AU625315B2 - Malaria-specific DNA sequences, expression products thereof, and the use thereof - Google Patents
Malaria-specific DNA sequences, expression products thereof, and the use thereof Download PDFInfo
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- AU625315B2 AU625315B2 AU27619/88A AU2761988A AU625315B2 AU 625315 B2 AU625315 B2 AU 625315B2 AU 27619/88 A AU27619/88 A AU 27619/88A AU 2761988 A AU2761988 A AU 2761988A AU 625315 B2 AU625315 B2 AU 625315B2
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- proteins
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Classifications
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- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
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Description
BEHRINGWERKE AKTIENGESELLSCHAFT 87/B 043J- Ma 664 SDr. Lp/Bn Malaria-specific DNA sequences, expression products thereof, and the use thereof An important step towards the development of a vaccine against malaria is the identification of protective antigens. In general, the antigens categorized as protective are those which have provided protection from P. falciparum infection administered intravenously in in vivo experiments in an animal model such as, for example, in Saimiri or Aotus monkeys. Only unsatisfactory protection experiments have hitherto been described in humans, but several isolated P. falciparum proteins have show,- a complete or partial 15 protective effect in an animal model. This applies both to protein fractions of 75kD and 100kD purified by gel electro- Sphoresis and to the protein bands of molecular weights 200kD, 140kD and 41kD purified by gel electrophoresis (L.H.
Perrin et al. (1984), Clin. exp. Immunol. 56, 67-72; L.H.
S. 20 Perrin et al. (1985), J. Clin. Invest, 75, 1718-1721; W.A. Siddiqui et al. (1987), Proc. Natl. Acad. Sci., USA 84, 3014-3018). Of the proteins which are specific for merozoites and have been prepared to date by biotechnologij 25 cal methods, a partial protective effect in immunization experiments with Saimiri or Aotus monkeys has been shown by a recombinant expression protein of the 5' repeat region of S the so-called RESA 155kD merozoite protein as well as by a synthetic oligopeptide of the 200kD merozoite surface pre- 30 cursor protein and by a combination of synthetic oligopepl tides of proteins of molecular weights 35kD, 55kD and 200kD. Recombinant proteins, prepared by genetic manipulation, of the above antigens, which display a protective effect in in vivo experiments with monkeys, are potential candidates for a malaria vaccine.
The aim of the investigations was to isolate coding sequences for the protective 41kD antigen band described by L. Perrin (1985 loc. cit.), to bring about the expression of the sequences, and to test the expression products for Ot S- 2 their protective effect in the monkey model. A specific antiserum against the 41kD antigen band was used to isolate from a genomic expression bank fifteen clones and to elucidate the structure of their insertions. The sequences of the clones 41-1 to 41-10 and 41-12 to 41-15, as well as 41-17, depicted in Tab. 1-15. Two clones (41-2 and 41-7) with very intense immunological reactions were used to isol ite mono-specific antibodies from the serum used for the screening. These antibodies react specifically in the Western blot with a merozoite antigen of 41kD.
In the Southern blot, a 3.0 kb EcoRI fragment and a 2.0 kb Sau3A fragment hybridized with the insert DNA of the clone 41-2. Both DNA fragments were isolated and sequenced.
The Sau3A fragment contains the complete coding region of 15 the 41-2 gene. This region contains no introns and codes for 184 amino acids with a molecular weight of 21512 D.
The 41-2 protein possesses a signal sequence and two further hydrophobic sections. No repetitive sequence portions are present. Western blot analysis of schizont proteins with rabbit antisera prepared against an expression product which contains 70 of the coding region produced a band of 29 kD. Furthermore, an mRNA of 1.6 kb was detected by Nortneri blot analysis.
9* o i 9 B i 99 9 9 a99 o On the other hand, the insert DNA of the clone 41-7 codes for the 41kD protein. Rabbit antisera prepared against a fusion protein of 41-7 unambiguously recognize a 41kD band in the Western blot. It was possible, by screening a genomic lambda gtll EcoRI* gene bank with the insert DNA of the clone 41-7, to identify a clone which contains a malaria-specific insert of 2.3kb. This was isolated and sequenced. It contains the complete coding region for a 41kD protein. The gene does not encode a signal sequence and contains neither introns nor repetitive sections. The derived amino acid sequence of the 41kD protein from P. falciparum is highly homologous with aldolases from muscle and Liver of mammals and with the aldolase 3 from Trypanosoma brucei. In contrast to the mammalian genome, only one aLdolase gene per genome was found ty Southern blot analyses for P. falciparum.
