Olive Pollen Allergens

Chapter 110

Olive Pollen Allergens: An Insight into Clinical, Diagnostic and Therapeutic Concepts of Allergy Eva Batanero, Rosalía Rodríguez and Mayte Villalba Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain

110.1  Introduction Type I allergy is a major public health problem that affects the quality of life of millions of children and adults, and its prevalence has dramatically increased in many countries throughout the last few decades, particularly in those that are industrialized, where it affects more than 25% of the population. This disorder is characterized by raised IgE antibody levels to otherwise harmless environmental antigens (the so-called allergens), which are responsible for the clinical symptoms such as asthma, eczema, rhino­ conjunctivitis or anaphylactic shock, due to the activation of cells in latter encounters between the allergen and the body (Figure 110.1). In Mediterranean countries and some areas of America, South Africa, Japan and Australia, olive (Olea europaea) pollen constitutes one of the most important causes of pollinosis (Liccardi et al., 1996). This species sheds its pollen in high concentrations (reaching a weekly average of 500 grains/m3 and exceptional daily peaks higher than 5000 grains/m3 in some areas of southern Spain) during the pollination season, leading to allergic symptoms from seasonal rhinoconjunctivitis to asthma in susceptible individuals. The onset of allergic symptoms in pollen­sensitive patients is often related to the number of airborne pollen grains. For olive, the threshold level of airborne pollen required to elicit clinical symptoms in sensitized patients is extremely high, around 400 grains/m3, compared to the 50 grains/m3 for patients allergic to grass pollens (Quiralte et al., 2007). However, the threshold level appears to vary among sensitive patients and during the pollination season. Over the last few years, intense efforts have been made to define the allergenic components (allergogram) of olive pollen (Rodríguez et al., 2007a), after pioneering studies in the 1980s (Blanca et al., 1983). The knowledge of the complete olive pollen allergogram would enable us to Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3

understand the mechanisms involved in the ­ development of pollinosis as well as to design novel strategies for an accurate diagnosis and a safer and more effective immunotherapy. Standard laboratory techniques have shown that olive pollen extract has a very complex and heterogeneous allergogram, containing at least 20 protein bands with allergenic activity (Figure 110.2). To date, ten allergens have already been identified, isolated and characterized – named Ole e 1 to Ole e 10 according to the recommendations published by the International Union of Immunological Societies. However, new allergens are ­ waiting to be detected and identified. In this sense, the advances in proteomic tools – e.g. two-dimensional electrophoresis in combination with mass spectrometry – have allowed the detection of two minor components of the extract and their identification as allergens with clinical significance (Rodríguez et al., 2007b), and their studies are being carried out at present: Ole e 11 and Ole e 12 (unpublished data). Several olive allergens have been reported as major allergens, because they exhibit prevalence higher than 50%, such as Ole e 1 and Ole e 4. However, the prevalence of olive pollen allergens (major versus minor allergens) are related with the levels of airborne pollen, and therefore, with the geographical area of the sensitized population (Rodríguez et al., 2001; Barber et al., 2007; Quiralte et al., 2007). While Ole e 1 seems to be the only relevant allergen involved in olive sensitization in areas of low/intermediate exposure, minor allergens (prevalence 50%) that rarely sensitized allergic patients in normally exposed areas, become major allergens (e.g. Ole e 6, Ole e 7, Ole e 9 and Ole e 10) in locations with high levels of exposure. According to this fact, it has been suggested that allergic patients from areas with extremely high levels of exposure exhibit different and more complex allergograms, as determined by molecular diagnosis, when compared with

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Section  |  II  Immunology and Inflammation

Figure 110.1  Mechanism of type I allergy. Allergy involves two temporally different processes: sensitization and provocation. In an initial exposure to the allergen of susceptible individuals, the uptake of allergen by professional antigen-presenting cells (APCs) leads to the activation of allergen-­specific T helper 2 (Th2) cells which produce key cytokines. These cytokines are involved in the class-switching of B cells to IgE synthesis. These antibodies specifically bind to a high-affinity receptor on effector cells (mast cells and basophils), resulting in allergic sensitization. Subsequent encounters with the allergen cause cross-linking of effector cell-bound IgE (provocation stage), leading to the cell activation and the rapid secretion of a wide array of mediators responsible for the allergic symptoms.

Figure 110.2  Allergogram of olive pollen. Olive pollen proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extract and stained with Coomassie blue (CB) or detected with individual sera from patient allergic to olive pollen (IgE). The IgE-binding patterns were selected as representative for variability and complexity of responses in individuals suffering from olive pollinosis. Ribbon diagram of 3-D structures of Ole e 6 (Treviño et al., 2004) and C-terminal domain of Ole e 9 (Treviño et al., 2008) are shown.

patients living in areas with lower pollen count. Regarding allergogram heterogeneity, 45 different allergograms were observed when eight olive pollen allergens were tested in 156 patients from Jaén with olive pollinosis (Quiralte et al., 2007). The main properties of olive pollen allergens are summarized in Table 110.1. Olive pollen allergens have been

classified into seven of the 29 pollen allergen families, according to their physicochemical properties. Olive pollen allergens reveal to be restricted to a small number of taxonomically diverse plant families such as Ole e 1, or are ubiquitous (pan-allergens) such as Ole e 2 (profilin) and Ole e 3 (polcalcin). Pan-allergens constitute families of homologous and structurally related proteins from different species

