ES2326399A1 - Olive pollen allergen-hypersensitivity diagnosis apparatus includes a mixture of extracts of pollens of various olive species, with or without modification - Google Patents

Abstract

The olive pollen allergens hypersensitivity diagnosis apparatus has a mixture of extracts of pollens of olive (Olea europea), wild olive tree and Fraxinus, Ligustrum, Phillyrea Forsytia, Jasminum and Syringa. An Independent claims is also included for a process that employs the mixture as raw extracts with or without modification or e.g. purification and optionally with other allergens and polymerized materials.

Description

Refinements introduced in the object of Spanish patent application No. P 200100995 for: "Kit for  diagnosis of hypersensitivity to allergens from pollen olive tree and its use ".

Field of the Invention

The invention falls within the sector doctor-pharmacist, especially in the field of clinical analysis and immunotherapy. In general, the invention It is related to a system for the diagnosis of pollen allergy of olive tree of a defined variety of olive tree based on the analysis of the reactivity of a subject against a set of extracts of individualized olive pollen from different defined olive varieties. From the diagnosis obtained customized hyposensitizing vaccines can be developed for its administration to a subject in order to treat said allergy to Pollen of one or several defined olive varieties.

Background of the invention

A wide reference to conventional systems for the diagnosis and treatment of olive pollen allergy can be found in Spanish patent application No. P200100995. Said Spanish patent application describes and claims a kit for the diagnosis of hypersensitivity against allergens of olive pollen characterized in that said kit includes a mixture of pollen extracts from various olive varieties ( Olea europaea ), olive oil, and others members of the Oleaceae family ( Fraxinus, Ligustrum, Phillyrea, Forsytia, Jasminum, Syringa ), specifically chosen among the varieties of a geographical area.

The olive tree ( Otea europaea L. ) is one of the most important crops in the Mediterranean area, where it has been cultivated for millennia. Extensive cultivation of this plant has led to the appearance of an extremely wide and varied germplasm. By way of illustration, it is worth mentioning that in the South of Spain alone more than 150 different cultivars have been identified whose territorial distribution has been characterized [Barranco D. Varieties and Patterns. In: Barranco D, Fernández-Escobar D, Rallo L, editors. The cultivation of the olive tree. Madrid: Junta de Andalucía / Ediciones Mundi-Prensa, 1997; 61-79; Barranco D, Rallo L. Olive varieties grown in Andalusia. Madrid: Ministry of Agriculture-Junta de Andalucía. 1984]. Until very recently, the characterization of olive cultivars has been carried out primarily according to morphological and agronomic parameters. However, the use of molecular techniques is emerging in order to make this varietal characterization much more reliable, quick and simple.

\hbox{no solo alergia al polen
de olivo de las  variedades de olivo cultivadas sino también a olivo
silvestre (acebuche).}

Olive pollen is one of the most important causative agents of seasonal allergy (polynosis) phenomena in humans, more specifically of type I allergies, in those countries where this plant is widely distributed. In fact, olive pollen allergy currently represents a public health topic. The symptoms associated with this allergy range from an irritant syndrome of the highways, with sneezing, rhinorrhea, obstruction of the nasal passages, pharyngitis with different clinical manifestations in the case of frequent allergic rhinitis and allergic conjunctivitis symptoms to the most severe corresponding to asthma. The expression "allergy to olive pollen" includes, in general,

 \ hbox {not only pollen allergy
from olive of cultivated olive varieties but also to olive
wild (olive).} 

Some of the olive pollen proteins (Ole e 1, Ole e 2, Ole e 3, Ole e 4, Ole e 5, Ole e 6, Ole e 7, Ole e 8, Ole e 9 and Ole e 10) are capable of binding to IgE antibodies humans, and therefore, can be listed as allergens. The Ole e 1 and Ole e 2 proteins, in particular, Ole e 1, are the Major allergens of olive pollen and are responsible for the vast majority of cases of allergy to olive pollen.

The diagnosis of hypersensitivity to olive pollen includes a series of specific tests, including so-called SPTs (skin prick tests), intradermal tests, RAST tests (radioallergosorbent test), tests for the determination of specific serum IgE , histamine release tests, and conjunctival, nasal or bronchial provocation tests [The Merck Manual of Diagnosis and Therapy. 17th Edition Beers MH and Berkow R Eds. Internet Edition: Medical Services USMEDSA NSHH. www.merck.com/pubs/mmanual/ ].

The treatment of patients with allergy to olive pollen through allergen immunotherapy (also called allergen vaccination, hyposensitization or desensitization) is a treatment of choice even when exposure to the allergenic agent is reduced (such is the case of numerous pollens), and therapeutic treatment be able to sufficiently reduce symptoms. These types of treatments are based on repeated injection of the allergen subcutaneously in gradually increasing doses getting long term a prevention of the progression of bronchial hyperreactivity.

Both the diagnosis and the treatment of allergy to olive pollen are currently based on utilization of protein extracts obtained from said pollen by various procedures Although most patients are allergic to a limited number of "major" allergens of the olive pollen, there are numerous different allergens in the same source. Actually, it can be assumed that any protein in the Allergenic material can potentially act as an allergen. Therefore, an important aspect to consider in the determination The quality of allergenic vaccines is that they have the as much complexity as possible in its composition. Another aspect to have given, given the complexity of the allergenic extracts and the variability of extraction procedures and material heading, lies in the standardization of the extracts that are going to be used. This standardization is carried out internationally according to recommendations of the World Health Organization (WHO) through the International Union of Immunology Societies, Allergen Standardization Subcommittee [WHO Expert Committee on Biological Standardization. Guidelines for the preparation and establishment of reference materials and reference reagents for biological substances WHO Technical Report Series No. 626, 1978].

