other cockroach species such as *P. fuliginosa* and *Blatta orientalis* and from other insects. The protein is most prevalent in the midgut, probably because the Bla g 1 gene is exclusively expressed by midgut cells. The presence of Bla g 1 in fecal particles makes this molecule, together with Bla g 2, a good marker of cockroach allergen exposure.

Group 2 Bla g 2 is an unusual (inactive) aspartic protease with strong allergenic properties. There is three times more Bla g 2 in cockroach feces compared with the whole extract. It was originally found to be the allergen with the highest IgE antibody prevalence among 5 cockroach allergens in a US population.⁷⁴⁶ The crystal structure shows that Bla g 2 has a bilobal shape.⁷⁴⁷ The antigenic structure of this allergen was analyzed by X-ray crystallography and site-directed mutagenesis, providing important information about function, key amino acids and carbohydrates determining epitopes and antigen-antibody interactions⁷⁴⁸,⁷⁴⁹ (Figure 62). Even a role for Bla g 2-associated glycans in allergen-induced immune reactions through basophil activation has been reported.⁷⁵⁰

Group 3 Allergens of this group show high homology to insect hemolymph proteins. Per a 3 induces IL-4 expression in PBMC from allergic patients and this correlates with skin reactivity and clinical symptoms.⁷⁵¹

<table><caption>TABLE 23 Clinically relevant cockroaches species</caption><thead><tr><td>Family</td><td>Genus/species</td><td>Common name</td><td>Geographic distribution</td></tr></thead><tbody><tr><td>Blaberidae</td><td>Leucophaea maderae</td><td>Madeira</td><td>Asia, Africa, America, Oceania</td></tr><tr><td>Ectobiidae</td><td>Blattella germanica</td><td>German</td><td>Mainly temperate dry zones</td></tr><tr><td>(formerly Blattellidae)</td><td>Blattella asahinai</td><td>Asian</td><td>Japan, tropical and subtropical</td></tr><tr><td></td><td>Supella longipalpa</td><td>Brown-banded</td><td>Tropical</td></tr><tr><td rowspan="6">Blattidae</td><td>Periplaneta americana</td><td>American</td><td>Mainly tropical and subtropical</td></tr><tr><td>Periplaneta australasiae</td><td>Australian</td><td>Cosmopolitan</td></tr><tr><td>Periplaneta brunnea</td><td>Brown</td><td>Mainly tropical</td></tr><tr><td>Periplaneta fuliginosa</td><td>Smoky brown</td><td>China, Russia, Korea, Japan, Australia and USA</td></tr><tr><td>Blatta orientalis</td><td>Oriental</td><td>America, United Kingdom, Germany</td></tr></tbody></table>
<figure><img src="image_151.png" /><figcaption>**FIGURE 61** Some allergen components from American and German cockroaches. Homologous molecules have the same color. Complete in Table 24</figcaption></figure>