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Table 110.1  Olive pollen allergens and their main features. Olive pollen allergens Allergen

MW (kDa)1

Pl2

Prevalence (%) Cross-reactivity

Ole e 1

16.3/18.5*

5.5–6.5*

55–90

Ole e 2

14-16**

5.1**

Ole e 3

9.2*

Ole e 4

Family

Recombinant expression

Oleaceae

Ole e 1-like

E. coli, P. pastoris

24

Pollens, foods and latex

Profilin

E. coli

4.3*

20–30

Pollens

Polcalcin

E. coli, A. thaliana

32**

4.6-5.1*

80

ND

Unknown

–

Ole e 5

16**

4.2*

35

ND

Cu/Zn-superoxide dismutase

E. coli

Ole e 6

5.8*

14.6**

10–55

Oleaceae (PD)

Unknown

P. pastoris

Ole e 7

10*

9*

473

Pollens (low) (PD)

Lipid transfer protein

–

Ole e 8

18.8*

4.5**

5

Oleaceae (PD)

Ca 2-binding protein

E. coli, A. thaliana

Ole e 9

46.4*

4.8–5.4*

653

Pollens, foods and latex

1,3--glucanase

P. pastoris (CtD and NtD)

Ole e 10

10.8*

5.8*

553

Pollens, foods and latex

Carbohydrate-binding module 43

P. pastoris, S. frugiperda

1 Molecular mass determined by mass spectrometry (*) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (**). For Ole e 1, molecular masses of non-glycosylated/glycosylated forms are shown. 2 Isoelectric point (pl) determined experimentally (*) or deduced from the amino acid sequence (**). 3 Data from allergic patients living in a region with high exposure to olive pollen. ND, not determined; PD, preliminary data.

responsible for extensive IgE cross-reactivity among a variety of allergic sources. Interestingly, the prevalence of panallergens Ole e 2 and Ole e 3 is usually low (around 20%), indicating that they might not be relevant olive allergens and that sensitization to them might be caused by other sources. Biochemical and molecular studies to characterize olive pollen allergens have shown that polymorphism is a general feature. Ole e 1, Ole e 5 and Ole e 7 present a high degree of polymorphism. For Ole e 9, a relatively low, although still significant, degree of polymorphism has been detected. Allergen polymorphism is closely related to the cultivar origin of olive pollen (Alché et al., 2007), as it has been described for other sources of plant allergens such as date palm (Phoenix dactylifera) or birch (Betula pendula) pollens and apple (Malus domesticus). In this context, it has been speculated that broad polymorphism could be involved in the physiology of the olive reproductive system, including the adaptation of the plant to different environmental conditions, the establishment of the compatibility system, and pollen performance (Alché

et al., 2007). Regarding the clinical implications of allergen polymorphism, the ­ differences in allergen composition in cultivars, particularly in Ole e 1, are responsible for important differences in allergenic activity (Alché et al., 2007). The concentration of Ole e 9 has also been reported to vary several hundred times between different pollen batches. Preliminary studies regarding the levels of expression for Ole e 2, Ole e 3, Ole e 5 and Ole e 6 in the major olive cultivars indicate the presence of significant differences (Alché et al., 2007). This is a major concern for clinicians since reliability of pollen extracts used for clinical purposes is required for an accurate diagnosis and effective and safe immunotherapy. Pollen extracts should imitate as much as ­possible the composition of the panel of allergens to which patients are normally exposed and are reactive. Therefore, the quantification of both major and minor allergens must be an integral part of the standardization of olive pollen extracts. Furthermore, it has been reported that olive pollen allergens are quickly released in different rates from pollen, having an impact on sensitization and the elicitation

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of allergic symptoms. High yields of Ole e 1, Ole e 6 and Ole e 7 are obtained after 15 minutes of pollen hydration in mild saline buffers, and others such as Ole e 3, Ole e 9 and Ole e 10 are completely extracted after 3 hours. This property facilitates the accessibility of the proteins once the pollen grains come in contact with respiratory mucosa, explaining the fast induction of allergic symptoms. Olive pollen allergy is a complex disease which could result from the interaction between environmental and genetic factors (Cárdaba et al., 2007; Quiralte et al., 2007). A strong association between HLA class II antigens DR7 and DQB2 and the IgE response to Ole e 1, Ole e 2, and Ole e 3 has been reported in unrelated populations. HLADR2 antigen is associated with the IgE response to Ole e 10. Other factors brought about by human civilization, including atmospheric pollution, exposure to tobacco, lifestyle related to diet and hygiene habits, may also have considerable effects on pollen allergy. Thus, olive pollen allergy represents an interesting model to study the allergic response; therefore, the identification of allergens of olive pollen will be required for the development of rational strategies for standardization, patient diagnosis and therapy which may increase the quality of life of allergic patients.

