WO2002072833A1

WO2002072833A1 - Cloning of the cdna that codes for cup s 1, the major allergen of cupressus sempervirens

Info

Publication number: WO2002072833A1
Application number: PCT/ES2002/000106
Authority: WO
Inventors: Rosalia RODRÍGUEZ GARCÍA; Maria Teresa VILLALBA DÍAZ; Rafael Monsalve Clemente; Eva Batanero Cremades
Original assignee: Universidad Complutense De Madrid
Priority date: 2001-03-09
Filing date: 2002-03-08
Publication date: 2002-09-19
Also published as: ES2180415B2; ES2180415A1

Abstract

The invention relates to the cloning of the recombinant DNA that codes for the cypress pollen (Cupressus sempervirens) Cup s 1 allergen, said allergens having a very similar structure to other Cupressaceae pollen allergens. The invention also relates to the polypeptides which result from the expression of said DNA and which can be used in the diagnosis and treatment of allergies.

Description

TITLE

Cloning of the cDNA that encodes Cup s 1, the major allergen of Cυpressus sempervirens.

OBJECT OF THE INVENTION

The present invention provides recombinant DNA molecules (SEQ ID NO: 1, 2, 3, 4, and 5) encoding polypeptides that present at least one allergenic epitope of those present in the major Cupressus sempervirens allergen, Cup s 1. The invention it also refers to the nucleotide sequences obtained by mutagenesis of the sequences SEQ ID NO: 1, 2, 3, 4, and 5, which encode both complete proteins and fragments that include one or more epitopes of the molecule.

STATE OF THE ART PRIOR TO THE INVENTION

Type I allergy is a disease that affects more than 20% of the population in industrialized countries. This condition is caused by allergens, present in both organisms and biological products -food, mites, insect poisons, pollens, fungi, mammalian epithelia-, as well as in synthetic materials. In most allergenic biological sources, allergens are proteins of molecular masses between 5 and 70 kDa. Symptoms that stem from allergy, such as rhinitis, conjunctivitis, or asthma, are caused by the release of cellular mediators, such as histamine, from basophils and mast cells, cells of the immune system. Said release is induced by the cross-linking of IgEs antibodies bound to the high affinity FcεRI receptors, which are in turn anchored to basophils and mast cells. IgE crosslinking is caused by the binding of the corresponding allergen, or a fragment of its own, through an IgE epitope (contained in said allergen, or in its fragment). The therapy currently being used to treat allergy involves hyposensitization of the patient by parental administration or oral doses of the allergen itself, or related allergoids. In practically all cases, an allergenic extract obtained using minimal manipulation from the natural biological source is administered, which implies a very complex mixture of proteins and other substances, in which the allergen -or allergens- can represent a minimum part of the total product used.

Indeed, at present, the protocols used for the diagnosis of allergy cases and their subsequent immunotherapy involve the use of allergenic extracts that are frequently not characterized, nor even standardized with respect to the most important allergens they may contain. Often, its administration does not provide a complete diagnosis, especially when the patient's hypersensitivity refers to allergens present in low concentration in said extracts. On the other hand, the immunotherapy carried out with these preparations is frequently ineffective and sometimes causes undesirable side effects that can be more serious than the allergic condition itself that is intended to be resolved. An alternative to the use of these extracts is the preparation of mixtures of the most significant allergens, obtained by isolation from their natural source. However, this route presents two important barriers. On the one hand, it implies a good knowledge of the allergenic components of the material that causes the allergy, at least of all its main allergens, and this data is frequently not available. On the other hand, the process of isolating known allergens from biological material is often difficult and does not provide the necessary quantities or the quality required for subsequent use, mainly due to the low levels at which it is already found. the great variability of the composition of these sources. All these problems are accentuated when the allergenic material is the pollen of a plant species. The large amount of pigments, carbohydrates, lipids and insoluble components make their handling very difficult. It goes without saying that its availability is also very low and of high economic cost, due to the difficulty of its collection. For all the above, the design of procedures for the production of allergens to be used in allergy diagnosis and immunotherapy protocols. Recombinant DNA production is envisioned as the most reproducible and effective way of obtaining well-defined allergenic polypeptides, not only for clinical use, but even for research.

