ALLERGENS OF ALDER POLLEN AND APPLICATIONS THEREOF

Abstract: This invention provides recombinant DNA molecules which code for polypeptides that exhibit the antigenicity of an Aln g I allergen of alder, Alnus sp., of a Cor a I allergen of hazel or of a Bet v I allergen of birch and other plants of the order Fagales, and for polypeptides comprising at least one epitope thereof, as well as nucleic acids which under stringent conditions hybridize with such DNA sequences or are derivable from such sequences by degeneracy of the genetic code. In addition, methods are described for using the polypeptides coded by these DNA molecules and their use in the diagnosis or therapy of allergic diseases.

Description: ALLERGENSOFALDERPOLLENANDAPPLICATIONSTHEREOF 1. F^ D QF T ffilNVENnO The invention provides recombinant DNA molecules which code for polypepti¬ des, and the polypeptides per se, that have at least one epitope of an Aln g I pollen allergen, or a Cor a l pollen allergen or a Bet v l pollen allergen of a tree of the order Fagaies, particularly alder, Alnus SQ.., or the entire Aln g I allergen protein, particu- larly hazel, Corylus sp.. or the entire Cor a I allergen protein, or particularly birch, Betula sp.. or the entire Bet v l allergen protein, and exhibit the same or similar anti- genicity as the Aln g I. the Cor a l or the Bet v I allergen. The invention also provi¬ des replicable microbial expression vehicles and microorganisms for use in processes for producing such allergenic polypeptides. Methods are provided for the diagnosis and therapy of allergic diseases using the synthetic polypeptides of the invention. 2. BACKGROUND OF THE INVENTION It has long been known that a type I allergy to pollen proteins is associated with symptoms such as itchy and reddened eyes, running nose, swollen eyelids, coughing and asthmatic conditions. In this respect, the pollens of early-flowering trees of the order Fagaies (e.g., birch, hazel, alder and hornbeam) hold an important position. Numerous studies have been carried out to identify and characterize the allergens of these pollens precisely (1 - 4). Progress with regard to the exact characterization of pollen allergens has been hindered by the heterogeneity of the pollen extracts current¬ ly in use. Some eight allergens of alder pollen elicit an IgE response in atopies and one of them, Aln g I. a 17 kD protein, reacts with a majority of the sera of allergic patients as the major allergen (5, 6). At least 10 % of the population suffers from pollen allergies at various times and to varying extent. These allergies are mediated by IgE antibodies which react with pollen proteins. The possibility exists for a therapy for pollen allergies by hypo sensitization, i.e., by the regular and slowly increasing administration of the proteins producing the allergy. Diagnostic methods for allergic diseases, such as radio-allergosorbent tes (RAST), paper radioimmunosorbent test (PRIST), enzyme-linked immunosorbent assay (ELISA), radioimmunoassays (RIA), immuno-radiometric assays (IRMA), luminescence immunoassays (LIA), histamine release assays, and IgE immunoblots depend greatly upon the availability of pure allergens. Protein extracts from pollen isolated from natural sources are difficult to standardize because preparations vary from batch to batch. For example, they may contain unwanted constituents, and/or certain proteins may be lost in the extraction procedure and be missing from the final preparation (7). Clearly, diagnostic tests which employ well defined allergens that can be reproducibly prepared would be superior to tests which employ raw pollen extracts with an insufficiently defined mixture of allergens and other components. Recombinant DNA production of allergenic polypeptides, or allergenic fragments thereof, would allow more reproducible preparations of allergens of defined content for standardized diagnostic and therapeutic methods. Allergens may be purified to homogenity from pollen by known protein/chemi¬ cal methods, for example, by means of affinity chromatography (8). These methods are relatively costly and require pollen as an expensive source for allergens. It would, therefor, be cheaper and more efficient to use recombinant DNA methods to produce an allergenic protein, or fragments of that protein. Hyposensitization has proved to be an effective therapy in allergic diseases. This therapy consists of parenteral or oral administration of allergens in increasing doses over a fairly long period of time. 3. SUMMARY OF THE INVENTION The present invention provides recombinant DNA molecules which contain a nucleotide sequence that codes for a polypeptide which exhibits the same or similar antigenic properties as the major allergen, Aln g I. Cor a l or Bet v l of trees of the order Fagaies, for example, of alder (Alnus sp... hazel (Corylus sp or birch fBetula sp or a polypeptide which comprises at least one epitope of such allergens. The invention provides the complete cDNA sequence of an Aln g I. a Cor a l or a Bet v l allergen and hence the complete deduced amino acid sequences. Additionally, the invention includes (a) nucleotide sequences which hybridize with such a cDNA se¬ quence under high stringency and encode a polypeptide having at least one epitope of an Aln g I. a Cor a l or a Bet v I allergen and (b) nucleotide sequences which can be derived from such allergenic polypeptides by degeneracy of the genetic code. This nucleotide sequence can be expressed as an Aln g I. a Cor a l or a Be v I allergen, or as a polypeptide which comprises at least one epitope thereof. In a preferred embodi¬ ment, this cDNA sequence contains the whole sequence or parts of the sequence set forth in the Sequence Listing as SEQ ID NO. 2 for Aln g I. as SEQ.ID NO.10, 13, 16 and 19 for Cor a l and as SEQ ID NO. 22 for Bet v l. As concerns their IgE binding, pollens of birch, alder, hazel and hornbeam possess similar major allergens which - so far as is known - exhibit a high degree of homology on the amino acid level. The present invention therefore relates not only to an Aln g l allergen of alder, or Cor a l of hazel or Bet v l of birch, but as well to Aln g I. Cor a I or Bet v I pollen allergens of other species which are coded by DNA allergen under stringent conditions