New mutant allergens

FIELD: biotechnology, medicine.

SUBSTANCE: invention relates to new recombinant allergens that represent mutants of allergens of the natural origin and comprising at least four mutations. Examples of recombinant allergens are allergens Bet v1 and Ves v1. The primary mutations in recombinant allergen are separated of one another by interval for at least 15 Å and is location is characterized by that at least one circle region of surface of size 800 Å doesn't comprise mutations. Recombinant allergens are used as a pharmaceutical agent as a component of pharmaceutical composition that represents vaccine against allergic response reactions. Invention describes methods for using recombinant allergens in pharmaceutical composition for producing the immune response in subject. Invention represents DNA sequences given in the invention claim that encode recombinant allergens, expressing vector comprising DNA and cell-host for providing the recombinant allergen. Also, invention describes methods for preparing pharmaceutical composition and recombinant mutant allergen. Using recombinant allergen allows decreasing the specific IgE-binding capacity as compared with IgE-binding capacity of the natural allergen. Invention can be used in medicine for preparing vaccine against allergic response reactions.

EFFECT: valuable medicinal properties of allergens.

33 cl, 62 dwg, 10 ex

 

The technical field to which the invention relates.

The present invention relates to new recombinant allergens, which are mutants exist in nature allergens. The invention relates also to compositions comprising a mixture of new recombinant mutant allergen. In addition, the invention relates to a method for producing such recombinant mutant allergen, as well as to pharmaceutical compositions, including vaccines containing recombinant mutant allergen. In an additional implementation of the present invention relates to methods of inducing immune responses in the subject, vaccination or treatment of a subject, and methods of producing the compositions according to the invention.

Prior art

Genetically predisposed individuals become sensitized (allergosorbent) to antigens, derived from various natural sources, to allergens encountered individuals. Allergic reaction occurs when a previously sensitized individual re-encounters the same or homologous antigen. Allergic responses range from hay fever, rhinoconjunctivitis, rhinitis and asthma to systemic anaphylaxis and death in response, for example, the stinging business bee or wasp or insect bites. The reactions which is immediate and can be caused by a number of atopic allergens, such as connections, available in grasses, trees, weeds, insects, food, drugs, chemical compounds and fragrances.

However, the responses do not occur when an individual encounters an allergen for the first time. Develops initial adaptive response, and usually there are no symptoms. But when produced antibodies and T-cells capable of interacting with the allergen, any subsequent exposure can provoke symptoms. Thus, allergic responses show that the immune response can cause significant pathological conditions that may pose a threat to life.

Antibodies involved in atopic allergies, belong primarily to the immunoglobulin E. IgE bind with specific receptors on the surface of mast cells and basophils. After the formation of the complex specific allergen with IgE bound to mast cells, cross-linking of receptors on the cell surface leads to the conduction of a signal through receptors and the physiological response of target cells. Degranulation is to release, among other things, histamine, heparin, a chemotactic factor for eosinophilic leukocytes, leukotrienes C4, D4 and E4, which cause long-lasting reduction in cells of smooth muscles of the bronchi. Result is that the effects can be systemic or local in nature.

Hypersensitivity reactions, mediated by antibodies, can be subdivided into four classes, denoted as type I, type II, type III and type IV. Allergic reactions type I represent the classical immediate hypersensitivity reaction, occurring within seconds or minutes after exposure to the antigen. These symptoms mediated by allergen-specific IgE.

Usually allergic reactions are observed in response to a protein allergen is present in, for example, pollen, house dust mites, wool and animal dander, poison and food.

In order to reduce or destroy allergic reactions, usually apply precisely controlled and repeated administration of allergen vaccines. Vaccination with allergen is usually performed by means of parenteral, intranasal or sublingual injection in increasing doses over a relatively long period of time, resulting in desensitization of the patient. The exact immunologic mechanism is not known, but induced differences in the phenotype of allergen-specific T cells, as expected, has a special significance.

Vaccination with allergen

The concept of vaccination is based on two fundamental characteristics of the immune system, called specificity and memory. Vaccination is primer is to the immune system of the recipient and with repeated exposure to similar proteins, the immune system will be able to more accurately respond to control infection, for example, infection by microbes. Vaccines are a mixture of proteins intended for use in vaccination, with the aim of developing such a protective immune response in the recipient. Protection should cover only the components present in the vaccine, and homologous antigens. Compared with other types of vaccination vaccination with allergen complicated by the existence of current immune response in allergic patients. This immune response is characterized by the presence of IgE, allergen-specific mediating the implementation of allergic symptoms when exposure to allergens. Thus, vaccination with allergen using allergens from natural sources has its inherent risk of side effects, have very significant consequences, threatening the patient's life.

Attempts to circumvent this problem can be divided into three categories. When practical measures often combine more than one category. The first category of measures includes the introduction of several small doses over a long period of time to achieve substantial accumulated dose. The second category of measures includes physical modification of allergens by including allergens in forming a gel substance such as aluminum hydroxide. The composition of the aluminum hydroxide has the effect of adjuvant and effect depot is La slow release of the allergen, reducing tissue concentration of the active components of the allergen. The third category of measures includes chemical modification of allergens to reduce allergenicity, i.e. binding of IgE.

The detailed mechanism after successful vaccination, allergen remains controversial. It, however, that T cells play a key role in the overall regulation of immune responses. In accordance with the present universal opinion of the relationship between two extreme phenotypes of T cells, Th1 and Th2 determines the allergic status of the individual. When the allergen stimulation of Th1 cells secrete interleukins dominated by interferon-γthat leads to protective immunity and to preserve the health of the individual. The Th2 cells, on the other hand, secrete predominantly interleukin 4 and 5, which leads to the synthesis of IgE and eosinophilia, and to the induction of Allergy in an individual. In vitro studies have demonstrated the ability to change the responses of T cells, allergen-specific, by loading peptides derived from allergens and contains epitopes that are associated with T-cells. Currently available approaches to new vaccines, allergens, therefore, based mainly on the effects on T cells to call areactively T cells (induction of anergy) or switching response phenotype Th2 to Th1 phenotype.

The epitopes bound is Yausa antibody (b-cell epitopes)

X-ray crystallographic analysis of complexes Fabantigen contributed to the understanding of the structure of epitopes that bind antibodies. In accordance with this type of analysis epitopes that bind antibodies, can be defined as a part of a surface antigen that includes atoms of 15-25 amino acid residues that are within reach of atoms antibodies, capable of direct interaction. The affinity of the interaction of antigen-antibody cannot be predicted only on the basis of enthalpy, which contribute to the strength of the van der Waals forces, hydrogen bonds or ionic bonds. The entropy associated with almost complete removal of water molecules from the surface interaction, gives similar largest energy contribution. This means that a perfect fit between the contours of the interacting molecules is a fundamental factor underlying the high affinity interactions of antigen-antibody.

In patent WO 97/30150 (reference 1) States that a panel of protein molecules and protein molecules are characterized by the distribution of specific mutations in the amino acid sequence compared to the parent protein. From the description it is clear that the invention applies to obtain analogues, which are modified compared to the parent protein, but they are being captured, the hydrolysis is conducted and presented to T cells in the same way, the parent protein (natural existing allergens). Thereby obtain a modified T-cell response. Library of modified proteins obtained by applying the methods denoted as PM (economical mutagenesis).

In patent WO 92/02621 (reference 2) describes recombinant DNA molecules, and molecules include DNA encoding a polypeptide having at least one epitope of the allergen trees kind of Fagales, and allergen selected from Aln g 1, Cor a 1 and Bet v 1. Described in this description recombinant molecules should all have the amino acid sequence or part of the amino acid sequence that corresponds to the sequence of natural existing allergen.

Patent WO 90/11293 (reference 3) include, among other things, to selected peptides of allergen pollen, ragweed and modified peptides pollen ragweed. Disclosed therein peptides have an amino acid sequence corresponding to the sequence or nature of an existing allergen, or existing natural isoforms.

Chemical modification of allergens

There have been several approaches to chemical modification of allergens. Approaches the beginning of the seventies include chemical joining of allergens to polymers and chemical cross-linking of the allergen is in using formaldehyde and so on to obtain the so-called "allergoids". The rationale of these approaches consisted in a randomized destruction of epitopes that bind IgE, by attaching a chemical ligand, resulting in decreased IgE binding while maintaining immunogenicity due to the increased molecular weight complexes. The disadvantages of obtaining "allergoids, associated with the difficulties of controlling the process of chemical cross-linkage and difficulties in the analysis and standardization of the resulting macromolecular complexes. "Allergodil" currently used in clinical practice, and due to the randomized destruction of epitopes that bind IgE, can be entered higher doses compared with conventional vaccines, but the parameters of safety and effectiveness is not improved compared to conventional vaccines.

More modern approaches to chemical modification of allergens is aimed at the complete destruction of the tertiary structure of the allergen, and is destroyed thus the binding of IgE and assumes an important target for therapeutic treatment is allergen-specific T-cell. Such vaccines contain synthetic peptides derived from the sequence of the allergen, which represents the minimal epitopes for T-cells, longer peptides representing St is related to T-cell epitopes, synthetic peptides derived from a larger sequence of the allergen, which represents the area of the immunodominant epitopes for T-cells, or molecules allergens, cut into two halves using recombinant methods. Another approach, based on this rational justification, is a proposal for the use of recombinant isoforms with low IgE binding". In recent years it has become clear that natural allergens are heterogeneous, containing isoalleles and options, which replaced approximately 25% of the amino acids. Some recombinant isoalleles, as found, are less effective at binding IgE, possibly due to the destruction of the tertiary structure.

Mutagenesis in vitro and vaccination allergen

Attempts to reduce the allergenicity using site-directed in vitro mutagenesis was undertaken using several allergens including Der f 2 (Takai et al., reference 4), Der p 2 (Smith et al., reference 5), 39 kDa allergen Dermatophagoides farinae (Aki et a.l, reference 6), phospholipase A2 poison bees (Förster et al., reference 7), Ara h 1 (Burks et al., reference 8), Ara h 2 (Stanley et al., reference 9), Bet v 1 (Ferreira et al., the link 10 and 11), profilin birch (Wiedemann et al., reference 12) and Ory s 1 (Alvarez et al., reference 13).

The rationale of these approaches again is based on allergen-specific T cells, however, at the same time when igenii risk of side effects, mediated by IgE, by reducing or destroying the binding of IgE with violations of the tertiary structure of recombinant mutant allergen. The rationale of these approaches does not include the concept of dominant epitopes that bind IgE, and does not include the concept of initiation of a new protective immune response, which also involves b cells in antibody production.

In article Ferreira et al. (reference 11) describes the use of site-directed mutagenesis with the aim of reducing the binding of IgE. Although the three-dimensional structure of Bet v 1 is mentioned in the article, the authors do not use the structure for predicting exposed to the solvent amino acid residues in relation to mutation, half of which is characterized by a low degree of exposure to the solvent. Rather, they apply the method developed to predict functional residues in proteins other than the understanding of the structure-based identification of conserved regions of the surface described in this description. Although the authors are really discussing conservatism tertiary structure α-carbon skeleton, this concept is not part of therapeutic strategy, but simply included to assess the binding of IgE in vitro. Moreover, the presented evidence is not adequate, since the normalization of the spectra of the CH prepyatstvuet the assessment denaturation share of the sample, that is a common problem. Described therapeutic strategy aimed at the induction of tolerance to the allergen-specific T cells and the initiation of a new immune response is not mentioned.

In article Wiedemann et al. (reference 12) describes the use of site-directed mutagenesis and peptide synthesis with the aim epitope characteristics of monoclonal antibodies. The authors are aware of the tertiary structure of the antigen and they use this knowledge to select exposed on the surface of amino acids for mutations. The applied algorithm can be estimated as described opposite the inventors of the present invention, as selected amino acid that differs from homologous sequences. Research shows that replacing superficially located amino acids is able to modify the binding characteristics of monoclonal antibodies, which is not surprising, given the known facts. The experiments described are not intended to assess the modulation of binding of polyclonal antibodies, such as serum IgE, allergic patients. In one of the included experiments really apply serum IgE and although this experiment is not suitable for quantitative analysis, the binding of IgE, obviously, did not influence made mutations.

In the article Smith et al. (reference 5) describes the use of the tell site-directed mutagenesis to map the epitopes of monoclonal antibodies and reduction of IgE binding. The authors are not known tertiary structure and they do not attempt to evaluate the conservatism of the tertiary structure α-carbon skeleton. Implemented algorithm does not guarantee that the amino acids selected for mutagenesis, really exhibited on the surface of the molecule. Only one of these mutants leads to a significant reduction in IgE binding. This mutant is characterized by lack of binding to all tested antibodies, which indicates a violation of the tertiary structure. The authors do not define therapeutic strategy, and the initiation of a new immune response is not mentioned.

In article Colombo et al. (reference 14) describes the study of the epitope that binds IgE, using site-directed mutagenesis and peptide synthesis. The authors used computer modeling of three-dimensional structure based on the crystal structure of the homologous protein, to illustrate the presence of the epitope on the surface of the molecule. The additional presence of the epitope on the excellent allergen having homology of the primary structure, is achieved by using synthetic peptides representing the epitope. therapeutic strategy is based on the treatment with the use of this synthetic peptide representing an epitope binding to monovalent IgE. Conservative area of the surface is activity between homologous allergens, as well as therapeutic concept initiation of a new protective immune response not mentioned.

In article Spangfort et al. (reference 15) describes a three-dimensional structure and conservative located on the surface area of the main allergen of birch. The article does not mention any major epitopes that bind IgE or site-directed mutagenesis or the purpose of therapeutic applications.

None of the studies described above, the binding of IgE was reduced when replacing exposed on the surface of amino acids while maintaining the tertiary structure α-carbon skeleton. The rationale of the above mentioned approaches do not include the concept of a dominant epitopes IgE binding and does not include therapeutic concept initiation of a new protective immune response.

In patent WO 99/47680 disclosed introduction artificial amino acid substitutions at certain critical positions while maintaining the tertiary structure α-carbon skeleton of the allergen. In particular, in patent WO 99/47680 disclosed recombinant allergen, which is unnatural mutant, leading from the beginning of the natural allergen, in which at least one exposed on the surface of a conservative amino acid residue of the epitope In cells replaced by another residue that is not in the same position in the amino acid posledovatelno and any known homologous protein within the taxonomic order which is specified natural allergen, and the indicated mutant allergen has essentially the same tertiary structure α-carbon skeleton, as specified natural allergen specific IgE binding with mutant allergen decreased compared to binding with the specified natural allergen.

Recombinant allergen, disclosed in the patent WO 99/47680, obtained by a) identifying amino acid residues in the natural allergen, which saved more than 70% identity in all known homologous proteins within the taxonomic order, which is specified natural allergen, b) determining at least one zone of conservative amino acid residues, in coordination United for at least 400 Å2the surface of three-dimensional molecules allergen, as determined by the accessibility to solvent constituting at least 20%, and at least one area comprises at least one epitope In cells, and (C) replacing at least one amino acid residue in the specified at least one zone to another amino acid, which is non-conservative in a particular position and essentially preserves the overall tertiary structure α-carbon skeleton of the molecule allergen.

a Brief description of the drawings

Figure 1 shows specific for the mutant oligonucleotide primers used for mutant number 1 Bet v 1. Mutated nucleotides are underlined.

Figure 2 shows two generally applicable primer (labeled "fully semantic" and "fully " antisense"), which was synthesized and used for all mutants.

Figure 3 shows the DNA and amino acid sequence of natural 1 allergen Bet v 1, and the number of mutations Bet v 1.

Figure 4 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool serum of patients allergic abioterrorism Bet v 1 and mutant Bet v 1 Glu45Ser.

Figure 5 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool serum of patients allergic abioterrorism Bet v 1 and mutant Bet v 1 Asn28Thr+Lys32Gln.

Figure 6 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool serum of patients allergic abioterrorism Bet v 1 and mutant Bet v 1 Pro108Gly.

7 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool serum of patients allergic abioterrorism Bet v 1 and mutant Bet v 1 Glu60Ser.

On Fig shows the CD spectra of recombinant and mutant three zones recorded at concentrations, nl is skih to equal.

Figure 9 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool serum of patients allergic abioterrorism Bet v 1 and mutant Bet v 1 in the three zones.

On figa-D shows availability for solvent individually aligned residues of the antigen 5 and the alignment of sequences of the antigen 5 hot (left panel). On the right panel of figure 10 shows the molecular surface antigen 5 with conserved antigen 5:s hot areas.

Figure 11 shows the sequence of primer corresponding to aminobenzo Ves v 5, leading from the beginning of the semantic chain. The lower sequence of the primer originates from the antisense chain.

On Fig shows two generally applicable primer (labeled "fully semantic" and "fully " antisense"), which was synthesized and used for all mutants.