Various studies have shown the existence of a high variability in the allergenic content of olive pollen (and consequently in extracts produced from said pollen) from various geographical distributions, and, in particular, from different olive varieties . In the vast majority of published studies, the work material used corresponds to mature pollen of undetermined origin. There is, however, a very limited number of jobs that have used olive pollen in which at least one discrimination has been made regarding the geographical distribution of the source of material. In these studies, clear differences have been observed in the serum reactivity of Spanish allergic patients against pollen extracts from California [Boluda L, Alonso C and Fernández-Caldas E. Purification, characterization, and partial sequencing of two new allergens of Olea europaea. J. Allergy Clin. Immunol 1998; 101: 210-216], as well as quantitative differences in the levels of certain allergens between extracts from various sources in Spain and also from California [Martínez, A, Asturias JA, Palacios R, Sanz ML, Sánchez G, Oehling A, Martínez J. Identification of a 36-kDa olive-pollen allergen by in vitro and in vivo studies. Allergy 1999; 54: 584-592]. In this sense, studies conducted in Israel [Waisel Y, Geller-Bernstein C, Keynan N, Arad G. Antigenicity of the pollen proteins of various cultivars of Olea europaea can be considered pioneers. Allergy 1996; 51: 819-825; and Geller-Bernstein C, Arad G, Keynan N, Lahoz C, Cardaba B, Waisel Y. Hypersensitivity to pollen of Olea europaea in Israel. Allergy 1996; 51: 356-359] taking as reference the use of olive pollen of different native and foreign varieties. These studies have shown clear differences in the quantitative / qualitative composition of allergens in these extracts as well as in the reactivity of patient sera.

The use of protein pollen extracts from olive tree commonly used for diagnostic purposes as well as of hyposensitization (vaccines) prepared from mixtures of olive pollen belonging to indeterminate varieties (not defined) of olive tree entails, on the one hand, diagnostic errors of allergic subjects, and, on the other hand, limited successes in Immunotherapy treatments of such subjects.

There is, therefore, the need to develop alternative systems of diagnosis and treatment of allergy to olive pollen that overcome the problems previously mentioned.

Compendium of the invention

The invention is based on the use of a set of individualized olive pollen extracts, where each of said olive pollen extracts is a pollen extract of olive tree from a defined variety of different olive tree and is separated from the rest of olive pollen extracts, in the diagnosis of hypersensitivity to allergens of olive pollen. From In this way, it is not only possible to identify the defined variety of olive tree whose pollen is the causative agent of the allergic process in a subject, eliminating or minimizing the risk of errors of diagnosis, but also facilitate the design of an immunotherapy customized more efficient since the subject in need of treatment you will be given the extract or pollen extracts adequate, thereby achieving a significant increase in the efficacy of therapeutic treatment.

The studies carried out by the inventors They are the basis for the development of an experimental kit for the diagnosis of hypersensitivity to olive pollen, capable to discriminate between defined olive varieties of major regional / regional diffusion causing allergy. From that diagnosis, the basis for the production of personalized hyposensitizing vaccines for use in immunotherapy that include olive pollen extracts from defined olive varieties that cause allergy in adequate proportions. Both aspects are closely related.

The teachings contained in this document can be applied in cases of subjects suspected of suffering allergy to olive pollen. In cases of symptomatic subjects with ambiguous diagnosis of allergy to olive pollen, as well as in cases of patients treated by immunotherapy without decreasing symptoms, it would also be advisable to make a new diagnosis based on the teachings contained in this document.

In one aspect, the invention relates to some improvements introduced in the object of the request Spanish Patent No. P200100995 for "Kit for the diagnosis of hypersensitivity to allergens of olive pollen and its use ", characterized in that said kit comprises a set of individualized olive pollen extracts, where each of said olive pollen extracts is an extract of olive pollen from a defined olive variety and is separated from the rest of olive pollen extracts.

In another aspect, the invention relates to a kit comprising a set of olive pollen extracts individualized, wherein each of said pollen extracts of Olive is an extract of olive pollen from a variety defined as a different olive tree and is separated from other extracts of olive pollen.

In another aspect, the invention relates to the use of said kit in (i) an in vivo or in vitro method for diagnosing allergy to olive pollen of a defined olive variety, or (ii) in the development of personalized vaccines , specific, of hyposensitization against allergens of olive pollen of defined varieties of olive.

In another aspect, the invention relates to an in vitro method for diagnosing allergy to olive pollen of a defined olive variety in a subject, which comprises determining, in a biological sample of said subject, the IgE titer against allergens present in each of the olive pollen extracts contained in said kit.

In another aspect, the invention relates to an in vitro method for (i) determining the course of the olive pollen allergy course of a defined olive variety in a subject, or for (ii) evaluating the efficacy of a treatment against allergy to olive pollen of a defined variety of olive in a subject, which comprises determining, in a biological sample of said subject, the IgE title and the IgG title, against allergens present in each of the extracts of Olive pollen contained in said kit.