or can be derived from such polypeptide allergens by degeneracy of the genetic code. Hybridization of a polynucleotide with another polynucleotide under stringent conditions requires at least a 60 % identity between such polynucleotides at the nu¬ cleic acid level. Such stringent conditions entail washing of hybridized nitrocellulose filters as follows: (a) For DNA/D A and DNA/RNA hybridizations: A temperature of 55 °C, a salt concentration of 150 mM NaCl and 15 mM Na 3 citrate at pH 7,0, and a SDS (So¬ dium Dodecyl Sulfate) detergent at a concentration of 0, 1 % (w/v). (b) For oligodeoxynucleotide/DNA hybridizations: A temperature of 55 °C, a salt concentration of 1M NaCl and 10 mM Na 3 citrate x 2H 2 O at pH 7,0, and a SDS (Sodium Dodecyl Sulfate) detergent at a concentration of 0,5 % (w/v). In this context "oligodeoxynucleotide" refers to an oligomer of a single-stranded DNA of up to 100 nucleotides in length. In addition, this invention provides expression plasmids that contain a nucleoti- de sequence as described above and host cells which harbor these expression plas¬ mids. This invention also provides compositions containing synthetic polypeptides which exhibit the antigenicity of parts or of the whole of an alder Aln g I allergen or of allergens of other plants which, because of a high degree (at least 50 ) of amino acid homology (9), exhibit antigenic cross-reactivity to parts or to all of an alder Aln g I allergen, i.e., antibodies or cellular antigen binding sites which are actually di¬ rected to alder Aln g I allergen are likewise able to bind to these molecules. These synthetic polypeptides include fusion and nonfusion polypeptides which contain a polypeptide portion that possesses the antigenicity of a part or of all of an alder poly- peptid which contain a polypeptide portion that possesses the antigenicity of a part or of all of an Aln g I. a Cor a I or a Bet v I allergen. The method for preparing such synthetic polypeptides comprises the steps of culturing of prokaryotic or eukaryotic host cells which contain an expression plasmid described above and purification of the synthetic polypeptide(s) from the culture. The term "synthetic" here alternatively includes polypeptides which are prepa¬ red by cloning and expression of the nucleotide sequences described here or by che¬ mical synthesis of polypeptides encoded by these nucleotide sequences. The synthetic polypeptides which are produced according to this invention exhi¬ bit antigenicity the same as or similar to the native allergen. As shown below, a cDNA clone coding for an alder Aln g I. a hazel Cor a I or a birch Bet v I can be used to produce a nonfusion polypeptide which reacts with IgE in the sera of allergic persons. It is therefore possible to use this polypeptide as an antigen in diagnostic tests (such as RAST, PRIST, ELISA, RIA, IRMA, LIA, histamine release assays and IgE immunoblots known in the art and referred to above), as a component of prophy- lactic or therapeutic agents in hyposensitization therapy, and as a component in any kind of in vivo diagnostic procedure such as bronchial, conjunctival, dermal, nasal and oral provocation and skin tests. In particular, the synthetic allergens can be used as diagnostic reagents in vitro and in vivo, since their antigenicity corresponds to that of the native Aln g I pollen allergens and they are therefore able to bind IgE of sera of persons suffering from Aln g I pollen allergy. In the same way, the antigenicity corresponds to that of the native Cor a l or Bet v l pollen allergens and they are also able to bind IgE of sera of sensitive or allergic patients. It is therefore one of the objects of the present invention to provide a method for the preparation of pollen allergens, in particular for ln g I, Cor a I or Bet v I allergens, so as to have this family of allergens available for diagnostic tests for de¬ tection of the corresponding allergy and, alternatively, for hyposensitization therapy. As main epitopes capable of modifying T-cell response the following amino acid sequences were found: GlyValPheAsnTyrGlu PhelleLeuAspGlyAspLysLeu AlalleSerSerValGluAsnlle GlyAsnGlyGlyProGlyThrlleLysLysIleSerPhe LysTyrValLysAspArgValAspGluValAsp LeuLeuArgAlaValGluSerTyrLeuLeuAlaHisSer. All these sequences are present in all said allergens, i.e. Aln g I. Cor a I and Bet v I. 4. BRIEF DESCRIPTION OF THE FIGURES The following figures and description aid in understanding the field and scop of the invention. FIG. 1 shows a cDNA (665 nucleotides, SEQ ID NO.l) encompassing th nucleotide sequence encoding an Aln g I allergen of alder. The cDNA sequenc consists of a coding region of 483 nucleotides (including the initation and terminatio codons), a 3' noncoding region of 162 nucleotides and a poly-A tail of 20 nucleotides. The deduced amino acid sequence of alder Aln g l polypeptide is indicated in FIG. under the respective codons. The complete protein has 160 (SEQ ID NO.3) amin acids (including the methionine of the initiation codon). FIG. 2 shows the nucleotide sequence of the BP-A primer (SEQ ID NO.4) tha was used for synthesis of the first cDNA strand. The recognition sequences of th restriction enzymes Bgiπ (nucleotides 19-24) and Hindlll (nucleotides 31-36) are un derlined. The sequence of T7 primer (nucleotides 4-17), which was used as primer fo the PCR amplification of Aln g l and is the constituent of BPA, is likewise indicated. FIGS. 3 - 10 show Immunoblot analysis of isoforms of the major hazel polle allergen Cor a I as recombinant non-fusion proteins, in particular FIGS. 3 - 6: An identical set of patients' sera was used to characterize the Cor a I isoforms (lanes 1 - 9). Lanes B: buffer control without addition of patients' sera. Lanes N: a pool of non-allergic normal human sera. IgE antibodies from the allergic patients' sera, which bound to the isoforms, was detected by 12S I labeled rabbit-anti human IgE. Each of the isoforms shows reacti¬ vity with IgE from allergic patients' sera. All isoforms were able to bind IgE, alt¬ hough their individual binding pattern may differ from patient to patient. FIG. 7: An identical set of experiments was performed using E.coli JM 105 transformed with the plasmid pKK 223.3 without any cDNA insertion. No bound IgE could be 5 detected. FIG. 8: Likewise the cDNA fragment whose sequence is shown in SEQ ID NO. 1 was ligated into the expression plasmid pKK 223.3. The protein corresponding to the coding region (see SEQ ID NO. 2 and SEQ ID NO.3) was expressed in E.coli JM 10 105 and tested with the identical set of patients' sera as above. rAln g I was able to bind IgE from these patients' sera in each case (lanes 1 - 9). In lanes B (buffer con¬ trol, no patients' sera) and N (a pool of sera from non allergic individuals) no binding could be observed. FIG. 9: 15 This represents the quality control of the patients' sera used in the above experi¬ ments. The very same set of sera was tested on separated and blotted proteins from an aqueous extract of birch pollen. IgE from every single serum bound strongly to the major allergen of birch pollen, Bet v l (lanes 1 - 9). No binding could be observed for the buffer control (lane B) and the pool of sera from non allergic individuals. 