CA2204362A1 - Recombinant alternaria alternata allergens - Google Patents

Abstract

The invention relates to the complete cDNA sequences of the Alternaria alternata allergenes Alt a 45 and Alt a 12. It has been possible in the course of molecular biological analysis of the allergens to identify Alt a 45 as a protein-disulphide isomerase. This protein is a 45 kD large protein to which 47 % of patients react and is thus an important primary allergen. Alt a 12 (to which 8 % of patients react) was identified as a ribosomal protein which is of interest because auto-antibodies for ribosomal proteins are found in patients suffering from lupus. It remains to be seen whether there is any connection between allergy to mould fungus and autoimmune diseases. Using this recombinant sequence and computer analysis, it has been possible to identify highly potent B- and T-cell epitopes and to use the peptides derived from the recombinant proteins for diagnosis of an allergy to Alternaria alternata mould fungus. The peptides has also proved suitable for both in vitro and in vivo allergen-specific stimulation of T-cells (proliferation, interleukin production), and for blocking the T-cells resulting in a tolerance of the allergen-specific T-lymphocytes.

Description

CA 02204362 1997-0~-02 Translation from German --Ref. # 4-35/hh _______________________ ~ WO 96/14407 PCT/AT95/00212 o ~
o z~ Recombinant Alternaria Alternata Allerqens Y ~
~ The invention relates to recombinant DNA molecules that zl code for polypeptides which possess the antigenicity of the allergens Alt a 45 and Alt a 12, or for peptides which have at least one epitope of these allergens.
~'~ In particular, the invention relates to the complete cDNA
~ sequences of Alt a 45 and Alt a 12 of Alternaria alternata, as O ~ well as the peptide sequences derived from these primary sequences which, in individuals allergic to fungi, lead to a pathologic immune response with overshooting of IgE
antibodies. Recombinant allergens or partial peptides having , an immunogenic action may be used not only for improving the R ~ diagnosis but also for in vivo or in vitro induction of immunotolerance or anergy of T lymphocytes.
p Immunologic mechanisms, which have been established in the course of evolution for providing protection against t: antigens in the environment, normally can differentiate " ~ between self and non-self. However, as is often the case in complex control mechanisms, the immune system is also subject to a certain error rate and when it breaks down an attack on the body's tissue takes place. There are four principal situations in which the body is attacked by its own immune system. These situations differ in the origin of the antigens which trigger the attack and in their mechanism and manifestation. The response may be triggered by environmental antigens, by an infectious agent (but also by harmless substances), by tissue antigens coming from another person and by antigens of the individual himself. The reaction is termed CA 02204362 1997-0~-02 .