110.2  Ole e 1 as a marker for sensitization to Oleaceae pollens Ole e 1 is the main allergen of olive pollen with a ­prevalence ranging from 55 to 90%, depending on the geographical areas. It is the most abundant protein of olive pollen, representing up to 20% of the total protein content of pollen in the most profuse varieties. Ole e 1 contributes significantly to the total allergenicity of the olive pollen extracts, and its concentration correlates well with the total allergenic potency of the extracts. Moreover, it also represents the main sensitizing agent within the protein family of Ole e 1-like proteins, and it is a diagnosis marker for sensitization to Oleaceae pollens (Palomares et al., 2006a). Ole e 1 is a polymorphic and acidic (isoelectric point, pI, 5.5–6.5) glycoprotein of 145 amino acid (aa) residues with a glycan at position asparagine-111 (N-glycan) (Villalba et al., 1993; Batanero et al., 1994). Because of the presence of the glycan, Ole e 1 exhibits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a pattern of multiple bands with two main components, a glycosylated form of 20 kDa (85% of the whole allergen) and a non-glycosylated variant of 18.5 kDa (10%). Two minor variants corresponding to the hyperglycosylated (22 kDa) and the 20 kDa-dimer (40 kDa) are frequently present in Ole e 1 preparations. Its single polypeptide chain contains six cysteine residues which are involved in three disulfide bridges: Cys19-Cys90, Cys22-Cys131, and Cys43-Cys78 (González et al., 2000).

Section  |  II  Immunology and Inflammation

Ole e 1 belongs to a large family of homologous proteins (Ole e 1-like), which are specifically expressed in pollen tissue, and it has been suggested to be involved in fertilization events: pollen hydration and/or pollen germination (Villalba et al., 1994; Alché et al., 2007). This is in agreement with Ole e 1 localization in the endoplasmic reticulum, pollen wall and tapetum, and in the outer region of the pollen exine. This family comprises both allergenic members such as Ole e 1, Fra e 1 (Fraxinus excelsior), Lig v 1 (Ligustrum vulgare), Syr v 1 (Syringa vulgaris) – members of Oleaceae family, Pla l 1(Plantago lanceolata), Che a 1 (Chenopodium album), Lol p 11 (Lolium perenne) and Phl p 11 (Phleum pratense), as well as non-allergenic members such as BB18 from Betula verrucosa. Other members of the family are known through their corresponding nucleotide sequences and their derived mature proteins have not been isolated or characterized; therefore, their potential allergenicity has not been explored: LAT52 (Lycopersicon esculentum), Zmc13 (Zea mays), OSPG (Oryza sativa), and putative proteins from Phalaris coerulenscens, Sambucus nigra and Arabidopsis thaliana. The position of the six cysteine residues is conserved in all members, suggesting similar three-dimensional (3-D) structures. In addition, all these proteins exhibit a putative N-glycosylation site, which seems to be conserved among members of the same taxonomic family. This fact could be related to a potential role for the N-glycan in modulation of the biological function and/or discrimination between species. A high degree of cross-reactivity has been described among Oleaceae pollens, and Ole e 1-like proteins are one of the molecules involved in such process (Rodríguez et al., 2001, 2007b). Recently, it has been demonstrated that epitopes of Ole e 1 are only present in Oleaceae pollens but not in unrelated ones (Palomares et al., 2006a). This could be explained by comparing the amino acid sequence of the members of this family: identity values ranging from 86% to 89% are obtained for the Oleaceae counterparts, whereas low but significant identities (32% to 39%) are displayed in the remaining members. Moreover, isoforms of Ole e 1 and Ole e 1-like allergens from Oleaceae, which differ only in a few amino acids, show differences in both IgE and IgG reactivity. Finally, ash pollen is an important cause of allergy in Central Europe, whereas privet and lilac (two ornamental plants) have been described as elicitors of allergic symptoms in conditions of local exposure (Liccardi et al., 1996). Thus, the study of Ole e 1-like allergens will provide knowledge of the molecular organization of allergenic epitopes which is invaluable in terms of allergen standardization and diagnostics as well as in the designing of novel allergen-specific immunotherapy. T-cell (Cárdaba et al., 2007) and B-cell (González et al., 2006) epitopes of Ole e 1 have been analyzed. Cárdaba et al. have defined the regions 91–102 and 109–130 of Ole e 1 as immunodominant T-cell epitopes, but display no IgE-binding capacity. At least four B-cell epitopes have

Chapter  |  110  Olive Pollen Allergens: An Insight into Clinical, Diagnostic and Therapeutic Concepts of Allergy

been defined by means of 12-aa overlapping ­ synthetic ­peptides and recombinant large fragments, being the C-terminal an immunodominant region and the tyrosine at position 141 a critical residue for IgE binding (González et al., 2006).