The clones 41-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, and 17 were detected on the basis of their cross-reactivities with the antiserum against the 41kD protein band.
They are suitable for the preparation of a vaccine. Expression of the insert DNAs of the clones 41-1 to 41-5 and 41-7, 41-10 and 41-14 was brought about in the vector pEX- 31, ;jnd the resulting fusion proteins were purified. A combination of an immunologically effective amount of three expression products (41-1, 41-2 and 41-3) protects Aot]is monkeys from P.falciparum infection.
Consequently, the invention relates to a) the purified and isolated DNA sequences of the clones 41-1 to 41-10, 41-12 to 41-15 and 41-17, as well as 41-2gen and 41-7gen, including their transcription pro- 20 ducts, b) DNA structures and vectors containing these sequences in whole or in part, c) pro- or eukaryotic cells transformed with such DNA, d) the polypeptides, or parts thereof, expressed by these 25 cells by reason of the transformation, 35 ei the amino acid sequences thereof, f) antibodies against the polypeptids under including the use thereof for passive immunization, for diagncsis and for purifying said polypeptides, g) vaccines against malaria which contain the amino acid sequences from alone or in combination, h) a process for the preparation, by genetic manipulation, of the polypeptides, or parts thereof, detailed under i) and the use of the said amino acid sequences for diagnosis.
Further embodiments of the invention are detailed in the examples and tables which follow and in the patent claims I 4 Examples E x am p L e 1 Example 1: Screening of a lambda gtll expression bank using the monospecific anti-41kD serum 10 PFU (plaque-forming units) of a genomic Lambda gt11 e'cpression Vb-a-rr (prepared from DNA of the P. falciparum strain T996) were screened with an antiserum against the 41kD antigen band Perrin et aL. (1985) loc. cit.) from the P. falciparum strain SGE2 by known methods Ozaki 15 (1986), J. Immun. Method. 89, 213-219; Promega Biotec (1986), Proto Blot Immunoscreening System, Technical Manual). The detection system used for this was an antirabbit IgG/alkaline phosphatase conjugate (Promega, Order No. P 3731).
S.The screening of the genomic lambda gtll gent bank with the antiserum against the 41kD protein band maJe it possible to identify two very intensely reacting clones (41-2 and 41-7) and thirteen other clones which reacted more 25 weakly (41-1, 41-3, 41-4, 41-5, 41-6, 41-8, 41-9, 41-10, 41-12, 41-13, 41-14, 41-15 and 41-17) and about 40 other very weakly reacting clones. The insert DNAs of the fifteen clones, which amount to 140bp to 650bp, were cut out with EcoRI and cloned into the EcoRI site of the vector pUC8 for further characterization.
Example 2: Sequencing of the insert fragments of clones 41-1 to 41-10, 41-12 to 41-15 and 41-17 The insert DNAs were sequenced by the dideoxy method using a primer and a reverse primer directly from the pUC8 plasmids Chen and P.H. Seeburg (1985), DNA 4, 165-170).
i I oil~
I
W 5 Tables 1-15 show the malaria-specific DNA sequences, and the amino acid sequences derived therefrom, of the clones 41-1, 41-2, 41-3, 41-4, 41-5, 41-6, 41-7, 41-8, 41-9, 41- 41-12, 41-13, 41-14, 41-15 and 41-17 in the only possible open reading frames. There are no overlaps or homologies whatever in these 15 sequence sections. The UWGC' (University of Wisconsin, Genetic Computer Group) pro. am was used to examine these 15 sequences for homologous sequence sections within the EMBL data bank. None of these 15 partial sequences or relatively large homo- 10 logous sections have hitherto been described. There is merely a 74 homology of nucleotides 1 to 134 in the se- S quence of the clone 41-10 with a partial sequence from S nucleotide 2144 to 2274 in the 140kD protein gene, as proposed in the Application DE-P 3,741,057. The sequence of 15 the clone 41-10 is also the only one which contains *9 repetitive sequence sections typical of P- falciparum proteins. The amino acid sequence of this clone includes three tetrapeptides of the sequence Pro-Ser-Glu-Ser, with the second serine residue in the second repeat being replaced by an asparagine residue, caused by a G-A transition. In addit ion, the sequence of the clone 41-7 from nucleotide 50 to 163 is 56 homologous with an aldolase mRNA from nucLeotide 218 to 331 of the rat (T.