Brief description of the figures

Figure 1 is a graph in which the series represented by columns shows the relative values of the majority allergic content of the olive tree (Ole e 1) in the pollen of ten model cultivars. These values have been obtained by densitometry in immunoblots of crude pollen protein extracts, tested with a specific anti-Ole monoclonal antibody and 1. The variety with the highest allergen content (Lechín) appears with a value of 100%, while the rest of cultivars have minor relative contents that reach only 17.9% in the Gorda] Sevillana variety. The average reactivity values (measurement of reaction area in mm2) of 30 patients diagnosed with allergy to olive pollen after being subjected to SPT (skin prick) tests are superimposed (in a continuous line) test) with diluted extracts of these cultivars. A good level of correlation can be observed between the relative allergen content of each cultivar and the average reactivity of the patients to the corresponding extracts, although this correlation is not absolute as can be seen from the interpretation of Figures 2
and 3.

Figure 2 is a graph depicting the individualized reactivity of three patients to SPTs using crude protein extracts from the ten cultivars proposed as a model. In all three patients, high reactivity to positive control (histamine) appears, as well as extracts from Olea europaea from commercial allergy evaluation kits in which the cultivars used as a source are not specified. In the case of patients # 1 and # 3, positive reactivity appears to various model cultivars, even with area values greater than those that appeared in relation to the commercial extract (eg, Loaime variety in the case of patient # 1, and Loaime and Lechín varieties in the case of patient # 3). On the contrary, the reactivity is generally reduced in all cases to the varieties Frantoio, Gordal and Arbequina. In the case of patient # 2, all cultivars produced less reactivity than the commercial extract.

Figure 3 is a graph depicting the individualized reactivity of three patients to SPTs using crude protein extracts from the ten cultivars proposed as a model. In all three patients, high reactivity to positive control (histamine) appears, but not to extracts of Olea europaea from commercial allergy evaluation kits, as they are patients undergoing prolonged immunotherapy. However, the maintenance of an important reactivity towards extracts of certain varieties can be appreciated.

Detailed description of the invention

In one aspect, the invention relates to some improvements introduced in the object of the request Spanish Patent No. P200100995 for "Kit for the diagnosis of hypersensitivity to allergens of olive pollen and its use ", characterized in that said kit comprises a set of individualized olive pollen extracts, where each of said olive pollen extracts is an extract of olive pollen from a defined olive variety and is separated from the rest of olive pollen extracts.

Therefore, in one aspect, the invention is relates to a kit, hereinafter kit of the invention, which comprises a set of olive pollen extracts individualized, wherein each of said pollen extracts of Olive is an extract of olive pollen from a variety defined as a different olive tree and is separated from other extracts of olive pollen.

The term "olive tree", as used herein, includes any member of the species Olea europaea L.

Likewise, the term "defined variety" of olive tree as used herein refers to any olive variety described and identified, currently known to those skilled in the art, or which may be described and identified later as the teachings of the present invention may be extended to said defined olive varieties that can be identified and cataloged after the date of filing of this patent application. In a particular embodiment, said defined variety is a defined olive variety in Spain, such as any of the defined olive varieties cataloged and described in the book: "Olive varieties in Spain" [Luis Rallo, Diego Barranco, Juan M. Caballero, Carmen del Rio, Antonio Martín, Joan Tous, Isabel Trujillo (editors). 2005. Olive varieties in Spain . Board of Andalusia, MAP and Mundi-Press Editions. Madrid], for example: alfafara, aloreña, arbequina, blanqueta, castellana, royal changlot, cornicabra, empeltre, farga, gordal sevillana, hojiblanca, piglet of Granada, piglet of Seville, chamomile cacereña, chamomile of Seville, morisca, morrut, picual , weevil, sevillenca, verdial de Badajoz, verdial de Huévar, verdial de Vélez-Málaga, Villalonga, argudell, callosita, lemon peak, vera, blue, corbella, gordalejo, tomatillo etc.

The term "whole", as used herein refers to at least one individualized olive pollen extract, from a defined variety of different olive, for example, 1, 2 or more individualized olive pollen extracts, coming from different olive varieties defined. The number of individualized olive pollen extracts, from different defined olive varieties, that may be present in the kit of the invention may vary within a wide range. By way of illustration, the kit of the invention may contain at least 1 or 2, typically at least 6, advantageously at least 12, preferably at least 24, more preferably at least 48, and, even more preferably, at least 98, individualized olive pollen extracts, each from a defined variety of different olive. Of the approximately 250 defined varieties of olive trees in Spain there are 24 main varieties, 24 secondary varieties and 50 diffused varieties, the remaining being very unrepresentative, as mentioned in the book "Olive varieties in Spain" (quoted above ).

As used herein, the term "individualized" refers to each of the extracts of olive pollen present in the kit of the invention (i) comes from a single defined olive variety and (ii) is separated (it is say, not mixed) of the rest of the olive pollen extracts included in the set of pollen extracts from the kit invention.

Individualized olive pollen extracts which may be present in the kit of the invention may Obtained by conventional methods known to experts in the matter from different defined olive varieties. There are different methods of obtaining pollen extracts from olive; some of these methods comprise the step of submitting the pollen to an extraction process in aqueous media, hydroalcoholics, saline solutions etc., with a previous step optional physical pollen disruption (for example, by ground in mortar with the addition of liquid nitrogen), by passive diffusion to the aqueous medium for more or less periods prolonged, re-extraction procedures sequential, subsequent dialysis, etc.