"allergic."
A principal feature in allergies is that an administered substance induces increased sensitivity or hypersensitivity, instead of protection. Some of these substances are toxins, others are harmless proteins.
Today four types of hypersensivity are distinguished:
Types I-IV. Types I-III are mediated by antibodies, while type IV is mediated by T lymphocytes.
Type I hypersensitivity is also termed immediate type hypersensitivity, because its effect appears within hours of antigen contact. In the initial sensitizing phase of this mechanism, antigen (allergen) gets into the body, is absorbed by antigen-presenting cells (APC) and processed. The processed antigen is then presented together with MHC II on the surface of APC to T helper cells (Th). The Th produce lymphokines which help B lymphocytes to differentiate into antibody-producing plasma cells. B lymphocytes recognize the allergen by their surface receptors and secrete IgE. They bind to receptors of mast cells and basophils. However, this initial binding has no evident effect on the cells.
However, when the allergen comes into the body once again, difficulties arise. The multivalent allergen binds to IgE and via other epitopes to additional IgE molecules, so that bridge formation (cross linking) between the IgE
molecules starts. Aggregation immobilizes the receptors and induces a signal transduction chain, which ultimately leads to degranulation of the mast cells. Degranulation leads to the production of prostaglandins and leukotrienes. The released substances are chiefly histamine and heparin. Histamine stimulates smooth muscle cells, vascular endothelial cells and nerve endings. Heparin exerts an inhibiting effect on thrombocytes. The allergic response is influenced by the nervous system in the acute as well as the late phase.
Neurotransmitters interact with the corresponding receptors on effector cells and activate these either through cAMP or CA 02204362 1997-0~-02 through cGMP. The target cells of the neurotransmitters are again the mast cells, the smooth muscles and epithelial and secretory cells.
Not all persons who are exposed to the allergen develop an allergy. Why? The primary cause of allergic states is believed to be a defect in the immune system.
Neonatal and postnatal serum immunoglobulin levels, particularly IgA, are very low. Allergic individuals often have fewer T lymphocytes (CD8+). Many allergic persons had an elevated IgE level at birth. Their basophils degranulate more readily. Some also have a defect in suppressor T cells. The IgE level increases when T cells decrease.
A strong argument against an immunologic defect lies in that the asthmatic state may also often be triggered without participation of the allergen. Advocates of this thesis argue that the primary cause of allergies is physiologic rather than immunologic. Perhaps the nerve endings in the smooth muscle, the secretory glands and the blood vessels of target organs are genetically hyperactive in allergic patients.
The allergy is triggered by characteristic changes after a second exposure to the same antigen. The quantity of antigen required for sensitization fluctuates considerably. Too small a dose produces no response and too great a dose may end in protection rather than sensitization. A desensitized state may be produced when a state of shock has been overcome. But the level of reactive IgE is restored after a few weeks.
The best treatment is avoidance of the allergen. Since one hundred percent avoidance is hardly achievable, additional possibilities lie in the use of antihistamines. Currently, so-called immunotherapy is also being tried. In this approach, an inability to respond to specific allergens is induced. For this purpose, the patient is repeatedly i~lln;zed with a mixture of allergens. One begins with low doses and slowly increases them until the patient no longer reacts.
Immunotherapy is successful in hay fever, insect bites and CA 02204362 1997-0~-02 allergic asthma. Treatment seems to induce a form of partial immunologic tolerance. However, desensitization of the patient is never absolute.
Up to now, however, neither the diagnosis nor the therapy of allergic diseases has been satisfactory. Molecular characterization of the chief allergens of Alternaria by means of cDNA cloning, sequencing, sequence comparison of the allergenic protein with protein databases, as well as production of recombinant allergens, will give more information about the in vivo function of proteins that trigger false immune reactions. This information is of interest for the following reasons:
1) High-purity recombinant allergens can be used for a more careful diagnosis, one that is better than can now be made with crude extracts.

2) At the same time the sequence of the allergens will help to define tolerogenic peptides, and possibly also to learn to understand the IgE class switch which takes place during ;r-lln;zation with the allergen.
For decades IgE-caused allergies, thus for example, also allergies to fungal spores, have been treated by hyposensitization (Bousquet et al., 1991). This treatment consists in administering allergenic extracts in the form of injections or by or application in aqueous form as drops in increasing doses, until a maintenance dose has been attained over several years. The result of this treatment is the achievement of tolerance to the allergens introduced, which manifests itself in a decrease of disease symptoms (~irkner et al., 1990). The problem with this type of treatment is the many adverse reactions which it engenders. In the course of hyposensitization therapy cases of anaphylactic shock have occurred during treatment. The problem here is the difficulty of standardizing the fungal protein isolates. By using peptides derived from allergens but devoid of anaphylactic effect it might be possible to administer higher doses without CA 02204362 1997-0~-02 risk, whereby a substantial improvement of hyposensitization could be achieved.
Alternaria alternata can be found practically everywhere in nature. Preferred sites or habitats of the fungus are various soil types, grain silos, rotted wood, but also living plants, compost heaps and bird's nests. When tomatoes are covered with black spots, they most likely originate from Alternaria. But it is not only in nature that Alternaria alternata may be found. Very often the fungus is encountered in humid indoor areas and on window frames. In general, warm temperatures and high atmospheric humidity favor the growth of the fungus.

a) Description of the allergenic proteins of Alternaria alternata by Western blotting For cloning the present allergens of Alternaria alternata, sera of 128 patients were available. To test the reactivity of the patients with fungus protein extract, Alternaria alternata (collection of Prof. Windisch, Berlin, No. 08-0203) was cultivated on solid medium (2% glucose, 2% peptone, 1% yeast extract). For the protein extraction the fungal mat was lifted after 3 days of growth at 28~C and broken up with liquid nitrogen. Separation of the extracted proteins was carried out on a denaturing polyacrylamide gel, which was subsequently blotted, incubated with patient serum and detected with 125I-labeled antihuman IgE. Expressed in percentages, the patients reacted to the allergenic proteins as follows:
Alt a 45 47%
Alt a 12 8%
As these numbers reveal, Alt a 45 is a principal allergen and Alt a 11 is a secondary allergen.

