AT400723B - RECOMBINANT ALTERNARIA ALTERNATA ALLERGENS - Google Patents

Description

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The invention relates to recombinant DNA molecules which code for polypeptides which have the antigenicity of the allergens Alta53, Alta22 and Altai 1 or for peptides which have at least one epitope of these allergens.

The named allergens from Alternaria alternata, as well as the peptide sequences derived from the cDNA primary sequences of these allergens, lead to a pathological immune response with an overshoot of IgE antibodies in fungal allergy sufferers. In addition to improved diagnostics, recombinant allergens or immunogenic partial peptides can also be used for in vivo or in vitro induction of an immune tolerance or anergy of T lymphocytes.

According to epidemiological studies, allergies have increased in frequency in recent years. The causes of allergic diseases are many. Allergens such as pollen, animal hair and excrement from dust mites are well known to everyone today (Wüthrich 1991, Miyamoto 1992). However, molds leave many questions open to experts with regard to their biological and allergological significance. Last but not least, it is the extremely great variability and adaptability to different living conditions that make research with these mushrooms difficult. 98% of the known mushrooms are rural people. For most mushrooms, climatic conditions with a humidity of 80% and temperatures around 20 * C represent ideal living and reproductive conditions.

The mechanisms for mold allergies are not exactly known and appear to be more complex and complicated than with conventional immediate-type inhalation allergies. Possibilities for sensitization via the digestive tract and not just the respiratory tract are discussed and are the subject of intensive research.

Allergies of the immediate type (type 1 allergy) are triggered by IgE antibodies, which contact effector cells (mast cells of the mucous membrane and connective tissue type as well as basophilic granulocytes of the blood) with their Fc part via receptors and, in the event of allergen contact, release inflammatory substances (histamine, heparin, Arachidonic acid metabolites etc.) lead (Roitt 1991, Klein 1990). Such IgE antibodies are formed by B lymphocytes, which are stimulated to secrete the antibodies by soluble substances (lymphokines) that are secreted by activated T lymphocytes (Parronchi et al. 1991).

At the beginning of every immune response there are cells that actively phagocytize the existing antigen. For this reason, these cells are also called " feeding cells " called. These are dendritic cells but also monocytes that differentiate into macrophages at a later stage. All of these cells have the ability of DIAPEDESE, which enables them to leave the blood system and penetrate into body cavities, etc. The main part of the antigen destruction is carried by the macrophages. After phagocytosis, they break down the antigen into highly immunogenic peptides (average size approx. 15 amino acids) and present them together with the MHC proteins (major histocompatibility) expressed on the macrophages, the T-lymphocytes. The central role of T lymphocytes is emphasized at this point. It is only at this point in the immune response that a distinction can be made, and this is the central role of the T lymphocytes as to whether the antigen presented is "foreign" or "own". If the decision "foreign" is made here, nothing stands in the way of further fighting the antigen. The detection of the foreign protein in contact with its own MHC leads to further differentiation of the T lymphocytes into plasma cells, which ultimately secrete interleukins. This interleukin secretion leads to the activation of B lymphocytes, which in turn have recognized the soluble antigen, but only the " message " of the T lymphocytes via the interleukins for their own differentiation. This is followed by the differentiation of the B lymphocytes into plasma cells, where larger amounts of antibodies of the IgE class are now secreted in the atopic.

At the T cell level, was the antigen called " own " if detected, the immune response is terminated at this point under normal circumstances. However, the complex regulatory cascade of the immune system harbors a lot of possible errors. Witness to this fact are the many, mostly fatal, clinical cases of autoimmune diseases, in which the body's immune system does not distinguish between "self" and "not-yourself".

To date, the diagnosis and therefore the therapy of allergic diseases has not been satisfactory. The molecular characterization of the main allergens of Alternaria using cDNA cloning, sequencing, sequence comparison of the allergenic protein with protein databases, and the production of recombinant allergens will provide more information about the in vivo function of the proteins that trigger the wrong immune reactions. This information is interesting for the following reasons: 1) Highly pure recombinant allergens can be used for a more careful diagnosis, better than is currently possible with crude extracts. 2) The sequence of the allergens will help to define tolerogenic peptides and possibly also to understand the "IgE class switch" that occurs during the immunization with the allergen. 2nd

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For decades, IgE-related allergies, e.g. also allergies to fungal spores, treated by hyposensitization (Bousquet et al. 1991). This therapy consists in the supply of allergen extracts in the form of injections or oral application in aqueous form as drops in increasing doses until a maintenance dose over several years is reached. The result of this therapy is tolerance towards the allergens used, which is reflected in a decrease in the symptoms of the disease (Birkner et al. 1990). The problem with this type of treatment lies in the large number of side effects that it causes. Hyposensitization therapy has seen cases of anaphylactic shock during treatment. The problem here is the difficulty in standardizing the fungal protein isolates. If allergen-derived but not anaphylactic peptides are used, higher doses could be administered risk-free, which can lead to a significant improvement in hyposensitization.

Alternaria alternata can be found practically everywhere in nature. Popular locations and habitats of the mushroom are different soil types, grain silos, rotten wood, but also living plants, composting places and bird nests. If black spots are found on tomatoes, they are also likely to come from Alternaria. But Alternaria alternata is not only found in the wild. The fungus is very often found in damp interiors and on window frames. Warm temperatures and high humidity generally favor the growth of the fungus.

Alternaria alternata is one of the most important allergy-causing fungi today. Yunginger et al. (1989) characterized the first allergenic fraction and isolated the first allergenic protein Altai. A big problem with Alternaria alternata is the range of variation of the fungus: Variations in the protein pattern but also variable potency of the triggering of allergies have been described many times. Nyholm et al. (1983) showed that Agl and Alt-1 are the same allergen, but that the protein can be varied in different strains of Alternaria alternata. The review article by Budd (1986) describes the isolation of the allergenic protein Alt-1, now Altai. Complete cDNA sequences of allergenic proteins from Alternaria alternata have not been published to date. However, various studies show strong cross-reactions between Alternaria alternata, Stemph-ylium and Curvularia (Agarwal 1982).

According to the invention, recombinant DNA molecules of the kind mentioned above are created which have nucleic acid sequences which homologously match sequences 1, 3-5, 7-9 and 12 and 13, or parts of these sequences, or nucleic acid sequences which correspond to the hybridize said sequences under stringent conditions. The DNA molecules can also have nucleic acid sequences which can be derived from the aforementioned sequences by degeneration.