Cupresaceal pollinosis has increased significantly in recent years, both in Spain [Subiza J. et al. (1995) J. Allergy Clin. Immunol. 96, 15-23] as in other Mediterranean countries [Mari A. et al. (1996) J. Allergy Clin. Immunol. 98, 21-31]. In fact, in recent years, several works have appeared that describe the detection of allergenic components in pollens of various species of the Pináceas group, which includes the Cupressaceae and Taxodiaceae family. In the case of cupresceae, several groups have highlighted the difficulty in standardizing the extracts of these pollens [Mari A. et al. (1996) J. Allergy Clin. Immunol. 98, 21-31; Ford SA et al (1991) Int. Arch. Allergy Api. Immunol. 95, 178-183] due to the fact that a small amount of protein is extracted, and that it is itself contaminated with hydrocarbon-type components and pigments that are difficult to remove. Cupressus sempervirens (common cypress, the most frequent in France) have been identified by Western Blot in some extracts [Ford SA et al (1991) Int. Arch. Allergy Appl. Immunol. 95, 178-183], of C. arizonica [Di Felice G. et al. (1994) J Allergy Clin Immunol. 94, 547-555] and another species from the cupresceae family, Juniperus oxicedrus [lacovacci P. et al. (1998) J. Allergy Clin. Immunol. 101, 755-761]. In Spain, Juniperus pollen counts are insignificant, but those of C.arízonica and C.sempervirens are increasingly higher and of great importance from a clinical point of view, and they have been described to have a very high cross-reactivity, identifying its main allergens as two glycoproteins of molecular mass 43 and 36 kDa. In species more taxonomically distant and not present in Spain, their main allergens have been studied and characterized, in some detail. This is the case of the Japanese cedar (Cryptomería japónica), belonging to the Taxodiaceae family, whose pollen is the most important cause of pollinosis in Japan, affecting 90% of atopic individuals, and whose main allergens, Cry j 1 and Cry j 2, have been characterized and cloned [Soné T. et al. (1994) Biochem. Biophys. Res. Commun. 199, 619-625; Namba M. et al. (1994) FEBS Letts. 353, 124-128]. Furthermore, these allergens have been reported to be cross-reactive with Cupressus spp pollens [Panzani R. et al. (1986) Ann. Allergy 57, 26-30]. Data on their antigenic structure are available for these allergens, having defined at least five different antigenic determinants and the binding of IgEs to some of these epitopes [Sakaguchi M. et al. (1997) Immunology 91, 161-166].

Recently, the main pollen allergens of species belonging to the Cupressaceae family have been characterized, such as the case of the Japanese cypress (Chamaecyparís obtusa) [Suzuki M. et al. (1996) Mol. Immunol. 33, 451-460; Mori T. et al. (1999) Biochem. Biophys. Res. Commun. 263, 166-171], of mountain cedar (Juniperus ashei), which is a cause of pollinosis in the state of Texas, USA. A [Midoro-Horiuti T. et al (1999) J. Allergy Clin. Immunol. 104, 608-612 and 613-617] and of Cupressus arízonica [Aceituno E. et al (2000) Clin. Exp. Allergy 30, 1750-8]. In the first case, two major allergens, Cha or 1 and Cha or 2, have been purified and cloned, while in the last two cases, the major allergens, Jun at 1 and Cup at 1, respectively, have been isolated and characterized, achieving expression in E.coli in a recombinant form, although the purified recombinant protein could not be obtained.

EXPLANATION OF THE INVENTION

Cloning of the cDNA that codes for Cup s 1, the major allergen of Cupressus sempervirens.

The present invention comprises determining the DNA sequence coding for the major cypress pollen allergen. By means of the process, object of the invention, molecules of Recombinant DNA encoding polypeptides that exhibit the antigenicity of Cup s 1, a cypress pollen allergen, as well as for polypeptides that contain at least one Cup s 1 epitope in their structure. The invention relates to DNA polynucleotide sequences that hybridize under restrictive conditions with those of this allergen -which implies an identity level of at least 60% among its nucleotide sequences-, or are derived from them by code degeneration genetic or mutagenesis.