b) Construction of the cDNA expression bank Total RNA was obtained by the acid guanidium-phenol CA 02204362 1997-0~-02 extraction method from fungus material cultivated by us.
Poly(A)plus enrichment was done with oligo(dT) cellulose obtained from Boehringer. The cDNA synthesis (first and second strand) was performed as described in the "Mânual des Lambda ZAP Systems" [Manual of the lambda ZAP system] of Stratagene Co. The cDNA was then provided with EcoRI (on the 3' side) and XbaI linkers (on the 5' side), ligated in predigested lambda-ZAP arms, and packaged. The titer of the primary bank was 900,000 clones.

c) Screening of the cDNA gene bank with patient sera, in VlVO excision, sequenclng The expression bank was screened by incubation of the "lifted" phage plaques with a serum mixture of 2 patients, for which it was known, from Western blotting, that they cover the spectrum of the detected antigens. Detection was again done with antihuman IgE RAST antibodies of Pharmacia Co. After secondary and tertiary screening 150 positive clones remained.
The two clones described were sequenced according to Sanger's method (Sanger, 1977).

d) Expression of the Alt a 45 and Alt a 12 cDNAs as B-galactosidase fusion protein The respective recombinant plasmids were transformed into the E. coli strain XLI-Blue strain and induced with IPTG
(isopropyl-B-D-thiogalactopyranoside). The E. coli total protein extract was then electrophoretically separated and blotted on nitrocellulose. The fusion protein was detected with serum IgE of patients allergic to fungus and with an iodine-labeled rabbit antihuman IgE antibody (Pharmacia, Uppsala, Sweden).
The ~-galactosidase portion of the fusion protein contains 36 amino acids, which corresponds to a molecular -CA 02204362 1997-0~-02 weight of 3800 daltons. Taking this "enlargement" into consideration shows precisely that the recombinant fusion proteins Alt a 45 and Alt a 12 likewise exhibit IgE binding.

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 by means of appropriate computer programs. Through these studies it is possible to define specific T and B-cell epitopes which have the capacity, for example, of stimulating T lymphocytes and inducing them to proliferate, but also (in the case of an exactly defined dose) of bringing the cells into a state of tolerance or nonreactivity (anergy) (Rothbard et al., 1991). Each of the epitopes determined will be cited in the description of the recombinant protein in individual figures.
The search for B-cell epitopes was carried out with the aid of the GCG (Genetics Computer Group) program. The determination is based on weighing the parameters hydrophilicity (Kyte-Doolittle), secondary structure (Chou-Fasman), surface localization (Robson-Garnier) and flexibility, whereby the antigenicity of partial peptides is calculated.
The principle of T-cell epitope prediction was carried out essentially according to the algorithm of Margalit et al.
(1987). The principle consists in looking for amphipathic helices according to primary sequence of the protein to be determined, flanked by hydrophilic regions. For relevant T-cell epitopes the calculated score must be greater than 10. In the case of the MHC II-associated peptides no consensus can be defined on the basis of either the sequence or the length of the peptide, as in the case of HLA-A2 (human leucocyte antigen) (MHC I)-associated peptides. In the case of HLA-A2-CA 02204362 1997-0~-02 associated peptides the length of the peptide is 10 amino acids, the second amino acid being a tyrosine and the last amino acid a leucine (Rammensee et al., 1993). The calculated epitopes will be separately cited in the description of the individual allergenic sequences.
Below, the cDNA sequences and the analyses carried out with them are presented in succession. Computer evaluation of the following sequences was done on an Ultrix-DEC 5000 work station using the GCG software package (= Wisconsin package:
the algorithms of this package were developed by the University of Wisconsin).
The DNA molecules according to the invention therefore have nucleic acid sequences which correspond in homologous fashion to the following sequences (1-6), or to partial regions of these sequences, or nucleic acid sequences which hybridize with the above nucleic acid sequences under stringent conditions. The degree of stringency is defined by 0.1 x SSC. The degree of homology should be more than 60%.

A. Alt a 45 The following sequence 1 shows the complete cDNA sequence of Alt a 45 beginning with the initiating ATG. The length of the cDNA is 1302 bp, which corresponds to a calculated molecular weight of 45904 daltons. Thus on the basis of the molecular weight the observed band in the Western blot at 45 kD correlates with the cloned and sequenced allergen. On the basis of analysis carried out so far, there is probably no signal peptide before the mature protein.