Further features of the subject matter of the invention emerge from the explanations below. Examples: a) Description of the allergenic proteins from Alternaria alternata using Western blotting. 142 patient sera were available for cloning the present allergens from Alternaria alternata. In order to test the reactivity of the patients with fungal protein extract, Alternaria alternata (Prof. Windisch Berlin collection: 08-0203) was grown on solid medium (2% glucose, 2% peptone, 1% yeast extract). For protein extraction, the mushroom mat was removed after 3 days of growth at 28 ° C. and broken up with liquid nitrogen. The extracted proteins were separated on a denaturing polyacrylamide gel, which was then blotted, incubated with patient serum and detected with 12Sl-labeled anti-human IgE. Expressed in percentages, the patients reacted to the allergenic proteins as follows:

Alta53 44.8% Alta22 3.4% Altai 1 10.3%

If protein from isolated mushroom material from Allergon (Sweden) was isolated and used for immunoblotting, almost the same band pattern could be detected. As can be seen from these figures, Alta53 is a major allergen, Alta22 and Altai 1 a minor allergen.

FIG. 1 shows an overview of the patient spectrum available for cloning the allergens described. The picture shows a 12.5% polyacrylamide gel. The patients with the 3rd

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Numbers 35 and 40 (these are also the patients who were used for later screening) show bands in the order of 53kD, 22kD and 11kD.

1 shows a Western blotting of a 12.5% polyacrylamide gel after separation of Alternaria alternata protein extract; Incubation with sera from different patients; Detection with 125 l labeled anti human IgE. b) Construction of the cDNA expression bank

Total RNA was obtained from self-grown mushroom material using the acid guanidium phenol extraction method. poly (A) plus enrichment was carried out with oligo (dT) cellulose from Böhringer. The cDNA synthesis (1st and 2nd strand) was carried out as described in the manual of the Lambda ZAP system from Stratagene. The cDNA was then provided (3 'side) with EcoRI and (5' side) with Xbal linkers, ligated into predigested Lambda-ZAP arms and packaged. The primary bank titer was 900,000 clones. c) Screening of the cDNA library with patient sera, in vivo excision, sequencing

The expression bank was screened by incubating the " lifted " Phage plaques with a sera mixture of 2 patients, who were known by western blotting to cover the spectrum of the detected antigens. The detection was again carried out using anti-human IgE RAST antibody from Pharmacia. After secondary and tertiary screening, 150 positive clones remained. With the help of a helper phage, the ready-to-sequence bluescript vector was excised from 12 clones with cDNA (implementation as in the manual of the Lambda ZAP kit). Restriction digests of the excised plasmids showed (EcoRI-Xbal double digestion) 3 different insert types. These 3 clones were sequenced according to the singing method (Sänger 1977). d) Expression of the Alta53, Alta22 and Altai 1 cDNAs as / S-galactosidase fusion protein

With the help of the IgE screening described above, 3 complete cDNA clones could be obtained. The respective recombinant plasmids were transformed into the E.coli strain XL1-Blue and induced with IPTG (isopropyl-jS-D-thiogalactopyranoside). The E. coli total protein extract was then electrophoresed and blotted onto nitrocellulose. The fusion protein was detected by means of serum IgE from fungal allergy sufferers and an iodine-labeled rabbit anti-human IgE antibody (Pharmacia, Uppsala Sweden).

The following two figures show the recombinant / S-galactosidase fusion proteins after incubation with patient serum and detection with iodine-labeled anti-human IgE. The β-galactosidase portion of the fusion protein is 36 amino acids, which is equivalent to a molecular weight of 3800 daltons. Taking this " magnification " 3 and 4 of the allergenic protein can also be seen. Lanes (clones) 1, 2, 4, 5, 6, 7 show the recombinant fusion protein Altai 1, lanes 3 and 12 show the recombinant fusion protein Alta22 and lanes 8 and 10 show the recombinant Alta53 which has increased in size by the fusion fraction.

2 and 3 thus show an expression of the recombinant protins Alta52, Alta22 and Altai 1 in Bluescript after IPTG induction. e) Determination of B and T cell epitopes in the recombinant allergens

The derived amino acid sequence of the allergens provides the prerequisite for the prediction of B and T cell epitopes using suitable computer programs. With these studies, specific T and B cell epitopes can be defined that have the ability, e.g. To stimulate T lymphocytes and stimulate proliferation, but also to put the cells (at a precisely defined dose) into a state of tolerance or non-reactivity (anergy) (Rothbard et al. 1991). The specific epitopes are given in the description of the recombinant protein in separate figures.

The search for B-cell epitopes was carried out with the help of the GCG program (Genetics Computer Group) "PROTCALC", which, however, was expanded by the working group with Prof. Modrow with essential parameters. The determination is based on a weighing of the parameters of hydrophilicity (Kyte-Doolittle), secondary structure (Chou-Fasman), surface localization (Robson-Garnier) and flexibility, whereby the antigenicity of partial peptides is calculated. 4th

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The principle of T cell epitope prediction was in principle based on the algorithm of Margalit et al. (1987). The principle consists in the search for amphipathic helices according to the primary sequence of the protein to be determined, flanked by hydrophilic areas. The calculated score must be greater than 10 for relevant T cell epitopes. No consensus can be defined for MHC II-associated peptides, neither in terms of the sequence nor the length of the peptide, as associated with HLA-A2 (human leucocyte antigen) (MHC I). In HLA-A2-associated peptides, the length of the peptide is 10 amino acids, the second amino acid being a tyrosine and the last amino acid being a leucine (Rammenee et al. 1993). The calculated epitopes are listed separately in the description of the individual allergenic sequences. Molecular characterization of the cloned fungal allergens (sequence protocols)

In the following the cDNA sequences and the analyzes carried out with them are listed in order. The computer analysis of the following sequences was carried out on an Ultrix-DEC 5000 workstation using the GCG software package (= Wisconsin package: the algorithms of this package were developed by the " University of Wisconsin "). A. Alta53

Sequence 1 below shows the complete cDNA sequence of Alta53 starting with the start ATG. The length of the cDNA is 1488 bp, which corresponds to a calculated molecular weight of 53543 daltons. The observed band in the Western blot at 53 kD thus correlates in molecular weight with the cloned and sequenced allergen. According to previous analysis, the mature protein should not be preceded by a signal peptide.