On Fig shows the DNA and amino acid sequence of the natural allergen Ves v 5, and two mutations Ves v 5.

On Fig shows the inhibition of binding of biotinylated recombinant Ves v 5 with serum IgE from a pool serum of patients allergic abioterrorism Ves v 5 and mutant Ves v 5 Lys72Ala.

On figa and shown In a theoretical model of the interaction between allergen and mast cells through cross-reaction with IgE.

On Fig shows the DNA and amino acid sequence of the natural allergen Der p 2.

On Fig schematically shows the primers used to generate mutations. (I) shows the sense and antisense primers. (II) shows the final recombinant protein carrying mutations in these provisions.

On Fig is shown an illustration of constructing mutant Bet v 1 and the list of used primers. Mutants contain from five to nine amino acids.

On Fig shown introduced point mutations on the surface of Bet v 1 (2628) and Bet v 1 (2637). In the mutant Bet v 1 (2628) was administered five primary mutations in one half of the Bet v 1, while the other half remained unchanged. In the mutant Bet v 1 (2637) was administered five primary and three secondary mutations in the other half, the first half remained unchanged.

On Fig shows the spectra of circular dichroism (CD) recombinant Bet v 1.2801 (wild type) and mutant Bet v 1 (2637)recorded at nearly equal concentrations.

On Fig shows the inhibition of binding of biotinylated recombinant Bet v 1.2801 (wild-type) with serum IgE from a pool serum of patients allergic abioterrorism Bet v 1.2801 and Bet v 1 (2628), Bet v 1 (2637) and a mixture of 1:1 Bet v 1 (2628) and Bet v 1 (2637).

On Fig shows the release of histamine basophilic cells of a person under the action of Bet v 1.2801 (wild-type), Bet v 1 (2628) and Bet v 1(2637).

On Fig shows the release of histamine basophilic cells of a person under the action of Bet v 1.2801 (wild-type), Bet v 1 (2628) and Bet v 1 (2637).

On Fig shows point mutations on the surface of Bet v 1 (2744).

On Fig shows point mutations on the surface of Bet v 1 (2753).

On Fig shows point mutations on the surface of Bet v 1 (2744) and Bet v 1 (2753).

On Fig shows the spectra of circular dichroism (CD) Bet v 1.2801 (wild-type) and Bet v 1 (2744), recorded at nearly equal concentrations.

On Fig shows the release of histamine basophilic cells of a person under the action of Bet v 1.2801 (wild type) and mutant Bet v 1 (2744).

On figa-D shows the release of histamine basophilic cells of a person under the action of Bet v 1.2801 (wild type) and mutant Bet v 1 (2744).

On Fig shows point mutations on the surface of Bet v 1 (2733).

On Fig shows the primers used for site-directed mutagenesis, Der p 2.

On figa-C shows an alignment of the sequence of Der p 2 with the other group 2 allergens of house dust mites.

On Fig shows the contours of the surface of the Der p 2 under four different angles.

On Fig shows the contours of the surface mutant of Der p 2 under four different angles.

On figa and B shows the alignment of the sequence of Der p 2 with the other group 1 allergens house dust mites.

On Fig shows the contours of the surface is Der p 1 under four different angles.

On Fig shows the contours of the surface mutant of Der p 1 under four different angles.

On figa-D shows the alignment of the sequence of Phl p 5 c other group 5 allergens of grass.

On figa and B shows the contours of the surface of Phl p 5, model A and model B, respectively, under four different angles.

On figa and B shows the contours of the surface of the mutant Phl p 5, model A and B respectively, under four different angles.

On Fig shows the proliferation of peripheral blood lymphocytes, expressed as stimulation index (SI), for different preparations Bet v 1.

On Fig-44 shows the profile of cytokines by T cells stimulated with various preparations Bet v. On Fig shows a patient with a Th0 profile on Fig profile of Th1 and Fig - Th2 profile.

The PURPOSE of the INVENTION

The rationale of the present invention

The present invention is based on the unique rationale. In accordance with this justification mechanism of successful vaccination allergen is not changing the current of the immune response of Th2 type, and preferably parallel to the initiation of a new immune response, which includes the recognition of B-cell epitope based on the tertiary structure, and the formation of antibodies. It is suggested that the immune response is a partially immune response of the Th1 type. This model is confirmed about what narushenie, what levels of specific IgE does not affect the successful vaccination and this successful treatment is often accompanied by a significant increase in allergen-specific IgG4. In addition, studies of biopsies of the nasal cavity before allergen load and after it did not show a decrease in T cells with a Th2-like phenotype, but there is preferably an increase of T cells with Th1-like phenotype. When the vaccine (or pharmaceutical composition) to impose other way, other than air, it is suggested that the immune response develops in a location physically separate from the current Th2 response, which provides the possibility of parallel existence of the two responses.

Another important aspect of the immune system is the recognition of the existence of so-called dominant epitopes that bind IgE. It is assumed that the dominant epitopes that bind IgE formed linked areas of the surface are dependent on the tertiary structure, large enough to accommodate the binding of antibody and conservative epitopes among isoalleles, variants and/or homologous allergens from related species. The existence of cross-reactive IgE can bind similar epitopes on homologous allergens, supported by the clinical observation that patients with allergies often real the display for a few closely related species, for example, alder, birch and hazel, many kinds of herbs or a few species of house dust mites of the genus Dermatophagoides. This is further supported by laboratory experiments showing the cross-reactivity of IgE between homologous allergens from related species and the ability of an allergen to inhibit the binding of IgE with homologous allergens (Ipsen et al., 1992, reference 16). It is well known that exposure and immune responses associated dose-dependent manner. Based on a combination of observational data, it is assumed that a conservative region of the surface presented to the immune system in higher doses than non-conservative region of the surface, which in result leads to the generation of IgE antibodies with higher affinity, hence the term "dominant epitopes that bind IgE".

In accordance with this rationale it is essential that the allergen had tertiary structure α-carbon skeleton, which is essentially the same as the natural allergen, thus providing a conservative topology of the surface areas surrounding the conservative zone, which are targets for mutagenesis designed to reduce the binding of IgE. Match these criteria allergen can be administered in relatively high doses that improves efficiency is the ratio of generation of a protective immune response without risk to safety.

Moreover, the invention is based on the fact that the trigger for allergic symptoms is cross-linking of the allergen with two specific IgE bound to the surface of effector cells, i.e. mast cells and basophils via high-affinity IgE receptor, FceRI. To illustrate the authors refer to Fig, which depicts theoretical model of allergen with epitopes that bind IgE. On the induction of the release of mediators of mast cell and, hence, on allergic symptoms effect mediated by allergen cross-linking of IgE bound to the surface of fat cells, compare figa. In the case presented on FIGU, two epitope is subjected to mutagenesis to reduce their binding ability with respect to IgE, and, consequently, mediated by allergen cross-linking is prevented. In this regard, it should be noted that the allergens usually include more than three b-cell epitopes. However, on the basis of theoretical model depicted in Fig, it can be assumed that the more epitopes, which are subjected to mutagenesis to destroy or reduce their binding ability with respect to IgE, the lower the risk mediated by allergen cross-linking and the resulting allergic symptoms.

However, in order for tanty allergen was able to cause a new immune response, including IgG response, the allergen must include at least one intact epitope. Preferably, the intact epitope represented the dominant epitope, such as mutant allergen should provide enhanced protection when applying for vaccination.

In conclusion, the inventive idea of the present invention is based on the recognition that the mutant allergen carrying mutations that reduce binding to IgE, in many B-cell epitopes, and at least one intact epitope must, on the one hand, to reduce mediated by allergen cross-linking and, on the other hand, to enable the induction of IgG response by binding ability, competing with IgE. Thus, the mutant allergen must be extremely advantageous allergen due to the fact that the risk of anaphylactic reactions should be substantially reduced.

The present invention is also based on the recognition that a vaccine containing a mixture of various mutated specified allergens, where ideally, many or all of the epitopes presented as intact, should be equally effective in their ability to induce protection against allergic symptoms, as well as natural an existing allergen, which occurred mutant allergens.

A brief statement of the substance of the invention

The present invention relates to the introduction of artificial amino acid substitutions in the number of certain critical provisions, i.e. epitopes that bind IgE, with the purpose of reduction of specific IgE-binding ability of each mutant epitope.

In the invention features a recombinant allergen, characterized in that it is a natural mutant of the existing allergen, where the mutant allergen has at least four primary mutations, each of which reduces specific IgE-binding ability of the mutant allergen compared to the IgE-binding ability of the specified existing natural allergen, where each primary mutation is a substitution of one exposed on the surface amino acid residue by another residue that is not found in the same position in the amino acid sequence of any known homologous protein within the taxonomic species of which is specified existing natural allergen, where each primary mutation separated from each the other primary mutation a period of at least 15 Å and where the primary mutations are located so that at least one circular area surface area 800 Å2does not contain the mutation.

Without assuming theory, the authors suggest that b-cell epitope may be distributed almost over the entire surface of the allergen. Moreover, there is experimental evidence that at least some of the epitopes are part of the cluster of epitopes, including a large number of overlapping epitopes. Therefore, theoretical basis of the present invention is that any exposed on the surface of the amino acid is a potential site for mutations, which can lead to a reduced ability to bind IgE.

Accordingly, the primary mutation is determined by their location relative to each other, i.e. they are distributed separately, so that was a confidence that they represent a mutation in a separate cluster of epitopes.

The present invention also features a composition that includes two or more of the above recombinant mutant allergen variants, where each variant is defined as having at least one major mutation that is absent in at least one of the other variants, where each variant is not present secondary mutations within a radius of 15 Å missing from each of the primary mutation. The composition preferably includes 2-12, more preferably 3-10, more preferably 4-8, and most preferably 5-7 options.

The present invention also proposes a method of obtaining the criminal code of the mentioned above recombinant allergen, characterized in that

a) existing natural allergen identify the number of amino acid residues that have at least 20% available for solvent;

b) choose at least four of the identified amino acid residue in such a way that each of the selected amino acid is separated from the other selected amino acid a period of at least 15 Å and that the selected amino acids are arranged in such a way that at least one circular area surface area 800 Å2does not contain the selected amino acids, and

(C) conduct initial mutation of each of the selected amino acids, which reduces specific IgE-binding ability of the mutant allergen compared to the binding ability of the specified existing natural allergen, where each primary mutation is a substitution of selected amino acid residue with another amino acid that does not exist in the same position in the amino acid sequence of any known homologous protein within a taxonomic species originates from the specified existing natural allergen.

In an alternative aspect the invention relates to a method for producing a recombinant allergen according to the invention, characterized in that the allergen receive is from DNA sequences obtained by DNA shuffling (molecular breeding), from DNA encoding the corresponding natural existing allergen.

Moreover, the invention relates to the above the recombinant allergen for use as pharmaceuticals.

In addition, the invention relates to the use of the above recombinant allergen to obtain a pharmaceutical for the prophylaxis and/or treatment of allergies.

Moreover, the invention relates to the aforementioned composition for use as pharmaceuticals.

The invention relates to the use of the above composition to obtain a pharmaceutical for the prophylaxis and/or treatment of allergies.

Further, the invention relates to the aforementioned pharmaceutical composition, characterized in that it includes the above recombinant allergen or the above composition optionally in combination with a pharmaceutically acceptable carrier and/or excipient and optional with adjuvant. The pharmaceutical composition in accordance with the invention can be in the form of a vaccine allergic reactions caused by natural existing allergen in patients suffering from allergies.

The invention relates to a way of generating an immune response in which object, includes introduction to the subject of the above recombinant allergen, the above composition or the above pharmaceutical compositions.

Further, the invention relates to vaccination or treatment of a subject comprising administration to the subject of the above recombinant allergen, the above composition or the above pharmaceutical compositions.

The invention relates to a method for producing the above pharmaceutical composition, comprising mixing the above recombinant allergen or above compositions with pharmaceutically acceptable substances and/or excipients.

Further, the invention relates to pharmaceutical compositions obtained by the above method.

The invention relates to a method for treatment, prevention or relief of allergic reactions in a subject comprising administration to the subject of the above recombinant allergen, the above composition or the above pharmaceutical compositions.

Further, the invention relates to DNA sequences encoding the allergen in accordance with the invention, its derivative, to part of the sequence, to its degenerate sequences or sequences with which it is hybridized in tough conditions is, where the specified derivative, partial sequence, a degenerate sequence or hybridization sequence encodes a peptide having at least one b-cell epitope.

The invention relates to an expression vector comprising DNA according to the invention.

Moreover, the invention relates to the cell host comprising the expression vector according to the invention.

Additionally, the invention relates to a method for producing a recombinant mutant allergen, comprising the stage of culturing the host cell in accordance with the invention.

Finally, the invention relates to the recombinant allergen according to the invention encoded by the DNA sequence in accordance with the invention, comprising at least one T-cell epitope capable of stimulating a clone of T-cells or T-cell line specific for natural existing allergen.

The mutants according to the invention should preferably be capable of stimulating allergen-specific T-cell lines in a similar way/in a similar extent when measured using the index of stimulation of T cells.

Detailed description of the invention

In a preferred implementation of the invention the primary mutation is edeleny intervals 20 Å preferably 25 Å and most preferably 30 Å.

It is assumed that the allergen includes a number of potential binding regions for specific IgE, where each area has a size of approximately 800 Å2moreover , the surface region includes a large number of overlapping epitopes. Thus, the allergen is the number of potential primary mutation region of the surface, divided by 800 Å2.

Preferably, a recombinant allergen according to the invention includes from 5 to 20, preferably from 6 to 15, more preferably from 7 to 12 and most preferably from 8 to 10, the primary mutations.

In a preferred implementation of the invention the surface area, not including mutations, is an area of 700 Å2preferably 600 Å2more preferably 500 Å2and most preferably 400 Å2.

In a preferred implementation of the invention the recombinant allergen includes a number of secondary mutations, each of which reduces specific IgE-binding ability of the mutant allergen compared to the binding ability of the specified existing natural allergen, where each secondary mutation is a substitution of one exposed on the surface of aminotic is now residue by another residue, which is not found in the same position in the amino acid sequence of any known homologous protein within the taxonomic species of which is specified existing natural allergen, where secondary mutations located outside of the specified circular region of the surface.

Secondary mutation can be localized in the immediate vicinity of the primary mutations, i.e. secondary mutation may be an additional mutation of the same epitope, which was mutated in primary mutations.

In a preferred implementation of the invention at least one exposed on the surface of amino acids that are replaced in existing natural allergen, is accessible to solvent than 20%, preferably above 30%, more preferably above 40%, and most preferably above 50%.

In another preferred implementation of the invention at least one exposed on the surface of amino acids that are replaced in existing natural allergen, is more conservative than 70%, preferably 80% and most preferably 90% identity in all known homologous proteins within a species of which is specified existing natural allergen.

Preferably, a recombinant allergen according to izaberete the receiving had the same tertiary structure α -carbon skeleton of the indicated existing natural allergen.

When comparing tertiary structures α-carbon skeleton of mutant molecules and natural existing allergen average root mean square deviation of atomic coordinates is preferably below 2 Å.

In the preferred implementation of the recombinant allergen according to the invention, each amino acid residue included in the mutant allergen, is not found in the same position in the amino acid sequence of any known homologous protein within a taxonomic genus, preferably subfamily, more preferably family, more preferably - superfamily, more preferably Legion, more preferably suborder and most preferably squad to that which is specified existing natural allergen.

In a preferred implementation of the invention the recombinant mutant allergen in accordance with the invention is not existing in the nature of the allergen.

Specific IgE binding with mutant allergen preferably reduced by at least 5%, preferably at least 10% compared with the natural existing itelligence or similar recombinant proteins in immunological those who tah serum, obtained from allergic patients that respond to specific IgE source, or pools.

Another way of estimating reduced IgE binding and reduced ability to mediate cross-linking of mutant is the ability of the mutant to trigger the release of histamine (HR). The release of histamine can be measured in several tests for determining the release of histamine. Reduced ability of the mutants to release histamine is due to a reduced affinity of specific IgE associated with the cell surface, and their reduced ability to facilitate cross-linking. HR preferably reduced by 5-100%, more preferably 25-100%, even more preferably 50-100%, and most preferably 75-100% for mutants according to the invention compared with existing natural allergens.

Usually circular area surface area 800 Å2not containing mutations that includes atoms of 15-25 amino acid residues.

Preferred recombinant allergen according to the invention differs in that it exposed on the surface amino acid residues are ranked in relation to accessibility to solvent, and the fact that one or more amino acids among the more solvent-accessible replaced.

More preferred R is combinatii allergen in accordance with the invention differs what is exposed on the surface of amino acid residues ranked on the degree of conservatism in all known homologous proteins within a species of which is specified existing natural allergen, and the fact that one or more amino acids among the more conservative replaced.