Mucai et al. (1986), J. Biol. Chem. 261, 3347-3354).
Example 3: Detection of the 41kD antigen using specific antibodies against the expression clones 41-2 and 41-7 The method of L.S. Ozaki (198i, loc. cit.) was used to isolate from the antiserum against the 41kD protein band antibodies which are directed specifically against the products of the expression clones 41-1, 41-2, 41-3, 41-7, 41-8 and Lambda gtll (control). To obtain schizonts, P.
falciparum was cultivated in human erythrocytes ,W.
Trager and J.B. Jensen (1976), Science 193, 673-675) and synchronized by treatment with sorbitol Lambros and Yle I I r: ;C -6 J.P. Vanderberg (1979), J. Parasitol. 65, 418-420). The schizonts were enriched to about 90% by flotation in Gelafundin(R' (Braun Melsungen) (in analogy to G. Pasvol et al. (1978), Ann. Trop. Med. Parasitol. 72, 87-88). The schizonts were removed by centrifugation, washed, heated at 100 0 C in SDS sample buffer for 5 min, treated with ultrasound, and frozen in aliquots.
Aliquots of the schizont solution were used for Western blot analysis of the abovementioned specific antibodies Johnson et al. (1984) Gene Anal. Tech. 1, In this the antibodies which were isolated using the expression clones 41-2 and 41-7 reacted very intensely with a 41kD antigen band from schizonts.
Reversing this technique is also possible. In such a system polyclonal or monoclonal antibodies specific for the expression clones of the instant invent-in can be used j to isolate the 41kD antigen or immunogenic parts thereof.
This can be achieved by affinity chromatography methods of the like known by a person skilled in the art.
Example 4: i e Cloning of a DNA fragment which contains the genetic information of the clone 41-2.
i 15 jg of genomic DNA of the P. falciparum strain FCBR, which had been obtained by lysis of schizont cultures S 25 followed by ethidium bromide/CsCl centrifugation Oquendo et al. (1986) Molecular and Biochemical Parasitology 18, 89- S101), were digested with the restriction enzyme EcoRI, blotted onto Gene Screen membranes (Dupont) in accordance j with the manufacturer's instructions, and then hybridized 1 30 with nick-translated insert DNA of the clone 41-2 with a specific activity of 107 to 10 8 q spm/ug. After the filter had been washed in 0.3xSSC (IxSSC 0.15 M NaCl, 0.015 M Na citrate) and 1% SDS (sodium dodecyl sulfate) at 65 0 C for 1 h, the filters were autoradiographed. This Southern blot 6a experiment was used to identify a genomic EcoRI fragment which is about 3kb in size and hybridizes with the insert DNA of the clone 41-2. 60 pg of P. falciparum DNA of the strain FCBR which had been cut with the restriction enzyme S EcoRI were fractionated in a preparative gel, and the region from 2.8kb to 3.2kb was cut out and electroeluted Perbal (1984), a Practi- 5*5*e5 S S S S
S.
S S DBM/JMW/CH (3:22) 7 cal Guide to Molecular Cloning). This DNA was cloned by the method of T.V. Huynh et al. (in DNA cloning Vol. I, ed D.M. Glover (1985), 49-88) into the vector lambda gtlO. 105 PFU of the resulting gene bank were screened with nicktranslated insert DNA of the clone 41-2 by known methods Maniatis et al. (1982), Molecular Cloning, A Laboratory Manual). This resulted in several phage clones which hybridized with the insert DNA of the clone 41-2. The phage DNA of one of these clones was isolated Davis et al. (1980), A Manual for Genetic Engineering, Advanced Bacterial Genetics) and digested with the restriction *6 enzyme EcoRI; a DNA fragment 3.0kb in size was purified by gel electrophoresis and subcloned into the EcoRI restriction site of the vector pUC18 (plasmid pUC 41-2gen). In 15 the subsequent Southern blot analysis Maniatis et al., loc. cit.), this 3.0kb EcoRI DNA fragment of pUC 41- 2gen hybridized with the insert DNA of the clone 41-2.