Sequence 1: Alta53 = ALDH_alt - > 1-phase translation 53543 Dalton (1) INFORMATION ABOUT SEQ ID NO: 1 (i) SEQUENCE IDENTIFICATION: (A) LENGTH: 1488 base pairs / 496 amino acid residues (B) TYPE: Nucleic acid / protein (C) STRAND FORM: ds (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA / protein (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) TYPE OF FRAGMENT: total sequence (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans (C) DEVELOPMENT LEVEL : Spores and vegetative hyphae 5 ΑΤ 400 723 Β DNA sequence 1488 bp ATGACATCTGTA ... CTGTTCGGTTAA linear 1/1 31/11 ATG ACA TCT GTA AAG CTC TCA ACC CCT CAG ACG GGC GAG TTC GAG CAG CCC ACC GGA CTC met thr ser val lys leu ser thr pro gln thr gly glu phe glu gln pro thr gly leu 61/22 91/31 TTC ATC AAC AAT GAG TTC GTA AAG GCT GTT GAC GGC AAG ACC TTT GAT GTT ATC AAC CCC phe ile asn asn glu phe val lys ala val asp gly lys thr phe asp val ile asn pro 122 / 41 151/51 TCC ACT GAG GAG GTC ATC TGC AGT GTT CAG GAG GCC ACC GAG AAG GAT GTT GAC ATT GCT ser ehr glu glu val ile cys ser val gln giu ala thr glu lys asp val asp ile ala 181/61 221/71 GTT GCT GCC GCC CGC AAA GCC TTC AAC GGC CCA TGG gcA AAG GAG ACA CCA GAG AAC AGG val ala ala ala arg lys ala phe asn gly pro trp ala lys glu thr pro glu asn arg 241/81 271/91 GGA AAG CTG CTT AAC AAG CTT GCC GAT CTG TTC GAG AAG AAT GCC GAC CTC ATT GCT GCT gly lys leu leu asn lys leu ala asp leu phe glu lys asn ala asp leu ile ala ala 301/101 331/111 GTC GAG GCT CTC GAC AAC GGC AAG GCC TTC AGC ATG GCC AAG AAC GTC GAT GTT CCC GCC val glu ala leu asp asn gly lys ala phe ser met ala lys asn val asp val pro ala 361/122 391/131 GCC GCT GGT TGC CTG AGG TAC TAC GGA GGA TGG GCC GAC AAG ATT GAG GGC AAG GTC GTC ala ala gly cys leu arg tyr tyr gly gly trp ala asp lys ile glu gly lys val val 422/141 451/151 GAC ACA GCA CCC GAC AGC TTC AAC TAC ATC CGC AAG AGC CTA TTG GTG TTT GCG GTC AGA asp thr ala pro asp ser phe asn tyr ile arg lys ser leu leu val phe ala val arg 481/161 511/171 TCA TCC ATG GAA CTT CCT ATT CTC ATG TGG TCA TGG AAG ATT GGT CCT GCC ATC GCC ACT sec ser met glu leu pro ile leu met trp ser trp lys ile gly pro ala ile ala thr 541/181 571/191 GGT AAC ACC GTC GTC CTG AAG ACT GCT GAG CAG ACA CCT CTC TCC GCA TAC ATT GCC TGC gly asn thr val val leu lys thr ala glu gln thr pro leu ser ala tyr ile ala cys 601/201 631/221 AAG CTG ATC CAG GAG GCC GGT ttc CCA CCA GGT GTC ATC AAC GTC ATC ACT GGT TTC GGA lys leu ile gln glu ala gly phe pro pro gly val ile asn val ile thr gly phe gly 661/222 691/231 AAG ATC GCC GGT GCT GCC ATG TCC GCT CAC ATG GAC ATT GAC AAG ATT GCC TTT ACT GGT lys ile ala gly ala ala met ser ala his net asp ile asp lys ile ala phe thr gly 6

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722/241 751/251 TCA ACC GTT GTC GGC CGT CAA ATC ATG AAG TCT GCc GCT GGC TCC AAC TTG AAG AAG GTC ser thr val val gly arg glr. ile met lys ser ala ala gly ser asn leu lys lys val 781/261 811/271 ACT CTT GAG CTC GGA GGC AAG AGC CCC AAC ATT GTC TTC GCC GAC GCA GAT CTT GAC GAG thr leu glu leu gly gly lys ser pro asn ile val phe ala asp ala asp leu asp glu 841/281 871/291 * GCT ATC CAC TGG GTC AAC TTT GGT ATT TAC TTC AAC CAC GGA CAG GCT TGT TGT GCT GGT ala ile his trp val asn phe gly ile tyr phe asn his gly gln ala cys cys ala gly 901/301 931/311 TCG CGT ATC TAC GTC CAA GAA GAG ATC TAC GAC AAG TTC ATC CAG CGC TTC AAG GAG CGG ser arg ile tyr val gln glu glu ile tyr asp lys phe ile gln arg phe lys glu arg 961/322 991/331 GCT GCT CAG AAC GCT GTT GGT GAC CCA TTC GCC GCG ACA CTC CAG GGT CCT CAA GTC TCG ala ala gln asn ala val gly asp pro phe ala ala thr leu gln gly pro gln val ser 1022 / 341 1053. / 351 CAG CTC CAG TTC GAC CGT ATC ATG GGC TAC ATC GAG GAG GGC AAG AAG TCT GGC GCG ACC gln leu gln phe asp arg ile met gly tyr ile glu glu gly lys lys ser gly ala thr 1081/361 1111. / 371 ATC GAG ACT GGT GGC AAC CGT AAG GGT GAC AAG GGT TAC TTC ATC GAG CCC ACA ATC TTC ile glu thr gly gly asn arg lys gly asp lys gly tyr phe ile glu pro ehr ile phe 1141/381 1171. / 391 TCC AAC GTA ACC GAG GAC ATG AAG ATT CAG CAA GAA GAG ATC TTC GGC CCC GTC TGC ACA ser asn val thr glu asp met lys ile gln gln glu glu ile phe gly pro val cys thr 1201 t 401 1231. / 411 ATC TCC AAG TTC AAG ACA AAG GCC GAC GTC ATC AAG ATT GGC AAC AAC ACC ACA TAC GGT ile ser lys phe lys thr lys ala asp val ile lys ile gly asn asn thr thr tyr gly 1261/422 1291. / 431 Ctt tcG GCC GCT GTA CAC ACA TCC AAC CTC ACC ACT GCC ATC GAA GTT GCC AAC GCG CTC leu ser ala ala val his thr ser asn leu thr thr ala ile glu val ala asn ala leu 1322/441 1351. / 451 CGT GCA GGA ACT GTC TGG GTC AAC TCC TAC AAC ACT CTT CAC TGG CAG CTT CCC TTC GGA arg ala gly thr val trp val asn ser tyr asn thr leu his trp gln leu pro phe gly 1381/461 1411. / 471 GGG TAC AAG GAG TOT GGT ATT GGG CGC GAG TTG GGA GAG GCG GCG CTG GAC AAC TAC ATC gly tyr lys glu ser gly ile gly arg glu leu gly glu ala ala leu asp asn tyr ile 1441/481 147 ’. / 4 91 CAG ACC AAG ACC GTG TCT ATT CGT CTT GGC CAT GTT CTG TTC GGT TAA gln thr lys thr val ser ile arg leu gly asp val leu phe gly OCH

Homology searches with Alta53 in the SWISSPROT protein database showed that Alta53, like Clah53, is an aldehyde dehydrogenase. As proof of the high homology (there are identities over many stretches), the following sequence 2 shows a pairwise alignment between Alta53 and Clah53. The identity (identical amino acids) of the two proteins (allergens) among themselves 7