Preferably the recombinant allergen according to the invention comprises from 1 to 4 secondary mutations in the primary mutation.

The preferred implementation of the invention is characterized by the fact that one or more substitutions by using site-directed mutagenesis.

Another preferred implementation of the invention is characterized by the fact that one or more substitutions by using random mutagenesis.

Additional preferred implementation of the invention is characterized by the fact that one or more substitutions by using DNA shuffling.

Recombinant allergens in accordance with the invention can be suitable mutant inhaled allergen originating, inter alia, from trees, grasses, plants, fungi, house dust mites, cockroaches, and animal hair and dandruff. Important allergens of pollen of trees, grasses and plants are those that come from taxonomic units Fagales, Oleales, and Pinales, including, among other things, birch (Betula), alder (Alus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), detachment Poales, including, among other things, the grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, and Secale, units Asterales and Urticales including, among other things, plants of the genera Ambrosia and Artemisia. Important inhaled allergens fungi are, among others, those that come from the genera Alternaria and Cladosporium. Other important inhaled allergens are derived from house dust mites of the genus Dermatophagoides that occur from cockroaches and which come from animals, such as cat, dog and horse. In addition, recombinant allergens in accordance with the invention may be mutants of allergens, poisons, including those that occur from stinging or biting insects such as those that result from taxonomic order Hymenoptera, including bees (superfamily Apidae), wasps (superfamily Vespidea) and ants (superfamily Formicoidae).

Specific allergic components include, for example, Bet v 1 (B. verrucosa, birch), Aln g 1 (Alnus glutinosa, alder), Cor a 1 (Corylus avelana, hazel) and Car b 1 (Carpinus betulus, hornbeam) detachment Fagales. Others are Cry j 1 (Pinales), Amb a 1 and 2, Art v 1 (Asterales), Par j 1 (Urticales), Ole e 1 (Oleales), Ave e 1, Cyn d 1, Dac g 1, Fes p 1, Hol 1 1, Lol p 1 and 5, Pas n 1, Phl p 1 and 5, Poa p 1, 2 and 5, Sec c 1 and 5, and Sor h 1 (different types of grass pollen), Alt a 1 and Cla h 1 (mushrooms), Der f 1 and 2, Der p 1 and 2 (house dust mites, D. farinae and D. pteronyssinus, respectively), Lep d 1 and 2 (Lepidoglyphus destructor; glue and storage), Bla g 1 and 2, Per a 1 (cockroaches, Blatella germanica and Periplaneta americana, respectively), Fel d 1 (cat), Can f 1 (dog), Equ c 1, 2 and 3 (horse), Apis m 1 and 2 (worker bee), Ves v 1, 2 and 5, Pol a 1, 2, and 5 (all OS) and Sol i 1, 2, 3 and 4 (ant Richter).

In one implementation of the recombinant allergen is a mutant Bet v 1. Amino acids that are potentially suitable for substitutions include amino acids

One or more primary or secondary substitutions can be selected from the group consisting of

and +160 N

where + means includes additional amino acid.

Examples of mutant Bet v 1 in accordance with the present invention are the following (brackets when they are used, indicate the primary and secondary mutations):

Mutant A:

Mutant B:

Mutant 2595 (example 2):

Mutant 2628 (example 4):

Mutant 2637 (example 4):

Mutant 2724:

Mutant 2733 (example 4):

Mutant 2753 (example 4):

Mutant 2744 + 2595:

is utant 2744 + 2628:

Mutant 2744 + 2595 + 2628:

In addition, all of the above mutants include one or more of the following substitutions:

In another implementation of the recombinant allergen is derived from allergen poison from taxonomic units Vespidae, Apidae and Formicoidae.

In an additional implementation of the recombinant allergen is derived from the Ves v 5. Amino acids that are potentially suitable for substitutions include amino acids

One or more primary and secondary substitutions can be selected from the group consisting of K29A, T67A, K78A, V84S, Yl02A, K112S, K144A, K202M and N203G.

In another implementation of the recombinant allergen is derived from the Der p 2. Amino acids that are potentially suitable for substitutions include amino acids

One or more primary and secondary substitutions can be selected from the group consisting of K6A, N10S, Kl5E, S24N, H30N, K48A, E62S, H74N, K77N, K82N, K100N and Rl28Q.

Examples of mutant Bet v 1, in accordance with the present invention, are the following.

Mutant A:

K6A, K15E, H30N, E62S.

Mutant B:

K6A, K15E, H30N, E62S, H74N, K82N.

Mutant C:

K6A, N10S, K15E, S24N, H30N, K48A, E62S, H74N, K77N, K82N, K100N and R128Q

Vaccines

Getting the vaccine is generally well what izvestno in the art. Vaccines are usually obtained in the form of injectables, either as liquid solutions or suspensions. The vaccine can also emulsify or to make so that it was suitable for intranasal, and oral administration, including hominids and sublingual administration. Consider immunogenic component (recombinant allergen, as here defined) may be appropriately mixed with excipients that are pharmaceutically acceptable and, in addition, is compatible with the active ingredient. Examples of suitable excipients include water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. The vaccine may further contain other substances, such as moistening agents, emulsifying agents, sautereau agents or adjuvants that enhance the effectiveness of the vaccine.

Most often, the vaccine is administered parenterally by subcutaneous or intramuscular injection. The compositions, which are suitable for administration by other means, include oral formulations and suppositories. Vaccine for oral administration can be appropriately composed with excipients commonly used for such compositions, such as mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, carbonate mage the Oia and the like, pharmaceutical quality. The composition may be formulated as solutions, suspensions, emulsions, tablets, pills, capsules, and compositions of continuous release, aerosols, powders or granular forms.

Vaccines are given in a way that was compatible with the dosage formulation and in such amount which would be therapeutically effective and immunogenic. The amount of active ingredient contained in the vaccine depends on the subject to the treatment of the subject, among other things, the ability of the immune system of a subject to respond to treatment, routes of administration, age and weight of the subject. Suitable dose ranges can vary in the range approximately from 0.0001 μg to 1000 μg.

As mentioned above, the enhanced effect can be obtained by adding to the composition adjuvants. Examples of such adjuvants include aluminum hydroxide and phosphate (alum) or calcium phosphate in the form of from 0.05 to 0.1 percent solution in phosphate buffered saline, synthetic polymers of sugars or polylacticacid (PLG)used as 0.25 percent solution. Can also be used a mixture of c bacterial cells such as C. parvum, endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil carrier, such as monooleate mA is Nida (Aracel A) or emulsion with 20 percent solution of perfluorocarbons (for example, Fluosol-DA)used as a block replacement. Can also be applied to oily emulsions, such as MF-59. Can also be applied to other adjuvants, such as complete and incomplete adjuvants's adjuvant, as well as the saponins, such as Qui1A, Qs-21 and ISCOM and RIBI.

Often to ensure that steps should be repeated introduction of the vaccine. Often the vaccine is administered in the form of the initial introduction and subsequent inoculation or other injections. The number of vaccinations is usually from 1 to 50, usually not more than 35 vaccinations. Vaccination is usually carried out in the interval from once a fortnight to once in two months during the period from 3 months to 5 years. It is assumed that it creates the desired prophylactic or therapeutic effect.

Recombinant allergen can be applied in the form of a pharmaceutical preparation that is suitable to provide some protection against allergic reactions in the season when there are symptoms (prevention). Typically, the treatment should be repeated every year to maintain the protective action. Particularly suitable for this purpose, the preparations made for intranasal, oral and sublingual application.

A method of obtaining a recombinant allergen according to the invention

As indicated above, the present invention t is the train relates to a method for producing a recombinant mutant allergen according to the invention, cf. p.

Exposed on the surface of amino acids, which are suitable for replacement in accordance with the present invention, can be identified on the basis of information about their accessibility to solvent (water), which reflects the degree of exposure on the surface. The preferred implementation of the method according to the invention is characterized by the ranking of the above identified amino acid residues in relation to accessibility to solvent and replacing one or more amino acids among the more solvent-accessible.

The second argument, which can contribute to the identification exposed on the surface of amino acids, which are suitable for replacement in accordance with the present invention, is the degree of conservatism of the amino acids in all known homologous proteins within a species of which is specified existing natural allergen. Alternatively, as the second option is used, the degree of conservatism in all known homologous proteins within a taxonomic genus, subfamily, family, superfamily, more preferably Legion, suborder, and most preferably squad to that which is specified existing natural allergen.

Accordingly, the preferred implementation of the method according to the but the invention is distinguished by the selection of the identified amino acid residues, who are conservative, with more than 70%, preferably more than 80% and most preferably more than 90% identity with all known homologous proteins within a species of which is specified existing natural allergen.

Moreover, particularly preferred implementation of the method according to the invention is ranking above identified amino acid residues in the degree of conservatism in all known homologous proteins within a species of which is specified existing natural allergen, and replacing one or more amino acids among more conservative.

Additional preferred implementation of the method according to the invention includes the selection of the identified amino acids to create the mutant allergen, which has essentially the same tertiary structure α-carbon skeleton, as specified natural existing allargando preferred implementation of the method according to the invention is characterized by the fact that substitution of amino acid residues is performed using site-directed mutagenesis.

An alternative preferred implementation of the method according to the invention is characterized by the fact that substitution of amino acid residues is performed using a dicing the NC.

Criteria replacement

For molecules, the tertiary structure of which is defined (for example, by x-ray crystallography or NMR, electron microscopy), the mutant carrying the substituted amino acid(you)should preferably satisfy the following criteria.

1. Full tertiary structure α-carbon skeleton of the molecule preferably is conservative. Conservatism is defined as the average value of the square root of the standard deviation of the atomic coordinates of the atoms in a comparison of the structures below 2 Å. This is important for two reasons: a) it is expected that the solid surface of the natural allergen is an overlapping continuum of potential epitopes that bind to the antibodies. The main part of the surface of the molecule does not undergo replacement(us), and, thus, its properties to induce the binding of the antibody is maintained, which is important for the development of new protective variants of antibodies directed to epitopes present in natural allergen; (b) Stability regarding retention periods, and when introduced into the body fluids.

2. Amino acids that are replaced are located preferably on the surface and thus available for binding antibodies. Amino acids located on the surface in the three-dimensional structure, usually have available the ability to solvent (water) at least 20%, suitable 20-80%, more suitable 30-80%. Accessibility to solvent is defined as the area of the molecule available for a sphere with a radius comparable to the solvent molecule (water, r=1,4 Å).

3. Each of the substituted amino acids is preferably in conservative areas, with areas larger than 400 Å2. Conservative zone is defined as consistently related subjects exposed on the surface of conservative amino acid residues and skeleton. Conservative amino acid residues determined using sequence alignment of all known (withdrawn) the amino acid sequences of homologous proteins within the same taxonomic species, genus, subfamily, family, superfamily, Legion, suborder or detachment. The position of the amino acids with identical amino acid residues in more than 70% of the sequences are considered as conservative. It is expected that conservative areas contain epitopes that are being targeted IgE majority of patients.

Conservatism tertiary structure α-carbon skeleton is best determined by obtaining identical structures using x-ray crystallography or NMR before and after mutagenesis. In the absence of structural data describing needlecases CD spectra of the mutant, or unanimiously data for example, antibody reactivity, it is possible to imagine the possible conservatism of the tertiary structure α-carbon skeleton, when compared with the data obtained in the analysis of molecules defined structurally.

4. Within conservative areas amino acids for mutagenesis should preferably be selected among the most accessible to solvent (water), preferably located near the center of conservative area.

5. Preferably a polar amino acid residue substituted another polar amino acid residue and non-polar amino acid residue replaced with other non-polar amino acid residue.

Order essentially preserve the three-dimensional structure of the allergen amino acid is intended to include, can be selected on the basis of comparison with protein, which is a structural homologue of the allergen, for example protein, which belongs to the same taxonomic unit that allergen, and which has no cross-reactions with the allergen.

DNA according to the invention

In the preferred implementation of the DNA sequence according to the invention is a derivative of a DNA sequence that encodes a natural existing allergen.

Preferably derived DNA obtained by site-directed or random mutagenesis is NC, coding existing natural allergen.

In a first particularly preferred implementation of the DNA sequence is a derivative of the sequence presented in figure 3, where the DNA sequence metirovan so that it encodes an allergen having at least four mutations selected from the group consisting of

In a second particularly preferred implementation of the DNA sequence is a derivative of the sequence presented on Fig, where the DNA sequence metirovan so that it encodes an allergen having at least four mutations selected from the group consisting of

In a third particularly preferred implementation of the DNA sequence is a derivative of the sequence presented on Fig, where the DNA sequence metirovan so that it encodes an allergen having at least four mutations selected from the group consisting of

DNA shuffling

Recombinant mutant allergen in accordance with the present invention can be obtained by using DNA sequences obtained by DNA shuffling (molecular breeding), sootvetstvujushej natural DNA. DNA shuffling may be performed in accordance with the techniques disclosed in the article Punnonen et al. (reference 25), and techniques disclosed in the mentioned articles, all of which are included in this description as the link.

Diagnostic test

Recombinant mutant allergen in accordance with the invention have diagnostic capabilities and advantages. Vaccines allergen prior art are based on natural extracts of an existing source of allergen, and, thus, represent a wide variety of isoforms. Individuals with allergies originally sensibiliser, and he has IgE to one or more isoforms present. Some of the isoforms may be related to allergic reactions of the individual with allergies due to homology and subsequent cross-react with isoform in respect of which the individual is allergic, while other isoforms may not be relevant, since they have no IgE binding epitopes to which the individual is allergic has specific IgE. Because of this heterogeneity specificity populations of IgE some isoforms may therefore be safe in the introduction, i.e. they do not lead to allergic response through IgE, whereas other isoforms can be harmful, causing n is desirable side effects.

Thus, the mutants according to the invention and compositions according to the invention, intended for therapeutic administration, may also be used for diagnostic test in vivo or in vitro to monitor the appropriateness, safety or outcome of treatment using such mutants or compositions. Diagnostic specimens, intended for use, include samples taken from the body, such as serum.

Thus, the invention also relates to a diagnostic test to assess the appropriateness, safety or outcome of treatment of a subject with the use of mutant allergen in accordance with the invention, or compositions in accordance with the invention, where containing IgE sample of the subject is mixed with the indicated mutant or a specified composition and assess the level of interaction between IgE specified pattern and the specified mutant. Assessment of the level of interaction between IgE specimen and the specimen can be carried out using known immunoassay.

Definition

In accordance with the present invention the expression "reduction of specific IgE-binding capacity in comparison with the IgE-binding ability of the specified existing natural allergen" means that the reduction is measured with statistical significance (p<0.05), at least one immunological the Ohm test using serum from the subject, allergic towards existing natural allergen. Preferably IgE-binding capacity is reduced by at least 5%, more preferably at least 10%.

The expression "exposed on the surface of the amino acid" means that the amino acid residue is located on the surface of the three-dimensional structure so that when the allergen is in solution at least part of at least one atom of amino acid residue available for contact with the surrounding solvent. Preferably, the amino acid residue in the three-dimensional structure was accessible to solvent (water) at least 20%, suitable at least 30%, more suitable at least 40% and most preferably at least 50%.

The expression "the accessibility to solvent" is defined as the area of the molecule available for a sphere with a radius comparable to the radius of the solvent molecules (water, r=1,4 Å).

The expression "exposed on the surface and exposed to solvent" is used interchangeably.

The expression "the taxonomic species of which is specified existing natural allergen" means species in the taxonomic system.

Further, the expression "specified mutant allergen, having essentially the same tertiary structure α-carbon skeleton as specified existing natural allergen", means for comparing structures the average value of the square root of the standard deviation of the atomic coordinates is preferably below 2 Å.

In accordance the present invention, the expression "replacement" means a deletion, substitution or addition of amino acids compared to the amino acid sequence of natural existing allergen.

The present invention will be further illustrated by the following non-limiting examples.

Example 1

Example 1 describes the preparation of recombinant mutant allergen with one and three primary mutations. Recombinant mutant allergen according to the invention, i.e. allergens, containing at least four primary mutations, can be obtained using similar methods.

Identification of common epitopes of allergens in pollen Fagales

The major allergen of birch pollen Bet v 1 is approximately 90% amino acid sequence identity with the major allergens of pollen taxonomically related trees, i.e. Fagales (for example, hazel and hornbeam), and in patients with Allergy to birch pollen is often manifested clinical symptoms of allergic cross-reactions to data homologous to Bet v 1 proteins.

Bet v 1 also shows approximately 50-60% sequence identity with allergic and proteins, available in some fruits (e.g. Apple and cherries) and vegetables (such as celery and carrots), and there are clinical indications cross-allergic reaction to Bet v 1 and the data associated with food proteins.

In addition, Bet v 1 exhibits significant similarity in sequence (20-40%) with a group of plant proteins called proteins related to pathogenesis (RP-10), however, reports of allergic cross-reactions to RP data-10 proteins are absent.