<table><caption>**TABLE 24** Cockroach allergenic molecules</caption><thead><tr><th>Species</th><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence of allergen-specific IgE among patients (%)</th><th>MW in kDa</th></tr></thead><tbody><tr><td rowspan="12">B. germanica</td><td>Bla g 1</td><td>Midgut microvilli protein-homologue</td><td>20-50</td><td>21-90</td></tr><tr><td>Bla g 2</td><td>Unusual aspartic protease</td><td>18-73</td><td>36</td></tr><tr><td>Bla g 3</td><td>Arylphorin/hemocyanin</td><td>22</td><td>78.9</td></tr><tr><td>Bla g 4</td><td>Lipocalin</td><td>11-47</td><td>21</td></tr><tr><td>Bla g 5</td><td>Glutathione S-transferase</td><td>39-73</td><td>23</td></tr><tr><td>Bla g 6</td><td>Troponin C</td><td>14-0</td><td>17</td></tr><tr><td>Bla g 7</td><td>Tropomyosin</td><td>16</td><td>33</td></tr><tr><td>Bla g 8</td><td>Myosin light chain</td><td>14</td><td>21</td></tr><tr><td>Bla g 9</td><td>Arginine kinase</td><td>25-53</td><td>40</td></tr><tr><td>Bla g 11</td><td>Alpha-amylase</td><td>29-73</td><td>57</td></tr><tr><td>Bla g 12</td><td>Chitinase</td><td>30</td><td>58</td></tr><tr><td rowspan="15">P. americana</td><td>Per a 1</td><td>Midgut microvilli protein-homologue</td><td>30 - 50, 100</td><td>26-51</td></tr><tr><td>Per a 2</td><td>Unusual aspartic protease</td><td>63</td><td>42</td></tr><tr><td>Per a 3</td><td>Arylphorin/hemocyanin</td><td>26-95</td><td>46-79</td></tr><tr><td>Per a 4</td><td>Lipocalin</td><td>15</td><td>17</td></tr><tr><td>Per a 5</td><td>Glutathione S-transferase</td><td>100</td><td>23</td></tr><tr><td>Per a 6</td><td>Troponin C</td><td>17</td><td>17</td></tr><tr><td>Per a 7</td><td>Tropomyosin</td><td>13-54</td><td>33</td></tr><tr><td>Per a 8</td><td>Myosin light chain</td><td>20</td><td>23</td></tr><tr><td>Per a 9</td><td>Arginine kinase</td><td>80-100</td><td>43</td></tr><tr><td>Per a 10</td><td>Serine protease</td><td>82</td><td>28</td></tr><tr><td>Per a 11</td><td>Alpha-amylase</td><td>83</td><td>55</td></tr><tr><td>Per a 12</td><td>Chitinase</td><td>64</td><td>45</td></tr><tr><td>Per a 13</td><td>Glyceraldehyde-3-phosphate dehydrogenase</td><td>3-3</td><td>36</td></tr></tbody></table>
<figure><img src="image_150.png" /><figcaption>FIGURE 61 Some allergen components from American and German cockroaches. Homologous molecules have the same color. Complete in Table 24</figcaption></figure>

<table><caption>TABLE 24 Cockroach allergenic molecules</caption><thead><tr><th>Species</th><th>Allergenic molecule</th><th>Biochemical name</th><th>Prevalence of allergen-specific IgE among patients (%)</th><th>MW in kDa</th></tr></thead><tbody><tr><td rowspan="12">B. germanica</td><td>Bla g 1</td><td>Midgut microvilli protein-homologue</td><td>20-50</td><td>21-90</td></tr><tr><td>Bla g 2</td><td>Unusual aspartic protease</td><td>18-73</td><td>36</td></tr><tr><td>Bla g 3</td><td>Arylphorin/hemocyanin</td><td>22</td><td>78.9</td></tr><tr><td>Bla g 4</td><td>Lipocalin</td><td>11-47</td><td>21</td></tr><tr><td>Bla g 5</td><td>Glutathione S-transferase</td><td>39-73</td><td>23</td></tr><tr><td>Bla g 6</td><td>Troponin C</td><td>14-0</td><td>17</td></tr><tr><td>Bla g 7</td><td>Tropomyosin</td><td>16</td><td>33</td></tr><tr><td>Bla g 8</td><td>Myosin light chain</td><td>14</td><td>21</td></tr><tr><td>Bla g 9</td><td>Arginine kinase</td><td>25-53</td><td>40</td></tr><tr><td>Bla g 11</td><td>Alpha-amylase</td><td>29-73</td><td>57</td></tr><tr><td>Bla g 12</td><td>Chitinase</td><td>30</td><td>58</td></tr><tr><td rowspan="15">P. americana</td><td>Per a 1</td><td>Midgut microvilli protein-homologue</td><td>30 - 50, 100</td><td>26-51</td></tr><tr><td>Per a 2</td><td>Unusual aspartic protease</td><td>63</td><td>42</td></tr><tr><td>Per a 3</td><td>Arylphorin/hemocyanin</td><td>26-95</td><td>46-79</td></tr><tr><td>Per a 4</td><td>Lipocalin</td><td>15</td><td>17</td></tr><tr><td>Per a 5</td><td>Glutathione S-transferase</td><td>100</td><td>23</td></tr><tr><td>Per a 6</td><td>Troponin C</td><td>17</td><td>17</td></tr><tr><td>Per a 7</td><td>Tropomyosin</td><td>13-54</td><td>33</td></tr><tr><td>Per a 8</td><td>Myosin light chain</td><td>20</td><td>23</td></tr><tr><td>Per a 9</td><td>Arginine kinase</td><td>80-100</td><td>43</td></tr><tr><td>Per a 10</td><td>Serine protease</td><td>82</td><td>28</td></tr><tr><td>Per a 11</td><td>Alpha-amylase</td><td>83</td><td>55</td></tr><tr><td>Per a 12</td><td>Chitinase</td><td>64</td><td>45</td></tr><tr><td>Per a 13</td><td>Glyceraldehyde-3-phosphate dehydrogenase</td><td>3-3</td><td>36</td></tr></tbody></table>
**FIGURE 62** Crystal structures of the cockroach allergens Bla g 2 in complex with Fab' of the monoclonal antibody 7C11, Bla g 1, Bla g 4 and Bla g 5 (Protein Data Bank accession numbers are 2nr6, 7jrb, 3ebk, and 4q5r, respectively). The heavy and light chains of the mAb 7C11 Fab' are shown in dark and light grays, respectively. The allergen molecules are shown from the N- (blue) to the C-termini (red). One of the two molecules in the Bla g 5 dimer is shown in gray.