0 FIG.10: Furthermore the same sera were tested on rBet v I and showed exactly the same strong reactivity with the recombinant nonfusion protein. FIG. 11: Inhibition experiment showing the capacity of rBet v I to bind IgE from tree 5 pollen allergic patients' sera and thus to prevent the IgE from further binding to the corresponding hazel pollen allergen Cor a I. 1 ml each of a 1 : 10 dilution of birch pollen allergic individuals' sera (1 - 5), of a serum pool of non allergic individuals (6), and buffer without the addition of serum (7) was incubated over night at 4°C with the addition of 5 μg of rBet v I (panel 1), 5 y of BSA (panel 2), or buffer only 0 (panel 3). These samples were used to probe a Western blot of SDS-PAGE-separated hazel pollen proteins. In the case where rBet v I had been added no IgE binding the 17kD Cor a l could be observed. The addition of bovine serum albumin (BSA) buffer without addition of a protein could not inhibit the binding of patients' IgE the hazel Cor a I. 5. EXAMPLES 5.1. Poly A + RNA isolation from pollen and synthesis of the first cDN strand: Polyadenylated (polyA+) mRNA was isolated from ripe alder pollen (Allerg AB, Engelsholm,Sweden) (1). Using this, the first strand of cDNA was synthesized follows: 2 μl lOx buffer (480 mM Tris (hydroxymet hyl) aminomethane (Tris), 60 m MgCl 2 , 400 mM KC1, pH 4,8) 2 μl 10 mM dithiothreitol (DTT) 1 μl primer BP-A (100 ng/μl, nucleotide sequence of FIG.2) (SEQ ID NO.4) 2 μl 25 mM deoxynucleoside triphosphates (dNTPs), i.e. 25 mM each of dAT dCTP, dGTP, dTTP (Pharmacia, Uppsala, Sweden) ll μl H 2 O 1 μl poly A +RNA (3 μg) 1 μl AMV reverse transcriptase (United States Biochemical Corporation (USB Cleveland, Ohio, USA) = 32 Units. This reaction, with a total volume of 20 μl, was incubated for 2 hours at 42 ° then diluted 1 : 1 with lx TE buffer (10 mM Tris, ImM ethylenediamine tetraacet acid (EDTA), pH 8,0) and stored at 4°C. 5.2 Polymerase chain reaction fPCR.: PCR was carried out on the hybrid RNA-DNA molecules prepared in Sectio 5.1. A mixture of the following two oligodeoxynucleotides was used as primer f the 5 '-end of the molecules: No. 2482 (SEQ ID NO.5) 5'- GTT TTC AAT TAC GAA GCG GAA AC -3' No.2490 (SEQ ID NO.6) 5'- GTT TTC AAT TAC GAA GCG GAG AC -3' The nucleotide sequences of these oligodeoxynucleotides were derived from the N-terminal amino acid sequence of alder Aln g I partially determined by Edman de¬ gradation and follwing the codon usage of birch (B. verrucosa.. T7 primer (SEQ ID NO.7) (Pharnmacia), which is likewise a constituent of the BP-A primer, was used as primer for the 3' end of the molecules. The following mixture was used for the reaction: 2,5 μl of the reaction mixture in Section 5.1 5,0 μl lOx PCR buffer (400 mM KC1, 10 mM MgCl 2 , 10 % gelatin, 100 mM Tris, pH 8,3) 2,0 μl T7 primer (SEQ ID NO.7) (Pharmacia) = 20 pmol 4,0 μl primer mix in equal parts of No. 2482 (SEQ ID NO.5) and 2490 (SEQ ID NO.6) = 100 pmol 2,5 μl 2 mM dNTPs (Pharmacia) l,5 μl l00 mM MgCl 2 32,5 μl H 2 O (to 50 l) Addition of 1 unit Taq DNA polymerase (USB). The reaction mixture was incubated for 30 seconds at 93°C, for 30 seconds at 55°C and for 1 minute at 72°C. This cycle was run through 30x in all. Finally, the reaction mixture was kept at 72°C for another 10 minutes. 5.3 Cloning of the PCR fragment and sequencing: The DNA fragment synthesized in Section 5.2 was isolated from a 1,5 % aga- rose gel by means of DEAE paper (10). This fragment was then kinased at the 5 '-end. a) Kinasing 10 μl DNA (= 500 ng Aln g l DNA) 2,5 μl lOx T4 polynucleotide kinase buffer (Boehringer, Mannheim, Germany) 7,0 μl ∞P- TP, 10 mCi/ml (Amersham, Little Chalfont, England 4,5 μJ H 2 0 1,0 μl polynucleotide kinase (Boehringer) The reaction mixture was incubated for 20 minutes at 37 β C. After that another addition of 1 μl polynucleotide kinase was made and the mixture was incubated for 60 minutes at 37°C. b) Klenow fill-in reaction: To the above reaction mixture was added: 1 μl 2mM dNTPS (Pharmacia) 1 μX Klenow Fragment (= 2 units) The kinased and filled-in DNA fragment was purified by way of a Nick™ Co¬ lumn (Pharmacia) and was then precipitated with ethanol and sodium acetate (9). c) Bgiπ digestion of fragment: Several restriction enzyme sites were added at the 3 '-end to the Aln g I sequen- ce through the use of the BP-A oligodeoxynucleotide (FIG.2; SEQ ID NO.4) in the PCR. The Bgiπ site in this sequence was selected for cleavage with the restriction enzyme, BgUI, to ligate the fragment in the corresponding Bgiπ site of pBluescript R plasmid (Stratagene, LaJolla, California, USA). Due to the Klenow reaction, blut ends had already been produced at the 5' -end of the sequence. All the DNA precipita- ted in Section 5.3b was dissolved in 2 μl lOx Bgiπ buffer (Boehringer). 