Example Sequence analysis of the clone pUC 41-2gen The plasmid DNA pUC 41-2gen was sequenced starting from the EcoRI edge sites and using a primer and reverse primer 25 Chen and P.H. Seeburg (1985) loc. cit.). From this S it was possible to determine about 250 bases from each of the ends of the 3.0kb EcoRI fragment. The sequence of one j of these ends is identical to the insert DNA of the cLone 41-2. To construct a restriction map, 0.5 pg samples of the isolated 3.0kb EcoRI DNA fragment were incubated with various restriction enzymes, fractionated by gel electrophoresis, blotted onto nitrocellulose and hybridized with nick-translated insert DNA of the clone 41-2 by known methods Maniatis, loc. cit.). The size of the restriction fragments to be hybridized made it possible to deduce the distance of various restriction cleavage sites from the EcoRI cleavage site, which is common to the two clones 41-2 and pUC 41-2gen. Based on the restriction map constructed in this way, restriction fragments of the clone S- 8 S pUC 41-2gen were isolated and subcLoned into the phage vectors M13mp18 and M13mp19 for sequencing Sanger et at. (1977), Proc. Natl. Acad. Sci. USA, 74, 5463-5467).
From this, the sequence was determined from the EcoRI restriction site, which belongs to the gene, in the direction towards the 5' end of the gene up to a Dral restriction cleavage site. Table 16 shows the DNA sequence and the derived amino acid sequence of this DraI-EcoRI DNA fragment comprising 1230bp of the clone pUC 41-2gen. The sequence from position 1036 to 1228 is identical to the insert DNA of 41-2. Since the sequence of the clone 41-2 and the genomic sequence of the clone pUC 41-2gen derive from different P. falciparum strains (strain T996 from Thailand and strain FCBR from Colombia), it appears that at Least this gene section is highly conserved. The open reading frame of this sequence begins in position 784 with an ATG start codon and terminates with a TTC codon which belongs to the EcoRI restriction cleavage site within the gene. This part codes for the 149 N-terminal amino 20 acids of the protein. The partial sequence of this gene has no repetitive sequence segments. The derived amino acid sequence begins with a sequence section of 18 amino acids, of which 4 are acidic and 5 are basic. This sequence section is followed by a hydrophobic part which 25 is composed of 11 residues and is flanked on both sides by acidic amino acid residues. This hydrophobic region might function as a signal sequence. The region with basic and S acidic amino acids in front of this putative signal I sequence is relatively long; however, regions of similar 30 Length have also been described for other P. falciparum proteins Triglia et al. (1987), The EMBO Journal 6, 1413-1419). The derived amino acid sequence was examined, using the UWGCG program, for hydrophilic regions and surface regions as well as for potential immunogenic epitope regions. .nis revealed three hydrophilic regions of the protein which are encoded by the nucieotide sequences of positions 890 to 907, 1079 to 1093 and 1151 to 1168.
The 5' non-coding region of the gene is extremely AT-rich d I 9 (AT content as has also been described for other P. falciparum genes Weber (1987), Gene 52, 103-109), and has in each case more than 15 stop codons in each of the three reading frames. Furthermore, a possible CAAT Lox is present in position 274, and a possible TATA box is located 64 nucleotides downstream of this. These structures might specify a possible promoter region for this gene.