Due to the complexity of the composition Chemistry of allergenic extracts of olive pollen, such as consequence, among other factors, of the variability of extraction methods and the starting material used, is it is convenient to proceed with the standardization of pollen extracts of olive trees that are going to be used. This standardization leads to out following the recommendations of the World Organization of the Health (WHO) through the International Union of Societies of Immunology, allergen standardization subcommittee [WHO Expert Committee on Biological Standardization. Guidelines for the preparation and establishment of reference materials and reference reagents for biological substances. WHO Technical Report Series No. 626, 1978]. Although this organization maintains different patterns, most producers of olive pollen extracts own Your own internal patterns for your own reference.

Therefore, in a particular embodiment, the individualized olive pollen extracts present in the kit of the invention are "standardized." The standardization of said extracts requires their characterization by in vitro procedures, such as, for example, analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), isoelectric focusing (IEF), cross-immunoelectrophoresis (ICD) ), etc. Likewise, the potency of each extract must be expressed in clinical efficacy units. Different comparative units of immunological potency have been established such as the so-called SQ (standardized quality), HEP (histamine equivalent prick), BU (biological unit) in Europe, and AU (allergy units) or BAU (bioequivalent allergy unit) units in States United. In a specific embodiment, the standardization of the individualized olive pollen extracts present in the kit of the invention is carried out by SDS-PAGE, CIE, ELISA and clinical trials, and, optionally, by quantifying the major allergen [eg, Ole and 1] and its immunological potency depending on the type of test to which the extract is intended (diagnostic / therapeutic, in vivo or in vitro ). The concentration of the major allergen, eg Ole e 1, in the extract may vary within a wide range and, in any case, may be established by one skilled in the art. By way of illustration, not limitation, the concentration of the major allergen, eg Ole e 1, in the extract can be up to 100 µg / ml (although higher concentrations can also be used if necessary).

Each of the olive pollen extracts individualized present in the kit of the invention can presented in the form of raw extracts, isolated, partially purified, adsorbed (e.g., on aluminum hydroxide, phosphate of calcium, etc.), or modified, in aqueous, oily solutions, depot, polymerized, encapsulated in liposomes, etc.

The set of olive pollen extracts individualized present in the kit of the invention may include olive pollen extracts from virtually any variety of olive tree, both native to a specific geographical area and native to other geographical areas, for example, varieties of local, regional, national, or foreign olive trees; no However, in a particular embodiment, the set of extracts of individualized olive pollen present in the kit invention comprises olive pollen extracts of most common olive varieties in a given geographical area. In general, due to applications both in diagnosis and in therapy of the kit of the invention, said determined geographical area will correspond to the geographical region where it is usually move the subject you want to know your hypersensitivity against the pollen of a certain defined olive variety; in this sense, in a specific embodiment, said kit will include the olive pollen extracts corresponding to the varieties defined olive trees of a given geographical area (which will correspond to the area where the subject under study) and, if desired, olive pollen extracts corresponding to the secondary olive varieties of said area geographical

The term "subject", as here uses, includes any animal likely to induce a immune response in response to an antigen, for example, a mammal, including the human being. In a particular embodiment, said subject usually resides in Andalusia and, the kit of the invention includes olive pollen extracts corresponding to the defined dominant olive varieties and, if desired, secondary, geographical area (Andalusia), such as, by example, Lucio, Picual, Loaime, Hojiblanca, Arbequina, Lechín de Seville, Verdial de Vélez-Málaga, Picudo, Chamomile of Seville, Gordal Sevillana, Cornicabra, Lechín de Granada, Sevillenca, and Acebuche.

The different olive pollen extracts individualized that constitute the set of pollen extracts olive oil from the kit of the invention are contained in appropriate containers or containers. Virtually any container type can be used, for example, jars, tubes, blisters, etc. The nature of the containers is not criticism as long as it does not adversely affect stability of pollen extract; by way of illustration, said containers They can be made of glass, glass, plastic, etc.

The kit of the invention further comprises, if want, positive controls (i.e. hydrochloric histamine), negative (the same buffer in which the dilutions of the extracts, distilled water, physiological serum, solutions glycerinated, pollen extracts from defined olive varieties With low / no reactivity, varieties pollen extracts irrelevant, etc.).

A distinctive feature of the kit invention versus other known kits, for example, kits for diagnosis of hypersensitivity to allergens from pollen Olive tree known, is that the different pollen extracts of olive tree that constitute the set of olive pollen extracts individualized of the kit of the invention are not mixed about with others but are separated from each other and maintain their individuality. Another distinctive feature is that, at know the defined olive variety from which each one comes of said olive pollen extracts, the kit of the invention can be used efficiently both for diagnostic purposes to identify the specific olive variety whose pollen causes a allergic reaction (hypersensitivity) in a subject (in fact, the kit of the invention allows detecting the olive variety against which pollen can be sensitized a subject which guarantees the diagnosis) as for therapeutic purposes by allowing selection the appropriate extracts for the design and development of the immunotherapy to be administered to the subject in need of treatment, thus facilitating the therapeutic treatment to be administered to the subject

In a practical application, the kit invention can be used in the diagnosis of pollen allergy of olive from a defined variety of olive. A subject may have allergy to olive pollen of one or more defined varieties of olive. The kit of the invention can be used to identify the variety or defined varieties of olive tree to whose pollen a subject he is allergic.

Therefore, in another aspect, the invention relates to the use of the kit of the invention in an in vitro or in vivo method for the diagnosis of allergy to olive pollen of a defined variety of olive in a subject. Said in vitro or in vivo methods for the diagnosis of olive pollen allergy of a defined olive variety are based on the use of conventional techniques known to those skilled in the art that will be mentioned below. In a particular embodiment, the kit of the invention comprises olive pollen extracts corresponding to the defined dominant or main olive varieties and, if desired, secondary, of the geographical area where the subject under study moves.