17 μl H 2 O and 1 μl Bgiπ (11 units) were added. The reaction mixture was incubated for 1,5 hours at 37°C. The fragment so cut was eluted from a 1,5% agarose gel by means of DEAE paper (10). d) Ligation of the DNA fragment in pBluescript- R KS+ plasmid: pBluescript R KS + plasmid (Stratagene) was selected as cloning vector and cut with the restriction enzymes EcoRV (supplies flush ends; the 5 '-end of the Aln g I fragment is ligated to these) and BamHI (supplies staggered ends compatible with BglH; the 3 '-end of the Aln g I fragment is ligated to these). The phosphate groups at the 5 '-ends of the plasmid were removed by alkaline phosphatase (12) to prevent non- specific religation of the vector. Ligation of Aln g I fragment in pBluescript R KS + plasmid: 20 ng DNA from Section 5.3c dissolved in 10 μl H 2 O 2,0 μl lOx ligation buffer (200 mM Tris, 50 mM MgCl 2 , 50 mM DTT, 500 / g/ ml bovine serum albumin; pH 7,6) 1,0 μl 10 mM ATP 3,0 μl pBluescript R KS+ cut with EcoRV and BamHI (= 50 ng) 4,0 μl H 2 O 1,0 μl T4 DNA ligase Boehringer (= 3 units) This reaction was incubated for 4 hours at room temperature. e) Transformation of competent E.coli host cells: Transformation was carried out in E.coli XL1 -Blue cells (Stratagene) (13). The selection of positive clones was carried out on ampicillin-containing (100 μg/ml) cul¬ ture plates by means of the blue-white indication system (14). f) Sequencing of Aln g I DNA: Sequencing of Aln g I DNA was carried out by means of a T7 sequencing kit (Pharmacia), according to the manufacturer's instructions. 5.4 Expression of Aln g I DNA and detection of IgE binding of the resulting proteins: a) The DNA insert from the pBluescript R KS + vector, which contains the co¬ ding sequence for Aln g I. was subjected to mutagenesis according to Kunkel et al (15). To complete the Aln g I sequence at the 5 '-end and provide it with the ATG codon and an additional EcoRI site, the following oligodeoxynucleotide was synthesi¬ zed (SEQ ID NO.8): S'-CTT CGT AAT TGA AAA CAC CCA TGA ATT CCG ATA CCG TCG A -3' and used for mutagenesis. This enabled the Aln g I sequence to be ligated, in the correct orientation, by means of the EcoRI site at the 5'-end and by means of the Hindiπ site at the 3' -end of the gene in the expression plasmid pKK 223-3 (Pharmacia). E.coli K12 JM105 cells (thi, rpsL, endA, sbcB15, hsdR4, delta (lacpro AB)/F', thraD36, proAB, lacI«Z delta M15) were transformed with this plas- mid. After protein synthesis was effected, the bacterial cells were harvested and bro- ken up with liquid nitrogen. The lysate was separated on a SDS polyacrylamide gel. Detection of recombinant Aln g I nonfusion protein was done by means of immuno- blot. IgE in the sera of allergic patients was bound by the recombinant Aln g I. De¬ tection of bound IgE was effected by 125 I-labeled antihuman IgE (Pharmacia). 5 b) The DNA insert in pBluescript R KS+ plasmid, which contains the sequence coding for Aln g I. was ligated by means of EcoRI linkers (Boehringer) in the expres¬ sion plasmids pEX A, pEX B and pEX C (16), which shift the reading frame of the insert one nucleotide each time. In this way, in one case the correct reading frame for Aln g I was obtained and the production of a recombinant Aln g I fusion protein was 10 induced. The capability of this recombinant Aln g l fusion protein to bind IgE in sera of patients allergic to alder pollen was shown by means of immunoblot. Detection of bound IgE was effected by 125 I-labeled antihuman IgE (Pharmacia). An analogous method was applied for the cloning and expressing of Cor a I. 15 5.5 Expression of Cor a I DNA and detection of IgE binding of the resulting protein The cDNA fragments whose sequences are shown in SEQ ID NO.9, 12, 15 and 18 were ligated into the expression plasmid pKK 223.3 (Pharmacia LKB Biotechnolo¬ gy, Uppsala, Sweden). The proteins corresponding to the coding region (see SEQ ID 20 NO.10, 13, 16 and 19) of these fragments were expressed in E.coli JM 105 transformed with the respective recombinant plasmids. Cultures were grown until the OD^ reached 0,4. Isopropyl-/3-D-galactopyranoside was then added to a final concentration of 0,5 mM and the cultures grown at 37°C over 3,5 hours for expression of recombi¬ nant non-fusion proteins. Bacterial cells were harvested by centrifugation, taken up in 25 50 mM Tris-HCl buffer, pH 7,5, containing 220 mM NaCl and the cells were disrup¬ ted by a freezethaw cycle. The supernatant containing the recombinant non-fusion proteins was loaded onto a 15 % SDS-PAGE. The separated proteins were transferred to a nitrocellulose filter. IgE-binding proteins were detected by the use of allergic patients' sera. 30 The results are shown in FIGS. 3 - 6. 5.6 Test of reaction of T-cell epitopes Peripheral blood was collected from birch pollen allergic patients who showed igE reactivity to Bet v I exclusively, as demonstrated by Western Blot. Peripheral mononuclear cells (PBMC; the white blood cell fraction containing the lymphocytes) were isolated by density gradient centrifucation. Allergen specific T-cells were enri¬ ched by culturing PBMC in presence of Bet v I. After a cloning procedure, T-cell clones (TCC) were proved to react with the complete Bet v l molecule by a prolifera¬ tion assay, showing that in presence of the specific allergen a proliferation occurs, which is at least 10-fold higher than the autoproliferative activity of the TCC, as measured by 3 H-Thymidine incorporation. Two Bet v I specific TCC isolated from atopic donors reacted in the same way with the above mentioned peptides as with the whole Bet v I molecule, proving that these peptides represent or contain the relevant T-cell epitopes. TCC+FC +PEPTIDE 31580* cpm 23309** cpm The present invention covers the use of the recombinant or synthetic polypepti¬ de allergens to treat a mammal using such polypeptides alone or in combination with any pharmaceutically acceptable carriers or diluents, in accordance with standard pharmaceutical practice. The method of treatment involves the administration of such a polypeptide aller¬ gen or parts thereof by any route of administration, that is bronchial, conjunctival, dermal, enteral, nasal, oral or vaginal. A range of from 1 picogram to 10 milligrams per application can be used. The diluents and carriers can be chosen by those skilled in the art according to commonly accepted galenic procedures. Like diagnostic me¬ thods, it requires pure and well defined allergens. The use of purified recombinant allergens or synthetic peptides would greatly reduce the risk of sensitizing patients to unwanted components. 7. REFERENCES The references cited in the above specification are: 1. Breiteneder, H. , Pettenburger, K_, Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, o., Breitenbach, K. (1989). The gene coding for the major birch pollen allergen, Set v I, is. highly homologous to a pea resistance response gene. EMBO J., £: 1935-1938. 