Molecular modeling suggests that patterns of allergens Fagales and food allergens and proteins RP-10 is almost identical to the structure of Bet v 1.

About the structural basis of antigenic cross-reactions of Bet v 1 was reported at Gajhede et al., 1996 (reference 17), where on the surface molecules of Bet v 1 was able to identify three areas that are common to known allergens pollen of trees. Thus, any IgE that recognizes zone information on Bet v 1, should be able to cross-react and bind with other major allergens of Fagales pollen and cause Allergy symptoms. Identification data the common areas was carried out after the alignment of all known amino acid sequences of the major allergens of pollen of trees in conjunction with the analysis of surface molecules Bet v 1, identified on the basis of the tertiary structure α-carbon skeleton, which coalsales link 17. In addition, the area covered by the antibody after binding, was defined as areas that have a certain minimum size (>400 Å2).

The choice of amino acid residues for site-directed mutagenesis

Amino acid residues for site-directed mutagenesis was chosen among the remaining available in specific areas of Bet v 1 and common areas, since it was expected that modifications will affect the binding of serum IgE in most patients exhibiting clinical cross-allergic reaction to pollen of trees.

The relative orientation and the percentage of exposure to the solvent of each amino acid residue in the corresponding area was calculated on the basis of their atomic coordinates. Residues having a low degree of exposure to solvent (<20%), were considered as irrelevant for mutagenesis because of the possible disturbance of structure or lack of interaction with the antibody. The remaining residues were ranked in accordance with the degree of their exposure to the solvent.

Sequence alignment

Sequences homologous to the desired sequence (Bet v 1 No. 2801, WHO IUIS of the Subcommittee on the nomenclature of allergens), were obtained from GenBank and database EMBL sequences using BLAST search (Altschul et al., reference 18). Took into consideration all of th is of the sequences, for BLAST which gave a probability of less than 0.1, and was one list that contains a single list of homologous sequences. They were aligned using CLUSTAL W (Higgins et al., reference 19) and the percentage identity was calculated for each position in the sequence when considering a complete list or only taxonomically related species. Just with Bet v 1 No. 2801 were homologous 122 sequence, of which 57 sequences originated from taxonomically related species.

Cloning of the gene encoding Bet v 1

RNA was obtained from Betula verrucosa pollen (Allergon, Sweden) by extraction with phenol and precipitation of LiCl. Affinity chromatography on oligo(dT)-cellulose was performed batchwise in abandonhouse tubes, and double-stranded cDNA was synthesized using a commercially available kit (Amersham). Encoding Bet v 1 DNA amplified by PCR and cloned. Briefly, PCR was performed using cDNA as template, and primers were designed to match the sequence of the cDNA in positions corresponding to aminobenzo Bet v 1 and the 3'-untranslated region, respectively. The primers were extended at the 5'-ends to create restriction sites (NcoI and HindIII) for directional cloning into pKK233-2.

Subclavian in pMAL-c

The gene encoding Bet v 1, then subcontinua is a hybrid vector of the protein, binding maltose, pMAL-c (New England Biolabs). Gene amplified by PCR and was subcloned into the same frame with a malE to create hybrid operons protein - binding protein maltose (MBP), - Bet v 1, in which MBP and Bet v 1 was divided by the site of proteolytic cleavage by factor Xaplaced to restore the true aminobenzoic sequence of Bet v 1 after splitting, as described in reference 15. Briefly, PCR was performed with the use of pKK233-3, containing an insert of the Bet v 1 as the matrix, and primers corresponding to the amino - and carbonsilicon protein, respectively. Promoter proximal primer was extended with 5'-end to create 4 codons encoding in a frame read the site of proteolytic cleavage by factor Xa. Both primers was further extended on the 5'-ends to create restriction sites (kpni restriction sites for cloning. Encoding Bet v 1 genes were subclinically using 20 cycles of PCR to reduce the frequency of PCR artifacts.

Mutagenesis in vitro

The in vitro mutagenesis was performed by PCR using recombinant pMAL-c with the insertion of a Bet v 1 as a matrix. Each mutant gene Bet v 1 were obtained using 3 PCR reactions using 4 primers.

Two specific mutations oligonucleotide primer was synthesized in accordance with each mutation, one for each DNA chain, see figure 1 and 2. Using Mut is nnogo nucleotide(s) as a starting point both primer was extended by 7 nucleotides on the 5'-end and 15 nucleotides at the 3'-end. Extending nucleotides were identical in sequence to the gene of Bet v 1 in the effector region.

Further synthesized and used for all mutants two generally applicable primer (labeled in figure 2 as "fully semantic" and "fully " antisense"). These primers had a length of 15 nucleotides corresponding to the sequence of fields in the vector pMAL-c, located at a distance of approximately 1 TPN above and below the Bet v 1. The upper sequence of the primer originates from the sense circuit, and the lower sequence of the primer originates from the antisense chain, see figure 2.

Conducted two independent PCR reactions, essentially in accordance with standard methods, except that spent only 20 temperature cycles to reduce the frequency of PCR artifacts. In each PCR reaction used the pMAL-c with the insertion of a Bet v 1 as a matrix, one specific for the mutation and one generally applicable primer in specific combinations.

Introduction four amino acid substitutions (Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly) in the mutant in the three zones was similar to the above-described stage sequential way. First introduced mutation Glu45Ser, then the mutation Pro108Gly and, finally, mutations Asn28Thr, Lys32Gln using pMAL-c with the insertion of a Bet v 1 No. 2801, Bet v 1 (Glu45Ser), Bet v 1 (Glu45Ser, Pro108Gly) as matrices, respectively. PCR products were cleaned by e is entropies agarose gel and electrosurgery with subsequent precipitation with ethanol. A third PCR reaction was carried out using the combined PCR products of the first two PCR reactions as matrix and both generally applicable primers. And again applied a standard PCR (20 cycles. The PCR product was purified using the methods of agarose gel electrophoresis and electroelution with subsequent precipitation with ethanol, cut by restriction enzymes (BsiWI/EcoRI) and directionally ligated into pMAL-c with insertion of Bet v 1, cut with the same enzymes.

Figure 3 shows a General view of all of the following 9 mutations Bet v 1: Thr10Pro, Asp25Gly, Asn28Thr + Lys32Gln, Glu45Ser, Asn47Ser, Lys55Asn, Glu60Ser (netnanny), Thr77Ala and Pro108Gly. Was also obtained additional mutant with four mutations (Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly). Of these five mutants were selected for further testing: Asn28Thr + Lys32Gln, Glu45Ser, Glu60Ser, Pro108Gly and mutant three zones Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly.

Sequencing of nucleotides

Determination of the nucleotide sequence of the gene coding Bet v1, conducted before and after sublimirovanny and after mutagenesis in vitro, respectively. Plasmid DNA from 10 ml of bacterial culture, growing to saturation overnight in LB medium with the addition of 0.1 g/l ampicillin, was purified on columns (Qiagen-tip 20 and sequenced with the use of the kit for sequencing DNA Sequenase version 2.0 (USB), in accordance with the recommendations of the suppliers.

Expression and purification of recombinant Bet v 1 and mutants

<> Recombinant Bet v 1 (Bet v 1 No. 2801 and mutants) were hyperexpression in Escherichia coli DH 5a in the form of a hybrid with maltose binding protein and was purified as described in reference 15. Briefly, recombinant E. coli cells were grown at 37°C to an optical density of 1.0 at 436 nm, after which the expression of a hybrid protein Bet v 1 was induced by adding IPTG. The cells were collected by centrifugation after 3 hours after induction, resuspendable in lytic buffer and destroyed by sonication. After ultrasonic treatment and additional centrifugation was isolated recombinant hybrid protein with amylose affinity chromatography and then were digested by incubation with factor Xa (reference 15). After FXa cleavage of recombinant Bet v 1 were isolated by the method of gel filtration and, if considered necessary, were subjected to additional cycle amylose affinity chromatography to remove trace quantities of maltose binding protein.

Purified recombinant Bet v 1 was concentrated by ultrafiltration to approximately 5 mg/ml and kept at 4°C. the Final yield of purified preparations of recombinant Bet v 1 ranged from 2 to 5 mg per liter of E. coli culture.

Preparations of purified recombinant Bet v 1 were the only bands after electrophoresis in polyacrylamide with DDS-Na and color silver with an apparent mol the molecular mass of 17.5 kDa. Sequencing of the N-Terminus showed sequence, expected on the basis of the cDNA nucleotide sequences, and quantitative amino acid analysis showed the expected amino acid composition.

Previously it has been shown (reference 15), recombinant Bet v 1 No. 2801 immunochemical indistinguishable from natural Bet v 1.

Immunoelectrophoresis using rabbit polyclonal antibodies

Seven mutant Bet v 1 was obtained in the form of recombinant protein Bet v 1, was purified as described above and tested for their ability to react with polyclonal rabbit antibodies, induced against Bet v 1, isolated from pollen of birch. In the analysis using immunoelectrophoresis (Rakitovo of immunoelectrophoresis) in the in vivo rabbit antibodies were able to precipitate all mutants, indicating that the mutants contained conservative α-carbon skeleton of the tertiary structure.

To analyze the impact on the reaction of polyclonal IgE person for subsequent analysis of selected mutants Glu45Ser, Pro108Gly, Asn28Thr + Lys32Gln and Glu60Ser.

Mutant Glu45Ser Bet v 1

Glutamic acid at position 45 shows a high degree of exposure to solvent (40%) and is located in the area of a surface molecule that is common to allergens Fagales (zone I). It was found that the serine residue located in position 45 of some homologous to Bet v 1 proteins PR-10 suggesting that that glutamic acid may be replaced by a serine without disturbing α-carbon skeleton of the tertiary structure. In addition, since the known sequences of allergens Fagales not contain a serine at position 45, the replacement of glutamic acid serine leads to a not existing in the nature of a molecule of Bet v 1.

The proliferation test of the T cells using recombinant mutant Glu45Ser Bet v 1

The analysis was carried out as described in Spangfort et al., 1996a. It was found that the recombinant mutant Glu45Ser Bet v 1 is able to induce proliferation of lines of T-cells from three different patients with Allergy to birch pollen with parameters of stimulation, similar to the indicators specific recombinant and natural protein.

Crystallization and structural analysis of recombinant Glu45Ser Bet v 1

Crystals of recombinant Glu45Ser Bet v 1 were grown using the vapor diffusion at 25°C, essentially as described by Spangfort et al., 1996b (reference 21). Glu45Ser Bet v 1 in a concentration of 5 mg/ml was mixed with an equal volume of a mixture of 2.0 M ammonium sulfate, 0.1 M sodium citrate, 1% (V/V) dioxane, pH of 6.0, and balanced against 100 volumes of a mixture of 2.0 M ammonium sulfate, 0.1 M sodium citrate, 1% (V/V) dioxane, pH of 6.0. After 24 hours of equilibration, the growth of crystals induced by application of the method of application described in reference 21, using crystals of recombinant Bet v 1 wild type is diluted.

After about 2 months the crystals were collected and analyzed using x-rays obtained with a rotating anode Rigaku, as described in reference 21, and the structure was determined by molecular replacement.

The structure of the mutant Bet v 1 Glu45Ser

The structural impact of mutations was found by determining the diffraction grown three-dimensional crystals of the protein Bet v 1 Glu45Ser with a resolution of 3.0 Å analysis using x-rays obtained with a rotating anode. Replacement of glutamic acid serine at position 45 was confirmed using a map of the electron density structure of Bet v 1 Glu45Ser, which also showed that the overall tertiary structure α-carbon skeleton is preserved.

Properties of the IgE binding mutant Bet v 1 Glu45Ser

Properties of the IgE binding mutant Bet v 1 Glu45Ser compared with those of recombinant Bet v 1 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from patients allergic to birch.

Recombinant Bet v 1 no. 2801 was biotinilated at a molar ratio of 1:5 (Bet v 1 no. 2801:Biotin). The inhibition test was carried out as follows: serum sample (25 μl) were incubated with anti-IgE on the solid phase, washed, resuspendable and additionally incubated with a mixture of biotinylated Bet v 1 no. 2801 (3.4 nm) and the mutant (0 to 28,6 nm). The amount of biotinylated Bet v 1no. 2801 associated with the solid phase was determined by measuring RLU after incubation with streptavidin labeled with acridine ester. The degree of inhibition was calculated as the ratio between the RLU obtained with the use of buffer and mutant as an inhibitor.

Figure 4 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool obtained from allergic patients, abioterrorism Bet v 1 and mutant Bet v 1 Glu45Ser.

There is a clear difference in the number of the corresponding recombinant proteins needed to achieve 50% inhibition of the binding of serum IgE present in the United whey. 50% inhibition of recombinant Bet v 1 is achieved at approximately 6.5 ng, whereas the corresponding concentration for the mutant Bet v 1 Glu45Ser approximately 12 ng. This shows that the point mutation introduced in the mutant Bet v 1 Glu45Ser, reduces the affinity for specific serum IgE approximately in 2 times. The maximum level of inhibition achieved under the action of the mutant Bet v 1 Glu45Ser, clearly below the achieved under the action of recombinant Bet v 1. This may indicate that after replacing Glu45Ser some of the specific IgE present in the United whey, not able to identify the mutant Bet v 1 Glu45Ser.

Mutant Bet v 1 Asn28Thr + Lys32Gln

Aspartate and lysine Polo is aniah 28 and 32, accordingly, exhibit a high degree of exposure to solvent (35% and 50%, respectively) and localized in the area of the molecular surface, which is common to allergens Fagales (zone II). Structural aspartate 28 and lysine 32 are located close to each other on the molecular surface and is most likely to interact via hydrogen bonds. It was found that some proteins PR-10, homologous to Bet v 1, the residues of threonine and glutamate occupy positions 28 and 32, respectively, which indicates that the aspartate and lysine can be substituted by threonine and glutamate, respectively, without disrupting the tertiary structure α-carbon skeleton. In addition, since none of the natural itelligence sequence does not contain threonine and glutamate at positions 28 and 32, respectively, replace, create not existing in nature molecule Bet v 1.

Properties of the IgE binding mutant Bet v 1 Asn28Thr + Lys32Gln

Properties of the IgE binding mutant Asn28Thr + Lys32Gln compared with those of recombinant Bet v 1 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from patients allergic to birch, as described above.

Figure 5 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool obtained from allergic patients, abioterrorism Bet v 1 mutant Bet v 1 Asn28Thr + Lys32Gln.

There is a clear difference in the number of the corresponding recombinant proteins needed to achieve 50% inhibition of the binding of serum IgE present in the United whey. 50% inhibition of recombinant Bet v 1 is achieved at approximately 6.5 ng, whereas the corresponding concentration for the mutant Bet v 1 Asn28Thr + Lys32Gln approximately 12 ng. This shows that point mutations introduced into the mutant Bet v 1 Asn28Thr + Lys32Gln, reduce the affinity for specific serum IgE approximately in 2 times.

The maximum level of inhibition achieved under the action of the mutant Bet v 1 Asn28Thr + Lys32Gln, clearly below the achieved under the action of recombinant Bet v 1. This may indicate that after replacement Asn28Thr + Lys32Gln some of the specific IgE present in the United whey, not able to identify the mutant Bet v 1 Asn28Thr + Lys32Gln.

Mutant Bet v 1 Pro108Gly

Proline at position 108 exhibits a high degree of exposure to solvent (60%) and localized in the area of the molecular surface, which is common to allergens Fagales (zone III). It was found that some proteins PR-10, homologous to Bet v 1, the glycine residue occupies position 108 that indicates that Proline may be replaced by glycine without disrupting the tertiary structure α-carbon skeleton. In addition, since none of the natural from llangennech sequence does not contain a glycine at position 108, substitution of Proline for glycine creates not existing in nature molecule Bet v 1.

Properties of the IgE binding mutant Bet v 1 Pro108Gly

Properties of the IgE binding mutant Bet v 1 Pro108Gly compared with those of recombinant Bet v 1 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from patients allergic to birch, as described above.

Figure 6 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool obtained from allergic patients, abioterrorism Bet v 1 and mutant Bet v 1 Pro108Gly.

There is a clear difference in the number of the corresponding recombinant proteins needed to achieve 50% inhibition of the binding of serum IgE present in the United whey. 50% inhibition of recombinant Bet v 1 is achieved at approximately 6.5 ng, whereas the corresponding concentration for the mutant Bet v 1 Pro108Gly is approximately 15 ng. This shows that a single point mutation introduced in Bet v 1 Pro108Gly, reduces the affinity for specific serum IgE approximately in 2 times.

The maximum level of inhibition achieved under the action of the mutant Bet v 1 Pro108Gly, slightly lower than achieved under the action of recombinant Bet v 1. This may indicate that after replacing Pro108Gly some of the specific IgE present Obyedinennaya serum, not able to identify the mutant Bet v 1 Pro108Gly.