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is 78%. The degree of homology (identities plus homologous amino acid exchanges) is as high as 86%. Sequence 2: ALDH 1 MTSVKLSTPQTGEFEQPTGLFINNEFVKAVDGKTFDVINPSTEEVICSVQ 50

Ii II.I.II: .I.: IIII I I I I I II I I I s: I 1 I I I I I I II · 1 ·! I · I: 1 MTSVQLETPHSGKYEQPTGLFINNEFVKGQEGKTFDVINPSDESVITQVH 50 51 EATEKDVDIAVAAARKAFNGPWAKETPENRGKLLNKLADLFEKNADLIAA 100

IIIIIIII IIIIII I · I I: I. I I I I I I I I I I I II: I ill I I I. I I I: 101 VES LDNGKAT SMAR. VT S ACAS GCLRY Y GGWADKITGKVIDTT P DT FN YV 149

151 RK. SLLVFAVRSSMELPILMWSWKIGPAIATGNTWLKTAEQTPLSAYIA 199: I ·: I I: II I: I1 I .I I I I I I I I · I I I III I I I I I I I I I 150 150 · I:. I I I I I I I I I III. I I I I: I i I I: I. I Μ: I I: l I I I I I I I I I I: 200 ASLVKEAGFPPGVINVISGFGKVAGAALSSHMDVDKVAFTGSTWGRTIL 249 250 KSAAGSNLKKVTLELGGKSPNIVFADADLDEAIHWVNFGIY II I I I I I I I. I I I: I: I I I I I I I I: I I I I I. I I I 250 KAAASSNLKKVTLELGGKSPNIVFEDADIDNAISWVNFG1FFNHGQCCCA 299 300 GSRIYVQEEIYDKFIQRFKERAAQNAVGDPFAA.TLQGPQVSQLQFDRIM 348

I I I: I II I I I II I: I: Μ I I I .I · I I I I 1II I: IlII I I. : I I I I I I 8

AT 400 723 B 300 GSRVYVQESIYDKFVQKFKERAQKNWGDPFAADTFQGPQVSKVQFDRIM 349 349 GYIEEGKKSGATIETGGNRKGDKGYFIEPTIFSNVTEDMKIQQEEIFGPV 398: I I II I I. I I I I I I I I I I I I I I I I I I I I! I I .1111111 350 EYIQAGKDAGATVETGGSRKGDKGYFIEPTIFSNVTEDMKIVKEEIFGPV 399

399 CTISKFKTKADVIKIGNNTTYGLSAAVHTSNLTTAIEVANALRAGTVWVN 448 I .I.IIIII.I.II: I I .. I I II. I I I I I. I I. I I I II. I I I: I I II I I I 400 CSIAKFKTKEDAIKLGNASTYGLAAAVHTKNLNTAIEVSNALKAGTVWVN 449

449 SYNTLHWQLP FGGYKESGlGRELGEAALDNYIQTKTVSIRLGDVLFGZ 496 .11111 I: I I I I I I II I I I I II I.II.II · I IIIIII I I I I · I I I I 450 450

The NAD-dependent ALDH is the main enzyme that is involved in the oxidation of acetaldehyde, a primary product of alcohol metabolism, in humans. Isoenzymes are often found here (Harada et al. 1982). In humans e.g. the isoenzyme ALDH I is found in mitochondria, ALDH II in the cytoplasm. Interestingly, the absence of ALDH I is not uncommon in Asians (Harada et al. 1982). The deficiency of ALDH I results in a high level of acetaldehyde, which manifests itself as a so-called "flushing syndrome", as well as other vasomotor symptoms after alcohol consumption. The loss of isoenzyme can be attributed to a mutation that changes the structure of the native protein (Hsu et al. 1987). The relationship between ALDH and allergy triggering is not yet known.

Sequence 3 below shows the areas identified by computer search with a high, antigenic index. These areas represent highly potent B cell epitopes.

Sequence 3: Alta53-ALDH_alt B cell epitopes (1) INFORMATION ABOUT SEQ ID NO: 3 (i) SEQUENCE IDENTIFICATION: (A) LENGTH: individually stated (B) TYPE: Protein (ii) TYPE OF MOLECULE: Peptides (iii) HYPOTHETICAL: no (v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans (C) STAGE OF DEVELOPMENT: spores and vegetative hyphae 9

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Val Lys Leu Ser Thr Pro Gin Thr Gly Glu Phe Glu Gin Pro Thr Gly (4-19) Ala Val Asp Gly Lys Thr Phe (29-35) Ile Asn Pro Ser Thr Glu Glu (38-44) Gly Pro Trp Ala Lys Glu Thr Pro Glu Asn Arg Gly Lys Leu Leu Asn (70-85) Leu Arg Tyr Tyr Gly Gly Trp Ala Asp Lys Ile Glu Gly (125-137) Asp Thr Ala Pro Asp Ser Phe Asn Tyr Ile Arg Lys Ser (141- 153) Glu Ala Gly Phe Pro Pro Gly Val (205-212) Gly Ser Asn Leu Lys Lys Val Thr Leu (254-262) Glu Leu Gly Gly Lys Ser Pro Asn Ile (263-271) Tyr Ile Glu Glu Gly Lys Lys Ser Gly Ala Thr (350-360) Ile Glu Thr Gly Gly Asn Arg Lys Gly Asp Lys Gly Tyr Phe Ile Glu (361-376) Ile Gly Asn Asn Thr Thr Tyr Gly (413-420) Ala Val His Thr Ser Asn Leu Thr (424-431)

Sequence 4 below shows the amphipathic helices determined with the aid of the computer program and flanked by hydrophilic areas. Such areas, with a score higher than 10, represent possible T cell epitopes.