The procedure object of the invention allows obtaining the recombinant Cupressus sempervirens allergen, Cup s 1, correctly folded and in adequate quantities for subsequent studies. Insertion of DNA into expression vectors and host cells is carried out in such a way as to contain a nucleotide sequence as described in ID SEQ NO: 1 to ID SEQ NO: 5, encoding Cup s 1, or modified sequences of these or fragments derived from them. The methods of preparing these polypeptides involve their recombinant production from the polynucleotide molecules mentioned above, in a culture system of prokaryotic or eukaryotic cells that contain the described expression vectors as a vehicle for DNA molecules.

Therefore, with the procedure object of the invention, recombinant Cup s 1 -an allergen of cupresacea pollen of great clinical importance- are available, in addition to antigenic fragments of this allergen with its own B and T epitopes, which will serve to be incorporated in the "in vivo" and "in vitro" tests to be carried out for the faithful diagnosis of hypersensitivity to this pollen, and to other pollens related phylogenetically with it. They may also be used in allergen preparations that are used to carry out the corresponding immunotherapy for the treatment of cupresacea pollen allergy.

Applications

Protein extracts obtained from pollen are currently used for the diagnosis and therapy of pollen allergy to cupres. This it implies a low reproducibility and a high content of contaminating molecules, of protein and non-protein origin, which can cause adverse side effects in treated patients. Having homogeneous molecules obtained by recombinant DNA techniques, in unlimited quantities, perfectly quantifiable and standardized, will considerably reduce all the aforementioned drawbacks. This technology will make these molecules available, as well as peptides or modified forms thereof that contain at least one of the allergenic epitopes. The use of recombinant Cup s 1 in diagnosis, in vivo by means of skin tests or in vitro by means of immunodetection techniques (ELISA, RAST), will make it possible to determine which allergens are responsible for the clinical symptoms of an individual and thus reduce the number of molecules required for your immunotherapy. Therefore, personalized immunotherapy will originate.

BRIEF DESCRIPTION OF THE FIGURES

The report presents figures that illustratively represent the following:

FIGURE 1. Oligonucleotides designed for the PCR amplifications that have resulted in obtaining the complete Cup s 1 sequence. The nucleotide sequence of each one is shown, its sense of reading considering it "sense" when it is in the sense of reading in which the translation is given, and "antisense" in the opposite sense. The amino acid sequence corresponding to Cup s 1 that is encoded in said oligonucleotide is also presented. Those nucleotides that include an ad hoc designed restriction site, as explained in the text, are underlined. FIGURE 2. Scheme of the Cup s cloning process 1. The steps described in the text are presented and the oligonucleotides used in each case are shown, according to the nomenclature of Figure 1.

METHOD OF CARRYING OUT THE INVENTION

For the cloning of the complete Cup s 1 sequence, the following steps were followed: (1) Isolation of total RNA from Cupressus sempervirens pollen. (2) Amplification from the messenger RNA of the 3 'region of the Cup s 1 coding cDNA molecule, using specific oligonucleotides from the amino terminal end of the mature natural molecule. Cloning of the obtained product and determination of its sequence, especially the C-terminal sequence of the Cup s 1 molecule, to design specific oligonucleotides of said end. (3) PCR amplification of the coding region corresponding to the 5 'end of Cup s 1 (including the signal peptide), cloning of the obtained product and determination of the sequence in this region, allowing the design of specific oligonucleotides of the same . (4) PCR amplification of the cDNA encoding the complete Cup s 1 molecule, using oligonucleotides designed according to the data obtained in the previous steps. 5) Cloning and determination of the complete sequence of said cDNA.