In another aspect, the invention relates to an in vivo method for the diagnosis of allergy to olive pollen of a defined variety of olive tree in a subject comprising applying to a subject an adequate amount of each of the pollen extracts of Individualized olive contained in the kit of the invention and evaluate the reaction in the subject.

For the implementation of this in vivo diagnostic method, any of the conventional in vivo tests such as, for example, Skin Prick Test (SPT), intradermal tests, nasal, oral, bronchial and / or conjunctival challenge tests ( ophthalmic), etc. [van Hage-Harnsten M, Pauli G. Provocation testing with recombinant allergens. Methods 2004; 32 (3): 281-91].

The invention also relates, in another aspect, to an in vitro method for the diagnosis of allergy to olive pollen of a defined variety of olive trees in a subject, which comprises determining, in a biological sample of said subject, the title of IgE against allergens present in each of the olive pollen extracts contained in the kit of the invention. This method also allows identifying the defined variety of olive tree whose pollen is the causative agent of allergy in a subject.

As used herein, the term "sample biological of a subject "includes any sample of the subject liable to contain immunoglobulins, for example, blood complete, serum or plasma, nasal and bronchial secretions, tears, breast milk, umbilical cord, etc.

In a particular embodiment, the kit of the invention comprises olive pollen extracts corresponding to the defined dominant olive varieties and, if desired, secondary, of the geographical area where the subject moves, so as olive pollen extracts corresponding to the varieties defined olive tree to which the subject presents hypersensitivity

In a particular embodiment, the implementation of this method comprises performing in vitro tests based on the use of techniques comprising the detection of the allergen (for example, by immunoassays, etc.) and the visualization of the complex formed by any method. appropriate (eg, colorimetric, fluorimetric, chemiluminescent, radioactive, etc.). The supports on which these techniques are carried out can be very varied and are known to those skilled in the art. Illustrative, non-limiting examples of such techniques include techniques known as immunoblots, immunodots, chemiluminescent immunoassays, RIST [radioimmunosorbent test], PRIST [paper radioimmuno-sorbent test], RAST [ radioallergosorbent test], ELISA (linked enzyme immunosorbent assay), RIA (radioimmunoassay), FEIA [fluorescent enzyme immune assay], etc., including the use of commercially available diagnostic systems ( eg, Phadebas RAST, CAP, ImmunoCAP, UniCAP, CAP FEIA, AlaSTAT, INMULITE, FAST, IgEquick, CMG Immunodot, etc.), produced or supplied by various commercial companies, such as Pharmacia Diagnostics, CMG Laboratories, DPC, etc., and / or the determination of T cell activation, basophil activation, histamine release, etc.

In a particular embodiment, said method comprises contacting a biological sample of said subject, for example, a serum sample, with each of the extracts, separately, of individualized olive pollen contained in the kit of the invention and determining the IgE antibody titer against the allergens present in said extracts. The determination of the immunoglobulin (IgE) titer against the allergens contained in the individualized olive pollen extracts contained in the kit of the invention can be carried out by conventional methods known to those skilled in the art. In a particular embodiment, the determination of the specific IgE titer comprises analyzing the allergen-immunoglobulin (IgE) reaction present in the subject's biological sample, visualizing the formation of said complex and determining the olive variety responsible for the allergy in the subject. The visualization of the complex can be carried out by conventional methods, for example, by colorimetric, fluorimetric, chemiluminescent, radioisotopic, optical density (absorbance) measurements, other associated techniques: planimetry, cell count, flow cytometry, etc. A general reference on these techniques can be found in "Respiratory Diseases. Utility of the Laboratory". Second edition. Editors: P. Ancic Cortez, T. Clark, R. Rodriguez-Roisin, R. Paredes Martinez. Faculty of Medicine, University of Chile and The British Council ( http://www.med.uchile.cl/otros/dra_{-}ancic/libro.html ).

To establish if a subject has an allergy, in general, the normal limits already established will apply. How is known, total serum IgE is normally present in quantities of the order of ng / ml, being increased in different  allergic conditions, in viral and parasitic diseases, and in certain immunodeficiencies The total serum IgE value is expressed normally in international units (IU) per ml (1 IU = 2.4 ng of protein). The level of IgE varies with age; in the blood of Normal newborn cord is internal at 0.5 IU / ml, and in the normal adult is around 20 IU / ml (usually less than 80 IU / ml). From 14 years of age it is considered abnormally high an IgE greater than 333 IU / ml (800 ng / ml). In atopic people the IgE level is usually 3 to 5 times higher. To determine if the allergy is specifically produced by olive pollen it uses diagnostic techniques such as those described above to determine the specific IgE levels against allergens of olive pollen As an example, some values are cited below commonly accepted to establish the degree of sensitivity of a patient using the CAP system marketed by Phanmacia, of Widespread:

one

The invention also relates, in another aspect, to an in vitro method for (i) determining the course of the olive pollen allergy course of a defined olive variety in a subject, or for (ii) evaluating the efficacy of a treatment against allergy to olive pollen of a defined variety of olive in a subject, which comprises determining, in a biological sample of said subject, the IgE title and the IgG title, against allergens present in each of the Olive pollen extracts contained in the kit of the invention.