Mutant Bet v 1 Glu60Ser (netnanny mutant)

Glutamic acid at position 60 shows a high degree of exposure to solvent (60%), but not located in the area of the molecular surface, which is common to allergens Fagales. It was found that some proteins PR-10, homologous to Bet v 1, the serine residue occupies the position 60, which indicates that glutamic acid may be replaced by a serine without disrupting the tertiary structure α-carbon skeleton. In addition, since none of the natural itelligence sequence does not contain a serine at position 60, the replacement of glutamic acid serine creates not existing in nature molecule Bet v 1.

Properties of the IgE binding mutant Bet v 1 Glu60Ser

Properties of the IgE binding mutant Bet v 1 Glu60Ser compared with those of recombinant Bet v 1 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from patients allergic to birch, as described above.

7 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool obtained from allergic patients, abioterrorism Bet v 1 and mutant Bet v 1 Glu60Ser. Unlike mutants Glu45Ser, Pro108Gly and Asn28Thr+Lys32Gln replacement of glutamic acid 60 serine does not exert any appreciable influence on the STS bind IgE. This indicates that replacement outside of certain common areas Fagales have only a minor effect on the binding of specific serum IgE, which confirms the concept that conservative area of the molecular surface of allergen include dominant epitopes IgE binding.

Mutant Bet v 1 in three zones

In the mutant Bet v 1 in the three zones of point mutations (Glu45Ser, Asn28Thr+Lys32Gln and Pro108Gly), introduced in three different common areas Fagales, described above, were injected simultaneously to create an artificial mutant carrying four amino acid substitutions.

Structural analysis of mutant Bet v 1 in three zones

The structural integrity of the purified mutant in the three areas analyzed using spectroscopy circular dichroism (CD). On Fig shows the CD spectra of recombinant and mutant three zones, recorded with close to equal concentrations. The overlap of the peak amplitudes and positions in the CD spectra of the two recombinant proteins shows that drugs include equal the content of secondary structures, which allows reasonable to assume that the tertiary structure α-carbon skeleton does not change with the introduction of amino acid substitutions.

Properties of the IgE binding mutant Bet v 1 in three zones

Properties of the IgE binding mutant Bet v 1 in the three zones were compared with those of recombinant Bet v 1 V test liquid-phase inhibition of IgE using pooled serum IgE, obtained from patients allergic to birch, as described above.

Figure 9 shows the inhibition of binding of biotinylated recombinant Bet v 1 with serum IgE from a pool obtained from allergic patients, abioterrorism Bet v 1 and mutant Bet v 1 in the three zones. In contrast to the single mutants described above, the curve of inhibition of the mutant in three areas does not remain parallel to one recombinant. This indicates that the change introduced in the mutant in three zones, changed the properties of the IgE binding and epitope profile compared to the recombinant. The lack of parallelism of the difficulty of quantifying the reduction in affinity of the mutant for the three zones to specific IgE serum.

50% inhibition of recombinant Bet v 1 is achieved at approximately 6 ng, whereas the corresponding concentration for the mutant Bet v 1 in the three zones is 30 ng, i.e. the affinity was reduced by 5 times. However, achieving 80% inhibition values equal to 20 ng and 400 ng, respectively, i.e. the reduction is 20-fold.

The proliferation test of the T cells using recombinant mutant Bet v 1 in three zones

Analysis was performed as described in reference 15. It was found that the recombinant mutant Bet v 1 in the three zones is able to induce proliferation of lines of T-cells from three different patients with allergies to pollen b the cuts with the parameters of the stimulation, similar to those of recombinant and natural allergen. This suggests that the mutant three zones can initiate a cellular immune response necessary for the production of antibodies.

Example 2

Example 2 describes the preparation of recombinant mutant allergen with one primary mutation. Recombinant mutant allergen according to the invention, i.e. allergens, including at least four primary mutations, can be obtained using similar methods.

Identification of common epitopes of the main allergen antigen 5 poison hot vulgaris

Antigen 5 is one of three proteins in the venom of wasps, which are known allergens in humans. Wasps include hornets, these OS and burrowing OS. Two other well-known allergen wasp venom are phospholipase A1and hyaluronidase. Antigen 5 from hot vulgaris (Ves v 5) was cloned and expressed as recombinant protein in the system of yeast (Monsalve et al., 1999, reference 22). Was recently determined three-dimensional crystal structure of recombinant Ves v 5 with a resolution of 1.8 Å (forthcoming). The main features of the structure are the presence of four β-chains and four α-helices organized in three stacked layers of a layer with the formation of "α-β-α sandwich. The match sequence is homologous the x antigen 5 allergens of various types of hot is approximately 90%, that assumes the presence of conserved regions of the molecular surface and epitopes of B-cells.

Identification and identification of the common areas was performed after alignment of all known amino acid sequence of the allergen antigen 5 hot, as described above for allergens pollen of trees, in combination with analysis of molecular surface antigen 5, the disclosed data on the three-dimensional structure of Ves v 5. Figure 10 shows the accessibility to solvent individually aligned residues of the antigen 5 and the alignment of sequences of the antigen 5 hot (left panel). On the right panel of figure 10 shows the molecular surface antigen with 5 colored conservative for antigen 5:s hot areas.

The choice of amino acid residues for site-directed mutagenesis

Amino acid residues for site-directed mutagenesis was chosen among the remaining available areas that are common to hot, since it was expected that modifications will affect the binding of serum IgE from the majority of patients showing clinical cross-allergic reaction hot.

The relative orientation and the percentage of exposure to the solvent of each amino acid residue in the corresponding area was calculated on the basis of their atomic coordinates. Residues having a low degree of exposure to the solvent, Russ is trevali as not suitable for mutagenesis because of the possible disturbance of structure or lack of interaction with the antibody. The remaining residues were ranked in accordance with the degree of their exposure to the solvent.

Cloning of the gene encoding Ves v 5

Total RNA was isolated from the poisonous acidic glands OS hot vulgaris, as described in Fang et al., 1988 (reference 23).

The synthesis of the first chain cDNA, PCR amplification and cloning of the gene Ves v 5 was performed as described by Lu et al., 1993 (reference 24).

Subclavian in pPICZαA

Encoding Ves v 5 gene was then subcloned into the vector pPICZαA (Invitrogen) for Sekretareva expression of Ves v 5 in Pichia pastoris. Gene amplified by PCR and was subcloned into the reading frame with the coding sequence for α-factor secretion signal Saccharomyces cerevisiae. In this construct αfactor cleaved in vivo by the system Kex2 Pichia pastoris during secretion of the protein.

Briefly, PCR was performed with the use of Ves v 5 as template and primers corresponding to the amino - and carboxylic protein, respectively. The primers were extended from the 5'end to generate restriction sites for cloning EcoRI and XbaI, respectively. The nucleotides encoding the cleavage site Kex2, this construct was at a distance of 18 nucleotides above aminobenzo protein, resulting in Ves v5 expressively with six additional amino acids, Glu-Ala-Glu-Ala-Glu-Phe, aminocore.

Insert pPICZαA-ves v 5 in P. pastoris

The vector pPICZαA with the insertion of a CGU is and Ves v 5 was linearizable using enzyme Sac I and inserted into the AOX1 locus of the genome of Pichia pastoris. The insert was produced by homologous recombination in cells KM71 Pichia pastoris in accordance with the recommendations of Invitrogen.

Mutagenesis in vitro

The in vitro mutagenesis was performed by PCR using recombinant pPICZαA with insert Ves v 5 as a matrix. Each mutant gene Ves v 5 was obtained using 3 PCR reactions using 4 primers.

Two specific mutations oligonucleotide primer was synthesized in accordance with each mutation, one for each DNA chain, see 11 and 12. Using mutant(e) nucleotide(s) as a starting point, both primer was extended at 6-7 nucleotides on the 5'-end and at 12-13 nucleotides at the 3'-end. Extending nucleotides were identical in sequence to the gene Ves v 5 in the effector region.

Further synthesized and used for all mutants two generally applicable primers (marked on Fig as fully "semantic" and "fully " antisense"). To ensure expression of the mutants Ves v 5 existing in nature aminocom.com one primer corresponding to aminobenzo protein, was extended on the 5'-end of the website Xho I. After insertion mutant genes Ves v 5 in the vector pPICZαA restored site of cleavage by the Kex2 protease amino acid immediately before the end of Ves v 5. The second primer sequence corresponded to the region of the vector pPICZαA, located at a distance of CA is approximately 300 BP below the gene Ves v 5. The sequence of primer corresponding to aminobenzo Ves v 5, stems from the sense circuit, and the lower sequence of the primer stems from antisense chain, see 11.

Conducted two independent PCR reactions essentially in accordance with standard techniques (Saiki et al., 1988), except that spent only 20 temperature cycles to reduce the frequency of PCR artifacts. Each PCR reaction was used pPICZαA with insert Ves v 5 as a matrix, one specific for the mutation and one generally applicable primer in specific combinations.

PCR products were purified using a "Concert Rapid PCR Purification System (Life Technologies). A third PCR reaction was carried out using the combined PCR products of the first two PCR reactions as matrix and both generally applicable primers. And again applied a standard PCR (20 cycles. The PCR product was purified using a "Concert Rapid PCR Purification System (Life Technologies), cut by restriction enzymes (XhoI/XbaI) and directionally ligated into the vector pPICZαA, restrictively the same enzymes. Figure 11 presents the General overview of all mutations Ves v 5.

Insertion mutants pPICZαA-Ves v 5 in P. pastoris

The inserted vector pPICZαA mutant genes Ves v 5 was linearizable restriction Sac I and inserted into the AOX1 locus of the genome of Pichia pastoris. Insert produced by homologous recombination in principle this is Kah KM71 Pichia pastoris in accordance with the recommendations of Invitrogen.

Sequencing of nucleotides

Determination of the nucleotide sequence of the gene encoding Ves v 5, was carried out before and after sublimirovanny and after mutagenesis in vitro, respectively.

Plasmid DNA from 10 ml of bacterial culture, growing to saturation overnight in LB medium with the addition of 0.1 g/l ampicillin, was purified on columns (Qiagen-tip 20 and sequenced with the use of the kit for sequencing DNA Sequenase version 2.0 (USB) in accordance with the recommendations of the suppliers.

Expression and purification of recombinant Ves v 5

Recombinant yeast cells of strain KM71 Pichia pastoris was grown in 500-ml flasks containing 100 ml of phosphate buffer with a pH of 6.0, containing nitrogen base yeast, Biotin, glycerin and histidine, at 30°With circular stirring at 225 rpm until A600nm 4-6. The cells were collected by centrifugation and resuspendable in 10 ml of the same buffer medium containing methanol instead of glycerol. Incubation was continued at 30°C for 7 days with daily addition of 0.05 ml of methanol.

The cells were collected by centrifugation and combined the culture fluid was concentrated by ultra-filtration. After dialysis against 50 mm ammonium acetate buffer, pH 4.6, the sample was applied on the column effects for cation-exchanger SE-53 for HPLC (Pharmacia), equilibrated with the same buffer. The column was swirbul the linear gradient mixture 0-1,0 M NaCl, 50 mm ammonium acetate. Eluruumis at approximately 0.4 M NaCl peak recombinant Ves v 5 was collected and were dialyzed against 0,02 N. acetic acid. After concentration to about 10 mg/ml purified Ves v 5 was stored at 4°C.

Crystallization of recombinant Ves v 5

Crystals Ves v 5 was grown by the method of vapor diffusion at 25°C. For crystallization 5 ál of Ves v 5 at a concentration of 5 mg/ml was mixed with 5 μl of a mixture of 18% PEG 6000, 0.1 M sodium citrate, pH 6.0 and balanced against 1 ml of 18% PEG 6000, 0.1 M sodium citrate, pH 6,0.

Data on x-ray diffraction were obtained at 100 K using crystals of native Ves v 5 and after the introduction of derivatives of heavy atoms and used to determine three-dimensional structure of Ves v 5, see figure 10 (manuscript in preparation).

Immunoelectrophoresis using rabbit polyclonal antibodies

Two mutant Ves v 5 was obtained in the form of recombinant proteins Ves v 5 and tested for their ability to react with polyclonal rabbit antibodies, induced against recombinant Ves v 5. In the analysis using Rakitovo of immunoelectrophoresis in the in vivo rabbit antibodies were able to precipitate recombinant Ves v 5, and both mutant, indicating that the mutants contained conservative α-carbon skeleton of the tertiary structure.

The inhibition is specific to porotocol IgE

Properties of binding IgE mutants Ves v 5 was compared with those of recombinant Ves v 5 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from patients allergic to wasp venom.

The inhibition test was performed as described above using biotinylated Ves v 5 instead of Bet v 1.

Mutant Lys72Ala Ves v 5

Lysine at position 72 shows a high degree of exposure to solvent (70%) and is located in the area of a surface molecule that is common to antigen 5 hot. The relative orientation and a high degree of exposure to solvent suggests that the lysine-72 may be replaced by an alanine residue without disturbing α-carbon skeleton of the tertiary structure. In addition, since none of the known natural sequences isoalleles not contains alanine at position 72, the substitution of lysine by alanine leads to the creation of not existing in the nature of a molecule of Ves v 5.

Properties of the IgE binding mutant of Ves v 5 Lys72Ala

Properties of the IgE binding mutant of Ves v 5 Lys72Ala compared with those of recombinant Ves v 5 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from patients allergic to birch described above.

On Fig shows the inhibition of binding of biotinylated recombinant Ves v 5 with serum IgE from a pool obtained from allergic patients, sky is tinylinkuni Ves v 5 and mutant Ves v 5 Lys72Ala.

There is a clear difference in the number of the corresponding recombinant proteins needed to achieve 50% inhibition of the binding of serum IgE present in the United whey. 50% inhibition of recombinant Ves v 5 is achieved at approximately 6 ng, whereas the corresponding concentration for the mutant Ves v 5 Lys72Ala is 40 ng. This shows that the point mutation introduced in the mutant Ves v 5 Lys72Ala, reduces the affinity for specific serum IgE approximately 6 times. The maximum level of inhibition achieved under the action of the mutant Ves v 5 Lys72Ala, significantly lower than achieved under the action of recombinant Ves v 5. This may indicate that after replacing Lys72Ala some of the specific IgE present in the United whey, not able to identify the mutant Ves v 5 Lys72Ala.

Mutant Tyr96Ala Ves v 5

The tyrosine at position 96 shows a high degree of exposure to solvent (65%) and is located in the area of a surface molecule that is common to antigen 5 hot. The relative orientation and a high degree of exposure to solvent indicate that tyrosine 96 may be replaced by an alanine residue without disturbing the three-dimensional structure. In addition, since none of the known natural sequences isoalleles not contains alanine at position 96, the replacement of tyrosine with alanine Veda is to create not existing in the nature of a molecule of Ves v 5.

Properties of the IgE binding mutant of Ves v 5 Tyr96Ala

Properties of the IgE binding mutant of Ves v 5 Tyr96Ala compared with those of recombinant Ves v 5 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from patients allergic to birch described above.

On Fig shows the inhibition of binding of biotinylated recombinant Ves v 5 with serum IgE from a pool obtained from allergic patients, abioterrorism Ves v 5 and mutant Ves v 5 Tyr96Ala.

There is a clear difference in the number of the corresponding recombinant proteins needed to achieve 50% inhibition of the binding of serum IgE present in the United whey. 50% inhibition of recombinant Ves v 5 is achieved at approximately 6 ng, whereas the corresponding concentration for the mutant Ves v 5 Tyr96Ala is 40 ng.

This shows that a single point mutation introduced in the mutant Ves v 5 Tyr96Ala, reduces the affinity for specific serum IgE approximately 6 times.

The maximum level of inhibition achieved under the action of the mutant Ves v 5 Tyr96Ala, significantly lower than achieved under the action of recombinant Ves v 5. This may indicate that after replacing Tyr96Ala some of the specific IgE present in the United whey, not able to identify the mutant Ves v 5 Tyr96Ala.

Example 3

The IDA is the certification and selection of amino acids to replace

For identification and selection exposed on the surface of amino acids suitable for replacement allergens Bet v 1, Der p 2 and Ves v 5, used the visibility settings for the solvent and the degree of conservatism.

Accessibility to solvent

The accessibility of the solvent was calculated using the programs InsightII, version 97.0 (MSI) and the radius of the probe of 1.4 Å (Connolly surface).

The internal cavity were excluded from the analysis by filling probes with application programs PASS (putative active sites with spheres). Then manually delete the probes on the surface.

Conservatism

Bet v 1:

3-dimensional structure based on data with registration number Z80104 (lbvl.pdb).

38 other sequences Bet v 1, is included in the analysis conservative residues include the following registration numbers:

Der p 2:

3-dimensional structure based on data with registration number P49278 (1a9v.pdb).