The cloning method followed, except in step (1), was performed using the "SMART-RACE cDNA amplification kit" system from Clontech. Figure 1 shows the oligonucleotides that have been necessary to carry out these amplifications and which are mentioned below, while Figure 2 shows the cloning process. In detail, the procedure followed was as follows:

RNA isolation This was achieved by modifying the method described by

Breiteneder [Breiteneder et al. (1988) Int. Archs. Allergy Applied Immunol. 87, 19-24]. The pollen used is obtained commercially, through the ALK-Abelló company. First, 3.5g of frozen pollen is ground in a mortar with liquid nitrogen. It is then homogenized in Polytron in a 1: 1 mixture of homogenization buffer and phenol / chloroform / isoamyl alcohol in 24: 24: 1 ratio (FCI). A ratio of pollen to total volume of 12% (w / v) was used. The homogenization buffer contains 150mM NaCI, 50mM Tris-HCI pH 8.0, 5mM EDTA, 5% SDS, and 2-mercaptoethanol is added to 100mM just prior to use. The homogenate is centrifuged 15 minutes at 4 ° C, 2900xg, the aqueous phase is recovered and it is re-extracted twice against the same FCI mixture and once more against chloroform. The RNA is then precipitated successively from the aqueous phase: once by adding 3M sodium acetate pH 5.2 to a final concentration of 0.3M followed by 2.5 volumes of ethanol; it is left at -20 ° C for 8 hours and then it is centrifuged at 10000xg, 10 minutes at 4 ° C. The second precipitation is carried out on the material settled in the previous stage, after allowing the ethanol to evaporate, and redissolving it in distilled water, by adding a volume of 4M lithium chloride (LiCI), leaving it for 2 hours in a bath of ice and spinning at 10000xg for 20 minutes. The settled material (specifically RNA) is redissolved and precipitated twice more using sodium acetate / ethanol before it can be used in the next step. In fact, the last precipitation is not carried out until the moment of using the RNA, keeping the samples at -80 ° C in the corresponding volumes of water and ethanol. About 80μg of total RNA are obtained from the 3.5g of initial pollen.

PCR amplifications and cloning of the complete Cup s 1 sequence

As previously indicated, the commercial "SMART-RACE" amplification system with different specific oligonucleotides was used. After a first stage of synthesis of the first strand (starting from 2μg of total RNA), a PCR amplification was carried out using a degenerate oligonucleotide called CUP1 (Figure 1), corresponding to N- terminal of the mature Cup s 1 protein, together with the "UPM" oligonucleotide mixture of the SMART RACE method (Figure 1). In order to obtain specific amplification of Cup s 1 sequences, the PCR method proposed by the "SMART-RACE" manual had to be modified (a temperature of 55 ° C was used in the hybridization stage of the previously denatured sequences, and 30 cycles of amplification were performed). The amplified 1.3 kb band was purified from a low melting point agarose gel and DNA was preferably extracted by the GENECLEAN II method (from BIO101). Said band was preferably cloned into the pSTBIue-1 vector (using the method described by the "Perfectly Blunt" kit from Invitrogen) and sequenced, obtaining data from the 3 'region of the specific sequence coding for Cup s 1, thus as from non-coding sequence regions, down to the poly-A tail corresponding to the messenger RNA. With these sequence data, several specific oligonucleotides were designed, which are made in this case degenerate, to try to obtain isoforms of the molecule, if any. The reason for this strategy is based on the high degree of polymorphism that generally exists among plant allergens. Therefore, for an amplification of the 5 'coding and non-coding region of Cup s 1, the GSP1 and nGSP1 oligonucleotides were designed (Figure 1), corresponding, respectively, to the 3' end of the Cup s 1 coding sequence (amino acids 359 to 367 thereof) plus the translation termination codon, and to an inner Cup s 1 region (corresponding to amino acids 349 to 356) but also located in the 3 'region of this coding sequence. In fact, nGSP1 better met the requirements suggested by the SMART-RACE method for amplification of the 5 'end, and it was with this that Cup s 1-specific amplification was achieved. The band obtained was cloned into vector pCR2.1 (from TOPO-TA Cloning method from Invitrogen), where its sequences are checked, especially those that contain the coding region of Cup s 1 at its 5 'end, including the signal peptide. Next, a specific oligonucleotide of this region, FUGSP2 (Figure 1). Using this oligonucleotide and FUGPS1, corresponding to the coding 3 'end of Cup s 1 including the translation termination triplet and containing a restriction site for subsequent subcloning of the Cup s 1 cDNA, PCR reactions were carried out " long distance "(from the English word Long Distance PCR of the SMART-RACE amplification method) through which amplified bands of the expected size (1.1 kb) were obtained. These were preferably cloned into the pCR2.1 vector with the same method used previously, to proceed with their sequencing. Up to five clones were sequenced with the complete sequence coding for Cup s 1 (sequences listed below as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO : 5), with slight differences from each other, which are attributed to the existence of isoforms encoded by different genes of the family that codes for Cup s 1. The corresponding encoded proteins are shown in sections SEQ ID NO: 6 to SEQ ID NO: 10. To carry out the sequencing of these DNAs completely and unequivocally, specific oligonucleotides (S_429 and A_506 in Figure 1) of internal regions of the coding sequence for this molecule were designed so that all the sequences with at least two different experimental determinations. In addition, and in order to carry out subsequent experiments to express the cDNA encoding Cup s 1 in the yeast Pichia pastoris or in other microorganisms, specific oligonucleotides called B_CS1 and D_CS1 were designed (Figure 1). Both possess restriction sites necessary to subclone these fragments in certain expression vectors, such as pPICZαA from Invitrogen. Primer B_CS1 encodes the 5 'end of Cup s 1 without its signal peptide so that, once inserted into the vector, it would be in phase with a signal peptide that can be processed by P. pastoris. Primer D_CS1, allows an amplification of the 3 'end of Cup s 1 without its usual stop codon so that, when inserted into the expression vector, it would be read with a polypeptide fragment ending in a group of six histidines (a "tag" of histidines), followed by a new stop codon. This latter oligonucleotide can facilitate purification of the recombinant protein.