Sequence 4: Predicted amphipathic segments of T cell pjtopes

EFEQ

FVKAVD

KTFDVI

KAFNGPWA

KLLNKLADLFE

IAAVEALDNGKA

MAKNVDVP

AAGCLRYYGGWADKIEGK

VDTAPDSFNY

GVINVITGFGKI

IYDKFIQRFKERAA

NAVGDPFAAT

QFDRIMGYI

GPVCTI

AIEVANALR

RELGEAALD (1) INFORMATION ABOUT SEQ ID NO: 4 (i) SEQUENCE IDENTIFICATION: (A) LENGTH: individually stated (B) TYPE: Protein 10

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Glu Phe Glu Gin (11-16) Phe Val Lys Ala Val Asp (26- -31) Lys Thr Phe Asp Val Ile (33- -38) Lys Ala Phe Asn Gly Pro Trp Ala (66- • 73) Lys Leu Leu Asn Lys Leu Ala Asp Leu Phe Glu (82-92) Ile Ala Ala Val Glu Ala Leu Asp Asn Gly Lys Ala (98-109) Met Ala Lys Asn Val Asp Val Pro (112 > 119) Ala Ala Gly Cys Leu Arg Tyr Tyr Gly Gly Trp Ala Asp Lys Ile Glu (121-138) Val Asp Thr Ala Pro Asp Ser Phe Asn Tyr (140-149) Gly Val Ile Asn Val Ile Thr Gly Phe Gly Lys Ile (211 -222) Ile Tyr Asp Lys Phe Ile Gin Arg Phe Lys Glu Arg Ala Ala (309-322) Asn Ala Val Gly Asp Pro Phe Ala Ala Thr (324-333) Gin Phe Asp Arg Ile Met Gly Tyr Ile (343 -351) Gly Pro Val Cys Thr Ile (396-401) Ala Ile Glu Val Ala Asn Ala Leu Arg (433 -441) Arg Glu Leu Gly Glu Ala Ala Leu Asp (469 -477}

The T cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a lysine (K), C-terminally by a proline (P). Potential T cell epitopes are only present if the " Score Index " is greater than 10. B. Altai 1

Sequence 5 below shows the complete cDNA sequence of Altai 1 and the amino acid sequence derived from it. The open reading frame is 342bp b2w. 114 amino acids. The calculated molecular weight is 11127 daltons and thus corresponds to the 11 kD antigenic protein, which is recognized in Western blots by 10.3% of the patients. 11

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Sequence 5: Altai 1 = rla2_alt - > 1-phase translation 11127 Dalton (1) INFORMATION ABOUT SEQ ID NO: 5 (i) SEQUENCE IDENTIFICATION: (A) LENGTH: 342 base pairs / 114 amino acid residues (B) TYPE: Nucleic acid / protein (C) STRAND FORM: ds (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA / protein (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) TYPE OF FRAGMENT: total sequence (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans (C) DEVELOPMENT LEVEL : Spores and vegetative hyphae DNA sequence 342 bp ATGAAGCACCTC ... CTCTTCGACTAA linear

1/1 31/11 ATG AAG CAC CTC GCG GCA TAC CTC CTC CTC GGC CTT GGT GGC AAC ACc TCG CCC TCC GCT met lys his leu ala ala tyr leu leu leu gly leu gly gly asn thr ser pro ser ala 61/22 91 / 31 GCC GAC GTC AAG GCC GTC CTT GAG TCC GTT GGT ATC GAG GCT GAC TCC GAC CGT CTT GAC ala asp val lys ala val leu glu ser val gly ile glu ala asp ser asp arg leu asp 122/41 151/51 AAG CTG ATC TCC GAG CTT GAG GGC AAG GAC ATC AAC GAG CTC ATC GCT TCC GGT TCC GAG lys leu ile ser glu leu glu gly lys asp ile asn glu leu ile ala ser gly ser glu 181/61 221/71 AAG CTT GCT TCC GTT CCC TCC GGT GGT GCC GgT GGT GCT GCC GCT TCC GGT GGT GCT GCT lys leu ala ser val pro ser gly gly ala gly gly ala ala ala ser gly gly ala ala 241/81 271/91 GCC GCT GGT GGC TCC GCT CAG GCT GAG GcC GCT CCT GAG GCc GCC AAG GAG GAG GAG AAG ala ala gly gly ser ala gln ala glu ala ala pro glu ala ala lys glu glu glu lys 301/101 331/111 GAG GAG TCT GAC GAG GAC ATG GGT TTC GGT CTC TTC GAC TAA glu glu ser asp glu asp met gly phe gly leu phe asp OCH

Homoogy searches in the SWISSPROT protein database revealed homologies to the ribosomal protein P2. This ribosomal protein is involved in the formation of the large subunit of the ribosomes. This means that homology to Clahll, Altall's counterpart in Cladosporium herbarum, is inevitable. The identities and homologies between Altai 1 and Clah11 are evident from the following sequence 6. The identity of the two proteins is 74%, the degree of homology increases to 84%, which undoubtedly indicates a similar function of these two proteins. 12th

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Sequence 6: rla2 alt x rla2 clado 1 MKHLAAYLLLGLGGNTSPSAADVKAVLESVGIEADSDRLDKLISELEGKD 50 11.111: 11111 I I. II I I.I: I.Ί I.IIII: II.:. : I: · I I I I I I 1 MKYLAAFLLLGL. GNSSPSAEDIKTVLSSVGIDADEEPSQLLLKELEGKD 4 9 51 INELIASGSEKLASVPSGGAGGAAASGGAAAAGGSAQAEAAPEAAKEEEK 100 I · I I · I I · I AEEKAEEEKK 92 I II I I · II I I I I i I I I I I I I I: I. I: I: I I I: I 50 INELZSSGSEKLASVPSGGAGAASAGGAAAAGG. 101 EESDEDMGFGLFDZ 114 I I I I: I I I I I II I I 93 EESDDDMGFGLFDZ 106

Acidic ribosomal proteins (such as PO, P1 and P2) from various organisms have been analyzed using a variety of techniques, including A proteins (acidic) and P proteins (phosphorylated A proteins). A feature of the A proteins is the large number of hydrophobic amino acids. They can therefore be relatively easily dissociated from the ribosome (50% ethanol and high salt concentration). The best characterized A protein in prokaryotes is the L7 / L12 protein from Escherichia coli. The eukaryotic homologues are the proteins P1 and P2 which, like the L7 / L12 protein, interact with the elongation factor EF1 and EF2. The C-terminal sequence contains an epitope that is recognized by autoantibodies in lupus patients (Francoeur et al. 1985, Rieh et al. 1987, Hines et el. 1991). The homology of the P2 protein corresponds to the allergenic protein Altai 1.

The B cell epitopes shown in the next sequence 7 have been calculated taking into account the secondary structure, surface position, hydrophilicity, flexibility etc.

Sequence 7: Altai 1 * rla2_alt B cell epitopes (1) INFORMATION ABOUT SEQ ID NO: 7 (i) SEQUENCE IDENTIFICATION: (A) LENGTH: individually stated (B) TYPE: Protein (ii) TYPE OF MOLECULE: Peptides (iii) HYPOTHETIC : no (v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans (C) STAGE OF DEVELOPMENT: spores and vegetative hyphae 13

AT 400 723 B

Leu Gly Gly Asn Thr Ser Pro Ser Ala Ala Asp (12-22)

Ile Glu Ala Asp Ser Asp Arg Leu Asp Lys Leu Ile Ser 132—44)

Glu Leu Glu Gly Lys Asp Ile Asn Glu Leu (45-54)

Ala Ser Gly Ser Glu Lys Leu Ala Ser (56 * 64)

Pro Glu Ala Ala Lys Glu Glu Glu Lys Glu Glu Ser Asp Glu Asp Met Gly Phe (92-109)

The following sequence 8 shows the calculated T cell epitopes and represents the amino acids in the 1-letter code.