6 other sequences Der v 2 included in the analysis conservative residues include the following substitutions:

Ves v 5:

3-dimensional structure based on data with registration number Q05110 (pdb coordinates are not published).

Another sequence of Ves v 5 in the analysis conservative residue contains one amino acid substitution M202K.

Results the ATA

Bet v 1:

59 amino acids with high exposure to the solvent:

57 amino acids with high exposure to the solvent and conservatism (>70%):

Produced 23 mutations:

Table 1 presents the list of amino acids Bet v 1 in descending order of exposure to the solvent. In column 1 presents the number of amino acids, starting with aminocore, column 2 shows the amino acid in one-letter abbreviation, column 3 presents the normalized indicator of exposure to the solvent, column 4 shows the percentage of known sequences containing the considered amino acid in this position.

Table 1: Bet v 1

Der p 2:

55 amino acids with a high degree of exposure to solvent:

54 amino acids with a high degree of exposure to solvent and conservatism (>70%):

Produced mutations:

K6A, K15E, H30N, E62S, H74N, K82N.

Table 2 presents re the Yan amino acids Der p 2 in descending order of exposure to the solvent. In column 1 presents the number of amino acids, starting with aminocore, column 2 shows the amino acid in one-letter abbreviation, column 3 presents the normalized indicator of exposure to the solvent, column 4 shows the percentage of known sequences containing the considered amino acid in this position.

Table 2: Der p 2

Ves v 5:

89 amino acids with a high degree of exposure to solvent:

88 amino acids with a high degree of exposure to solvent and conservatism (>70%):

9 produced mutations:

K29A, T67A, K78A, V84S, Y102A, K112S, K144A, K202M, N203G.

Table 3 provides a list of amino acids Ves v 5 in descending order of exposure to the solvent. In column 1 presents the number of amino acids, starting with aminocore, column 2 shows the amino acid in one-letter abbreviation, column 3 presents the normalized indicator of exposure to the solvent, column 4 shows the percentage of known sequences containing the considered amino acid in this position.

Table 3: Ves v 5

Example 4

This example describes the fabrication and characterization of recombinant mutant allergen Bet v 1 according to the invention, i.e. allergens with reduced affinity binding of IgE, which includes at least four primary mutations.

The choice of amino acid residues for site-directed mutagenesis Bet v 1

The availability of amino acid residues Bet v 1 for solvent shown in example 3, table 1. The degree of conservatism of amino acids based on sequence alignment performed using the ExPaSy Molecular Biology Server (http://www.expasy.ch/) with the application of the algorithm Clusta1W of BLAST search, where as the introductory sequence is used amino acid sequence of Bet v 1.2801 wild type. The alignment includes 67 sequences of allergens (39 sequences Bet v 1, 11 sequences Car b 1, 6 sequences Cor a 1 and 13 sequences Aln g 1) within the squad Fagales (Bet v 1: Betula verrucosa; Car b 1: Carpinus betulus; Cor a 1: Corylus avellana; Aln g 1: Alnus glutinosa). As for shown in the examples mutant recombinant allergens Bet v 1, the replacements for the OST the Cove targets was the identity of amino acids ≥ 95%.

As described in example 1 for the site-directed mutagenesis of selected amino acid residues with a high degree of exposure to the solvent and a high degree of conservatism among pollen allergens related species. Amino acid residues with a low degree of exposure to solvent (<20%) were considered as unsuitable for mutagenesis because of possible violations of the tertiary structure, or lack of interaction with antibodies.

All entered the remains are in the relevant provisions of the isoforms of a group of plant proteins called linked with pathogenesis (PR-10) proteins. Molecular modeling suggests that the tertiary structure of allergens Fagales and proteins PR-10 is close to coincidence. Bet v 1 is considerable overlap sequence (20-40%) with proteins PR-10. However, there are no reports of allergic cross-reactions in relation to these proteins PR-10. Thus, the replacement of the highly conserved and exposed to the solvent amino acids of Bet v 1, the amino acid in the corresponding position in the protein PR-10 gives the mutant protein Bet v 1 with unchanged tertiary structure α-carbon skeleton, but with reduced affinity IgE binding.

Mutagenesis in vitro

The in vitro mutagenesis was performed by PCR using recombinant pMAL-c with the insertion of a Bet v 1 in catastematic. The preparation of recombinant mutant allergen, comprising from five to nine primary mutations included two stages of PCR, stage I and stage II. First, each single mutation (or mutations, if they were next to each other in the DNA sequence) were injected sequentially in the DNA sequence of Bet v 1.2801 or derivatives Bet v 1.2801 using sense and antisense specific mutations oligonucleotide primers adapted for each mutation(mutations) within the sense and antisense oligonucleotide primers adapted to above or below neighboring mutations or N-end/C-end of the Bet v 1, as schematically illustrated Fig (I). Secondly, the products of the PCR reaction (I-PCR were purified, mixed and used as matrices for additional PCR reaction (II) with oligonucleotide primers that are adapted to the N-end and C-end of the Bet v 1, as schematically illustrated in Fig (II). PCR products were purified by agarose gel electrophoresis and purification of PCR gel (Life Technologies) with subsequent precipitation with ethanol, the cutting restriction enzymes (SacI/EcoRI or SacI/XbaI) and directionally ligated into pMAL-c, cut with the same enzymes.

On Fig shows the synthesized oligonucleotide primers and a schematic illustration of constructing mutant Bet v 1, containing the t five to nine primary mutations. Mutant amino acids are preferably selected from the group consisting of amino acids, characterized by a high degree of exposure to solvent and conservatism, as described in example 3. Mutant Bet v 1, is identified in parentheses, include the following primary and secondary mutations:

Mutant Bet v 1 (2628): Tyr5Val, G1u45Ser, Lys65Asn, Lys97Ser, Lysl34Glu.

Mutant Bet v 1 (2637): Ala16Pro, (Asn28Thr, Lys32Gln), Lys103Thr, Pro108Gly, (Leu152Lys, Alal53Gly, Ser155Pro).

Mutant Bet v 1 (2733): (Tyr5Val, Lysl34Glu), (Asn28Thr, Lys32Gln), Glu45Ser, Lys65Asn, (Asn78Lys, Lys103Val) Lys97Ser, Pro108Gly, Arg145Glu, (Aspl56His, +160Asn)

Mutant Bet v 1 (2744): (Tyr5Val, Lysl34Glu), Glu42Ser, Glu45Ser), (Asn78Lys, Lys103Val), Lysl23Ile, (Aspl56His, + 160Asn).

Mutant Bet v 1 (2753): (Asn28Thr, Lys32Gln), Lys65Asn, (Glu96Leu, Lys97Ser), (Pro108Gly, Asp109Asn), (Aspl25Tyr, Glul27Ser), Argl45Glu.

Sequencing of nucleotides

Determination of the nucleotide sequence of the gene encoding Bet v 1, was carried out before and after sublimirovanny and after mutagenesis in vitro, respectively. Plasmid DNA from 10 ml of bacterial culture, growing to saturation overnight in LB medium with the addition of 0.1 g/l ampicillin, was purified on columns (Qiagen-tip 20 and sequenced with the use of the kit for sequencing Ready reaction dye terminator cycle and fluorescent sequencing machine AB PRISM 377, both from (Perkin Elmer) in accordance with the recommendations of the suppliers.

Expression and purification of recombinant Bet v 1 and mutants

Recombinant Bet v 1 (Bet v 1 No. 2801 and mutants) were hyperexpression in Escherichia coli DH 5; in the form of a hybrid with maltose binding protein and was purified as described in reference 15. Briefly, recombinant E. coli cells were grown at 37°C to an optical density of 0.8 at 600 nm, after which the expression of a hybrid protein Bet v 1 was induced by adding IPTG. The cells were collected by centrifugation after 3 hours after induction, resuspendable in lytic buffer and destroyed by sonication. After ultrasonic treatment and additional centrifugation was isolated recombinant hybrid protein with amylose affinity chromatography and then were digested by incubation with factor Xa (reference 15). After FXa cleavage of recombinant Bet v 1 was isolated by gel filtration and subjected to additional cycle amylose affinity chromatography to remove trace quantities of maltose binding protein.

Purified recombinant Bet v 1 was concentrated by ultra-filtration to approximately 5 mg/ml and kept at 4°C. the Final yield of purified preparations of recombinant Bet v 1 was between 2 and 5 mg per liter of cell culture of E. coli.

Preparations of purified recombinant Bet v 1 were the only bands after electrophoresis in polyacrylamide with DDS-Na and color silver with an apparent molecular mass of 17.5 kDa.

Previously it has been shown (reference 15), recombinant Bet v 1 No. 2801 immunochemical and indistinguishable from natural Bet v 1.

Mutant Bet v 1 (2628) and Bet v 1 (2637)

On Fig shown introduced point mutations on the molecular surface of Bet v 1 (2628) and Bet v 1 (2637). In the mutant Bet v 1 (2628) five primary mutations were introduced in half of the Bet v 1 when saving the other half unchanged. In the mutant Bet v 1 (2637) five primary and three secondary mutations were introduced in the other half while maintaining the left half unchanged. Thus, mutations in the mutant Bet v 1 (2628) and mutant Bet v 1 (2637) affect different half of the surface of Bet v 1, respectively.

Crystallization and structural analysis of recombinant Bet v 1 (2628) mutant protein

Crystals of recombinant Bet v 1 (2628) were grown using the vapor diffusion at 25°C, essentially as described by Spangfort et al., 1996b (reference 21). Bet v 1 (2628) at a concentration of 5 mg/ml was mixed with an equal volume of a mixture of 2.2 M ammonium sulfate, 0.1 M sodium citrate, 1% (V/V) dioxane, pH 6.3, and balanced against 100 volumes of a mixture of 2.2 M ammonium sulfate, 0.1 M sodium citrate, 1% (V/V) dioxane, pH 6.3. After 24 hours of equilibration, the growth of crystals induced by application of the method of application described in reference 21, using crystals of recombinant Bet v 1 wild type as a seed.

After about 4 months the crystals were collected and analyzed using x-rays obtained with a rotating anode Rigaku, as described in reference 21, and the structure definition is Lyali using molecular replacement.

The structure of the mutant Bet v 1 (2628)

Structural effects of the mutations were found by determining the diffraction grown three-dimensional crystals of the protein Bet v 1 (2628) with a resolution of 2.0 Å analysis using x-rays obtained with a rotating anode. Replacement Tyr5Val, Glu45Ser, Lys65Asn, Lys97Ser, Lys134Glu confirmed using a map of the electron density structure of Bet v 1 (2628), which also showed that the overall tertiary structure α-carbon skeleton is preserved.

The structure of the mutant Bet v 1 (2637)

The structural integrity of the purified mutant Bet v 1 (2637) were analyzed using spectroscopy circular dichroism (CD). On Fig shows the CD spectra of recombinant Bet v 1.2801 (wild type) and mutant Bet v 1 (2637), recorded with close to equal concentrations. The overlap of the peak amplitudes and positions in the CD spectra of the two recombinant proteins shows that the two drugs include equal the content of secondary structures, which allows reasonable to assume that the tertiary structure α-carbon skeleton is not changed by the introduction of amino acid substitutions.

Properties of the IgE binding mutant Bet v 1 (2628) and Bet v 1 (2637)

Properties of the IgE binding Bet v 1 (2628) and Bet v 1 (2637), and a mixture of 1:1 Bet v 1 (2628) and Bet v 1 (2637) were compared with those of recombinant wild-type Bet v 1.2801 in the test liquid-phase inhibition of IgE using pooled serum IgE obtained from the patient is s allergic to birch.

As described in example 1, recombinant Bet v 1.2801 was biotinilated at a molar ratio of 1:5 (Bet v 1 no. 2801:Biotin). The inhibition test was carried out as follows: serum sample (25 μl) were incubated with anti-IgE on the solid phase, washed, resuspendable and additionally incubated with a mixture of biotinylated Bet v 1.2801 and this mutant, or a mixture 1:1 of both mutants. The amount of biotinylated Bet v 1.2801 associated with the solid phase, was determined on the basis of a measurement RLU after incubation with streptavidin labeled with acridine ester. The degree of inhibition was calculated as the ratio of RLU values obtained with the use of buffer and mutant as an inhibitor.

On Fig shows the inhibition of binding of biotinylated recombinant Bet v 1.2801 with serum IgE from a pool obtained from allergic patients, abioterrorism Bet v 1.2801 and Bet v 1 (2628), Bet v 1 (2637) and a mixture of 1:1 Bet v 1 (2628) and Bet v 1 (2637).

There is a clear difference in the number of the corresponding recombinant proteins needed to achieve 50% inhibition of the binding of serum IgE present in the United whey. 50% inhibition of recombinant Bet v 1.2801 is achieved at approximately 5 ng, whereas the corresponding concentration for the mutant Bet v 1 (2628) is approximately 15-20 ng. This shows that the point mutation introduced in m is tant Bet v 1 (2628), reduces the affinity for specific serum IgE approximately 3-4 times.

The maximum level of inhibition achieved under the action of the mutant protein Bet v 1 (2628), clearly below the achieved under the action of recombinant Bet v 1. This may indicate that some of the specific IgE present in the United whey, not able to identify mutant protein Bet v 1 (2628) due to the introduced point mutations.

50% inhibition under the action of Bet v 1 (2837) is achieved at approximately 400-500 ng, indicating that the mutant Bet v 1 (2837) point mutation reduces the affinity for specific serum IgE in 80 to 100 times compared to Bet v 1.2801. Big difference in IgE binding is further supported by a clear difference in the slope of the inhibition curve obtained with the mutant protein Bet v 1 (2837) and inhibition curve obtained for Bet v 1.2801. The difference in slopes indicates that the reduction of binding of IgE due to the distinct pattern of epitope mutant Bet v 1.2801.

In addition to the tests of inhibition with a separate modified allergens tested a mixture of 1:1 Bet v 1 (2628) and Bet v 1 (2637) with the same molar concentration of Bet v 1, and that each of the samples of Bet v 1 (2628) or Bet v 1 (2637), respectively, and testing showed complete (100%) the ability to inhibit binding of IgE to rBet v 1.2801. Ability to alemu inhibition of IgE binding provides a clear indication of the all active epitopes present on Bet v 1.2801, was present in the mixture of allergens 1:1. Additional proof of this is comparable to the slope of the two curves of inhibition for Bet v 1.2801 and mixtures of allergens. About reduced interaction with IgE sample with a mixture of allergens indicates the necessity of using a fourfold compared to Bet v 1.2801 concentration of the mixture of allergens to achieve 50% inhibition of IgE binding.

The proliferation test of the T cells using mutant recombinant allergens Bet v 1

Analysis was performed as described in reference 15. And Bet v 1 (2628), and Bet v 1 (2637) mutant proteins were able to induce proliferation of lines of T-cells of patients with Allergy to birch pollen with parameters of stimulation, similar to the indicators specific recombinant and natural protein. This suggests that each of the mutant protein Bet v 1 (2628) and Bet v 1 (2637) can initiate a cellular immune response necessary for the production of antibodies.

Test release of histamine by basophils person

Test release of histamine the basophilic leukocytes were performed as follows. Each patient with Allergy to birch pollen took heparinized blood (20 ml)was left at room temperature and used within 24 hours. Twenty-five microlitres heparinised C is through blood were made in the wells for micrometrology, coated glass fibers (Reference Laboratory, Copenhagen, Denmark), and incubated with 25 Microlitre allergen or anti-IgE for 1 hour at 37°C. After that, the tablets were washed and hindering the analysis of substances have been removed. Finally, using spectrophotofluorimeter measured associated with microfibers histamine.

Properties of the release of histamine mutant protein Bet v 1 (2628) and Bet v 1 (2637)

Data on the release of histamine shown in Fig and 23. Tested the effectiveness of the mutant protein Bet v 1 (2628) and Bet v 1 (2637) in the induction of the release of histamine by basophils person in two patients with Allergy to birch pollen. In both cases, the curves of the release of histamine induced mutant allergens, clearly shifted to the right compared with the curve release induced Bet v 1.2801. The shift indicates that the effectiveness of Bet v 1 (2628) and Bet v 1 (2637) reduced 3 - to 10-fold.

Mutant Bet v 1 (2744) and mutant Bet v 1 (2753)

In the same way designed Bet v 1 (2744) and Bet v 1 (2753) for use in mixed allergenic vaccine. In these mutant allergens point mutations distributed across the surface, causing the form shown in Fig and 25, and the newly created to have an impact on different areas in the two molecules, respectively, as shown in Fig. However, the data is modified allergens could also be applied RA the individual as a vaccine with a single allergen.