110.3  Ole e 2 and Ole e 10, two allergens associated with asthma Quiralte et al. (2007) reported that Ole e 2 and Ole e 10 show a statistically significant association with asthma. Ole e 2, an acidic (pI 5.1) 16 kDa protein, belongs to the well-known pan-allergen family of profilins (Ledesma et al., 1998a). Its molecular and immunological properties did not differ from those of other profilins. Ole e 2 exhibits polymorphism, with important implications for the 3-D structure of the molecule (Alché et al., 2007). Profilins contain a large number of members from plant and animal tissues which are involved in the assembly of actin filaments, being recognized as pan-allergens from fruits, vegetables, pollens and latex. Interestingly, the comparison of plant profilins with homologous from fungi and vertebrates revealed that they form a highly conserved family with sequence identities between 70% and 85% among each other, but low identities (30% to 40%) compared with non-allergenic profilins from other eukaryotes, including human beings. This explains their implication in cross-­reactivity between profilins of different vegetable sources, and their low clinical relevance (around 20% of prevalence). However, because of sensitivity to thermal denaturalization and gastric proteolysis, they are involved in the oral allergy syndrome (OAS), whose symptoms, limited to the oropharyngeal mucosa, are elicited after eating raw foods. Ole e 10, a Cys-rich small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from olive pollen in high-exposure areas (Barral et al., 2004a). Ole e 10 shows identity with deduced sequences from Arabidopsis thaliana genes (42–46% identity), with the non-catalytic C-terminal domain of plant 1,3--­glucanases (27–53% identity) such as Ole e 9, and with Cys-box domains from three families of 1,3--glucanosyltransferases involved in yeast development: Epd1, Gas-1p and Phr2 families (23% identity) (Barral et al., 2004a). However, it is important to remark that Ole e 10 is an independent protein that defines a novel family of carbohydrate-binding modules (CBMs), so-called CBM43 (Barral et al., 2005a). The ability of Ole e 10 to bind specifically 1,3--glucans, its localization within the mature pollen grain inside Golgiderived vesicles and its co-­localization with callose (1,3-glucans) in the growing pollen tube suggest a role for this protein in the pollen tube wall re-­formation during germination (Barral et al., 2005a). Regarding its allergenic activity, Ole e 10 is an allergen per se that can act as a primary

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sensitizer: it defines a new family of pan-allergens that shows notable intra- and interspecies cross-reactivity, and is a powerful candidate for pollen-latex-fruit syndrome (Barral et al., 2004a). Although it is a relevant allergen, Ole e 10 exhibits low intrinsic antigenicity because no specific IgG antibodies were elicited after different attempts to immunize animals with pure antigen (Barral et al., 2004a). This fact, added to its biological activity and retardation release from pollen after hydration allow us to speculate that this allergen could be expelled from the pollen linked to particles such as 1,3--glucans – it has been shown that this glycan promotes an airway allergic response in humans. In this way, the allergenic potential of Ole e 10 could be increased, contributing to eliciting asthma in allergic patients.

110.4  Ole e 3 and Ole e 8:   Ca2-binding allergens Ole e 3 and Ole e 8 are two Ca2-binding proteins (CaBPs) of the EF-hand family. Ole e 3 is a small (9.2 kDa) and acidic protein (pI 4.3) with a single polypeptide chain which does not contain cysteines (Batanero et al., 1996). Ole e 3 belongs to the widespread polcalcin family, which is characterized by its specific expression in pollen and the presence of two EFhand motifs (Ledesma et al., 1998b). Polcalcins belong to the buffering type CaBP subfamily and may have a role as inhibitors of cytoplasmic streaming of Ca2 in growing pollen tubes (Ledesma et al., 1998b). The reported prevalence for this family of pan-allergens varies between 5–46% and it is around 20–30% for Ole e 3. Members of this family show low or no polymorphism and sequence identities ranging from 64% to 92%, which explains their strong cross-reactivity. Thus, diagnosis of polcalcin­sensitized patients could be achieved whatever polcalcin used, whereas for immunotherapy, the polcalcin that acts as the primary sensitization agent should be used. Ole e 8 (18.75 kDa and pI 4.5) seems to be the first member of a new family of CaBPs with four EF-hand motifs present in the pollen tissue: it is a calcium-sensor protein which should display a regulatory function and perhaps may be involved in signal transduction pathways (Ledesma et al., 2000). This allergen is present at very low levels in the pollen (0.02–0.05% of total protein), and shows low prevalence (5%) (Ledesma et al., 2002a). In addition, all sera reactive to Ole e 8 also recognize Ole e 3 (Ledesma et al., 2000). Ole e 8 shows a low sequence identity with Ole e 3 and other CaBPs out of the EF-hand sites. Moreover, this allergen seems not to have counterparts with conserved amino acid sequence in other taxonomically non-related allergenic pollens, as significant crossreactivity was observed only with Oleaceae (Ledesma et al., 2002a).

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It is important to note that EF-hand motifs of the Ca2binding allergens have a non-significant role in their IgE and IgG epitopes, and cross-reactivity; however, their IgEbinding capacity is affected by the conformational change induced by the binding/releasing of Ca2 ions (Ledesma et al., 2002b). This property would allow designing hypo­ allergenic derivatives to be used in immunotherapy.