Example 6: Isolation of the complete gene for 41-2 The sequence analysis of the clone pUC 41-2gen revealed a Sau3A cleavage site 947 bp away from the EcoRI cleavage site. Southern blot analysis of the genome identified a Sau3A fragment which is 2.0 kb in size and hybridizes with I the insert DNA of the clone 41-2 (cf. Example Hence this Sau3A fragment ought to have about 1050 bp of the genetic information of the 41-2 gene in the 3' direction 20 from the EcoRI site, 60 pg of DNA from the strain FCBR Swhich has been cut with the restriction enzyme Sau3A were fractionated in a ;reparative gel, and DNA fragments of 1.8 kb to 2.2 kb were isolated. This DNA was cloned into the Xhol site of the vector lambda ZAP as stated by the 25 manufacturer (Stratagene). 10 PFU of this gene bank were screened with nick-translated insert DNA of the S clone 41-2, and about 40 phage clones were identified ***see (cf. Example By automatic excision using the Stratagene method, a bluescript vector (pSK 41-2gen) was isolated from one of these phage clones. Restriction of this plasmid DNA with KpnI and EcoRI resulted in the isolation of two DNA fragments of 950 bp and 1050 bp, of which the 950 bp fragment hybridized in the Southern blot with the insert DNA of the clone 41-2. The plasmid pSK- 41-2 DNA was sequenc.ed using a primer and reverse primer Chen and P.H. Seeburg (1985) Loc. cit.). The sequence determined using the primer is identical to the sequence of the insert DNA of the plasmid pUC 41-2gen from the Sau3A site in the 3' direction. The 1050 bp EcoRI DNA fragment of the plasmid pSK 41-2gen was subcloned into the EcoRI site of the vector pKS Maniatis et aL., Loc. cit.).
A restriction map of this DNA fragment was constructed.
Based on this map, restriction fragments were subcloned into the bluescript vectors and, after ssD r-reparation, sequenced (Stratagene instruction manual). This entailed the sequence being completely determined from the EcoRI restriction site of the 41-2 gene in the 3' direction as far as the Sau3A site. Table 17, which is a continuation of Table 16, shows the DNA sequence and the derived amino acid sequence of this EcoRI-Sau3A DNA fragment, which comprises 1050 bp, of the clone pSK 41-2gen. This gene section now codes for only 35 additional amino acids until a TAG stop codon supervenes. The 3' non-coding region of the gene is extremely AT-rich (AT content 84.7 and contains more than 11 stop codons in each of the three reading frames. The S1 mapping technique Ausubel et al. (1987), Current Protocols in Molecular Biology, Harvard Medical School, Boston) revealed no evidence what- 20 ever of an intron-exon structure of this gene. Finally, Northern blot analysis Maniatis et al.,.loc. cit.) detected an mRNA of the schizont stage with a size of 1.6 kb. It is therefore necessary to assume that the 41-2 gene has only a single coding section of 552 bp (AT
S.
25 content 73 The latter codes for an antigen of 21512 Dalton, which has a signal sequence (cf. Example but no repetitive sections. Besides the signal sequence, this antigen has two further hydrophobic sections in amn;r acid positions 73 to 85 and 130 to 147, which may have a 30 membrane-anchoring function.
S
Example 7: Expression of the inserts of the clones 41-1 to 41-5 and 41-7, 41-10 and 41-14 in the vector pEX31 The insert fragments of the clones 41-1 to 41-5, 41-7, 41-10 and 41-14 were, after restriction with EcoRI, isolated by gel electrophoresis, ligated into a vector pEX3lb which had 11 been digested with the restriction enzyme EcoRI and dephosphorylated Strebel et al. (1986) Journal of Virology 57, 983-991), and transformed into competent C600 bacteria containing the plasmid pCI857 Remaut et al.
(1981), Gene 15, 81-93). Individual colonies were examined by SDS-PAGE for expression of the Plasmodia-specific DNA sequences as MS2 polyermase fusion proteins. Induction was effected by increasing the temperature by the method of H.
Kupper et al. (in Y. Ikeda and T. Beppu Proceedings of the Fourth InternationaL Symposium on Genetics of Industrial Microorganisms (1982), ,oto Kodansha Ltd., Tokyo).
Expression of aLL 8 fragments was possible in high yield.