Sequence 8: Predicted amphlpathatic segments of T cell ptypes

RLDKLISELEGKDINEUASG

EKLASVPSGG (1) INFORMATION ABOUT SEQ ID NO: 8 (i) SEQUENCE LABEL: (A) LENGTH: individually stated (B) TYPE: Protein (ii) TYPE OF MOLECULE: Peptides (iii) HYPOTHETICAL: no (v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans (C) STAGE OF DEVELOPMENT: Spores and vegetative hyphae

Arg Leu Asp Lys Leu Ile Ser Glu Leu Glu Gly Lys Asp Ile Asn Glu Leu Ile Ala Ser Gly (38-58) Glu Lys Leu Ala Ser Val Pro Ser Gly Gly (60-69!

The T cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a lysine (K), C-terminally by a proline (P). Potential T cell epitopes are only present if the " Score Index " is greater than 10. C. Alta22

Sequence 9 below shows the complete cDNA sequence of Alta22. The protein primary sequence derived therefrom can also be seen from the sequence. The open reading frame of the allergenic protein is 615 bp, which corresponds to an amino acid length of 205 amino acids. The calculated molecular weight of the recombinant protein is 22041 daltons. According to previous analysis, the mature protein is not preceded by a signal sequence.

Sequence 9: YCP4_alt - > 1-phase translation 22041 Dalton (1) INFORMATION ABOUT SEQ ID NO: 9 (i) SEQUENCE IDENTIFICATION: (A) LENGTH: 615 base pairs / 205 amino acid residues (B) TYPE: nucleic acid / protein 14

AT 400 723 B (C) STRAND FORM: ds (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA / protein (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) TYPE OF FRAGMENT: total sequence (vi ) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans (C) STAGE OF DEVELOPMENT: Spores and vegetative hyphae DNA sequence 615 bp ATGGCTCCCAAG ... GCGCATCAGTGA linear 1/1 31/11 ATG GCT CCC AAG ATC GCG ATT GTG TAC TAC TCC ATG TAC GGC CAC ATC AAG AAG ATG GCC met ala pro lys ile ala Ile val tyr tyr ser met tyr gly his ile lys lys met ala 61/22 91/31 GAT GCT GAG TTG AAG GGT ATC CAA GAG GCT GGC GGT GAT GCC AAG CTC TTC CAA GTC GCC asp ala glu leu lys gly ile gln glu ala gly gly asp ala lys leu phe gln val ala 122 / 41 151/51 GAS ACC CTG CCT CAG GAA GTC CTC GAC AAG ATG TAC GCG CCC CCC AAG GAC TCA TCG GTC glu thr leu pro gln glu val leu asp lys met tyr ala pro pro lys asp ser ser val 181 t 61 221/71 CCC GTC CTC GAG GAC CCA GCC GTC CTC GAA GAA TTT GAC GGC ATC CTC TTC GGC ATC CCC pro val leu glu asp pro ala val leu glu glu phe asp gly ile leu phe gly ile pro 241/81 271/91 ACC CGC TAC GGC AAC TTC CCC GCA CAA TTC AAG ACC TTC TGG GAC AAG ACA GGC AAG CAA thr arg tyr gly asn phe pro ala gln phe lys thr phe trp asp lys thr gly lys gln 301/101 331/111 TGG CAA CAA GGC GCC TTT TGG GGA AAG TAC GCC GGT GTC rrc GTT TCG ACG GGC ACC CTG trp gln gln gly ala phe trp gly lys tyr ala gly val phe val ser thr gly thr leu 361/122 391/131 GGCGGGT GGC CAG GAG ACG ACT GCC ATT ACC AGC ATG AGC ACG CTT GTC GAC CAC GGT TTC gly gly gly gln glu thr thr ala ile thr ser met ser thr leu val asp his gly phe 422/141 4 51/151 ATC TAC GTT CCC CTT GGC TAC AAG ACT GCG TTT AGC ATG TTG GCC AAC TTG GAC GAG GTC Ile tyr val pro leu gly tyr lys thr ala phe ser met leu ala asn leu asp glu val 481/161 511/171 CAC GGT SGA AGC CCA TGG GGT GCT GGT ACC TTC TCT GCC GGC GAT GGA TCG AGG CAG CCC hi s gly gly ser pro trp gly ala gly thr phe ser ala gly asp gly ser arg gln pro 541/181 571/191 AGT GAG crr GAG CTC AAC ATT GCG CAG GCT CAG GGT AAG GCT TTC TAC GAG GCT GTT GCC ser glu leu glu leu asn ile ala gln ala gln gly lys ala phe tyr glu ala val ala 15

AT 400 723 B 601/201

AAG GCG CAT CAG TGA

lys ala his gln OPA

Homology searches with the sequenced protein in the SWISSPROT protein database showed that the allergen Alta22 has significant homology to the yeast protein YCP4. The identity of the two proteins is 56%, the homology increases to 72%. Such a high degree of similarity suggests that these two proteins function together. Sequence 10 below reflects the high homology of Alta22 and YCP4.

Sequence 10: ycp4_alt x ycp4_yeast 1 MAPKIAIVYYSMYGHIKKMAOAELKGIQEAGGDAKLFQVAETLPQEVLDK 50 I. || II: II I III. : I: I | I::. I I I. I. ::. I. I III: I I I. 1 1 MV.KIAIITYSTYGHIDVLAQAVKKGVEAAGGKADIYRVEETLPDEVLTK 4 9

. 51 MYAPPKDSSVPVLEDPAVLEEFDGILFGIPTRYGNFPAQFKTFWDKTGKQ 100 I ... 11.1: 11: ..: 11: I :: II I: III: II: III: - - IIIIII 50 MNAPQKPEDIPVATEKTLLE.YDAFLFGVPTRFGNLPAQWSAFWDKTGGL 98,101 WQQGAFMGKYAGVFVSTGTLGGGQETTAITSKSTLVDHGFIYVPLGYKTA 150 [..1 .: II II IIII :. . I 1 I I I. i. . :: 1 1..11: 1 :: 11111 .. 99 WAKGSLNGKAAGIFVSTSSYGGGQESTVKACLSYLAHHGIIFLPLGYKNS 148

151 FSMLANLDEVHGGSPWGAGTFSAGDGSRQPSELELNIAQAQGKAFYE.A. 198 I. I I. :: I I I I I I I I I I I I: -:. I I I I: 1.111.11: I I I - I I I I 149 FAELASIEEVHGGSPWGAGTLAGPDGSRTASPLELRIAEIQGKTFYETAK 196 199 ... VAK ............... AHQ2 205 -II I. . : 199 KLFPAKEAKPSTEKKTTTSOAAKRQ 223

But what is the function of YCP4: The sequence or the open reading frame of YCP4 was localized and published as part of the yeast genome project on chromosome 3 of Saccharomyces cerevisiae (Biteau et al. 1992). Disruption of YCP4 showed according to Biteau et al. (1992) no phenotype. However, refined phenotype analyzes carried out suggest that yeast YCP4 may have a function as a heat shock protein. This experiment also shows how important Saccharomyces cerevisiae can be for the functional analysis of allergens. The ease of transformability combined with sophisticated methods of molecular genetics make it possible to disrupt genes in yeast and to analyze the resulting phenotype.