Sequence 1: Alt a 45 45904 daltons (1) INFORMATION ON SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1302 base pairs/434 amino acid groups (B) TYPE: Nucleic acid/protein (C) STRAND FORM: ds (D) TOPOLOGY: Linear (ii) NATURE OF MOLECULE: cDNA to mRNA/protein (iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) NATURE OF FRAGMENT: Total sequence (vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternata (C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae DHA sequence 1302 b.p. ATGACCAAGCAG ... GACGAGTTGT M linear ATG ACC AAG CAG GCT CTC CCC GCC GTC TCC GAA GTC ACC M G GAC ACA CTC GAG GAG TTC
~et thr lys gln ala leu pro ala val ser glu val thr lys asp thr leu glu glu phe 61 / Zl 91 / 31 AAG ACC GCC GAC M G GTC GTC CTC GTC GCC TAC TTC GCC GCC GAC GAC M G GCC TCC M C
lys thr ala asp lys val val leu val ala tyr phe ala ala asp asp lys ala ser asn GAG ACC TTC ACC TCG GTC GCC M C GGT CTC CGT GAC M C TTC CTC TTC GGT GCC ACC M C
glu thr phe thr ser val ala asn gly leu arg asp asn phe leu phe gly ala thr asn GAC GCT GCT CTG GCC M G GCT GAG GGT GTC AAG CAG CCC GGT CTC GTC TGT ACA AGT CCT
asp ala ala leu ala lys ala glu gly val lys gln pro gly leu val cys thr ser pro TCG ACG ACG GCA AGG ACG TCT TCA CCG AGA CCT TCG ATG CGG ACG TAT CCG CGA CTT CGC
ser thr thr ala arg thr ser ser pro arg pro ser met arg thr tyr pro arg leu arg 3~1 / 101 331 / 111 AAG GTC GCC TCC ACA CCC CTC ATT GGT GAG G~T GGC CCC GAG ACC TAC GCC GGA TAC ATG
lys val ala ser thr pro leu ile gly glu val gly pro glu thr tyr ala gly tyr met GCC GCT GGC ATT CCC CTC GCA TAC ATC TTC GCC GAG ACT CCC GAG G M CGT GAG GAG m ala ala gly ile pro leu ala tyr ile phe ala 9lu thr pro glu glu arg glu glu phe 4Zl / 141 451 / 151 GCC AAG GAG CTG M G CCC CTC GCT CTC M G CAC Mr, GGC GAG ATC M C TTC GCT ACC ATC
ala lys glu leu lys pro leu ala leu lys his lys gly glu ile asn phe dla thr ile 481 / 161 Sll / 171 GAC GCC M G TCC TTC GGC CAG CAC GCT GGC M C CTT M C CTC AAG GTC GGC ACC TGG CCC
asp ala lys ser phe gly gln his ala gly asn leu asn leu lys val gly thr trp pro CA 02204362 1997 - 0~ - 02 541 / 181 571 t 191 GCT TTC GCT ATC CAG CGC ACC GAG AAG M C GAG AAG TTC CCT ACG M C CAG GAG GCC AAG
ala phe ala ile gln arg thr glu lys asn glu lys phe pro thr asn gln glu ala lys ATC ACC GAG M G GAG ATT GGC M G TTC GTT GAC GAC TTC CTC GCT GGC M G ATT GAC CCT
ile thr glu lys glu ile qly lys phe val asp asp phe leu ala gly lys ile asp pro 661 /. 221 691 / 231 AGC ATC AAG TCT GAG CCC ATT CCC GAA TCC MT GAC GGT CCC GTA ACT GTC GTC GTT GCC
ser ile lys ser glu pro ile pro glu ser asn asp gly pro val thr val val val ala CAC M C TAC M G GAT GTC GTC ATT GAC MC GAC M G GAC GTT CTC GTT GAG T~C TAC GCC
his asn tyr lys asp val val ile asp asn asp lys asp val leu val glu phe tyr ala CCC TGG TGC GGT CAC TGC M G GCT CTT GCT CCC AAG TAC GAG GAG CTC GGC CAG CTC TAC
pro trp cys gly his cys lys ala leu ala pro lys tyr glu glu leu gly gln leu tyr GCT TCC GAC GAG CTC TCC M G TTG GTG ACC ATT GCC M G GTT GAC GCT ACT CTC M C GAC
ala ser asp glu leu ser lys leu Yal thr ile ala lys Yal asp ala thr leu asn asp GTT CCC GAC GAG ATC CAA GGT TTC CTA CCA TCA AGC CTC TTC CCG CTG GCA AGA AGG ATG
val pro asp glu ile gln gly phe leu pro ser ser leu phe pro leu ala arg arg met CCC CAG TCG ACT ACT CTG GTT CCG CAC TGT CGA GGA TCT CGT CCA GTT CAT CGA AGA GAA
pro gln ser thr thr leu val pro his cys arg gly ser arg pro val his arg arg glu CGG CTC ACA CAA GCT AGC GCC AGC GTT GGC G M GCT GTT GAA GAT GCT ACC GAG TCC GCC
arg leu thr gln ala ser ala ser val gly glu ala val glu asp ala thr glu ser ala AAG GCC AGT GCC TCT TCC GCC ACA GAC TCT GCT GCC TCA GCT GTA TCA GAA GGC ACC GAG
lys ala ser ala ser ser ala thr asp ser ala ala ser ala Yal ser glu gly thr glu ACG GTC AAG TCT G~T GCG TCT GTC GCT TCC GAC TCA GCC TCT TCC GCC GCT TCC GAG GCT
thr Yal lys ser gly ala ser val ala ser asp ser ala ser ser ala ala ser glu ala ACC M G TCT GTC M G TCT GCC GCG TCC GAG GTT ACC M C TCT GCC TCG TCG GCT GCG TCA
thr lys ser val lys ser ala ala ser glu val thr asn ser ala ser ser ala ala ser GAG GCT TCA GCT TCG ~CC TCA AGC GTC M G GAC GAG TTG T M
glu ala ser ala ser ala ser ser val lys asp glu leu OCH