110.5  Ole e 7, a non-specific lipid transfer protein, and its   clinical significance Ole e 7 is a basic protein (pI  9) with molecular mass around 10 kDa (Tejera et al., 1999). Although it is a minor allergen, its prevalence increases up to 47% in populations exposed to high levels of olive pollen. Interestingly, a large number of adverse reactions are recorded in patients sensitized to Ole e 7 as well as to Ole e 9; these patients are less tolerant to immunotherapy at the recommended allergen doses (Barber et al., 2007). Ole e 7 belongs to the non-specific lipid transfer proteins (nsLTP) family of around 9 kDa ubiquitously distributed through the plant kingdom, and whose members show sequence identities from 47% to 92% (Salcedo et al., 2007). All of them conserve a pattern of eight cysteines, forming four disulfide bridges which are essential for the compact fold of nsLTPs in the characteristic all--type structure and, therefore, the lipid-binding and allergenic properties of these proteins. Interestingly, other major groups of plant food allergens, the 2S albumins and cereal -amylase/trypsin inhibitors, show 3-D structures close to nsLTP fold; these three groups constitute the prolamin superfamily. nsLTPs bind different types of lipids and are involved in plant defense mechanisms against phytopathogenic bacteria and fungi – becoming members of the ­pathogenesis-related 14 (PR-14) protein family – and probably in the assembly of hydrophobic protective layers of surface polymers such as cutin. Several members of the nsLTP family have been identified as relevant allergens in plant foods and even latex and pollens (Salcedo et al., 2007). Because of their ubiquitous distribution in different plant species and tissues, nsLTPs have been proposed as a novel pan-allergen with a potential spectrum of cross-reactivity comparable to that reported for profilins. Interestingly, nsLTP sensitization shows an unexpected pattern throughout Europe, with high prevalence in the Mediterranean area, but a low incidence in Northern and Central Europe. Genetic factors, differences in dietary habits and level and composition of pollen exposure could account for such differences. Food nsLTPs are considered to be ‘true’ food allergens that sensitize directly via the oral route and are responsible for the induction of severe symptoms in many patients. These features seem to be related to their high resistance

Section  |  II  Immunology and Inflammation

to both heat treatment and digestive proteolyis. Moreover, cross-reactivity among food nsLTPs allergens from botanically related and unrelated species has been described (Salcedo et al., 2007). However, concerns exist about whether pollen nsLTP allergens can act as a primary sensitization agent itself via the respiratory tract leading to food allergy because of cross-reactivity to food nsLTPs. Three different types of pollen nsLTP allergens can be defined according to their cross-reactivity with those from foods and their prevalence among pollinic patients (Salcedo et al., 2007). Regarding Ole e 7, our preliminary data indicate that it does not cross-react with foods in spite of the association of this allergen with plant-derived food anaphylaxis reported by Florido et al. (2002): only a very limited cross-reactivity with related and unrelated pollens has been observed (unpublished data). Future studies will help to clarify the different routes of sensitization and geographic patterns of sensitization to nsLTPs.

110.6  Ole e 9 and pollen-latex-fruit syndrome Ole e 9 is a 1,3--glucanase belonging to the PR-2 protein family whose enzymatic activity has been shown (Huecas et al., 2001). It exhibits low sequence identity (32–39%) to long 1,3--glucanases from plants. It is an acidic (pI 4.8– 5.4) and polymorphic glycoprotein (46.4 kDa) composed of two structurally and immunologically well-defined domains which are connected by a segment of 10–15 aa (Palomares et al., 2003, 2005). The N-terminal domain (NtD) of 334 aa contains the 1,3--glucanase activity, and its 3-D modeling fits well to a triose-phosphate isomerase (TIM)-barrel structure common to all known 1,3--­glucanases. The C-terminal domain (CtD), with around 100 aa, is a CBM43 which shows sequence identity with 1,3--glucanases from plant tissues, the Epd1/Gas-1p/Phr2 protein families and Ole e 10 (Palomares et al., 2003). Its capacity to bind 1,3-glucans suggests a role in the binding of the substrate (Rodríguez et al., 2007b). Disulfide bridges of the molecule have been determined at positions Cys14-Cys76, Cys33-Cys94, and Cys39-Cys48 (Palomares et al., 2003). Recently, its 3-D structure has been resolved, representing a novel type of allergen folding which consists of two parallel -helices, a small antiparallel -sheet and a 3–10 helix turn, all connected by long coil segments (Figure 110.2) (Treviño et al., 2008). Moreover, the CtD-epitope mapping shows that B-cell epitopes are mainly located on the loops (Treviño et al., 2008). Ole e 9 is a major allergen in populations living in highly exposed areas with a prevalence of 65% (Huecas et al., 2001). The ubiquity of 1,3--glucanases in higher plants suggests that they could be involved in the ­ pollenlatex-fruit syndrome. This is supported by a study showing the involvement of NtD in cross-reactivity among

Chapter  |  110  Olive Pollen Allergens: An Insight into Clinical, Diagnostic and Therapeutic Concepts of Allergy

pollens, vegetable foods and latex (Palomares et al., 2005). Moreover, it has been shown that NtD may be a useful marker of disease in diagnosis of pollen-allergic patients at risk of developing allergic symptoms to other vegetable sources (Palomares et al., 2006b). CtD has been described as a marker for patients who could develop asthma (Palomares et al., 2006b). Recently, Ole e 9 has been identified as the causative agent of occupational rhinitis in a researcher (Palomares et al., 2008a).