In a particular embodiment, the kit of the invention comprises olive pollen extracts corresponding to the defined dominant olive varieties and, if desired, secondary, of the geographical area where the subject moves, so as olive pollen extracts corresponding to the varieties defined olive tree to which the subject presents hypersensitivity

The implementation of this method includes the performance of in vitro tests based on the use of RIST, PRIST, RAST, ELISA, RIA, FEIA, etc. techniques, including the use of commercially available diagnostic systems (eg, Phadebas RAST, CAP, ImmunoCAP, UniCAP, CAP FEIA, AlaSTAT, INMULITE, FAST, IgEquick, CMG Immunodot, etc.), produced or supplied by various commercial companies, such as Pharmacia Diagnostics, CMG Laboratories, DPC, etc., and / or the determination of T cell activation, basophil activation, histamine release, etc., as previously mentioned.

In a particular embodiment, said method comprises contacting a biological sample of said subject, for example, a serum sample, with each of the extracts, separately, of individualized olive pollen contained in the kit of the invention and determine the titer of IgE antibodies and IgG, against the allergens present in these extracts. The IgG determination is especially interesting for evaluate the follow-up of the therapies applied to the subject in treatment. In this way, this method can be used to determine the course of the course of allergy in the subject (optionally undergoing treatment), as well as to evaluate the efficacy of an anti-allergic treatment administered to a subject suffering from allergy to olive pollen.

The determination of the immunoglobulin titer (IgE and IgG) against the allergens contained in the extracts of individualized olive pollen contained in the kit of the invention can be carried out by conventional methods known by the person skilled in the art, as previously mentioned, for example, analyzing complex formation allergen-immunoglobulin (IgE or IgG) present in the biological sample of the subject.

The use of the kit of the invention in an in vitro method to (i) determine the evolution of the course of allergy to olive pollen of a defined variety of olive in a subject, or to (ii) evaluate the efficacy of a treatment against allergy to olive pollen of a defined variety of olive in a subject, constitutes an additional aspect of this invention.

A feature of the present invention is which allows the combined design of diagnostic methods of the allergy to olive pollen of a defined variety of olive tree e custom desensitizing immunotherapies comprising, by one part, the discriminatory diagnosis of olive varieties and, Moreover, the preparation of customized vaccines for immunotherapy

Discriminatory diagnosis of varieties

The set of olive pollen extracts from defined varieties mentioned throughout this description It forms the basis of an experimental kit for diagnosis discriminatory to olive varieties. In fact the patients symptomatic suspected of having type I hypersensitivity to aeroallergens (e.g., olive pollen) are checked in a first approach using an SPT test with a kit that would contain common allergenic extracts, among which would be found various mixtures of olive pollen extracts corresponding to the dominant olive varieties in the geographical area where the subject under study usually moves (e.g., the community autonomous where you live, work, rest, etc.). After this first approximation, and in the case that the result was positive, would proceed to determine the reactivity against the various individualized olive pollen extracts, contained in the kit of the invention, coming from defined olive varieties, both  dominant as secondary, present in the geographical area by where the subject under study moves in order to adjust in the highest degree possible the variety (s) defined of Olive tree responsible for hypersensitivity. For this you can have an SPT test, intradermal tests, or any other the specific serum IgE determination tests previously mentioned, for example, RAST tests, release tests of histamine, conjunctival provocation tests, oral, nasal or bronchial with slight modifications, etc. Obviously, said kit it will include both positive controls (i.e. hydrochloric histamine, etc.), as negative controls (the same buffer in which they are made dilutions of extracts, distilled water, physiological serum, glycerinated solutions, varieties of olive pollen extracts With low / no reactivity, olive pollen extracts from irrelevant varieties, varieties of olive pollen extracts from other geographical areas far away from the geographical area where the subject moves, etc.).

Preparation of personalized vaccines for immunotherapy

Once the diagnosis is established with the highest possible accuracy, the composition of the vaccine would be customized corresponding for those patients in whom immunotherapy be the treatment of choice according to medical parameters well defined (e.g., impossibility of physical isolation from allergenic agent, maintenance of symptoms even in patients treated with medications, prevention of occurrence or exacerbation of asthma, etc.). These vaccines should contain the adequate number of total comparative power units immunological, although distributed proportionally for each of olive pollen extracts corresponding to varieties defined allergic olive trees for each patient in based on its reactivity to the tests and tests performed with diagnostic purposes The immunotherapy protocol would follow the recommendations already established for olive pollen extracts commercial, of which there is already extensive clinical experience.