CA 02204362 1997-0~-02 Homology searches with Alt a 45 in the SWISSPROT protein database have revealed that Alt a 45 is a protein disulfide isomerase (PDI). Multiple alignment with PDI sequences of several organisms has reflected the high homology of Alt a 45 to PDI sequences. The established consensus shows amino acid identities straight through all organisms. A central motif of homology is defined by the sequence "EFYAPWCGHCK." The function of protein disulfide isomerase (PDI) is support in the folding process of proteins. The exact mechanism is not yet known. The active site of PDI is similar to that of thioredoxin. The high consensus between homologous proteins is found from bacteria to plants and on up to higher mammals. The PDI protein is found in the lumen of the endoplasmatic reticulum.

Sequence 2: Alt a 45: B-cell epitopes (1) INFORMATION ON SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually (B) TYPE: Protein (ii) NATURE OF MOLECULE: Peptides (iii) HYPO~ CAL: No (v) NATURE OF FRAGMENT: N terminus to C terminus (vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternata (C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae CA 02204362 1997-0~-02 Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr Ala Asp Lys (11-25) Phe Ala Ala Asp Asp Lys Ala Ser Asn Gly Glu Thr Phe Thr ier Val Ala (32-47) Ser Pro Ser Thr Thr Ala Arg Thr Ser Ser Pro Arg Pro Ser Het Arg Thr TyrPro Arg Leu Arg Lys Val Ala (79-io3) Phe Ala Gly Glu Thr Pro Gly Glu Gly Glu Arg Gly Glu Gly Glu Phe Ala LysGly Glu Leu Lys Pro Leu Ala Leu (130-149) Gln Arg Thr Gly Glu Lys Asn Gly Glu Lys Phe Pro Thr Asn Gln Gly Glu AlaLys Ile Thr Gly Glu Lys Gly Glu Ile Gly Lys Phe Val Asp Asp (185-212) Asp Pro Ser Ile Lys Ser Gly Glu Pro Ile Pro Gly Glu Ser Asn Asp Gly ProVal Thr (219-236) Ala Arg Arg Het Pro Gl n 5er Thr Thr Leu Val Pro His Cys (317-330) Arg Gly Ser Arg Pro Yal His Arg Arg Gly GlU Arg Leu Thr Gln Ala Ser AlaSer Val Gly Gly Glu Ala (331-352) CA 02204362 1997-0~-02 Sequence 3: Predicted amphipathatic segments = T-cell epitopes Flags Hidpoints Angles Score .
K 9:22 85:120 33,4 YSEVTKOTLEEFKT
41:50 85:105 23.4 K P 66:71 125:135 11.9 KAEGVK
P 82:85 115:125 6.3 K P 96:101 115:120 16.0 YPRLRK
P 110:114 80:90 6.8 P 116:120 85:1~5 10.4 K P 141:145 100:125 9.4 AKELK
P 178:182 130:135 8.1 K P 204:216 80:100 31.5 KEIGKFYDDFLAG
K P 257:267 95:110 28.B EFYAP~CGHCK
275:281 110:125 15.2 K 283:293 90:130 25.6 DELSKLVTIAK
P 295:309 85:105 39.9 DATLHDVPDEIQGFL
K 345:361 90:125 45.9 ASASYGEAVEDATESAK
~ 364:3BS B5:135 49.3 ASSATDSAASAYSEGTETYKSG
K 391:413 80:115 73.2 DSASSAASEATKSVKSAASEVTH