<figure><img src="image_152.png" /><figcaption>Bla g 2</figcaption></figure>

<figure><img src="image_153.png" /><figcaption>Bla g 1</figcaption></figure>

<figure><img src="image_154.png" /><figcaption>Bla g 4</figcaption></figure>

<figure><img src="image_155.png" /><figcaption>Bla g 5</figcaption></figure>

**Group 4** Bla g 4 and Per a 4 are lipocalins. These molecules are very stable, and their structure consists of a C-terminal α-helix and a β-barrel enclosing an internal hydrophobic cavity that binds small ligands such as retinoids, glucocorticoids, and pheromones (Figure 62).

**Group 5** Bla g 5 is a sigma class glutathione S-transferase (GST) that has a high IgE response in cockroach sensitized individuals (Figure 62). Cross-reactivity with GSTs of several sources (for example, Der p 8 and Tyr p 8) is known. However, lack of significant IgE cross-reactivity among GSTs from cockroach, mite and Ascaris was found in a temperate US population.⁷⁰⁸

**Group 6** The allergens of this group are homologues to insect troponin C and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium-binding domains. Interestingly, IgE binding to Bla g 6 has proven to be calcium dependent indicating that IgE preferably binds to one of the conformers.⁷⁵²

**Group 7** Invertebrate tropomyosins are important pan-allergens among dust mites, chironomids, silverfish, crustaceans, nematodes and mollusks. Tropomyosins from *B. germanica* and *P. americana* have been described. IgE-binding frequency to cockroach tropomyosins are very different in some populations and this may reflect differences in environmental conditions (see chapter C05).

**Group 8** Bla g 8 shares 81-84% amino acid sequence identity with the myosin light chain of several insects and the shrimp *Litopenaeus vannamei*. The myosin regulatory light chains are small acidic polypeptides noncovalently bound to the neck region of the myosin head, which regulate the interaction of the myosin head with actin.

