Mayte Villalba

The CREATE Project : development of certified reference materials for allergenic products and validation of methods for their quantification

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE‐binding potencies as their focus. Unfortunately, each company is using their own in‐house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Understanding patient sensitization profiles in complex pollen areas: a molecular epidemiological study

Background:  Allergy diagnosis in patients exposed to multiple pollen species is complex and misdiagnosis is often a cause for unsuccessful specific immunotherapy.

IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e 1

BACKGROUND Pollen from olive trees (Olea europaea ) is a cause of pollinosis and an aggravating of asthma in Mediterranean regions. Recently, Ole e 1, the major allergen from olive tree pollen, has been isolated and its amino acid sequence has been elucidated. It is a glycoprotein whose carbohydrate moiety is involved in an IgE-binding epitope responsible for cross-reactivity among plant glycoproteins. However, the allergenicity of the free carbohydrate side chains remains to be clarified. OBJECTIVE The purpose of this study was to isolate the main carbohydrate component of Ole e 1 allergen and analyze its IgE-binding and histamine-release capabilities. METHODS Deglycosylation treatment of Ole e 1 with PNGase F and gel exclusion chromatography were used to isolate the main sugar component of the allergen. Sera of patients who are allergic to olive pollen and sera sensitive to Ole e 1 have been used in dot blotting assays of IgE binding to the isolated carbohydrate. Heparinized whole blood obtained from patients sensitive to Ole e 1 were stimulated by the free carbohydrate; the resulting histamine release was measured. RESULTS The main sugar component of Ole e 1 has been isolated. Free carbohydrate was able to bind IgE from sera of patients allergic to olive pollen; the sera of 65% of these patients contained anticarbohydrate reacting IgE, and 100% of those patients were sensitive to Ole e 1. The free carbohydrate promoted in vitro histamine release from basophils of sensitized patients. CONCLUSION The carbohydrate moieties of allergenic glycoproteins can constitute significant determinants on the binding to IgE of the sera from patients who are hypersensitive and can be responsible for inducing histamine release from blood cells.

Exosomes from Bronchoalveolar Fluid of Tolerized Mice Prevent Allergic Reaction

Exosomes are nanovesicles originating from multivesicular bodies that are secreted by a variety of cell types. The dual capability of exosomes to promote immunity or to induce tolerance has prompted their clinical use as vehicles for vaccination against different human diseases. In the present study, the effect of allergen-specific exosomes from tolerized mice on the development of allergen-induced allergic response was determined using a mouse model. Mice were tolerized by respiratory exposure to the olive pollen allergen Ole e 1. Exosome-like vesicles were isolated from bronchoalveolar lavage fluid of the animals by the well-established filtration and ultracentrifugation procedure, characterized by electron microscopy, Western blot, and FACS analyses, and assessed in a prophylactic protocol. To this end, BALB/c mice were intranasally treated with tolerogenic exosomes or naive exosomes as control, 1 wk before sensitization/challenge to Ole e 1. Blood, lungs, and spleen were collected and analyzed for immune responses. Intranasal administration of tolerogenic exosomes inhibited the development of IgE response, Th2 cytokine production, and airway inflammation—cardinal features of allergy— and maintained specific long-term protection in vivo. This protective effect was associated with a concomitant increase in the expression of the regulatory cytokine TGF-β. These observations demonstrate that exosomes can induce tolerance and protection against allergic sensitization in mice. Thus, exosome-based vaccines could represent an alternative to conventional therapy for allergic diseases in humans.

Cross-reactivity between the major allergen from olive pollen and unrelated glycoproteins: Evidence of an epitope in the glycan moiety of the allergen ☆ ☆☆ ★ ★★

Ole e 1, the major allergen from olive pollen, is a glycoprotein containing a single Asn-linked glycan moiety. Rabbit antiserum against this protein has been obtained; and its immunologic cross-reactivities in Western blotting with ascorbate oxidase, horseradish peroxidase, bromelain, ovalbumin, and honeybee venom phospholipase A2 have been studied. Ascorbate oxidase, peroxidase, and bromelain are recognized by the Ole e 1 antiserum. When these three proteins are deglycosylated by periodate treatment, such an immunologic reaction does not occur. The relative affinities of these proteins have been analyzed by direct and inhibition ELISA experiments. A commercially available antibody against horseradish peroxidase has also been considered in these studies. This antibody reacts with Ole e 1 but not with the periodate-deglycosylated allergen. Horseradish peroxidase, bromelain, and ascorbate oxidase are recognized by the IgE of sera from patients who are hypersensitive to olive tree pollen. This binding is also abolished by periodate treatment. The results are interpreted in terms of the presence of an epitope in the carbohydrate moiety of Ole e 1, which would contain a xylose involved in recognition by both IgE and IgG antibodies.