110.7  Other allergens from olive pollen: Ole e 4, Ole e 5 and Ole e 6 Ole e 4 is a partially characterized polymorphic allergen with an apparent molecular mass of 32 kDa and an acidic pI (4.6–5.1) (Boluda et al., 1998). However, it is not clear whether Ole e 4 is a genuine allergen or a proteolytic degradation product of Ole e 9 since all of the peptides obtained for the protein aligned with segments of Ole e 9. Ole e 5 (16 kDa) is the Cu/Zn-superoxide dismutase (SOD) of olive pollen, with an 80–90% sequence identity with Cu/Zn-SODs from other plants (Boluda et al., 1998; Butteroni et al., 2005). SODs catalyze the dismutation of superoxide anions into molecular oxygen and hydrogen peroxide. It has been suggested that pollen antioxidant systems such as Ole e 5 could play a role in pollen–stigma interactions and defense because of the constitutive accumulation of reactive oxygen species/H2O2 in angiosperm stigmas (Alché et al., 2007). Ole e 5 is a minor allergen with prevalence around 35%; however, it could be involved as a putative cross-reactive allergen in the pollen-latex-fruit syndrome due to the ubiquity of this enzyme family and its sequence identity with the allergenic Cu/Zn-SOD from tomato fruit. Moreover, it has been suggested that the allergenic Mn-SOD from latex (Hev b 10) could be involved in cross-reactivity with SODs from related and unrelated species. Ole e 6 is a small (5.83 kDa), acidic (pI 5.8) and Cysrich allergen whose prevalence is very dependent on the degree of pollen exposure, ranging from 10% to 55% (Batanero et al., 1997). It displays a peculiar twice-repeated cysteine motif (Cys-X3-Cys-X3-Cys) which is also present in the amino acid sequence deduced (107 aa) from Tap1, a stamen-specific gene from snapdragon (Antirrhinum majus) (Batanero et al., 1997). The 3-D structure of Ole e 6 has been resolved and consists of two parallel -helices joined together by three disulfide bridges (Figure 110.2) (Treviño et al., 2004). Ole e 6 shows 59% sequence identity with a Cys-rich pollen surface molecule (Ntp-CysR, 63 aa) from Nicotiana tabacum expressed almost exclusively in developing anthers and highly abundant in pollen. It has been suggested that Ntp-CysR could interact with pistil factors. Concerning cross-reactivity, an Ole e 6-like protein has been reported in ash pollen (Rodríguez et al., 2007b),

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and it is expected that homologous allergens exist in other Oleaceae members.

110.8  The role of N-glycans in olive pollen allergy Even though allergologists are sceptical about the clinical significance of glycan-related IgE reactivity, increasing numbers of reports have demonstrated that glycans, as part of an allergen, can elicit IgE antibodies in susceptible subjects. Many of these glycoepitopes behave as pan-epitopes, leading to extensive cross-reactivity among pollens, plant foods and insect venoms because of the presence of common monosaccharide components at specific positions along the chain (Altmann, 2007). Thus, they are so-called ‘crossreactive carbohydrate determinants’. The N-glycan of Ole e 1 is involved in antigenic and allergenic activities, being responsible for cross-reactivity between Ole e 1 and non-related glycoproteins (Batanero et al., 1994, 1999; van Ree et al., 2000). The primary structure of the N-glycan of Ole e 1 has been determined and the presence of glycoforms (different but close related glycan structures at a single N-glycosylation site) has been described (Figure 110.3) (van Ree et al., 2000). Ole e 1 has one major ‘complex’ N-glycan (GlcNAcMan3XylGlcNAc2) and one major ‘high mannose’ N-glycan (Man7GlcNAc2). A minor ‘complex’ N-glycan having an (1,3)-fucose residue attached to the proximal glucosamine residue has also been detected (Batanero et al., 1999; van Ree et al., 2000).

Figure 110.3  N-glycans of Ole e 1. Structures of the major N-glycans isolated from Ole e 1 were determined by 1H NMR and MALDI-TOF. Minor (1,3)-fucosylated N-glycans were also detected. n and n ­indicate  (1,n) and (1,n) linkages (n  2–6).

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It was demonstrated that both (1,2)-xylose and (1,3)fucose are involved in IgE binding, making the involvement of ‘high mannose’ structures in IgE binding unlikely (Batanero et al., 1999; van Ree et al., 2000). It has been suggested that both monosaccharides could account for the immunogenicity of plant glycans in humans since they are absent in human N-glycans. In addition, the free N-glycan of Ole e 1 is able to induce in vitro histamine release from basophils of olive-sensitized patients, confirming the allergenic character of this glycan (Batanero et al., 1999). The role of the N-glycan of Ole e 9 in the allergenicity of the molecule has not been studied so far.