(1) INFORM~TION ON SEQ ID NO: 3 (i) SEQUENCE CHARAC~ERISTICS:
(A) LENGTH: Listed individually (B) TYPE: Protein (ii) NATURE OF MOLECULE: Peptides (iii) HYPOTHETICAL: No (v) NATURE OF FRAGMENT: N terminus to C terminus (vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternata (C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae Val Ser Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr (9-22) Lys Ala Gly Glu Gly Yal Lys (66-71) Tyr Pro Arg Leu Arg Lys (96-101) Ala Lys Gly Glu Leu Lys (141-145) Lys Gly Glu Ile Gly Lys Phe Val Asp Asp Phe Leu Ala Gly (204-216) Gly Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys (257 267) Asp Gly Glu Leu Ser Lys Leu Val Thr Ile Ala Lys (283-293) Asp Ala Thr Leu Asn Asp Val Pro Asp Gly Glu lle Gln Gly Phe Leu (2g5-309) Ala Ser Ala Ser Val Gly Gly Glu Ala Val Gly Glu Asp Ala Thr Gly Glu Ser Ala Lys (345-361) Ala Ser Ser Ala Thr Asp Ser Ala Ala Ser Ala Val Ser Gly Glu Gly Thr Gly Glu Thr Val Lys Ser Gly (364-385) Asp Ser Ala Ser Ser Ala Ala Ser Gly Glu Ala Thr Lys Ser Val Lys Ser Ala Ala Ser Gly Glu Yal Thr Asn (391-413) CA 02204362 1997-0~-02 The T-cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a lysine (K) and C-terminally by a proline (P) (= flags).
Potential T-cell epitopes are present only when the score index is greater than 10.

B: Alt a 12 The following sequence 4 shows the complete cDNA sequence of Alt a 12 and of the amino acid sequence derived therefrom.
The open reading frame comprises 333 bp or 111 amino acids.
The calculated molecular weight is 11728 daltons and thus correlates with the antigenic protein observed at 45 kD, which is recognized in the Western blot of 8% of the patients.

Sequence 4: Alt a 12 11728 daltons (1) INFORMATION ON SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 333 base pairs/111 amino acid groups (B) TYPE: Nucleic acid/protein (C) STRAND FORM: ds (D) TOPOLOGY: Linear (ii) NATURE OF MOLECULE: cDNA to mRNA/protein (iii) HYPOTHETICAL: No (iv) ANTISENSE: No (v) NATURE OF FRAGMENT: Total sequence (vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternata (C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae DNA sequence 333 b.p. ATGTCTACCTCC ... CTCTTCGACTAA linear ATG TCT ACC TCC GAG CTC GCC ACC TCT TAC GCC GCT CTC ATC CTC GCT GAT GAC GGT GTC
~et ser thr ser glu leu ala thr ser tyr ala ala leu ile leu ala asp asp gly val CA 02204362 1997-0~-02 \
, 61 ~ 21 91 / 31 ~C ATC AC~ GCC GAC MG C~ CM TCC CTC ATC MG GCC GCA AAG ATC. G~ G GrC G~G
asp ile thr ala asp lys leu gln ser leu ile lys ala ala lys ile ~lu glu val glu 121 ~ 41 151 / 51 CCC ATC TGG ACG ACC CTG rrc GCC MG GCT CrT GAG GGC MG G~T GrC MG G~C ~G ~A
pro ile trp thr thr leu phe ala lys ala leu glu gly lys asp val lys asp leu leu CTG MC GTC GGC TCA GGC GGC GGT GCr GCC CCG CTG CCG G~G gCg cTG crc CTG CGC TG~
leu asn val gly ser gly gly gly ala ala pro leu pro glu ala leu leu leu arg trp CGT GCI GCT G~T GCC GCA CCA GCT GCr G4G G4G MG MG G~ G MG GAG GAG TCG
arg ala ala asp ala ala pro ala ala glu glu lys lys glu glu glu lys glu glu ser 64C G~G G~C ATG GGC TTC GGT crc rrc G~C TM
asp glu asp net gly phe gly leu phe asp OCH