Structural analysis of the mutant protein Bet v 1 (2744)

The structural integrity of the purified mutant Bet v 1 (2744) were analyzed using spectroscopy circular dichroism (CD). On Fig shows the CD spectra of recombinant Bet v 1.2801 (wild type) and mutant Bet v 1 (2744), recorded with close to equal concentrations. The overlap of the peak amplitudes and positions in the CD spectra of the two recombinant proteins shows that the two drugs contain an equal number of secondary structures, which allows reasonable to assume that the tertiary structure α-carbon skeleton does not change with the introduction of amino acid substitutions.

Properties of Bet v 1 (2744) on the release of histamine

Data from five experiments with the basophilic leukocytes from five different patients with Allergy to birch pollen in the release of histamine shown in figa-D. Tested the effectiveness of the mutant protein Bet v 1 (2744) in the induction of the release of histamine by basophils person. Curves release for mutant allergens clearly shifted to the right compared with the curve of the release of Bet v 1.2801, which indicates that the efficiency of Bet v 1 (2744) in respect of the release of histamine reduced 3-5-fold.

Mutant Bet v 1 (2733)

Was designed and recombinante downregulation of mutant Bet v 1 (2733) with nine primary mutations. The distribution of point mutations in Bet v 1 (2733) providing the em save multiple areas of the surface, components >400Å2, unchanged. On Fig shown introduced point mutations on the molecular surface of Bet v 1 (2733).

Example 5

This example describes the cloning of the gene encoding Der p 2, from Dermatophagoides pteronyssinus and design of mutants with reduced affinity IgE binding.

Amplificatoare PCR products of the synthesis of the first chain cDNA total RNA Dermatophagoides pteronyssinus were received from Dr. Wendy-Anne Smith and Dr. Wayne Thomas (TVW Telethon Institute for Child Health Research, 100 Roberts Rd, Subiaco, Western Australia 6008). When amplification of the library first chain cDNA Der p 2 was selectively amplified using specific to Der p 2 primers. The PCR fragments were then cloned into the Bam website HI pUC19 (New England BioLabs). DNA sequencing Der p 2-specific semantic vector (5'-GGCGATTAAGTTGGGTAACGCCAGGG-3') and antisense (5'-GGAAACAGCTATGACCATGATTACGCC-3') primers.

Total identified seven distinct isoforms of Der p 2, labeled ALK-101, ALK-102, ALK-103, ALK-104, ALK-113, ALK-114 and ALK-120. The clone, named ALK-114 was chosen as a starting point for creating a low-affinity to IgE mutants due to its high degree of identity of the NMR structure of Der p 2 with the database under the registration number 1A9V. Compared with ALK-114 6 other natural isoforms include the following replacements:

ALK-101: M76V.

ALK-102: V40L, T47S.

ALK-103: M111L, D114N.

ALK-104: T47S, M111L, D114N.

ALK-113: T47S.ALK-120: V40L, T47S, D114N.

Inserting Der p 2 in pGAPZ± -A

The gene encoding Der p 2 (ALK-114), then inserted into the vector pGAPZα-A (Invitrogen) for Sekretareva expression of Der p 2 in the yeast, Pichia pastoris. Gene amplified using sense primer OB27 (5'-GGAATTCCTCGAGAAAAGAGATCAAGTCGATGTCAAAGATTGTGCC-3') and antisense primer OB28 (5'-CGTTCTAGACTATTAATCGCGGATTTTAGCATGAGTTCG-3')corresponding to the amino - and carbonsilicon polypeptide Der p 2, respectively. The primers were extended at the 5'-end to include restriction sites Xho I and Xba I, respectively. The restriction site Xho I merges with the first codon Der p 2 in reading frame nucleic acid sequence that encodes a cleavage site KEX2 (LYS-ARG) pGAPZα-A. Conducted one round of PCR amplification in a volume of 100 microliters (ál): 0.1 mg DNA templates ALK-114, 1 X buffer Expand polymerase (available from Boehringer Mannheim), and 0.2 millimolar (mm) each of the four dNTP and 0.3 micromolar (μm) sense and antisense primers and 2.5 units of Expand polymerase (available from Boehringer Mannheim). DNA amplified in the 25 cycles: 95°C for 15 seconds, 45°C for 30 seconds, 72°C for 1 minute followed by 1 cycle at 72°C for 7 minutes. The obtained PCR fragment ALK-114 from 475 base pairs was purified using QIAquick purification procedure spin (available from Qiagen). Then the purified DNA fragment was digested Xho I and Xba I, gel purified and ligated with similarly digested pGAPZα-A. PR is the product of the ligation reaction was used to transform strain DH5α E.coli, which gave plasmid pCBo06.

The nucleotide sequence of Der p 2 was confirmed by DNA sequencing before and after cloning and subsequent mutagenesis in vitro (see below).

The sequence of Der p 2

SEQ ID NO 1 corresponds to the nucleic acid sequence of Der p 2 (ALK-114):

SEQ ID NO 2 corresponds to the derived amino acid sequence of Der p 2 (ALK-114):

Introduction pGAPZα-A-Der p 2 in P. pastoris

Vector pCBo06 was linearizable using restriction enzyme Avr II and transformed competent strain of P. pastoris X-33, as described in the Invitrogen. Selected recombinant cells resistant to 100 micrograms per milliliter (μg/ml) zeocin, and colonies were purified on fresh tablets YPD containing 100 μg/ml of zeocin.

Expression and purification of recombinant Der p 2

250 µl of YPD medium (1% yeast extract, 2% peptone, 2% glucose)containing 100 μg/ml of zeocin, inoculable night culture of recombinant yeast cells expressing Der p 2. The culture was grown at 30°C for 72 hours to achieve optimal expression of Der p 2. The cells were collected by centrifugation and the resulting culture supernatant was saturated with 50% ammonium sulfate. After centrifugation at 3000xg for 30 minutes the supernatant was saturated with 80% ammonium sulfate. After the second centrifugation the precipitate resuspendable 150 millimolar (mm) NH 4HCO3and fractionally on a column for gel filtration Superdex 75, equilibrated with the same buffer. Der p 2 was elyuirovaniya in the form of a main peak corresponding to the expected molecular mass. Elution Der p 2 was monitored by electrophoresis in Na-DDS PAG followed by painting silver and analysis by Western blot turns with the use of Der p 2-specific polyclonal antibodies.

The choice of amino acid residues for site-directed mutagenesis

The choice of amino acid residues for mutagenesis was based on the identification of residues with high exposure to the solvent and high conservatism among allergens from house dust mites (Der p 2/f 2 and Eur m 2) and storage mites (Tyr p 2, Lep d 2, Gly d 2). Amino acid residues with high exposure to the solvent identified visually by analyzing the molecular surface NMR structure of Der p 2 (#1.9 1A9V.pdb). For mutagenesis were selected twelve amino acid residues: K6A, N10S, K15E, S24N, H30N, K48A, E62S, H74N, K77N, K82N, K100N and R128Q.

Site-directed mutagenesis

The following describes the construction of recombinant mutant allergen with a single primary mutations and their multiple combinations.

Expression plasmids encoding mutant Der p 2 were obtained using pCBo06 as DNA templates. PCR reaction was performed using semantic and antimyeloma the primers, including specific mutations. Pairs of primers used in PCR reactions to generate specific mutations are listed on Fig on which mutations are shown in bold, and the restriction sites used in the next stage of cloning are underlined. To construct the mutant K6A, K15E, H30N, H74N and K82N the PCR reactions were carried out in strict accordance with the description in the section "Cloning of Der p 2 in pGAPZα-A". Purified fragments GWH digested on the created sites for restriction enzymes (see Fig), was gel purified, ligated into similarly digested pCBo06 and used to transform E. coli DH5α.

Mutation E62S caused by using the alternative method of mutagenesis PCR described to create mutant Bet v 1 in example 1. Synthesized two specific mutations oligonucleotide primer covering these mutations (OB47 and OB48 shown in Fig). Two additional primers, applied at the stage of secondary amplification were OB27 and OB28, as described in "Inserting Der p 2 in pGAPZα-A".

The obtained mutant allergens characterized using similar methods as described in example 4, for example spectroscopy circular dichroism (CD), crystallization, measurements of the properties of the IgE binding, release of histamine, the ability to stimulate proliferation of T-cells, etc.

Use the 6

Mutant recombinant mite allergens (Der p 2) with improved security for specific Allergy vaccination

In this example, the application of the concept of the present invention to allergens of the house dust mite is illustrated one allergen, Der p 2. Manipulation with other allergens of the house dust mite can be performed using equivalent methods.

The creation of mutant recombinant molecules Der p 2

SEQ ID NO. 3 shows the nucleotide and deduced amino acid sequence of clone Der p 2-ALK-G, which is an isoform of the wild type.

SEQ ID NO. 3: nucleotide and deduced amino acid sequence of Der p 2-ALK-G:

On Fig shows the alignment of the sequence produced at the ExPaSy Molecular Biology Server (http://www.expasy.ch/) using the ClustalW algorithm with a BLAST search using the amino acid sequence of Der p 2-ALK-G shown in SEQ ID NO. 3, as the introductory sequence. The alignment includes sequences from species of house dust mite, i.e. Der p 2, Der f 2 and Eur m 2. On Fig amino acid residues identical to the amino acids in the same position in the protein Der p 2-ALK-G, highlighted in black letters on a gray background. Non-identical amino acids are printed in black on a white background.

Image of the surface structure

Amino acid p is coherence, representing group 2 allergens of the house dust mite, have similarity above 85%, and part of the molecular surface of a conservative (grayed area), see Fig.

On Fig shows the contours of the surface, shown at 4 different angles when applying the sequence of the protein Der p 2-ALK-G on the published NMR structure PDB:1A9V, structure 1 of 10 contained in the PDB file.

For mutation choose conservative amino acids with high exposure to the solvent, distributed in space along the full surface at distances in the range of 25-30 Å. The following sections provide the following information: list of amino acids, which are considered as suitable for mutation (A), the list of generated mutants (B) and the DNA sequence representing the generated mutants (C). On Fig example shows the contours of the surface of the specimen 1. Gray color indicates a conservative amino acid residues. Black color shows amino acid residues selected for mutation.

A. a List of the amino acids selected for mutation.

K15, S24, H30, R31, K48, E62, H74, K77, K82, K100, R128.

B. the List of generated mutants.

C. the Nucleotide sequence mutants.

Example 7

Mutant recombinant mite allergens (Der p 1) with improved security for specific Allergy vaccination

In this example, the application of the concept of the present invention to allergens of the house dust mite is illustrated one allergen, Der p 1. Manipulation with other allergens of the house dust mite can be performed using equivalent methods.

The creation of mutant recombinant molecules Der p 1

SEQ ID NO. 4 shows the nucleotide and deduced amino acid sequence of clone Der p 1-ALK, which is an isoform of the wild type.

SEQ ID NO. 4: nucleotide and deduced amino acid sequence of Der p 1-ALK

On Fig shows the alignment of the sequence produced at the ExPaSy Molecular Biology Server (http://www.expasy.ch/) using the ClustalW algorithm with a BLAST search using the amino acid sequence of Der p 1-ALK shown in SEQ ID NO. 4, as the introductory sequence. The alignment includes sequences from species of house dust mite, i.e. Der p 1, Der f 1, Eur m 1. On Fig amino acid residues identical to the amino acids in the same position in the sequence of the protein Der p 1-ALK, highlighted in black is Okami on a gray background. Non-identical amino acids are printed in black on a white background.

Image of the surface structure

Amino acid sequences representing the group 1 allergens of the house dust mite, have some similarities, and some part of the molecular surface of a conservative (grayed area), see Fig. On Fig shows the contours of the surface, shown at 4 different angles when applying the sequence of the protein Der p 1-ALK on the model of the molecular structure of Der p 1.

For mutation choose conservative amino acids with high exposure to the solvent, distributed in space along the full surface at distances in the range of 25-30 Å. The following sections provide the following information: list of amino acids, which are considered as suitable for mutation (A), the list of generated mutants (B) and the DNA sequence representing the generated mutants (C). On Fig example shows the contours of the surface of the specimen under number 11. Gray color indicates a conservative amino acid residues. Black color shows amino acid residues selected for mutation.

A. a List of the amino acids selected for mutation.

E13, P24, R20, Y50, S67, R78, R99, Q109, R128, R156, R161, P167, W192

B. the List of generated mutants.

p>

C. Nucleotide sequence of the mutants.

Example 8

Mutant recombinant allergens of grass (Phl p 5) with improved security for specific Allergy vaccination

In this example, the application of the concept of the present invention to allergens of grass pollen illustrates one allergen, Phl p 5. Manipulation with other allergens of grass pollen can be performed using equivalent procedures.

The creation of mutant recombinant molecules Phl p 5

SEQ ID NO. 5 shows the nucleotide and deduced amino acid sequence of clone Phl p 5.0103, which is an isoform of the wild type.

SEQ ID NO. 5: nucleotide and deduced amino acid sequence of Phl p 5.0103:

On Fig shows the alignment of the sequence produced at the ExPaSy Molecular Biology Server (http://www.expasy.ch/) using the ClustalW algorithm with a BLAST search using the amino acid sequence of Phl p 5.0103 shown in SEQ ID NO. 5, as the introductory sequence. The alignment includes the sequence of the group 5 allergens of grass species, i.e. Phl p 5, Poa p 5, Lol p 5, Hol p 5, Pha a 5, Hor v 9 and Hor v 5. On Fig amino acid residues identical to the amino acids in the same position in the protein sequence Phl p 5.0103 highlighted in black letters on a gray background. Non-identical amino acids are printed in black on a white background.

Image of the surface structure

Amino acid sequences representing the group 5 allergens of grass pollen, have some similarities, and some part of the molecular surface of a conservative (grayed area), see Fig. On Fig shows the contours of the surface, shown at 4 different angles when applying protein sequence Phl p 5.0103 on the model of the molecular structure of Phl p 5. The structural model includes models of the two halves of the molecule, model A (amino acids 34-142), shown in figa, and model B (amino acids 149-259), shown in figv.

For mutation select amino acids with high exposure to the solvent, distributed in space along the full surface at distances in the range of 25-30 Å. The following sections provide the following information: list of amino acids, which are considered as suitable for mutation (A), the list of generated mutants (B) and the DNA sequence representing the generated mutants (C). On Fig A and B in the example shown the contours of the surface of the specimen 1 on mo the fir-trees A and B, respectively. Gray color indicates a conservative amino acid residues. Black color shows amino acid residues selected for mutation.

A. a List of the amino acids selected for mutation.

I45, R66, E133, R136, I137, D186, F188, K211, P214, Q222, P232, L243, Q254

B. the List of generated mutants.

C. Nucleotide sequence of the mutants.

Example 9

The reaction of T-cells on recombinant and mutant Bet v 1

Objective: to investigate the response of T-cells in vitro mutant allergens in terms of proliferation and cytokine production.

Ways:

In the following study used PBL (peripheral blood lymphocytes) of patients with allergies.

As described in a previously published Protocol (26), of PBL with natural purified bet v 1 was allocated eight bet v 1-specific line T cells to maintain T-cells presenting multiple isoforms of bet v 1.

Ten PBL and eight lines of T-cells stimulated by the extract of birch (Bet v), purified natural bet v 1 (nBet v 1), recombinant Bet v 1 (rBet v1 or wild type; 27) and four different mutant forms of rBet v 1 (is written in another place): 2595, 2628, 2637, 2744, 2773. It was later found that the mutant 2637 partially deployed, and he will not be discussed.

Briefly, in round-bottom 96-well plate was added PBL 2×105on the hole. Added various samples of birch in three different concentrations in four Parallels, and the cells were allowed to grow for 6 days. On day 6 were selected half of the cell volume (100 μl) from each well with the highest concentration of birch on the determination of cytokine production. To the wells were added radioactively labeled thymidine. The next day (day 7) cells collected on the filter. The filter was added to scintillation fluid and radioactivity was measured by scintillation counter.

Similarly, in round-bottom 96-well plate was added T-cells in the amount of 3×104T-cells per well and stimulated trained autologous PBL (1×105cells per well) and 3 different concentrations of different samples of birch. After 1 day, cells from each well with the highest concentration of birch were collected for determination of cytokine production. To the wells were added radioactively labeled thymidine. On day 2 the cells collected on the filter and the radioactivity was measured as described for PBL.

The supernatant of the four parallel samples were combined and cytokines were measured using CBA (ball set for cytokines) from Becton Dickinson.

Results

Ten PBL cultures showed specific stimulation birch. In General, PBL proliferation in response to various samples of birch was similar, although it was possible to see variations. 3 PBL nBet v 1 stimulated the proliferation better than rBet v 1 and mutants. Mutant samples birch stimulated PBL is almost identical with rBet v 1 (Fig). On Fig shows the stimulation indexes for the above preparations Bet v 1. The stimulation index calculated as proliferation (pulses/min pulses / min) stimulated sample (highest concentration), divided by proliferation (imp/min) in the control environment. PPD refers to purified protein derivative mucobacterium tuberculosis, which served as positive control.