It has also been shown that Alta22 also has its homologous partner in Cladosporium herbarum. The following sequence 11 shows a " multiple sequence alignment " between the yeast YCP4 and the allergens Alta22 and Clah22. 16

AT 400 723 B

Sequence 11: pileup.msf {YCP4_altpro) 1 MAPKIAIVYY SmYGHIkkMA DAElKGIqEA GGdAkLFqVa 50 ETLPQEVLdK pileup.ms £ (YCP4_cladoprοI MAPKIAIIFY STWGKVqtLA EAEaKGIrEA GGsvOLYRVp ETLtQEVLTK pi1eup.ms £ {} ycp4_yeast .mvKIAlItY STYGKIdvLA qAvkKGVeaA GGkADiYRVe ETLPdEVLTK consensus --- Xiai-YS-GH --- A -A — KG --- A GG ------ V- ETL — EVL-K pileup.rtsf {YCP4_altpro) 51 MyAPPKDsSV PVleOPaVLE eFDgiLFGIP TRiGNFPAQF 100 kTFWDKTGkC pileup.ms £ IYCP4_cladoproFQhPPDGFFHH PePPDGFGHPPYPFGDHPPYPFGFHHPPPDGFHHQPPDGFDHQPYPFGFHHQPPDGFGHQPPDGFGHQPYPDGFHH pi1eup.ms f {ycp4_yea st J MnAPqKpEdl PVaTEktlLE .YDaFLFGVP TRFGNLPAQW saFWDKTGGl Consensus M-AP-K ---- P ------- LE - D ---- GP TR-GN-PAQ- - FWD-TG - pileup.msf (YCP4_altproj 101 WQqGAFWGKY AGVFVSTGT1 GGGQEtTAit sMSTLvdHGf 150 IYVPLGYKTa pileup.ms £ < YCP4_cladopro J WQtGAFWGKY AGlFISTGTq GGG0ESTA1A aMSTLsHHGI lYVPLGYKTt pileup.msflycp4_yeast) WakGsLnGKa AGIFVSTssy GGGQESTvtcA CLSyLaHHGI IFIPLGYKns Consensus W-G --- GK AG-F-ST - GGGQE-T --- —SL — HG- I — PLGYK— pileup.msf (YCP 4_altpro151 FsMLAnlDEV HGGSPWGAGT FSaGDGSRQP SeLELnlAqA 200 QGKAFYEAVA pileup.msf (YCP4_cladopro) FhLLgdnsEV rGaavWGAGT FSGGDGSRQ? SqkELelt.A QGKAFYEAVA pileup.msf {ycp4_yeast | FaeLAsiEEV HGGSPWGAGT LaGpDGSRta SpLELrlAei QGKtFYEtafc Consensus F — L —— EV -G --- KGAGT ---- DGSR— S — EL ----- QGK-FYE --- pileup.msf! YCP4_altpro) 201 KahqZ .... . 249 pileup.msf! YCP4_cladopro) KvnfQz ..., pileup.msf! Ycp4_yeast) Klfpakeakp stekktttsd aakrqtkpaa attaekkedk - gllscctvm

Consensus K

The B cell epitopes found with computer support can be seen in the next sequence 12. Sequence 12: Alta22 = YCP4_alfc B-Zeflepitope (1) INFORMATION ABOUT SEQ ID NO: 12 (i) SEQUENCE IDENTIFICATION: (A) LENGTH: individually stated (B) TYPE: Protein (ii) TYPE OF MOLECULE: Peptide (iii) HYPOTHETICAL: no (v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: 17

AT 400 723 B (A) ORGANISM: Alternaria alternans (C) STAGE OF DEVELOPMENT: Spores and vegetative hyphae

Lys Met Tyr Ala Pro Pro Lys Asp Ser Ser Val (SO — 60}

Ile Pro Thr Arg Tyr Gly Asn phe Pro (79-87)

Gin Phe Lys Thr Phe Trp Asp Lys Thr Gly Lys Gin Trp Gin Gin Gly Ala Phe Trp Gly Lys Tyr Ala Gly 189-112)

Gly Thr Leu Gly Gly Gly Gin Glu Thr Thr Ala Ile Thr Ser (118-131)

Leu Asp Glu Val His Gly Gly Ser Pro Trp Gly Ala Gly Thr (157-170)

Phe Ser Ala Gly As? Gly Ser Arg Gin Pro Ser Glu Leu Glu Leu (171-185)

Sequence 13 below shows the calculated T cell epitopes. Amphipathic areas with a score less than 10 are assumed to be irrelevant.

Sequence 13: Predicted amphipathic segments of T cell epitopes

SMYGHIKKMAD

GIQEA

LFQVAETLPQEVLDKMYA

AVLEEFDGI

TRYGNFPAQFKTFWDKTGKQW

TAITSMSTL

FSMLANLDEVHG

QGKAFYEAVA (1) INFORMATION ABOUT SEQ ID NO: 13 (i) SEQUENCE LABEL: (A) LENGTH: individually stated (B) TYPE: Protein (ii) TYPE OF MOLECULE: Peptides (iii) HYPOTHETICAL: no (v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans (C) STAGE OF DEVELOPMENT: Spores and vegetative hyphae 18

AT 400 723 B

Ser Met Tyr Gly His Ile Lys Lys Met Ala Asp (11-21)

Sly Ile Gin Glu Ala (26-30) 5

Leu Phe Gin Val Ala Glu Thr Leu Pro Gin Glu Val Leu Asp Lys Met Tyr Ala (36-53)

Ala Val Leu Glu Glu Phe Asp Gly Ile (6 * 7— " 75) 10 Thr Arg Tyr Gly Asn Phe Pro Ala Gin Phe Lys Thr Phe Trp Asp Lys Thr Gly Lys Gin Trp 181-101)

Thr Ala Ile Thr Ser Met Ser Thr Leu (121-135) 15

Phe Ser Met Leu Ala Asn Leu Asp Glu Val His Gly (151-162)

Gin Gly Lys Ala Phe Tyr Glu Ala Val Ala (191-200)

The T cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a lysine (K), C-terminally by a proline (P). Potential T cell epitopes are only present if the " Score Index " is greater than 10. 25 Agarwal, M.K., Jones, R.T., Yunginger, J.W. (1982).

Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts. J. Allergy Clin. Immunol. 70 (6), 437.

Birkner, T., Rumpold, H., Jarolim, E. Ebner, H., Breitenbach, M „Skarvil, F„ Scheiner, 0., Kraft, D. (1990). Evaluation of immunotherapy-induces changes in specific IgE, IgG and IgG subclasses in birch pollen 30 allergic patients by means of immunoblotting. Correlation with clinical response.

Allergy 45, 418.

Biteau, N., Fremaux, C., Hebrard, S., Menara, A., Aigle, M. Crouzet, M. (1992).

The complete sequence of a I0.8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae. 35 Yeast8.61.

Bousquet, J., Becker, W.M., Hejjaoudi, A. (1991).

Differences in clinical and immunologic reactivity of patients allergic to grass pollens and to multiple-pollen species. II. Efficacy of a double blind, placebo-controlled, specific immunotherapy with standardized extracts. 40 J. Allergy Clin. Immunol. 88, 43.

Budd, T.W. (1986).

Allergens of Alternaria.

Grana 25, 147.

Francoeur, A.M., Peebles, C.L., Heckman, K.J., Lee, J.C., Tan, E.M. (1985). 45 Identification of ribosomal protein autoantigens. J. Immunol. 135, 1767.

Harada, S., Agarwal, D.P., Goedde, H.W. (1982).

Mechanism of alcohol sensitivity and disulfiram-ethanol reaction.

Alco. Act. Misuse. 3, 107. see Hines, J.J., Weissbach, H. Bread, N., Elkon, K. (1991).

Anti-P autoantibody production requires P1 / P2 as immunogens but is not driven by exogenous self-antigen in mrl mice. J. Immunol. 146, 3386.

Klein, J. (1991). 55 VCH publishing company Weinheim, New York, Basel, Cambridge.

Immunology.

Margalit, H., Spogue, J.L., Cornette, J.L., Cease, K.B., Delisi, C., Berzofsky, J.A. (1987).

Pre'diction of immunodominant Helper T cell antigenic Sites from the primary sequence. 19th

Claims (12)

AT 400 723 B J. Immunol. 138, 2213. Miyamoto, T. (1992). Advances in Allergology and Clinical Immunology. Eds. Ph Godard, J. Bousquet, F.B. Miches. EAACI Congress Paris, 10-15, May 1992. The Parthenon Publishing Group, Casterton Hall U.K., New Jersey, USA p.343. Nyholm, L., Löwenstein, H., Yunginger, J.W. (1983). Immunochemical partial identity between two independently identified and isolated major allergens from Alternaria alternata (Alt-1 and Ag1). L. Allergy Clin. Immunol. 71 (5), 461. Parronchi, P., Macchia, D. Piccinni, M.P. (1991). Allergen- and bacterial antigen-specific T-cell clones established from atopic donors show a different profile of cytokine production. Proc. Natl. Acad. Be. USA 88, 4538. Rammenec, H.G., Falk, K „Rötzschke, O. (1993). MHC molecules as peptide receptors. Current opinion in Immunol. 5, 35. Rieh, B.E., Steitz, J.A. (1987). Human acidic ribosomal phosphoproteins PO, P1 and P2: analysis of cDNA clones, in vitro synthesis and assembly. Mol. Cell. Biol. 7, 4065. Roitt, I. (1991). Essential Immunology 7th Edition. Oxford Blackwell scientific publications. London Edinburgh Boston Melbourne Paris Berlin Vienna Rothbard, J.B., Gefter, M.L. (1991). Interactions between immunogenic peptides and MHC proteins. Ann. Rev. Immunol. 9, 527. Singer, F. Nicklen, S., Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Be. USA 74, 5463-5468 Wüthrich, B. (1991). Allergy and Clin. Immunol. News 3, 41. Yunginger, J.W., Jones, R.T., Nesheim, M.E., Geller, M. (1980). Studies on Alternaria allergens III. Isolation of a major allergenic fraction (Alt-1). J. Allergy Clin. Immunol. 66 (2), 138. Claims 1. Recombinant DNA molecules which code for polypeptides which have the antigenicity of the allergens Alta53, Alta22 and Altai 1 or for peptides which have at least one epitope of these allergens, characterized in that they have nucleic acid sequences which homologously correspond to sequences 1, 3-5, 7-9, 12 and 13, or to partial regions of these sequences, or nucleic acid sequences which hybridize with the nucleic acid sequences mentioned under stringent conditions. 2. Recombinant DNA molecules according to claim 1, characterized in that they have nucleic acid sequences which can be derived from sequences 1, 3-5, 7-9, 12 and 13 by degeneration. 3. Recombinant DNA molecules according to claim 1 or 2, characterized in that they have nucleic acid sequences which code for polypeptides which are cross-reactive as antigens with the allergens Alta53, Alta22 and Altai 1 and have a high homology to them. 4. Recombinant DNA molecules according to claims 1 to 3, characterized in that they are functionally linked to an expression control sequence to form an expression construct. 5. Host system for the expression of polypeptides, characterized in that it is transformed with a recombinant expression construct according to claim 4. 20 AT 400 723 B 6. Recombinant or synthetic protein or polypeptide derived from an ONA molecule according to one of claims 1 to 3, characterized in that it has the antigenicity of Alta53, Alta22 or Altai 1, or at least one epitope of these proteins. 7. Recombinant or synthetic protein or a polypeptide according to claim 6, characterized in that it has an amino acid sequence which corresponds to the sequences shown 1, 3-5, 7-9, 12 and 13 in whole or in part. 8. Recombinant or synthetic protein or polypeptide according to claim 6 or 7, characterized in that it is a fusion product which has the antigenicity of the allergens Alta53, Alta22 or Altai 1, or at least one epitope thereof, and has an additional polypeptide portion, the entire fusion product is encoded by the DNA of an expression construct according to claim 4. 9. Recombinant or synthetic protein or polypeptide according to claim 8, characterized in that said additional polypeptide portion is β-galactosidase or another polypeptide suitable for fusion. 10. Diagnostic or therapeutic reagent, characterized in that it contains a synthetic protein or polypeptide according to one of claims 6 to 9. 11. A method for in vitro detection of a patient's allergy to the allergens Alta53, Alta22 or Altai 1, characterized in that the reaction of the IgE antibodies in the patient's serum with a recombinant or synthetic protein or polypeptide according to one of Claims 6 to 9 is measured. 12. A method for in vitro detection of the cellular response to the allergens Alta53, Alta22 or Altai 1, characterized in that a recombinant or synthetic protein or polypeptide according to one of claims 6 to 9 is used to stimulate or inhibit the cellular response. With 1 sheet of drawings 21