110.9  Recombinant olive pollen allergens as diagnostic and therapeutic reagents Many of the disadvantages associated with the use of allergen extracts from biological sources for diagnosis and treatment of allergy – e.g. difficult allergen standardization, risk of new sensitization and anaphylactic side-effects and endotoxin contamination – can be overcome with the use of recombinant allergens. Various heterologous systems are currently used for the production of recombinant allergens equalling the natural ones as defined molecules in consistent quality and high amount. Most of the olive pollen allergens have been obtained using these methods. Ole e 2, Ole e 3, Ole e 5 (as a fusion protein with glutation S-transferase, GST) and Ole e 8 allergens have been produced in the bacteria Escherichia coli as soluble highyield recombinant proteins (Asturias et al., 1997; Ledesma et al., 1998b, 2000; Butteroni et al., 2005), whereas the production of recombinant Ole e 1 as a fusion protein with GST in this system rendered a poor quality product: it was mainly obtained as insoluble inclusion bodies (Villalba et al., 1994). The yeast Pichia pastoris has been used as a successful expression system for olive pollen allergens with posttranslational modifications (such as glycosylation) and complex folding (including disulfide bridges): Ole e 1 (Huecas et al., 1999), Ole e 6 (Barral et al., 2004b), and the NtD and CtD of Ole e 9 (Palomares et al., 2003, 2005) have been obtained with high yields and they maintain their intrinsic properties. For Ole e 10, better yields and lower degradation of the soluble and functional recombinant protein have been achieved using baculovirus in host insect cells (Spodoptera frugiperda) system (Barral et al., 2006) than with the yeast P. pastoris (Barral et al., 2005b). Finally, Ole e 3 and Ole e 8 have also been produced in stable transgenic plants of Arabidopsis thaliana (Ledesma et al., 2006). These transgenic plants could constitute important tools to design edible vaccines for allergy. Recombinant allergens allow us for the first time to determine the precise sensitization profile (allergogram) of patients, which is a prerequisite to select the allergens for

Section  |  II  Immunology and Inflammation

patient-tailored immunotherapy. It is well established that a panel of a few recombinant allergens is sufficient to diagnose most pollen-allergic patients because of the extensive cross-reactivity. Thus, the first step for this procedure is the selection of the most relevant allergens which contain most of the important B- and T-cell epitopes and represent the originally sensitizing agents within a cross-reacting group, e.g. for Oleaceae pollinosis Ole e 1, the main sensitizing agent of Ole e 1-like family, could be use as diagnostic marker (Palomares et al., 2006a). Moreover, recombinant allergens can be engineered to produce hypoallergens or hypoallergenic derivatives that exhibit reduced or no allergenic activity but preserved T-cell epitopes and immunogenicity for safer forms of immunotherapy. Several recombinant DNA strategies, as well as synthetic peptide chemistry, have been employed to convert allergens into hypoallergenic derivatives: fragments, oligomers, point or deletion mutants, hybrids and T- or Bpeptides. In vitro and in vivo evaluation of hypoallergens is then required to identify the best vaccines for immunotherapy before clinical application. Clinical trials performed with hypoallergenic derivatives as well as recombinant wildtype allergens, have shown that these molecules can be used in immunotherapy in the near future. Hypoallergenic derivatives of Ole e 1 have been engineered based on the disruption of the immunodominant IgE epitope of the C-terminal of the molecule by producing one point and two deletion mutants (Marazuela et al., 2007). In addition, a peptide T of Ole e 1 has been designed based on epitope mapping studies (Marazuela et al., 2008a). To select the most suitable derivatives for immunotherapy they have been tested in vitro and in vivo (Marazuela et al., 2007, 2008a). Finally, recombinant allergens can be used to study the properties of olive pollen allergens, e.g. determination of the 3-D structures of Ole e 6 (Treviño et al., 2004) and CtD of Ole e 9 (Treviño et al., 2008) (Figure 110.2), assignment of disulfide bridges of Ole e 1 (González et al., 2000) and Ole e 9 CtD (Palomares et al., 2003), epitope mapping of Ole e 1 (González et al., 2006) and Ole e 9 (Palomares et al., 2003, 2005; Treviño et al., 2008), or epidemiological studies (Palomares et al., 2006b; Barber et al., 2007; Quiralte et al., 2007). So far, Ole e 1 is the only olive pollen recombinant allergen used in prick-test with patients in a study performed a few years ago, since current Spanish legislation does not allow the use of recombinant molecules on humans, although they may improve diagnosis and treatment of olive pollinosis.

110.10  New concepts for specific immunotherapy using Ole e 1 as   a model Currently, allergen-specific immunotherapy is the only curative treatment available for allergy. Even though this treatment can offer protection, it has several disadvantages including

Chapter  |  110  Olive Pollen Allergens: An Insight into Clinical, Diagnostic and Therapeutic Concepts of Allergy

long duration, anaphylactic side-effects and limited efficacy. Mucosal tolerance induction with nasal vaccines based on free or encapsulated hypoallergenic derivatives is a promising alternative strategy to conventional immunotherapy. A mouse model of IgE sensitization to Ole e 1 mimicking the human B- and T-cell responses has been established for preclinical testing of new vaccines against allergy (Marazuela et al., 2008a). Four prophylactic approaches were conducted to investigate whether nasal tolerance induction with vaccines based on Ole e 1 or derivatives could prevent sensitization in mice. In a first approach, low doses of a nasal vaccine based on a deletion mutant of Ole e 1 were able to protect mice against sensitization to the allergen (Rodríguez et al., 2007a). Recently, Marazuela et al. (2008a) have demonstrated that prophylactic i.n. administration of a peptide T of Ole e 1 may substitute for the whole protein in protecting mice against subsequent sensitization to the allergen. Moreover, specific protection for the long term was maintained. In a third study, it was shown that i.n. administration of micrograms of the peptide T of Ole e 1 encapsulated in poly