Here homology searches in the SWISSPROT protein database revealed homologies to ribosomal proteins. The allergenic protein Alt a 12 is of interest not only because of its property as an allergen of Alternaria alternata. Ribosomal proteins, here especially the human ribosomal proteins Pl and P2, have been described in the literature as autoantigens (Francoeur et al., 1985, Rich et al., 1987, Hines et al., 1991). 20% of patients with lupus erythematosus have autoantibodies (anti-rRNP) to components of ribosomes, in particular autoantibodies to the ribosomal proteins PO (38 kD), Pl (16 kD) and P2 (15 kD). Human autoantibodies cross-react with similar proteins, which means that epitopes that have been strongly preserved in evolution are recognized. The basis of immunologic cross-reactivity is provided by the C-terminal 17-amino-acid group region KEESEESD(D/E)DMGFGLFD.
Whether sensitization by Alt a 12 which has taken place in childhood and youth correlates with an autoimmune disease appearing in adulthood needs precise PX~;n~tion. However, administration of ribosomal proteins failed to produce any CA 02204362 1997-0~-02 , autoimmune disease in mice (Hines et al., 1991). Other ribosomal proteins (Cla h 11 and Alt a 11) likewise have already been identified as allergens (Achatz et al., 1994).
The B-cell epitopes shown in the next sequence 16 were calculated taking secondary structure, surface position, hydrophilicity, flexibility, etc. into consideration.

Sequence 5: Alt a 12: B-cell epitopes (1) INFORMATION ON SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually (B) TYPE: Protein (ii) NATURE OF MOLECULE: Peptides (iii) HYPOTHETICal: No (v) NATURE OF FRAGMENT: N terminus to C terminus (vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternata (C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae Ala Asp Asp Gly Val Asp (16-21) Thr Ala Asp Lys Leu Gln Ser Leu (23-30) Ala Lys Ile Gly Glu Gly Glu Val Gly GlU Pro Ile Trp Thr (34-44) Ala Leu Gly GlU Gly Lys Asp Val Lys Asp (50-58) Val Gly Ser Gly Gly Gly Ala Ala (63-70) Trp Arg Ala Ala Asp Ala (80-85) Ala Pro Ala Ala Gly Glu Gly Glu Lys Lys Gly GlU Gly GlU Gly GlU Lys Gly Glu Gly Glu Ser Asp Gly Glu Asp Het Gly (105) Sequence 6: Predicted A rhir~thatic segments = T-cell epitopes Fl~g~ ~idpo~nt~ Anqle~ score _ _ _ _ _ _ _ _ _ _ _ _ _ K P 26:37 90:135 27.6 KLaSLIKAAKIE
K 39:49 80:115 22.0 YEPII~TTLFAK
K 52:55 80:135 6.4 (1) INFORMATION ON SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually (B) TYPE: Protein (ii) NATURE OF MOLECULE: Peptides (iii) HYPOTHETICAL: No (v) NATURE OF FRAGMENT: N terminus to C terminus (vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternata (C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae Lys Leu Gln Ser Leu lle Lys Ala .Ila Lys lle Gly Glu (26-37) Yal Gly Glu Pro lle Trp Thr Thr ,eu Phe Ala Lys (39-49.) The T-cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a lysine (K) and C-terminally by a proline (P). Potential T-cell epitopes are present only when the score index is greater than 10.

J

6 . Ref erences Achatz. G., Oberkofler, H., Lechenauer, E., Simon, B., Unger, A., Kandler, D., Ebner, C., Prillinger, H., Kraft, D., Breit~nbach~ M. (1994).
Molecular cloning of major and minor allcrgens of Alternaria alternata and Cladosporium herbarum.
Mol. Immunol. in press.

Birkner, T., Rumpold, H., Jarolim, E. Ebner, H., Breitenbach, M., Skarvil, F., Scheiner, O., Kraft, D. (19g0).
Evaluation of immunotherapy-induces changes in specific IgE, IgG and IgG subclasses in birch pollen allergic patients by means of immunoblotting. Correlation with clinical response.
Allergy 45, 418.

Bousquet, J., Becker, W.M., He~aoudi, A. (1991).
Differences in clinical and immunologic reactivity of patients allergic to grass pollens and to multiple-pollen spe~ies. II. Efficacy of a double blind, placebo-controlled, specific immunotherapy with standardized extracts.
J. Allergy Clin. Immunol. 88, 43.