**Group 9** Per a 9 and Bla g 9 were identified as major allergens in Thai and US patients, respectively.⁷⁵³,⁷⁵⁴ Arginine kinase homologues have also been reported in the shrimp *Penaeus monodon* (Pen m 2), *D. pteronyssinus* (Der p 20) and the Indian meal moth *Plodia interpunctella* (Plo i 1). There is evidence suggesting that arginine kinase is an invertebrate pan-allergen.⁷⁵⁵

(Pen m 2), *D. pteronyssinus* (Der p 20) and the Indian meal moth *Plodia interpunctella* (Plo i 1). There is evidence suggesting that arginine kinase is an invertebrate pan-allergen.⁷⁵⁵

**Group 10** Per a 10 is a serine proteases isolated from P. americana and an important allergen in Indian allergic patients.⁷⁵⁶ Other important allergens are also serine proteases (Der f 3, Der p 3, Der p 6 and Der p 9), but cross-reactivity between cockroach and mite serine protease is unlikely due to low amino acid identities (32-41%) among these molecules.

**Group 11** Bla g 11 shares 56% sequence identity with pig α-amylase and with group 4 mite allergens Blo t 4 (50%), Der p 4 (50%) and Eur m 4 (47%). Bla g 11 seems to be an important novel allergen because the recombinant α-amylase inhibited 55% of specific IgE of German cockroach extract.⁷⁵⁷ The Per a 11 allergen has been described in China.⁷⁵⁸

**Group 12** These allergens are chitinases, essential for digestion of chitin. Per a 12 and Bla g 12 have been reported in Chinese and US populations, respectively, with very different IgE prevalence (Table 24).⁷⁵⁸,⁷⁵⁹ Their amino acid identities with the house dust mite chitinases Der p 15 and Der f 15 are low (~35%).

**Group 13** Allergens from this group are glyceraldehyde-3-phosphate dehydrogenases and have only been reported for *P. americana* as Per a 13.

## 4 – Sensitization to individual molecules and its clinical relevance

Cockroach allergens are strong inducers of sensitization and asthma⁷⁶⁰,⁷⁶¹ and cockroach allergy is an important risk factor for emergency room visits and hospital admissions. Most characterized
**FIGURE 62** Crystal structures of the cockroach allergens Bla g 2 in complex with Fab' of the monoclonal antibody 7C11, Bla g 1, Bla g 4 and Bla g 5 (Protein Data Bank accession numbers are 2nr6, 7jrb, 3ebk, and 4q5r, respectively). The heavy and light chains of the mAb 7C11 Fab' are shown in dark and light grays, respectively. The allergen molecules are shown from the N- (blue) to the C-termini (red). One of the two molecules in the Bla g 5 dimer is shown in gray.

<figure><img src="image_151.png" /><figcaption>Bla g 2</figcaption></figure>

<figure><img src="image_152.png" /><figcaption>Bla g 1</figcaption></figure>

<figure><img src="image_153.png" /><figcaption>Bla g 4</figcaption></figure>

<figure><img src="image_154.png" /><figcaption>Bla g 5</figcaption></figure>

**Group 4** Bla g 4 and Per a 4 are lipocalins. These molecules are very stable, and their structure consists of a C-terminal α-helix and a β-barrel enclosing an internal hydrophobic cavity that binds small ligands such as retinoids, glucocorticoids, and pheromones (Figure 62).

**Group 5** Bla g 5 is a sigma class glutathione S-transferase (GST) that has a high IgE response in cockroach sensitized individuals (Figure 62). Cross-reactivity with GSTs of several sources (for example, Der p 8 and Tyr p 8) is known. However, lack of significant IgE cross-reactivity among GSTs from cockroach, mite and Ascaris was found in a temperate US population.⁷⁰⁸

**Group 6** The allergens of this group are homologues to insect troponin C and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium-binding domains. Interestingly, IgE binding to Bla g 6 has proven to be calcium dependent indicating that IgE preferably binds to one of the conformers.⁷⁵²

**Group 7** Invertebrate tropomyosins are important pan-allergens among dust mites, chironomids, silverfish, crustaceans, nematodes and mollusks. Tropomyosins from *B. germanica* and *P. americana* have been described. IgE-binding frequency to cockroach tropomyosins are very different in some populations and this may reflect differences in environmental conditions (see chapter C05).