Therefore, the use of the kit of the invention in the elaboration of personalized vaccines, specific according to diagnosis, of hyposensitization against allergens of olive pollen of defined varieties of olive tree, constitutes an additional aspect of this invention. Said vaccines comprise one or a mixture of pollen extracts of defined olive varieties together with one or more pharmaceutically acceptable excipients in order to treat said pollen allergy of one or several defined olive varieties. Said mixture of pollen extracts of defined olive varieties will include the therapeutically appropriate amounts of each of the constituent extracts of said mixture; that is, said vaccines contain the appropriate number of total comparative units of immunological potency, distributed proportionally for each of the pollen extracts corresponding to the defined olive varieties causing the allergy for each patient based on their reactivity to the tests and tests performed for diagnostic purposes. Additionally, said vaccine may contain one or more allergenic extracts from sources other than olive pollen that have been prescribed by the specialist, and / or a pharmaceutically acceptable adjuvant giving rise to a "mixed" or "multivalent" vaccine. By way of illustration, not limitation, said mixed vaccine may contain, in addition to the extract or mixture of pollen extracts of defined olive varieties, one or more different allergens, for example, one or more allergenic extracts of pollens (eg, allergenic extracts of grasses, cupresceae, chenopodiaceae, oceans other than O. europaea , plantains, etc.), one or more allergenic extracts of mites, etc., as well as combinations of such allergenic extracts of pollens and mites; in this way, customized mixed vaccines can be designed, made "to measure" of what the subject in treatment needs, for example, mixed vaccines comprising, in addition to the extract or mixture of pollen extracts of defined olive varieties, one or more allergenic extracts of mites, or mixed vaccines that comprise, in addition to the extract or mixture of pollen extracts of defined olive varieties, one or more allergenic extracts of pollens (eg, grasses, cypress, etc.), or mixed vaccines comprising, in addition to the extract or mixture of pollen extracts of defined olive varieties, one or more allergenic extracts of mites and one or more allergenic extracts of pollens (eg, grasses, cypress, etc.), etc.

In a particular embodiment, said varieties defined olive trees come from one or several geographical areas determined (in general, these geographical areas will be the corresponding to the geographical areas where they usually moves the subject in need of treatment).

Said vaccine may be administered by any appropriate route (e.g., oral, subcutaneous, topical, etc.), for which excipients and vehicles will be used pharmaceutically acceptable necessary for the formulation of the Pharmaceutical form of administration chosen. A review of the different pharmaceutical forms of drug administration and of its preparation can be found in the book "Pharmacy Treaty Galénica ", by C. Faulí i Trillo, 1st Edition, 1993, Luzán 5, S.A. of Editions. By way of illustration, not limitation, said vaccine may be in the form of injectable solutions, forms pharmaceuticals suitable for sublingual administration, powders, granules, pearls, tablets, capsules, syrups, emulsions, suppositories, eye drops, mists, sprays, creams, liposomes, gels, etc.

The following examples illustrate the invention and they should not be considered in a limiting sense of it.

       \ vskip1.000000 \ baselineskip
    

Example one

Characterization of heterogeneities in the composition of allergens in olive pollen of different varieties

The richness of the olive gennoplasma (around 150 different cultivars in the Community of Andalusia, 250 in Spain and 3,000 in the world) made the inventors think about the presence of important heterogeneities in both the composition quantitative as qualitative of olive pollen allergens in Different varieties of olive. In order to analyze these differences in the varieties Picual, Loaime, Lucio, Frantoio, Gordal, Chamomile, Arbequina, Hojiblanca, Picudo and Lechín, se they used SDS-PAGE techniques and immunoblots that were tested with specific monoclonal antibodies against Ole e 1, the major allergen of olive pollen (Figure 1), as well as Raw sera from allergic patients. They have also been made different clinical tests to determine the reactivity of various patients with allergy diagnosed to olive pollen versus Raw protein pollen extracts of the ten olive varieties previously mentioned by SPT tests. The results are collected in Figures 1, 2 and 3.

The results obtained show the following conclusions:

1) important differences have been detected in ten olive varieties of different origins and distribution regional regarding the levels of the major allergen Ole e 1; relative allergen values range between 100% for variety with higher levels of allergenic protein (Lechín de Granada) and 17.9% in the Gordal Sevillana variety (Figure 1);

2) the patients' average SPT reactivity Allergic against protein extracts of each variety is also very variable and offers a certain correlation with the levels Absolute allergen absolute Ole e 1 present in each of them (Figure 1);

3) in a large number of cases, patients show exacerbated reactivity against extracts of certain particular varieties (Figure 2) regardless of quantitative levels of majority allergen present in these varieties; Y

4) in 10% of patients undergoing prolonged immunotherapy with commercial vaccines does not appear reactivity to olive pollen extracts from kits commercial diagnosis, although an important one is maintained reactivity against extracts of certain varieties (Figure 3).

       \ vskip1.000000 \ baselineskip
    

Example 2

A 46-year-old male patient, born and resident in Granada with a history of rhinitis and seasonal conjunctivitis, diagnosed with bronchial asthma using spirometry and bronchodilator test. After suppression of antihistamine treatment, the patient is subjected to SPT tests with an experimental kit that contains a set of olive pollen extracts, individualized, standardized, where each of said olive pollen extracts is an extract of pollen from Olive from a defined variety of different olive and is separated from the rest of olive pollen extracts. Said set of olive pollen extracts includes the most common olive pollen extracts in the region where the patient usually resides and works and, in particular, olive pollen extracts corresponding to the dominant varieties in Andalusia (Picual, Hojiblanca, Lechín de Sevilla, Manzanilla de Sevilla, Verdial de Huevar, Gordal Sevillana, Verdial de Velez-Málaga, Aloreña, Lechín de Granada, Manzanilla Prieta) as well as the secondary varieties of the Granada region "La Vega" (Lucio, Loaime and Cornezuelo) . The standardization of these extracts has been carried out previously based on studies of SDS-PAGE, CRIE and clinical trials, quantifying their content in the majority allergen Ole e 1 in 50 µg / ml and their immunological potency in 100 BU (biological units) / ml. The SPT test thus performed shows positive reactivity assessed according to the following
table:

2

The patient is considered a candidate for immunotherapy based on the impossibility of physical isolation of the allergenic source, low response to drug treatment and the progressive increase of symptoms. The reactivity study to SPT suggests the following composition of extracts for treatment, from concentrated extracts of each of the varieties:

3

The immunotherapy administration schedule it is carried out according to standards established by the Subcommittee of EAACI immunotherapy, until maintenance doses are reached according to patient tolerance.