2. Zpsen, H. and H nsen, O.C. (1990). Physicochemical and imaunological characteristics of allergens. In: Epitopes of atopic allergens. Sehon, A.H., Kraft, D. Kunkel, G. (eds) UCB, Brussels, Belgium, pp 3 7 8. 3. Rumpold, H., Rohac, K., Bohle, B., Breitenbach, M., Scheiner, O., Kraft, D. " (1990) . The relationship of.Set v I epitopes recognized by patients' IgE and monoclonal anti-Bet v I antibodies. In: Epitopes of atopic allergens. -Sehon, A. H., Kraft, D., Kun el, G. (eds) UCB, Brussels, Belgium, pp 26-28. 4. Valenta, R. ; Breiteneder, H. , Pettenburger, K. , Breitenbach, K. , Scheiner, 0. , Kraft, D. (1990) . RNA- and DNA-sequence similarities of the major allergens of birch, alder, ' hazel and hornbeam pollens. In: Epitopes ' of atopic allergens. Sehon, A.H. , . Kraft, D. , Kunkel, G. (eds) UCB Brussels, Belgium, pp 73-76. 5. Plorvaag, E. and Elsayed, s . (1984) . Comparative studies on tree pollen allergens. VIII . Immunological properties of the alder (Alnus inσana; pollen extract. Int. Arch. Allergy Appl. Immunol. 7 .: 300-308. 6. Plorvaag, E. , Elsayed, S. , Apold, J. (1982) . Comparative studies on tree pollen allergens . II. Isolation of alder (Alnus incana) pollen allergens: purification and some characteristics of the. major allergen pi 4.78. Int. Arch. Allergy Appl. Immunol. 6J.I 49-56. 7. Thomas, W.R., Chua, K.Y., Greene, W.K., and Stewart, G.A. (1990). Recombinant mite allergens. In: Epitopes of atopic allergens. Sehon, A.H., Kraft, D., and Kunkel, G. (eds). UCB Institute of Allergy, Brussels. 8. Jarolim, E. , TejJ l, M. , Rohac, M. , Schler a,. G. , Breitenbach, K. , Scheiner, O. , Kraft, D. , and Rumpold, H. (1989) . Konoclonal antibodies against birch pollen allergens; characterization by im unoblotting and use for single step affinity purification of the major allergen £≤£vi. int. Arch. Allergy Appl. Immunol. ££: 54-60. ' 9. Needleman, S.B. , Wunsch, CD. (1970) . A general method applicable to the search for similarities in- the amino acid sequence of two proteins. J. MolT Biol. ££: 443-453. 10. Ausubel, P.M. (ed) (1987) . current Protocols in Molecular Biology. Green Publishing Associates and Wiley Interscience: John Wiley and Sons, New York. Unit 2.6. 11. Ibid. , Unit 2.1. 12. Ibid. ' , Unit 3.10. 13. Ibid. , Unit 1.8. 14. Ibid. , Unit 1. 15. Kunkel, T.A., Roberts, J.D. Zakour, R.A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. In: Methods in Enzy ology Vol. 154. Wu, R. and Grossman, L. (eds) Academic Press, Inc. pp 367-382. 16. Nagai, K., Thψgersen, H.C. (1984). Generation of 6-globin by. sequence-specific proteolysis of a hybrid protein produced in Esherichia coli . Nature 1S£: 810-812. SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: Dr. Heimo Breiteneder Mag. Arnold Reikerβtorfer Dr. Rudolf Valenta Mrs. Dr. Karin Hoffmann - Sommergruber Dr. Michael Breitenbach Dr. Dietrich Kraft Dr. Helmut Rumpold Dr. Otto Scheiner (ii) TITLE OF INVENTION: ALLERGENS OF ALDER POLLEN AND APPLICATIONS THEREOF (iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Pennie _ Edmonds (B) STREET: 1155 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: USA (F) ZIP: 10036 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 07/683,831 (B) FILING DATE: ll-APR-91 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Harry C. Jones, III (B) REGISTRATION NUMBER: 20,280 (C) REFERENCE/DOCKET NUMBER: 6530-009 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (212) 790-9090 (B) TELEFAX: (212) 869-9741/8864 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 665 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( ii) MOLECULE TYPE : cDNA of toRNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: (A) ORGANISM: Alder (Alnus sp.) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATGGGTGTTT TCAATTACGA AGCGGAAACC CCCTCCGTTA TCCCAGCGGC TCGGCTGTTC 60 AAGGCCTTTA TCCTTGATGG CGATAAGCTC CTTCCAAAGG TTGCACCTGA AGCTGTTAGC 120 AGTGTTGAGA ACATTGAAGG AAATGGAGGG CCTGGAACCA TCAAGAAGAT CACCTTTCCC 180 GAAGGCAGCC CTTTTAAGTA CGTAAAGGAG AGGGTTGATG AGGTTGATCG CGTAAACTTC 240 AAATACAGCT TCAGCGTGAT CGAGGGTGGT GCCGTGGGCG ACGCACTGGA GAAGGTCTGT 300 AACGAGATCA AGATAGTGGC AGCCCCTGAT GGAGGATCCA TCTTGAAGAT CAGCAACAAG 360 TTCCACACCA AAGGCGACCA TGAGATAAAT GCAGAGCAGA TTAAGATTGA AAAAGAAAAG 420 GCCGTGGGAC TTCTCAAGGC CGTTGAGAGC TACCTCTTGG CACACTCTGA TGCCTACAAC 480 TAAATTCTGC CTAATTTTGA TCAGCTTGCA TGTGTTCTTG TCAAGCCATA AATACTGCTT 540 AACTTCGTCT TGCTAATAAA TGAAGCTGTT GTAGTCGTTT ATGAGTACGT AATAATGACA 600 CCAAACATAT GGAGCCAATT GCTTATGAAT AGAAGTTAAG TTCTTAAAAA AAAAAAAAAA 660 AAAAA 665 (3) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 480 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: (A) ORGANISM: Alder (Alnus sp.) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGGGTGTTT TCAATTACGA AGCGGAAACC CCCTCCGTTA TCCCAGCGGC TCGGCTGTTC 60 AAGGCCTTTA TCCTTGATGG CGATAAGCTC CTTCCAAAGG TTGCACCTGA AGCTGTTAGC 120 AGTGTTGAGA ACATTGAAGG AAATGGAGGG CCTGGAACCA TCAAGAAGAT CACCTTTCCC 180 GAAGGCAGCC CTTTTAAGTA CGTAAAGGAG AGGGTTGATG AGGTTGATCG CGTAAACTTC 240 AAATACAGCT TCAGCGTGAT CGAGGGTGGT GCCGTGGGCG ACGCACTGGA GAAGGTCTGT 300 AACGAGATCA AGATAGTGGC AGCCCCTGAT GGAGGATCCA TCTTGAAGAT CAGCAACAAG 360 TTCCACACCA AAGGCGACCA TGAGATAAAT GCAGAGCAGA TTAAGATTGA AAAAGAAAAG 420 GCCGTGGGAC TTCTCAAGGC CGTTGAGAGC TACCTCTTGG CACACTCTGA TGCCTACAAC 480 (4) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 160 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: No (vi) ORIGINAL SOURCE: (A) ORGANISM: Alder (Alnus βp. ) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Gly Val Phe Asn Tyr Glu Ala Glu Thr Pro Ser Val lie Pro Ala 1 5 10 15 AlaArg Leu Phe Lys Ala Phe lie Leu Asp Gly Asp Lys Leu Leu Pro 20 25 30 Lys Val Ala Pro Glu Ala Val Ser Ser Val Glu Asn lie Glu Gly Asn 35 40 45 Gly Gly Pro Gly Thr lie Lys Lys lie Thr Phe Pro Glu Gly Ser Pro 50 55 60 Phe Lys Tyr Val Lys Glu Arg Val Asp Glu Val Asp Arg Val Asn Phe 65 70 75 80 Lys Tyr Ser Phe Ser Val lie Glu Gly Gly Ala Val Gly Asp Ala Leu 85 90 95 Glu Lys Val Cys Asn Glu lie Lys lie Val Ala Ala Pro Asp Gly Gly 100 105 110 Ser lie Leu Lys lie Ser Asn Lys Phe His Thr Lys Gly Asp His Glu 115 120 125 lie Asn Ala Glu Gin lie Lys lie Glu Lys Glu Lys Ala Val Gly Leu 130 135 140 Leu