**Group 8** Bla g 8 shares 81-84% amino acid sequence identity with the myosin light chain of several insects and the shrimp *Litopenaeus vannamei*. The myosin regulatory light chains are small acidic polypeptides noncovalently bound to the neck region of the myosin head, which regulate the interaction of the myosin head with actin.

**Group 9** Per a 9 and Bla g 9 were identified as major allergens in Thai and US patients, respectively.⁷⁵³,⁷⁵⁴ Arginine kinase homologues have also been reported in the shrimp *Penaeus monodon* (Pen m 2), *D. pteronyssinus* (Der p 20) and the Indian meal moth *Plodia interpunctella* (Plo i 1). There is evidence suggesting that arginine kinase is an invertebrate pan-allergen.⁷⁵⁵

(Pen m 2), *D. pteronyssinus* (Der p 20) and the Indian meal moth *Plodia interpunctella* (Plo i 1). There is evidence suggesting that arginine kinase is an invertebrate pan-allergen.⁷⁵⁵

**Group 10** Per a 10 is a serine proteases isolated from P. americana and an important allergen in Indian allergic patients.⁷⁵⁶ Other important allergens are also serine proteases (Der f 3, Der p 3, Der p 6 and Der p 9), but cross-reactivity between cockroach and mite serine protease is unlikely due to low amino acid identities (32-41%) among these molecules.

**Group 11** Bla g 11 shares 56% sequence identity with pig α-amylase and with group 4 mite allergens Blo t 4 (50%), Der p 4 (50%) and Eur m 4 (47%). Bla g 11 seems to be an important novel allergen because the recombinant α-amylase inhibited 55% of specific IgE of German cockroach extract.⁷⁵⁷ The Per a 11 allergen has been described in China.⁷⁵⁸

**Group 12** These allergens are chitinases, essential for digestion of chitin. Per a 12 and Bla g 12 have been reported in Chinese and US populations, respectively, with very different IgE prevalence (Table 24).⁷⁵⁸,⁷⁵⁹ Their amino acid identities with the house dust mite chitinases Der p 15 and Der f 15 are low (~35%).

**Group 13** Allergens from this group are glyceraldehyde-3-phosphate dehydrogenases and have only been reported for *P. americana* as Per a 13.

## 4 – Sensitization to individual molecules and its clinical relevance

Cockroach allergens are strong inducers of sensitization and asthma⁷⁶⁰,⁷⁶¹ and cockroach allergy is an important risk factor for emergency room visits and hospital admissions. Most characterized
allergens are from *B. germanica* and *P. americana*, although homologous from other species have been purified. Satinover S et al. found that *Bla* g 2 and *Bla* g 5 have the highest IgE prevalences among five cockroach allergens tested in US patients,⁷⁴⁶ but there are important differences in the profiles of IgE reactivity among individual patients and populations worldwide. Three recent studies in US cohorts, using a larger set of 8-10 cockroach allergens, highlight that other allergens (*Bla* g 3, *Bla* g 6, *Bla* g 9, *Bla* g 11) also show high prevalence among subjects highly sensitized to cockroach.⁷⁵⁴,⁷⁵⁹,⁷⁶² One of the main findings from these studies is that there are no immunodominant cockroach allergens for B and T cell reactivity.

In a study performed in Taiwan to determine whether sensitization to different cockroach allergenic components correlates with different clinical manifestations and severities, eight *P. americana* allergens (Per a 1 through Per a 7 and Per a 9) were evaluated. IgE binding to Per a 2 was more frequent in patients with persistent asthma than in patients with rhinitis only, suggesting that this allergen could be a marker for more severe airway disease. Also, IgE to Per a 9 was strongly associated with rhinitis.⁷⁶³

The availability of cloned and purified allergens is allowing further investigation of their particular effects on the immune responses and the possibilities to be used as reagents for component-resolved diagnosis (CRD) and markers of severity and response to treatment. A recent study reported unique B and T cell reactivity patterns to 10 cockroach allergens per subject, without correlation with clinical phenotype/disease severity.⁷⁵⁹ The B cell reactivity to an expanded set of 8 cockroach allergens was compared in the URECA cohort between subjects with asthma and rhinitis, and subjects without these diseases. Recognition of more cockroach allergens with higher allergen-specific IgE levels was associated with asthma and rhinitis.⁷⁶²