Claims

1. Recombinant DNA molecules characterized by SEQ ID NO: 1,2,3,4 and

5, which encode polypeptides with allergenic activity that possess at least one common allergenic epitope with the main Cupressus sempervirens allergen.

2. Recombinant DNA molecules, or modifications thereof, according to claim 1, encoding polypeptides with at least one Cup s 1 epitope in their structure, linked to an additional polypeptide, or chemically or enzymatically modified.

3. Recombinant DNA molecules characterized by presenting at least 60% identity with those described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, containing a nucleotide sequence encoding a polypeptide with identical or reduced allergenic (hypoallergenic) activity with respect to Cup s.

4. Recombinant polypeptides from Cupressus sempervirens Cup s 1 allergen, or fragments of said polypeptides, according to claim 1, characterized by the translated sequences and presented in SEQ ID NO:

6, 7, 8, 9 and 10, which show the antigenicity of at least one epitope of Cup s l.

5. Polypeptides or fragments of said Cupressus sempervirens pollen polypeptides with at least one Cup s 1 epitope according to previous claims, producing recombinants linked to an additional polypeptide.

6. Pollen polypeptides of Cupressus sempervirens, according to previous claims, that are chemically or enzymatically modified.

7. A eukaryotic expression vector that contains any of the nucleotide sequences characterized by SEQ ID NO: 1, 2,3,4 or 5, according to claims 1, 2 and 3, and that allows their recombinant production, this vector being expression preferably pPICZalpha.

8. A eukaryotic host organism, transformed with the construct formed by an expression vector according to claim 7 and the DNA sequences characterized by SEQ NO: 1,2,3,4 and 5 of claims 1, 2 and 3, which serve to produce the recombinant polypeptides encoded by those sequences.

9. Use of the molecules described in claims 1,2,3,4,5 and 6, and produced according to claims 7 and 8, in the "in vitro" diagnosis of allergy.

10. Application of the recombinant molecules of claims 1,2,3,4,5 and 6, and produced according to claims 7 and 8, for the design of vaccines for allergy immunotherapy.

11. Application of the recombinant molecules of claims 4-6 to produce peptides containing at least one T epitope capable of acting as vaccines.

12. Application of the recombinant molecules of claims 4-6 for the production of hypoallergenic recombinant isoforms that have decreased or nullified binding to IgE for the treatment of allergies.