Lys Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn 145 (5) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1inear (ii) MOLECULE TYPE: Other nucleic acid-synthetic (iii) HYPOTHETICAL: Yes (ix) FEATURE: (D) OTHER INFORMATION: Primer for reverse transcription (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TTTAATACGA CTCACTATAG ATCTCCCGGG AAGCTTTTTT TTTTTTTTTT 50 (6) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid-synthetic (iii) HYPOTHETICAL: Yes (vi) ORIGINAL SOURCE: (C) INDIVIDUAL/ISOLATE: 2482 (ix) FEATURE: (D) OTHER INFORMATION: Primer for polymerase chain reaction (PCR) utilized at the 5' end of Aln g I mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTTTTCAATT ACGAAGCGGA AAC 23 (7) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid-synthetic (iii) HYPOTHETICAL: Yes (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 2490 (ix) FEATURE: (D) OTHER INFORMATION: Primer for polymerase chain reaction utilized at the 5' end of Aln g I mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTTTTCAATT ACGAAGCGGA GAC 23 (8) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1inear (ii) MOLECULE TYPE: Other nucleic acid-synthetic (iϋ) HYPOTHETICAL: Yes (ix) FEATURE: (D) OTHER INFORMATION: Primer for polymerase chain reaction (PCR) utilized at the 3' end of Aln g I mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AATACGACTC ACTA 14 (9) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid-synthetic (iii) HYPOTHETICAL: Yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CTTCGTAATT GAAAACACCC ATGAATTCCG ATACCGTCGA 40 INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 619 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel (Corylus εp. ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 9 INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 480 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA Of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (Vi) ORIGINAL SOURCE: (A) ORGANISM: hazel { Corylus sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 10 INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS: Cor a I 5 (c) (A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: not applicable (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel {Corylus sp. ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Met Gly Val Phe Asn Tyr Glu Val Glu Thr Pro Ser Val He Pro 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ala Ala Arg Leu Phe Lys Ser Tyr Val Leu Asp Gly Asp Lys Leu 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 He Pro Lys Val Ala Pro Gin Ala He Thr Ser Val Glu Asn Val 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Glu Gly Asn Gly Gly Pro Gly Thr He Lys Asn He Thr Phe Gly 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Glu Gly Ser Arg Tyr Lys Tyr Val Lys Glu Arg Val Asp Glu Val 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Asp Asn Thr Asn Phe Thr Tyr Ser Tyr Thr Val He Glu Gly Asp 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 Val Leu Gly Asp Lys Leu Glu Lys Val Cys His Glu Leu Lys He 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 Val Ala Ala Pro Gly Gly Gly Ser He Leu Lys He Ser Ser Lys 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 Phe His Ala Lys Gly Asp His Glu He Asn Ala Glu Glu Met Lys 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Gly Ala Lys Glu Met Ala Glu Lys Leu Leu Arg Ala Val Glu Thr 151 152 153 154 155 156 157 158 159 160 Tyr Leu Leu Ala His Ser Ala Glu Tyr Asn INFORMATION FOR SEQ ID NO: 12 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 742 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel (Coryl s sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 12 INFORMATION FOR SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 480 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel {Corylus sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 13 INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: not applicable (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel {Corylus sp. ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Met Gly Val Phe Asn Tyr Glu Val Glu Thr Pro Ser Val He Pro 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ala Ala Arg Leu Phe Lys Ser Tyr Val Leu Asp Gly Asp Lys Leu 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 He Pro Lys Val Ala Pro Gin Ala He Thr Ser Val Glu Asn Val 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Glu Gly Asn Gly Gly Pro Gly Thr He Lys Asn He Thr Phe Gly 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Glu Gly Ser Arg Tyr Lys Tyr Val Lys Glu Arg Val Asp Glu Val 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Asp Asn Thr Asn Phe Lys Tyr Ser Tyr Thr Val He Glu Gly Asp 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 Val Leu Gly Asp Lys Leu Glu Lys Val Cys Ser Glu Leu Lys He 106 107 108 109 110 111 112 113 114 115 116 117 ' 118 119 120 Val Ala Ala Pro Gly Gly Gly Ser He Leu Lys He Ser Ser Lys 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 Phe His Ala Lys Gly Asp His Glu He Asn Ala Glu Glu Met Lys 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Gly Ala Lys Glu Met Ala Glu Lys Leu Leu Arg Ala Val Glu Thr 151 152 153 154 155 156 157 158 159 160 Tyr Leu Leu Ala His Ser Ala Glu Tyr Asn INFORMATION FOR SEQ ID NO: 15 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 655 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel (Corylus sp. ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 15 INFORMATION FOR SEQ ID NO: 16 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 480 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel (Corylus sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ATGGGTGTTT TCAATTACGA GGCTGAGACC AAGGCTGTTC AAGTCCTATG TCCTTGATGG TTGCACCTCA AGCTATTACC AGCGTTGAAA CCTGGAACCA TCAAGAATAT CACCTTTGGC CGTGAAGGAG AGGGTTGATG AGGTTGACAA ACACCGTGAT CGAGGGTGAT GTCCTGGGTG CACGAGCTGA AGATAGTGGC AGCCCCTGGT CAGCAGCAAG TTCCACGCCA AAGGTGACCA TGAAGGGTGC CAAAGAAATG GCCGAGAAAC TACCTATTGG CACACTCTGC TGAATACAAC INFORMATION FOR SEQ ID NO: 17 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: not applicable (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel (Coryl s sp. ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Met Gly Val Phe Asn Tyr Glu Ala Glu Thr Thr Ser Val He Pro 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ala Ala Arg Leu Phe Lys Ser Tyr Val Leu Asp Gly Asp Lys Leu 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 He Pro Lys Val Ala Pro Gin Ala He Thr Ser Val Glu Asn Val 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Glu Gly Asn Gly Gly Pro Gly Thr He Lys Asn He Thr Phe Gly 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Glu Gly Ser Arg Tyr Lys Tyr Val Lys Glu Arg Val Asp Glu Val 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Asp Asn Thr Asn Phe Thr Tyr Ser Tyr Thr Val He Glu Gly Asp 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 Val Leu Gly Asp Lys Leu Glu Lys Val Cys His Glu Leu Lys He 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 Val Ala Ala Pro Gly Gly Gly Ser He Leu Lys He Ser Ser Lys 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 Phe His Ala Lys Gly Asp His Glu He Asn Ala Glu Glu Met Lys 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Gly Ala Lys Glu Met Ala Glu Lys Leu Leu Arg Ala Val Glu Thr 151 152 153 154 155 156 157 158 159 160 Tyr Leu Leu Ala His Ser Ala Glu Tyr Asn INFORMATION FOR SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 860 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel {Corylus sp. ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 18 ATGGGTGTTT TCAATTACGA GGTTGAGACC CCCTCCGTTA TCTCAGCGGC 50 AAGGCTGTTC AAGTCCTATG TCCTTGATGG CGATAAGCTC ATCCCAAAGG 100 TTGCACCTCA AGCTATTACC AGCGTTGAAA ACGTTGGAGG AAATGGAGGG 150 CCTGGAACCA TCAAGAATAT CACCTTTGGC GAAGGCAGCC GTTACAAGTA 200 CGTGAAGGAG AGGGTTGATG AGGTTGACAA CACAAACTTC AAATATAGCT 250 ACACCGTGAT CGAGGGTGAT GTCCTGGGTG ACAAGCTGGA GAAAGTCTGC 300 AGCGAGCTGA AGATAGTGGC AGCCCCTGGT GGGGGATCCA CCTTGAAGAT 350 CAGCAGCAAG TTCCACGCCA AAGGTGACCA TGAGATTAAT GCAGAGGAG 400 TGAAGGGTGC CAAAGAAATG GCCGAGAAAC TTTTAAGGGC GGTTGAGACC 450 TACCTATTGG CACACTCTGC TGAATACAAC TAAATATCGT CTTGTGTCTT 500 CGCCAATAAT AACTTGTACG TGGCTTTCAT GTTTTTTTTT AAAAAACTTT 550 GTTTACTTGC TAATAAAGGA GCTTGCGGTT GTGTTCATCT GCTTGCTGAA 600 ATCGATGTTG TAACTCGGAA GAATGCAAAC TGAATGTTGT ATTACTTTTT 650 GCATATATAC AAATAATGGA AAGGATAACA TCATTGAAGT TCAAAAAAAA 700 GAAAAAAAAA AGCTTTTTTT χχτχιχχχχτ τχχχχιχχχχ TTTTTTGTCA 750 ATTTTAACCC GATACTGATA CTCAAAAATG CAAGAGAGTT TCCGCATAAG 800 CACAATTTGT TTATGTTGAC TTAATACATT ATAAGCAAAA AAAAAAAAAA 850 AAAAAAAAAA 860 INFORMATION FOR SEQ ID NO: 19 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 480 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel (Corylus sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 19 INFORMATION FOR SEQ ID NO: 20 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: not applicable (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: hazel (Corylus sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Met Gly Val Phe Asn Tyr Glu Val Glu Thr Pro Ser Val He Ser 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ala Ala Arg Leu Phe Lys Ser Tyr Val Leu Asp Gly Asp Lys Leu 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 He Pro Lys Val Ala Pro Gin Ala He Thr Ser Val Glu Asn Val 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Gly Gly Asn Gly Gly Pro Gly Thr He Lys Asn He Thr Phe Gly 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Glu Gly Ser Arg Tyr Lys Tyr Val Lys Glu Arg Val Asp Glu Val 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Asp Asn Thr Asn Phe Lys Tyr Ser Tyr Thr Val He Glu Gly Asp 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 Val Leu Gly Asp Lys Leu Glu Lys Val Cys Ser Glu Leu Lys He 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 Val Ala Ala Pro Gly Gly Gly Ser Thr Leu Lys He Ser Ser Lys 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 Phe Hie Ala Lys Gly Asp His Glu He Asn Ala Glu Glu Met Lys 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Gly Ala Lys Glu Met Ala Glu Lys Leu Leu Arg Ala Val Glu Thr 151 152 153 154 155 156 157 158 159 160 Tyr Leu Leu Ala His Ser Ala Glu Tyr Asn INFORMATION FOR SEQ ID NO: 21 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 672 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: birch (Betula sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 21 INFORMATION FOR SEQ ID NO: 22 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 480 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA of mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: birch (Betula sp . ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 22 INFORMATION FOR SEQ ID NO: 23 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: not applicable (v) FRAGMENT TYPE: not applicable (vi) ORIGINAL SOURCE: (A) ORGANISM: birch (Betula sp. ) (vii) IMMEDIATE SOURCE: (A) POLLEN FROM ALLERGON AB, ENGELHOLM, SWEDEN (viii) POSITION IN GENOME: not applicable (ix) FEATURE: not applicable (x) PUBLICATION INFORMATION: not applicable (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Met Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val He Pro 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Ala Ala Arg Leu Phe Lys Ala Phe He Leu Asp Gly Asp Asn Leu 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Phe Pro Lys Val Ala Pro Gin Ala He Ser Ser Val Glu Asn He 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Glu Gly Asn Gly Gly Pro Gly Thr He Lys Lys He Ser Phe Pro 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Glu Gly Phe Pro Phe Lys Tyr Val Lys Asp Arg Val Asp Glu Val 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 Asp His Thr Asn Phe Lys Tyr Asn Tyr Ser Val He Glu Gly Gly 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 Pro He Gly .Asp Thr Leu Glu Lys He Ser Asn Glu He Lys He 106 107 108 109 110 111112 113 114 115 116 117 118 119 120 Val Ala Thr Pro Asp Gly Gly Ser He Leu Lys He Ser Asn Lys 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 Tyr His Thr Lys Gly Asp His Glu Val Lys Ala Glu Gin Val Lys 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Ala Ser Lys Glu Met Gly Glu Thr Leu Leu Arg Ala Val Glu Ser 151 152 153 154 155 156 157 158 159 160 Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn

Claims: CLAIMS 1. A recombinant DNA molecule, comprising a DNA coding for a polypeptide having at least one epitope of an allergen of trees of the order Fagaies, the allergen is selected from the group Aln g I. Cor a l or Bet v l. 2. A recombinant DNA molecule according to claim 1, wherein the allergen Aln g I r Cor a l or Bet v l is selected from the group consisting of birch, alder , hazel and hornbeam. 3. A recombinant DNA molecule according to claim 1, wherein the epitopes of the allergens having an amino acid sequence selected from the following group GlyValPheAsnTyrGlu PheϋeLeuAspGlyAspLysLeu AlalleSerSerValGluAsnlle GlyAsnGlyGlyProGlyThrlleLysLysIleSerPhe LysTyrValLysAspArgValAspGluV alAsp LeuLeuArgAlaValGluSerTyrLeuLeuAlaHisSer 4. A recombinant DNA molecule according to claim 1 having at least 60% identity to the nucleotid sequence as shown in SEQ ID No. 2. 5. A recombinant DNA molecule according to claim 1, wherein the allergen is an Aln l allergen of alder. 6. A recombinant DNA molecule according to claim 1, 2, 3, 4 or 5 which co¬ des for a polypeptide having the entire amino acid sequence of an Aln l allergen. 7. A recombinant DNA molecule according to claim 5, which codes for a po¬ lypeptide having all or part of the amino acid sequence as defined in the Sequence Listing by SEQ ID NO:3. 8. A recombinant DNA molecule according to claim 1, wherein the allergen is a Cor a l allergen of hazel. 9. A recombinant DNA molecule according to claim 1 or 8, which codes for a polypeptide having the entire amino acid sequence of a Cor a l allergen. 10. A recombinant DNA molecule according to claim 1 having at least 60% identity to the nucleotid sequence as shown in SEQ ID NOs. 10, 13, 16, and 19. 11. A recombinant DNA molecule according to claim 8 which codes for a poly¬ peptide having all or part of the amino acid sequence as defined in the Sequence Li- sting by SEQ ID NOs. 11, 14, 17, and 20. 12. A recombinant DNA molecule according to claim 1 wherein the allergen is a Bet v l allergen of birch. 13. A recombinant DNA molecule according to claim 1 having at least 60% identity to the nucleotid sequence as shown in SEQ ID No. 22. 14. A recombinant DNA molecule according to claim 12, which codes for a polypeptide having all or part of the amino acid sequence as defined in the Sequence Listing by SEQ ID NO. 23. 15. A recombinant DNA molecule according to claim 3 which codes for one of the epitopes of the allergens as listed in claim 3. 16. A polypeptide having at least one epitope of an Aln g I. a Cor a l or a Bgt v I allergen showing the same or a similar capacity to bind IgE from tree pollen allergic individual's sera. 17. A replicable prokaryotic or eukaryotic expression vehicle capable, in a transformant prokaryotic or eukaryotic host organism, of being replicated and of directing expression of a DNA of claim 1 to 15 to produce said polypeptides. 18. A prokaryotic or eukaryotic host organism transformed with an expression vehicle capable, in said host organism, of being replicated and of directing expression of a DNA of claim 1 to 15 to produce said polypeptides. 19. A host organism according to claim 18, wherein the organism is Escheri- chia coli. 20. A method for producing a polypeptide having at least one epitope of an Aln g I. a Cor a I or a Bet v I allergen, comprising culturing a prokaryotic or eukaryotic host organism containing an expression vehicle capable, in said host organism, of being replicated and of directing expression of a DNA of claim 1 to 15 to produce said polypeptides. 21. A composition comprising a polypeptide having at least one epitope of an Aln g I. a Cor a l or a Bet v l allergen and produced by a method according to claim 20. 22. A composition comprising a polypeptide having at least one epitope of an Aln g I allergen and produced by a chemical synthesis according to amino acid se¬ quence as defined in the Sequence Listing by SEQ ID NO. 3. 23. A composition comprising a polypeptide having at least one epitope of a Cor a I allergen and produced by a chemical synthesis according to amino acid se- quence as defined in the Sequence Listing SEQ ID NOs.11, 14, 17 and 20. 24. A composition comprising a polypeptide having at least one epitope of a Bet v l allergen and produced by chemical synthesis according to the amino acid sequence as defined in the Sequence Listing by SEQ ID No. 23. 25. An isolated allergenic peptide of pollen of trees of the order Fagaies, ha- ving at least one of the epitopes with amino acid sequence listed in claim 3. 26. A peptide according to claim 6, 7, 8, 9, 11, 12, 14, or 25, capable of mo¬ difying in a sensitive individual to whom it is administered, an allergic response to a pollen of a tree of the order Fagaies. 27. A peptide according to claim 26 capable of modifying T-cell response to a pollen of trees of the order Fagaies. 28. An isolated peptide of the claim 6, 7, 8, 9, 11, 12, 14 or 25 capable of interfering with an allergic response. 29. A method for detecting IgE antibodies comprising contacting serum of a mammal with a composition according to claim 21, and detecting any immunological reaction between IgE antibodies in the serum and said polypeptide having at least one epitope of an Aln g I. a Cor l or a Bet v l allergen. 30. A method for detecting allergic reactions to an Aln g I. a Cor a l or a Bet v I allergen, comprising challenging a mammal with a composition according to claim 21 so as to elicit bronchial, conjunctival, dermal, nasal or oral provocation of said mammal, and detecting any immunological reaction between said tissues and said polypeptides. 31. A method for detecting in vitro a cellular reaction to an Aln g I. a Cor a I or a Bet v I allergen , comprising contacting mammalian cells with a composition according to claim 21, and detecting any reaction between said cells and said poly¬ peptide. 32. A method for treating a mammal afflicted with a pollen allergy, comprising administering an effective amount of a composition according to claim 21 to hyposen- sitize said mammal to an Aln g I. a Cor a l or a Bet v I allergen.

Inventors: BREITENBACH MICHAEL, BREITENEDER HEIMO, EBNER CHRISTOF, FERREIRA FATIMA, HOFFMANN-SOMMERGRUBER KARIN, KRAFT DIETRICH, REIKERSTORFER ARNOLD, RUMPOLD HELMUT, SCHEINER OTTO, VALENTA RUDOLF, BREITENBACH, MICHAEL, BREITENEDER, HEIMO, EBNER, CHRISTOF, FERREIRA, FATIMA, HOFFMANN-SOMMERGRUBER, KARIN, KRAFT, DIETRICH, REIKERSTORFER, ARNOLD, RUMPOLD, HELMUT, SCHEINER, OTTO, VALENTA, RUDOLF
Applicants: BIOMAY BIOTECH PROD, BIOMAY BIOTECHNIK PRODUKTIONS- UND HANDELSGESELLSC
Application year: 1991
Main classification: C12N15
IPC codes: A61K38, A61K39, C07K14, C07K16, C12N15, C12P21, G01N33