## Clinical relevance

* Cockroach allergens are strong inducers of sensitization and asthma

* Sensitization to cockroach allergens needs to be further investigated in asthmatic patients worldwide

* In some places co-exposure to cockroaches and mite allergens occurs

* CRD could help to detect genuine sensitization to mite and cockroach allergens

In the US cockroach allergy was first recognized as a risk factor for asthma in 1979 in Kansas City, MO.⁷⁶⁴ Subsequently, it became clear that this group of insects, but particularly, the German cockroach, *Blattella germanica*, was an important cause of sensitization in many large cities.⁷⁶⁵⁻⁷⁶⁷ However, even within the US there are large regional variations related to climate and housing conditions.

Notably, the large Inner City Asthma Study published in the New England Journal in 1997 found that among children cockroach allergy and exposure to cockroach allergens were more relevant for asthma morbidity than dust mite.⁷⁶⁸ On the other hand, it is important to realize that that study was largely carried out among children living in apartments in Chicago and New York, where the winters are very dry, which impairs growth of dust mites. By contrast, in most southern towns, including Atlanta, Wilmington DE, and Dallas, particularly in areas of single housing, the pattern of sensitization is consistent to both cockroach and dust mite allergens.⁷⁶⁵⁻⁷⁶⁷ A further element of this pattern was that cockroach infestation is not common in single homes, as distinct from large apartments, unless there are at least eight months of the year where these insects can live outdoors. Thus, it is easy to see why cockroach infestation of houses and sensitization of children to cockroach allergens are significant factors in towns from tropical or sub-tropical areas and completely absent in Scandinavia and rare in suburban areas of northern states in the USA and in most northern areas of Europe. In Southern China and Korea, sensitization to cockroach allergens has been reported, sometimes associated with co-sensitization to shrimp and moth.⁷⁶⁹ The important practical fact is that home interventions to reduce exposure is possible. Since mite and cockroach co-exposure is common, a differential CRD of sensitization might be necessary. In some populations (e.g., from Colombia), there is cross-reactivity between *Bla* g 5 and other Glutathione-transferases (GST), such as Der p 8 and or Asc I 13. In these areas, the high correlation between IgE antibodies to *Bla* g 5 and *Ascaris lumbricoides* GST (Asc I 13) suggests the presence of cross-reactivity between these molecules. However, the frequency of sensitization to Asc I 13 and *Bla* g 5 in a tropical Caribbean population is only around 23% and, in comparison to that of the mite allergen Der p 2, the strength of the IgE response to these allergens was low, which makes it difficult to assess cross-reactivity.⁷⁷⁰ By contrast, no significant cross-reactivity was found among cockroach, mite and *Ascaris* GST allergens in temperate areas of the US. This result is consistent with low amino acid sequence identity at the level of the allergen molecular surfaces, despite sharing a similar three-dimensional structure.⁷⁰⁸ The clinical importance of potential cross-sensitization between helminth and cockroach GSTs should be further investigated.

*Bla* g 7 and Per a 7, two of the cockroaches' tropomyosins, are pan-allergens and positive correlations between shrimp, cockroach, and dust mite IgE levels have been described. In this study, high exposure to cockroach in the home showed significant correlation to higher IgE levels to cockroach and shrimp but not to mite. Sensitization rates to tropomyosins, including the ones from mites and cockroaches, are low in the US and Europe and high in tropical countries,⁷⁷¹,⁷⁷² more likely because of cross-reactivity with helminth tropomyosins.⁷⁷³⁻⁷⁷⁵ Arginine kinases have been described as allergens not only in seafood and other sources but also in cockroaches (Per a 9) and mites (Der p 20). Figure 63 shows predicted (thin lines) and experimentally confirmed (solid lines) cross-reactivity of *B. germanica*. Potential species-specific components are also shown (no lines).