Example 8: Purification of the expression products SCultures of transformed bacteria were each shaken vigorously in 1 L of LB medium containing 50 pg/ml f ampi- 20 cillin and 25 pg/ml kanamycin at 28 0 C for 20 h. Addition of 4 L of LB medium which had been heated to 420C was followed by renewed shaking at 42 0 C for 4 h. The bacteria w ere removed by centrifugation, resuspended in 200 ml of phosphate-buffered saline (PBS) and disrupted mechanically.
25 The soluble proteins were removed by centrifugation, and the sediments which contained the expression products were washed twice with PBS and then washed successively with increasing urea concentrations (from 1 M to about 5 M) until the fusion proteins dissolved. Subsequently, 9 30 dialysis was carried out with decreasing urea concentra- S tions until the urea concentration wnich sufficed to keep the expression products in solution was reached. This process resulted in a purity of 60-80%.
Example 9: Detection of the antigen encodpd i 41-2gen It was, not possible, using rabbit antisera directed against
L
a I I I I 12 the expression product of the clone 41-2, unambigucusly to detect malaria antigen in the Western blot with schizont proteins. Therefore the aim was to express a larger DNA fragment of the gene 41-2. For this purpose, the restriction enzymes ALuI and EcoRI were used to isolate a 388 bp fragment of the 41-2 gene which contains 70 of the coding region. This fragment was subcloned in the Smal and EcoRI sites of the plasmid pUC18. The restriction enzyme E:oRI was used to isolate from this plasmid a 401 bp fragment, which was cloned into the EcoRI site of the vector pEX31b. After transformation it was possible to identify bacterial colonies which express a fusion protein of 26 kD (cf. Example This was purified (cf. Example 8) and used for immunizing rabbits. The antisera recognized, in the Western blot with schizont proteins, a 29 kD antigen which is encoded by the 41-2 gene. The difference between Sthe calculated molecular weight of 22 kD and the molecular weight of 29 kD in SDS polyacrylamide gels can be explained by secondary modification. Thus, the protein has an N-gly- 20 cosylation site in the asparagine residue in position 166.
Confirmation that the clone 41-2 encodes a very small antigen is obtained from Northern blot analysis. Thus, there was detected in the schizont stage of P.falciparum a 25 mRNA of 1.6 kb which hybridizes with the insert DNA of the clone 41-2. Northern blot analysis was carried out by known methods Maniatis et al., Loc. cit.).
Example Assignment of antigens of other clones Antisera against the purified fusio:- teins of the clones 41-1, 41-3, 41-4, 41-5, 41-7 and 41-10 were used to identify the corresponding antigens by Western blot analysis with schizont proteins. In this, the antisera against the fusion proteins of the clones 41-1, 41-3 and 41-4 did not allow unambiguous identification. The insert DNA of the clone 41-5 can be assigned to a 96 kD antigen. A group of liall offill. l 13 three 96 ko antigens of P.falciparum has been described Jouin et aL. (1987), Inf. Imm. 55, 1387-1392). Antisera directed against an expression product of the clone 41-10 recognize two antigens of 113 and 140 kD. The reaction with, the 113 kD antigen was identified as a crossreaction with the SERPI antigen (cf. Example 2) which is identical to a protective antigen. 41-10 thus encodes a 140 kD protein of P.falciparum.
It is common to all these genes that the antigens, or parts the,'eof, encoded by them, react with a serum which is directed against a 41 kD protein band which has a protective action. Only the clone 41-7, which, together with the clone 41-2, has the strongest reactivity with the antiserum against the 41 kD protein band, can be unambiguously 3ssigned to a 41 kD protein.
In the Western blot with schizont proteins, antisera directed against the fusion protein of the clone 41-7 unambigui 20 ously recognize the 41 kD protein band, which is also recognized by the starting serum. This clone thus appears to encode a subfragment of the 41 kD antigen. Confirmation that the investigated clones harbor partial sequences of various genes was moreover obtained by Southern blot analyses for 25 the clones 41-1, 41-2, 41-3, 41-7, 41-10, 41-14 and 41-15.
Example 11: Preparation of a genomic lambda gtll gene bank 2 pg of DNA from the P.faLciparum strain FCBR were incubated at 370C overnight with 14 units of the restriction enzyme EcoRI in 10 mM tris-HCl (pH 10 mM MgCL 2 1 mM dithiothreitol and 40 glycerol, and fractionated by gel electrophoresis. Under these conditions, the restriction enzyme shows EcoRI asterisk activity, resulting in the formation of DNA fragments from about 50 bp to 10 kb.