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(lactide-co-glycolide) (PLG) microparticles as carrier vaccines prevented subsequent sensitization to the allergen (Marazuela et al., 2008b). In a previous work, PLG microparticles were described as a suitable vehicle vaccine for Ole e 1 that elicits a specific Th1-type response in mice, thus becoming a promising concept for allergy vaccine. During the last few years, exosome-based vaccines have been proposed as a novel strategy for treatment of human diseases including allergy. Exosomes are nanovesicles which are released in the extracellular environment by a variety of cell types and showed immunomodulatory properties. Our group has observed that intranasal administration of tolerogenic exosomes protects mice against sensitization to Ole e 1 (Prado et al., 2008) (Figure 110.4). In this respect, although the four mentioned approaches (using the same ­prophylactic protocol) suppress the most important clinical features of allergy – specific-IgE antibodies in serum, Th2-response and airway inflammation – exosomes have advantages over the previous reported vaccines. They are acellular and stable structures containing a wide array of cellular proteins, some

Figure 110.4  Intranasal pretreatment with tolerogenic exosomes protects mice against allergic sensitization. Exosomes were isolated from bronchoalveolar lavage fluid (BALF) from mice that were tolerized by respiratory exposure to Ole e 1. Exosomes isolated from naïve mice were used as controls. These exosomes were assayed as a preventive vaccine in a mouse model of allergy induced by intraperitoneal (i.p.) sensitization to Ole e 1 followed by airway allergen challenge. Mice were intranasal (i.n.) pretreated for 3 consecutive days with tolerogenic exosomes one week prior to sensitization/challenge with the allergen, and the allergic response was analyzed. Pretreatment with tolerogenic exosomes inhibit both airway inflammation and specific-IgE production. (A) Representative lung section stained with hematoxylin-eosin of tolerogenic exosomes-pretreated mice shows a reduced inflammatory cell infiltration ­compared to sham-pretreated mice that received control exosomes. Magnifications,  20 (ExoTol and ExoCon),  10 (naïve). (B) Serum IgE levels were determined by ELISA. Data are expressed as means  standard error (n  15 mice/group) from three independent experiments. *p  0.001. (Based on data from Prado et al., 2008). ExoTol, mice pretreated with tolerogenic exosomes; ExoCon, animals pretreated with control exosomes; Naïve, no-treated mice.

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of which modulate immune responses. Since exosomes are natural antigen-transferring units between immune cells, they allow cross-presentation and contribute to amplify immune responses reducing the dose of antigen required to induce an immune response. Finally, ‘exosome display technology’ permits manipulation of their protein composition and tailoring for different functions. These studies emphasize the high potential of nasal vaccines against allergy. Despite the clinical relevance to test the therapeutic effects of these vaccines, the possibility of using prophylactic vaccines for early prevention in atopic individuals or children at risk has been proposed.

110.11  Olive fruit: a new source of olive allergens Olive fruit is consumed either directly or through processed products as olive oil. Allergic reactions to olive fruit and its derived products have rarely been documented in literature: few cases of contact dermatitis on workers caused by olive oil, olive-induced urticaria and anaphylactic shock to olive fruits have been reported. Recently Palomares et al. (2008b) have described an olive oil mill worker suffering from occupational asthma (OA) who was sensitized to olive fruit particles by mucosal exposure. Respiratory symptoms were improved when he was away from the workplace, but relapsed at work. A 23-kDa protein, which shows homology to thaumatin-like proteins (TLPs) from plant foods and pollen, has been isolated and identified as the major causative allergen of OA to olive fruits. TLPs belong to the PR-5 family, which play a role in the plant defense system against pathogens or adverse environmental factors. In recent years, TLPs have been recognized as a new class of pan-allergens in food and pollens which could be involved in the OAS.

Summary points Olive pollen is one of the main causes of allergy in Mediterranean countries and some areas of America, South Africa, Japan and Australia. l Ten allergens – named Ole e 1 to Ole e 10 – have been isolated, characterized, and many of them obtained as recombinant molecules. l Recombinant proteins can be used to study allergen properties and the mechanism of allergy and immunotherapy. Moreover, recombinant allergens may improve diagnosis of allergic patients. Finally, hypoallergenic allergen derivatives have been in vivo and in vitro evaluated as vaccines for improved immunotherapy of allergy. l A mouse model of allergy to olive pollen that mimics the features of allergic patients has been established. l Preclinical studies in mice suggest the potential use of nasal vaccines for the treatment of allergy. l

Section  |  II  Immunology and Inflammation

Olive fruit has been identified as a new source of allergens, causing occupational asthma.

l

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