The European Union CREATE Project: A model for international standardization of allergy diagnostics and vaccines

Allergen measurements are used extensively in the formulation of allergy diagnostics and vaccines, yet no purified international allergen standards are available for calibration purposes. The aims of the European Union CREATE project were to develop international standards with verifiable allergen content. Purified natural and recombinant allergens were analyzed by means of SDS-PAGE, mass spectrometry, circular dichroism spectra, and small-angle x-ray scattering. IgE reactivity was assessed by means of direct RAST, RAST inhibition, immunoblotting, and basophil histamine release with sera from 961 allergic patients. Three recombinant allergens, rBet v 1, rPhl p 5a, and rDer p 2, were structurally indistinguishable from their natural counterparts and showed excellent IgE reactivity suitable for use as certified reference materials. A second tier of allergens (rPhl p 5b, rOle e1, rDer p 1, rDer f 1, and rDer f 2) was identified that could provide suitable candidates for certified reference materials with minor improvements to the recombinant proteins. Only rPhl p 1 was considered unsuitable as a reference material. Quantitative ELISAs were identified that accurately measured each allergen, except for rPhl p 1. The CREATE project has provided a major step forward in allergen standardization and provides a model for the development of a comprehensive panel of international reference preparations that will harmonize allergen measurements worldwide.

Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.

The Spectrum of Olive Pollen Allergens

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Among the high number of protein allergens detected in this pollen, 8 – Ole e 1 to Ole e 8 – have been isolated and characterized. Ole e 1 is the most frequent sensitizing agent, affecting more than 70% of the patients suffering of olive pollinosis, although others, such as Ole e 4 and Ole e 7, have also been shown to be major allergens. In this context, the prevalence of many olive pollen allergens seems to be dependent on the geographical area where the sensitized patients live. Some of the olive allergens have been revealed as members of known protein families: profilin (Ole e 2), Ca2+-binding proteins (Ole e 3 and Ole e 8), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). No biological function has been demonstrated for Ole e 1, whereas Ole e 4 and Ole e 6 are new proteins without homology to known sequences from databases. cDNAs encoding for Ole e 1, Ole e 3 and Ole e 8 have been overproduced in heterologous systems. The recombinant products were correctly folded and exhibited the functional activities of the natural allergens. In addition to the Oleaceae family, other species, such as Gramineae or Betulaceae, contain pollen allergens structurally or immunologically related to those of the olive tree. This fact allows to detect and evaluate antigenic cross-reactivities involving olive allergens. The aim of this research is the development of new diagnostic tools for olive pollinosis and new approaches to improve the classical immunotherapy.

1,3‐β‐glucanases as candidates in latex–pollen–vegetable food cross‐reactivity

Background 1,3‐β‐glucanases (group 2 of pathogenesis‐related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens.

Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen

BACKGROUND Olive tree (Olea europaea) pollen is an important cause of pollinosis in countries of the Mediterranean area and California. OBJECTIVE The aim of this study was to identify and purify a new allergen of olive tree pollen. METHODS Detection of a pollen allergen was done with individual allergic sera by immunoblotting and ELISA tests. Two allergenic fractions were isolated from olive pollen extract by using gel filtration and reverse-phase HPLC. Molecular characterization was achieved by acid hydrolysis and amino acid analysis, as well as by mass spectrometry. Sequencing of the N-terminal end of the allergen was carried out by Edman degradation of the polypeptide chain. Allergenic characterization was performed with sera from subjects with olive allergy by means of ELISA and immunoblotting after SDS-PAGE. RESULTS The new allergen Ole e 7 exhibits a high degree of polymorphism. Its molecular mass is in the range of 9875 d to 10,297 d. Twenty-one amino acid residues from the N-terminal end of 2 isoforms of the allergen have been sequenced revealing no homology with proteins contained in database banks. Ole e 7 has an average frequency of about 47% in patients with olive allergy. The strategy of purification of Ole e 7 can be useful on the isolation of new allergens. CONCLUSIONS A new olive pollen allergen of clinical significance has been purified and characterized, contributing to the study of the complete allergogram of the olive tree pollen.