After four years of treatment, the patient manifests a subjective improvement, corroborated by new SPT with a kit that contains a set of olive pollen extracts, individualized, standardized, of the different varieties defined olive tree most common in the region where usually the patient resides and works, observing a decrease in reactivity in all varieties tested.

       \ vskip1.000000 \ baselineskip
    

Example 3

Patient man, 36 years. Principal diagnostic: Rhinoconjunctivitis and extrinsic bronchial asthma by sensitization to olive pollen based on commercial skin test (olive ++++, without other relevant reactivity) using a mixture of extracts of Olive pollen from different olive varieties. Currently in 4th year of immunotherapy well tolerated with vaccine custom composed of olive pollen extracts from Indeterminate varieties The treatment has been carried out following strictly the guidelines recommended by the manufacturer. Clinically stable and subjectively improved, although it maintains upper airway symptoms. Does not present breathlessness of effort. It requires occasional bronchodilators and presents alterations of the night rest, especially in times of high content pollen in the atmosphere (March-June). Asymptomatic the rest of the year.

A new skin test using a kit commercial diagnosis (based on a mixture of pollen extracts of olive trees from different olive varieties) reveals a significant decrease in the response to pollen extracts from olive (+) and lack of significant reactivity to others aeroallergens However, a skin test (SPT) using a kit experimental that contains a set of pollen extracts from olive, individualized, standardized, where each of these Olive Pollen Extracts is an olive pollen extract from a defined variety of different olive and is separated from the rest of olive pollen extracts. Said set of  olive pollen extracts includes olive pollen extracts of locally well-represented varieties [corresponding to dominant varieties in Andalusia (Picual, Hojiblanca, Lechín de Seville, Camomile of Seville, Verdial de Huevar, Gordal Sevillana, Verdial de Velez-Málaga, Aloreña, Lechín from Granada, Manzanilla Prieta) as well as the varieties secondary schools of the Granada region "La Vega" (Lucio, Loaime and Ergot)] still shows hyperreactivity (++++) to extracts from pollen from the Loaime and Manzanilla cultivars of Seville, although virtually zero reactivity to the rest of the extracts tested. After prolongation of immunotherapy treatment with a vaccine customized (50% of each of the two reactive extracts) for two consecutive years, the application of  bronchodilators in no time.

Claims (13)

1. Refinements introduced in the object of Spanish patent application No. P200100995 for "Kit for the diagnosis of hypersensitivity to allergens against olive pollen and its use", characterized in that said kit comprises at least one extract of olive pollen individualized, wherein each of said olive pollen extracts comes from a defined variety of different olive and is separated from the rest of olive pollen extracts. 2. A kit comprising at least one extract of individualized olive pollen, where each of these Olive pollen extracts come from a defined variety of different olive tree and is separated from the rest of pollen extracts from olive. 3. Kit according to claim 2, wherein said individualized olive pollen extracts are Standardized 4. Kit according to claim 2, wherein individualized olive pollen extracts come from different defined olive varieties present in a certain area geographical 5. Kit according to claim 2, wherein individualized olive pollen extracts come from varieties defined olive trees of a specific geographical area and, if desired, of secondary olive varieties of said area geographical 6. Use of a kit according to any of claims 2 to 5, in an in vivo or in vitro method for diagnosing allergy to olive pollen of a defined olive variety. 7. Use according to claim 6, wherein said in vivo method for diagnosing allergy to olive pollen of a defined olive variety in a subject comprises the use of Skin Prick Test (SPT), intradermal tests, or nasal, oral, bronchial or conjunctival provocation tests. 8. An in vitro method for diagnosing allergy to olive pollen of a defined variety of olive in a subject, which comprises determining, in a biological sample of said subject, the IgE titre against allergens present in each of the extracts of olive pollen contained in a kit according to any of claims 2 to 5. 9. Method according to claim 8, comprising performing in vitro tests selected from immunoblots, immunodots, chemiluminescent immunoassays, RIST [radioimmunosorbent test], PRIST [paper radioimmuno-sorbent test (paper radioimmunoabsorption test) ], RAST [radioallergosorbent test], ELISA (linked enzyme immunosorbent assay), RIA (radioimmunoassay), FEIA [fluorescent enzyme immune assay], T cell activation determination, activation of basophils, histamine release, and combinations thereof. 10. An in vitro method to (i) determine the course of the olive pollen allergy course of a defined olive variety in a subject, or to (ii) evaluate the efficacy of a treatment against olive pollen allergy of a defined variety of olive in a subject, which comprises determining, in a biological sample of said subject, the IgE title and the IgG title, against allergens present in each of the olive pollen extracts contained in the kit of the invention. 11. Use of a kit according to any of claims 2 to 5, in an in vitro method for (i) determining the evolution of the course of allergy to olive pollen of a defined variety of olive in a subject, or for (ii ) evaluate the efficacy of a treatment against allergy to olive pollen of a defined variety of olive in a subject. 12. Use of a kit according to any of the claims 2 to 5, in the preparation of vaccines custom, specific, hyposensitization versus olive pollen allergens of defined olive varieties. 13. Use according to claim 12, wherein said personalized vaccine comprises, in addition to said set of individualized olive pollen extracts, one or more extracts allergens from one or more sources other than olive pollen.