Francoeur, A.M., Pe~bles, C.L., He~krn~n, K.J., Lee, J.C.. Tan, E.M. (1985).
Identification of ribosomal protein autoantigens.
J. Immunol. 135,1767.

Hines, J.J., Wei~sbach, H., Brot, N., Elkon, K. (1991).
Anti-P ~lto~ntibody production requires Pl/P2 as immunogens but is not driYen byexogenous self-antigen in mrl mice.
J. Immunol.. 146, 3386 Margalit, H., Spogue, J.L., Cornette, J.L., Cease, K.B., Delisi, C., Berzofsky, J.A.
(1987).
Prediction of immunodominant Helper T cell antigenic sites from the primary sequence.
J. Immunol. 138, 2213.

Rammensee, H.G.. Falk, K., Rotzschke. 0. (1993).

MHC molecules as peptide receptors.
Current Opinion in Immunol. 5. 35.

Rich, B.E., Steitz, J.A. (19~7).
Human acidic ribosomal phosphoproteins P0, Pl and P2: analysis of cDNA clones, in vitro synthesis and asscmbly.
Mol. Cell. Biol. 7, 4065.

Rothbard, J.B., Gef~er, M.L. (1991).
Interactions betwcen immunogcnic peptides and MHC proteins.
Ann. Rev. Immunol. 9, 527.

Sanger, F., Niclclen, S., Coulson. A.R. (1977).
DNA sequencinE with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA 74, 5463-5468

Claims (12)

claims:

1. Recombinant DNA molecules that code for polypeptides which have the antigenicity of the allergens Alt a 45 and Alt a 12, or for peptides which have at least one epitope of these allergens, having nucleic acid sequences that code for peptides which correspond to the sequences SEQ ID NO. 1 to 6, or hybridize with the aforementioned sequences under stringent conditions.

2. Recombinant DNA molecules according to claim 1, which have nucleic acid sequences that can be derived by degeneration from the amino acid sequences SEQ ID NO. 1 to 6.

3. Recombinant DNA molecules according to claim 1 or 2, which have nucleic acid sequences that code for polypeptides which, as antigens, are cross-reactive with allergens Alt a 45 (SEQ ID NO. 1) and Alt a 12 (SEQ ID NO. 4).

4. Recombinant DNA molecules according to claims 1 to 3, which are functionally connected with an expression control sequence to form an expression construct.

5. Host system for the expression of polypeptides, which is transformed by a recombinant expression construct according to claim 4.

6. Recombinant or synthetic protein or polypeptide coded by a DNA molecule according to any one of claims 1 to 3, which has the antigenicity of Alt a 45 (SEQ ID NO. 1) or Alt a 12 (SEQ ID NO. 4), or at least of one of the epitopes (SEQ ID NO.
2, 3 or 5, 6) of these proteins.

7. Recombinant or synthetic protein or a polypeptide according to claim 6, which has an amino acid sequence that wholly corresponds to the amino acid sequences SEQ ID NO. 1 to 6, or has such a degree of similarity that they cross-react immunologically.

8. Recombinant or synthetic protein or polypeptide according to claim 6 or 7, which constitutes a fusion product that possesses the antigenicity of the allergens Alt a 45 (SEQ
ID NO. 1) or Alt a 12 (SEQ ID NO. 4) or at least of one epitope (SEQ ID NO. 2, 3 or 5, 6) thereof and has an additional polypeptide part, the entire fusion product being coded by the DNA of an expression construct according to claim 4.

9. Recombinant or synthetic protein or polypeptide according to claim 8, wherein said additional polypeptide part is .beta.-galactosidase or another polypeptide suitable for fusion.

10. Diagnostic or therapeutic reagent which contains a synthetic protein or polypeptide according to any one of claims 6 to 9.

11. Process for in vitro detection of the allergy of a patient to allergens Alt a 45 (SEQ ID NO. 1) or Alt a 12 (SEQ
ID NO. 4), wherein the reaction of the IgE antibodies in the patient's serum with a recombinant or synthetic protein or polypeptide according to any one of claims 6 to 9 is measured.

12. Process for in vitro detection of a cellular reaction to the allergens Alt a 45 (SEQ ID NO. 1) or Alt a 12 (SEQ ID
NO. 4), wherein a recombinant or synthetic protein or polypeptide according to any one of claims 6 to 9 is used for stimulation or inhibition of the cellular reaction.