The DNA regvon between 500 bp and 7 kb was electroe uted and cloned by the method of T.V. Huynh et t (1985; in 14 DNA cloning Vol. I, a practical approach, ed. D.M. Glover) into the vector Lambda gt1l. A gene bank of 5 x 105 recombinant phage clones was prepared and was amplified.
Example 12: Isolation and sequencing of the 41-7 gene Since the clone 41-7 actually codes for a subfragment of a 41 kD protein (cf. Example 10), the aim is to isolate the complete gene. For this purpose, the genomic lambda gtll EcoRI* gene bank (cf. Example 11) was screened by known methods Maniatis et al., loc. cit.) with nick-translated insert DNA of the clone 41-7. This resulted in three lambda gt11 clones from each of which it was possible to isolate, using the restriction enzymes EcoRI and Sail, S an insert 3.3 kb in size. The malaria-specific portion of tte insert amounts to 2.3 kb. A restriction map of this DNA S. fragment was constructed. Based on this, subfragments 20 were cloned into the bluescript vectors (Stratagene) for Ssequencing. It was possible to elucidate the complete DNA sequence of this malaria-specific fragment which is 2.3 kb in size. Table 18 shows the DNA sequence of the 41-7 gene, j with the amino acid sequence derived therefrom. The gene i 25 has no introns. 525 base-pairs of the 5' non-coding region I were ascertained (AT content 84.2 as were 772 basepairs of the 3' non-coding region (AT content 84.2 The central section comprises 1086 base-pairs (AT content 64.4 and codes for 362 amino acids. The calculated :i .30 molecular weight of 39314 D for this gene agrees well with A .l-l the molecular weight of 41 kD determined by gel electrophoresis. This gene is transcribed into an mRNA which is 2.4 kb in size, as was ascertained by Northern blot analysis by known methods Maniatis et al. Loc. cit.). It was possible to deduce by Southern blot analysis (cf.
Example 4) that only one copy of this gene exists in each P.falciparum genome. It was also found that this gene is conserved in various P.falciparum strains (FCBR from Colombia, Palo Alto from Uganda, SGE2 from Zaire, ItG 2
GI
I~ 15 from Brazil, and FVOR from Vietnam). In addition, the malaria-specific DNA sequence of the clone 41-7 (strain T996 from Thailand) is identical to the partial sequence from position 464 to 729 of the gene isolated from the FCBR strain. Thus the clone 41-7 codes for the 88 Nterminal amino acids of the 41 kD protein.
It is evident from the derived amino acid sequence that the 41 kD protein contains no signal sequence and no repetitive sections. The UWGCG (University of Wisconsin, Genetic Computer Group) program was used to examine this amino acid sequence for homologous proteins within the NBRF protein data bank. It was found from this that the 41 kD protein is 66 homologous with human liver aldolase Mukai et al., (1985), Nucleic Acid Res. 13, 5055-5069), 66 homologous with rat liver aldolase Tsutsami et al, (1984) J. Biol. Chem. 259, 14572-14575), 68 homologous with rabbit muscle aldolase Tolan et at. (1984), J.
Biol. Chem. 259, 1127-1131) and 61 homologous with the 20 aldolase from Trypanosoma brucei CLayton (1985) EMBO J. 4, 2997-3003). The 41 kD protein thus appears to be the P.falciparum aldolase.
i Example 13: Experimental protection in an animal model: immunization Sof Aotus lemurinus griseimembra (karyotype VI) i This experiment was carried out to test the described i\ 30 expression products for their efficacy in inducing protective immunity in monkeys susceptible to P.falciparum.
The vaccine used in the experiment was a combination of the expression products of the immunologically strongly reacting clones 41-1, 41-2 and 41-3.
1. Design of experiment 6 Aotus monkeys of the abovementioned species (body weight 1,000-1,500 g, male and female animals bred by Behringwerke 16 AG) were randomized and assigned to 2 groups each of 3 animals.