Cytokine production dominated by IFN-gamma were increased in proportion to the proliferation of PBL. There was no noticeable signs of movement Th1/Th2 (Fig-44). On Fig shows a patient with a Th0 profile on Fig profile of Th1 and Fig - Th2 profile. Production of cytokines measured in PG/ml are shown as bars, and the ratio of IL-5/IFN-gamma represented by the lower dashed line (Y-axis on the right). Proliferation, measured as counts/min, shown on the Y-axis to the right in a solid line in the pulse/min as background controls included medium and MBP (binding protein maltose).

With the exception of one, all eight selected on nBet v 1 lines T-cells are equally x is well proliferated in response to all samples birch. Four lines of T-cells by the ratio of IL-5 and IFN-gamma secretively cytokines by type Th0 (Th2>5, 5>Th0>0,2, 0,2>Th1). Three T-cell lines secretively cytokines Th1 and one T-cell line secretarial Th2 cytokines. The ratio of IL-5/IFN-gamma did not change under the action of various samples of birch.

Conclusion

All cultures of PBL and 7/8 T-cell lines, which showed specific stimulation in response to nBet v 1, also responded to rBet v 1 and mutants. These data suggest that stimulation of T-cells only isoform Bet v 1 or data 4 mutant can substitute a mixture of individual isoforms detected in preparations of natural allergens.

Thus, vaccines based on recombinant allergens or data 4 mutants, should be directed to existing population of T-cells specific for Bet v 1.

Example 10

Induction is specific for Bet v 1 IgG antibodies and blocking antibodies after immunization with recombinant and mutant protein Bet v 1

In this section, the term "blocking antibodies" refers to antibodies other than IgE antibodies person who is capable of contact with the allergen and prevent the binding of IgE antibodies person with this allergen.

The ability of recombinant protein wild-type Bet v1 2227 (rBet v 1) and the mutant protein Bet v 1 2595, 2628, 2744 and 2773 to induce a Bet v 1-specific IgG antibodies and the lock is the following antibodies were tested in experiments with the immunization of mice.

Mice BALB/cA (8 in each group) were immunized with intraperitoneal injections of recombinant protein wild-type Bet v1 2227 or four mutant proteins. Mice were immunized four times with an interval between doses of 14 days. Different proteins conjugatively with 1.25 mg/ml Alhydrogel (gel aluminum hydroxide and 1.3%, pH 8.0-8.4 and, Superfos Biosector). Mice were immunized with either 1 μg of protein per dose, or 10 μg of protein per dose. Blood samples were collected by phlebotomy from orbit at 0, 14, 35, 21, 49, and 63 days.

The level of specific IgG antibodies were analyzed by direct ELISA using tablets for micrometrology covered with rBet v 1 and biotinylated antibodies rabbit against mouse IgG (Jackson) as developing antibodies. Immunization with the recombinant protein wild-type Bet v1 2227 or four mutant proteins caused a strong r Bet v 1-specific IgG response. This observation shows that the four mutant protein is able to induce antibodies that effectively cross-react with the wild-type protein Bet v1 2227.

To assess the induction of blocking antibodies serum samples obtained from patients allergic to birch pollen, incubated with paramagnetic beads coated with monoclonal antibody to mouse IgE against man. After incubation the beads were washed and resuspendable in the buffer or diluted samples (1:100) serum of unimmunized is ISA (control) or mice immunized as described above. Then to the mixture of beads and serum antibodies mouse was added biotinylated r Bet v 1. After incubation the beads were washed and bound biotinylated r Bet v 1 were determined using labeled with acridine streptavidin. Incubation of beads with serum from non-immunized mice did not affect the binding of r Bet v 1 with balls. In contrast, incubation of beads with serum from mice immunized with recombinant protein wild-type Bet v1 2227 or four mutant proteins significantly reduced the binding of r Bet v 1 with balls that indicates the presence of specific in relation to the Bet v 1 blocking antibodies in serum samples. So, on day 63 one or more serum samples from all groups immunization with a high dose (10 μg/dose) were able to reduce the binding of r Bet v 1 with balls more than 80%. These data show that the four mutant protein is able to induce antibodies that can act as a specific against Bet v 1 blocking antibodies.

Links

1. Recombinant allergen, which is a mutant allergen of natural origin, g is e mutant allergen has at least four mutations, each of which reduces specific IgE-binding ability of the mutant allergen compared to the IgE-binding ability of the specified allergen of natural origin, where each mutation is a substitution of one exposed on the surface amino acid residue by another residue that is not found in the same position in the amino acid sequence of any known homologous protein within a taxonomic species, of which the specified allergen of natural origin, characterized in that at least four mutations (primary mutations) are separated from each other by a gap of at least 15Å and these primary mutations are located so that at least one circular area surface area 800Å2does not contain mutations.

2. Recombinant allergen according to claim 1, in which the primary mutations are separated by intervals of 20Åpreferably 25Åand most preferably 30Å.

3. Recombinant allergen according to claim 1 or 2, which, in addition to at least four primary mutations, contains at least one additional mutation (secondary mutation), which is separated from any primary mutations a period of at least 15Åwhere each secondary mutation reduces specific IgE-binding sposobnostyami.html allergen compared to the binding ability of the specified allergen of natural origin, where each secondary mutation is a substitution of one exposed on the surface amino acid residue by another residue that is not found in the same position in the amino acid sequence of any known homologous protein within a taxonomic species, of which the specified allergen of natural origin, where the secondary mutations are located outside of the specified circular area.

4. Recombinant allergen according to any one of claims 1 to 3, where at least one exposed on the surface of amino acids to be replaced in the allergen of natural origin, is accessible to solvent than 20%, preferably above 30%, more preferably above 40%, and most preferably above 50%.

5. Recombinant allergen according to any one of claims 1 to 4, where at least one exposed on the surface of amino acids to be replaced in the allergen of natural origin, is conservative with more than 70%, preferably 80% and most preferably 90% identity in all known homologous proteins within a species, which is specified allergen of natural origin.

6. Recombinant allergen according to any one of claims 1 to 5, which essentially has the same tertiary structure α-carbon skeleton, as specified allergen prirodoohoronnogo, and where the average value of the square root of the standard deviation of the atomic coordinates between the specified recombinant allergen and the specified allergen of natural origin is less than 2Å.

7. Recombinant allergen according to any one of claims 1 to 6, in which each amino acid residue to be incorporated in the mutant allergen, is not found in the same position in the amino acid sequence of any known homologous protein within a taxonomic genus, preferably subfamily, more preferably family, more preferably superfamily, more preferably Legion, more preferably suborder and most preferably squad to that which is specified allergen of natural origin.

8. Recombinant allergen according to any one of claims 1 to 7, characterized in that the specific binding of IgE mutant allergen reduced by at least 5%, preferably at least 10%.

9. Recombinant allergen according to any one of claims 1 to 8, characterized in that the specified circular area of the surface includes atoms of amino acid residues 15-25.

10. Recombinant allergen according to any one of claims 1 to 9, characterized in that the exposed surface amino acid residues are ranked in relation to accessibility to solvent and one or b is more amino acids among the more solvent-accessible replaced and that exposed on the surface amino acid residues are not necessarily ranked in the degree of conservatism in all known homologous proteins within a species, which is specified allergen of natural origin, and one or more amino acids among the more conservative replaced.

11. Recombinant allergen according to any one of claims 1 to 10, where the mutant allergen is not existing in the nature of the allergen.

12. Recombinant allergen according to any one of claims 1 to 11, containing from 5 to 20, preferably from 6 to 15, more preferably from 7 to 12 and most preferably from 8 to 10 primary mutations, and from 0 to 4 secondary mutations in the primary mutation.

13. Recombinant allergen according to any one of claims 1 to 12, characterized in that one or more of the substitutions performed using site-directed mutagenesis and/or DNA shuffling.

14. Recombinant allergen according to any one of claims 1 to 13, characterized in that it is a mutant of inhaled allergen, preferably allergen pollen, more preferably allergen pollen originating from the taxonomic detachment of Fagales, Oleales or Pinales, most preferably Bet v1.

15. Recombinant allergen Bet v1, which is a mutant allergen Bet v1 of natural origin, where the mutant allergen Bet v1 has at least is our mutation each of which reduces specific IgE-binding ability of the mutant allergen compared to the IgE-binding ability of the specified allergen Bet v1 of natural origin, where each mutation is a substitution of one exposed on the surface amino acid residue by another residue that is not found in the same position in the amino acid sequence of any known homologous protein within a taxonomic species, of which the specified allergen Bet v1 of natural origin, characterized in that at least four mutations (primary mutations) are separated from each other by a gap of at least 15Å and these primary mutations are so that at least one circular area surface area 800Å2does not contain mutations, and where one or more substitutions selected from the group consisting of V2, D72, E87, K-129, E-60, N-47, K-65, P-108, N-159, D-93, K-123, K-32, D-125, R-145, D-109, E-127, Q-36, E-131, L-152, E-6, E-96, D-156, P-63, H-76, E-8, K-134, E-45, T-10, V-12, K-20, S-155, H-126, P-50, N-78, 119, V-2, L-24, E-42, N-4, A-153, I-44, E-138, G-61, A-130, R-70, N-28, P-35, S-149, 103, Y-150, H-154, N-43, A-106, K-115, R-14, Y-5, K-137, E-141, E-87, E-73.

16. Recombinant allergen according to any one of claims 1 to 14, characterized in that it is a mutant allergen pollen originating from the taxonomic squad Poales, Asterales or Urticales.

17. Recombinant allerg is N. according to any one of claims 1 to 14, characterized in that it is a mutant cockroach allergen or allergen house dust mite, preferably mutant of mite allergen originating from Dermatophagoides.

18. Recombinant allergen according to any one of claims 1 to 14, characterized in that it is a mutant animal allergen, preferably animal allergen originating from cats, dogs or horses.

19. Recombinant allergen according to any one of claims 1 to 13, characterized in that it is a mutant of venom allergen, preferably mutant allergen poison, derived from the taxonomic order Hymenoptera, more preferably mutant allergen poison from taxonomic squad Vespidae, Apidae and Formicoidae, and most preferably mutant Ves v5.

20. Recombinant allergen Ves v1, which is a mutant allergen Ves v1 of natural origin, where the mutant allergen Ves v1 has at least four mutations, each of which reduces specific IgE-binding ability of the mutant allergen compared to the IgE-binding ability of the specified allergen Ves v1 of natural origin, where each mutation is a substitution of one exposed on the surface amino acid residue by another residue that is not found in the same position in the amino acid sequence of any known homologous protein in PR the Affairs of the taxonomic species, originates from the specified allergen Ves v1 of natural origin, characterized in that at least four mutations (primary mutations) are separated from each other by a gap of at least 15Å and these primary mutations are located so that at least one circular area surface area 800Å2does not contain mutations, and where one or more substitutions selected from the group consisting of

K-16, K-185 K-11, K-44, K-210, R-63, K-13, F-6, 149, 128, E-184 TO K-112, F-157, E-3, K-29, N-203 N-34, K-78, K-151, L-15, L-158, Y-102, W-186, 134, D-87, K-52, T-67, T-125, 150, Y-40, Q-48, L-65, K-81, Q-101, Q-208, K-144, N-8, N-70, N-104, Q-45, K-137, K-159, E-205, N-82, A-111, D-131, K-24, V-36, N-7, M-138 T-209 V-84, K-172, V-19, D-56, P-73, G-33, T-106, N-170, L-28, T-43, Q-114, C-10, K-60, N-31, K-47, E-5, D-145, V-38, A-127, D-156, E-204, P-71, G-26, Y-129 D-141, F-201, R-68, N-200, D-49, S-153, K-35, S-39, Y-25, V-37, G-18, W-85 and I-182.

21. The use of recombinant allergen according to any one of claims 1 to 20 as a pharmaceutical agent.

22. The pharmaceutical composition is a vaccine for allergic reactions caused by allergen of natural origin in patients suffering from allergies where the specified pharmaceutical composition includes a pharmaceutically acceptable carrier and/or excipient and optional adjuvant, and two or more recombinant mutant allergen variant according to any one of claims 1 to 20, where each option is defined as having the nd at least one primary mutation, which is missing at least one of the other variants, where each variant is not present secondary mutation within a radius of 15Å from each of the missing primary mutations, and the said composition comprises 2 to 12, preferably 3-10, more preferably 4-8, and most preferably 5-7 options.

23. The way of generating an immune response in a subject and/or vaccination of a subject to prevent or alleviate allergic reactions, including introduction to the subject recombinant allergen according to any one of claims 1 to 20.

24. The way of generating an immune response in a subject and/or vaccination of a subject to prevent or alleviate allergic reactions, including introduction to the subject the composition according to item 22.

25. A method of obtaining a pharmaceutical composition according to item 22, which includes mixing the recombinant allergen according to any one of claims 1 to 20 with pharmaceutically acceptable substances and/or excipients.

26. The DNA sequence encoding a recombinant allergen according to any one of claims 1 to 20, its derivative, its partial sequence, its degenerate sequence or a sequence that hybridizes with her in harsh environments, where the specified derivative, partial sequence, a degenerate sequence or hybridizers sequence code the shape peptide, containing at least one b-cell epitope.

27. The DNA sequence for p, where the sequence is a derivative of the sequence shown in figure 3, where the DNA sequence metirovan so that it encodes the allergen containing at least four mutations selected from the group consisting of

V2, D72, E87, K-129, E-60, N-47, K-65, R-108, N-159, D-93, K-123, K-32, D-125, R-145, D-109, E-127, Q-36, E-131, L-152, E-6, E-96, D-156, P-63, H-76, E-8, K-134, E-45, T-10, V-12, K-20, S-155, H-126, P-50, N-78, 119, V-2, L-24, E-42, N-4, A-153, I-44, E-138, G-61, A-130, R-70, N-28, P-35, S-149, K-103, Y-150, H-154, N-43, A-106, K-115, R-14, Y-5, K-137, E-141, E-87, E-73.

28. The DNA sequence for p, where the sequence is a derivative of the sequence shown in Fig, where the DNA sequence metirovan so that it encodes the allergen containing at least four mutations selected from the group consisting of

K-16, K-185 K-11, K-44, K-210, R-63, K-13, F-6, 149, 128, E-184 TO K-112, F-157, E-3, K-29, N-203 N-34, K-78, K-151, L-15, L-158, Y-102, W-186, 134, D-87, K-52, T-67, T-125, 150, Y-40, Q-48, L-65, K-81, Q-101, Q-208, K-144, N-8, N-70, N-104, Q-45, K-137, K-159, E-205, N-82, A-111, D-131, K-24, V 36, N-7, M-138, T. 209, V-84, K-172, V-19, D-56, P-73, G-33, T-106, N-170, L-28, T-43, Q-114, C-10, K-60, N-31, K-47, E-5, D-145, V-38, A-127, D-156, E-204, P-71, G-26, Y-129, D-141, F-201, R-68, N-200, D-49, S-153, K-35, S-39, Y-25, V-37, G-18, W-85 and I-182.

29. The DNA sequence for p, where the sequence is a derivative of the sequence shown is as Fig, where the DNA sequence metirovan, so that it encodes the allergen containing at least four mutations selected from the group consisting of

R-128, D-129, H-11, H-30 S-1, K-77, Y-75, R-31, K-82, K-6, K-96, K-48, K-55, K-89, Q-85, W-92, I-97, H-22, V-65, S-24, H-74, K-126, L-61, P-26, N-93, D-64, I-28, K-14, K-100, E-62, I-127, E-102, E-25, P-66, L-17, G-60, R-95, E-53, V-81, K-51, N-103, Q-2, N-46, E-42, T-91, D-87, N-10, M-111, C-8, H-124, I-68, P-79, 109 and R-128, D-129, H-11, H-30, S-1, K-77, Y-75, R-31, K-82, K-6, K-96, K-48, K-55, K-89, Q-85, W-92, I-97, H-22, V-65, S-24, H-74, K-126, L-61, P-26, N-93, D-64, I-28, K-14, K-100, E-62, I-127, E-102, E-25, P-66, L-17, G-60, P-95, E-53, V-81, K-51, N-103, Q-2, N-46, E-42, T-91, D-87, N-10, M-111, C-8, H-124, I-68, P-79, K-109 and C-15.

30. Expressing a vector comprising the DNA according to any one of p-29.

31. A host cell comprising expressing a vector according to item 30, to obtain a recombinant mutant allergen.

32. A method of obtaining a recombinant mutant allergen, comprising the stage of culturing the host cell according p.

33. Recombinant allergen according to any one of claims 1 to 20 or encoded by the DNA sequence according to any one of p-29, comprising at least one T-cell epitope capable of stimulating T-cell clone or T cell line specific allergen of natural origin.



 

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