Fusion proteins of the clones 41-1, 41-2 and 41-3 were dissolved in 3 M urea in PBS and mixed in the ratio 1:1:1 (final concentration: 300 pg of protein/mi). 3 animals were immunized subcutaneously 3 x, at intervals of 3 weeks, each time with 1 ml of the combined fusion proteins. A admixture of 50% AL(OH) 3 /45% lecithin/5% saponin to the antigen was used as adjuvant.
3 animals in the infection control group likewise each received an injection of 3 M urea in PBS adjuvant i without antigenic component in accordance with the abovementioned scheme.
0* In order to ensure that the experimental P. falciparum infections of the animals were as near the same severity i as possible, all the monkeys were splenectomized eight 20 days after the last immunization (increased susceptibility).
On day 67 after the 1st vaccination, all 6 animals were li infected intravenously with 5 x 10 parasitized erythro- S cytes. The strain chosen for challenge was P. falciparum 25 Palo Alto (Uganda) which was adapted in vitro to Aotus erythrocytes and was transferred directly from a splenectomized donor animal parasitemia) to the experimental a animals. This strain is distinguished by high infectioj sity compared with other P. falciparum isolates. It is 30 also of interest to mention that this strain is heterologous in terms of provenance and serotype to the strain T996 (Thailand) used for obtaining the immunization artigens.
Physiological, parasitological, serological and clinicochemical parameters were examined regularly during the entire course of the study (before and after immunization and after the challenge).
ii
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17 2. Results No pathological changes in any of the physiological (clinical manifestations, temperature, weight) or clinicochemical (erythrocytes, hematocrit, ESR, serum enzymes GPT and GOT) parameters investigated were seen throughout the immunization period. Additional drug-safety investigations (acute subcutaneous toxicity in the mouse, local tolerability in the monkey in accordance with the specifications of the European Pharmacopoeia) demonstrated adequate safety and tolerability of the vaccine preparation used.
2.1 Parasitemia
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C
S
The main parameter for assessing the value of an induced protection is the detection under the microscope of parasitized erythrocytes in the peripheral blood of the experimental animal.
A few parasitized erythrocytes (Less than 1 per thousand) were detectable in the Giemsa-stained blood smear from the non-immunized animals as early as 7-10 days after the infection. The appearance of parasites in the immunized 25 animals was delayed to 10-15 days after infection, and they reached maximum parasitemias of 1-2% and controlled the infection spontaneously. There was one intercurrent death of an animal from pneumonia.
Whereas one animal in the non-immunized group was able itself to control a maximum parasitemia of it was necessary for the two other animals to be treated with mefloquine (Hoffmann La Roche) after a parasitemia of >10% had been reached, in order to prevent the infection taking a lethal course. The Palo Alto strain used for the challenge proved in preceding infection experiments to be chloroquine-resistant.
Figure 1 shows, on the left, the course of the parasitemia in Aotus monkeys af ter immun izat ion wi th a combinat ion ft. vaccine composed of fusion proteins of the c~ones 41-1, V41-2 and141-3 and, on the right, that of the controL (non immunized anima~.s).
0@ 04 6.
6 0 So
AW*
,19 TABLE 1: NucLeotidle sequence of the maLaria-specific DNA inse-rt of the c~one 41-1, and derived am 4 no acid sequence TOA AT CTTTATGTATTTTTAAA AAGAATAA4 A&AASGTCATZAAATALT7CA L S er L e a Cv s1 leF h e L, a I nA 9Am n rL y sL y s VaI S~ Ee r S e- A s nAs n EerF'heLew 1 ems pF he~rgAsmE erHsTrEr,' s I I e~r1e tL E-tT hrG I U;' IrL\ Q heE nsrI YI LeuLeuA.rLvsGI I u11eGI nMet~s Q 1 uerQ-2 nG I uro: 0 go Z 51: ZKC 20,:l;LLL~HSr,, ErSFLLft. C S.7GA TGO4S TTGT T5 TC G r oS rGI 5e ri L s'LreG u G Iov.G rVI LYsr L eLYEGI Lves 5 G A ,'SI U> LUTrT ft. ftAT ft ft ftr C 20 TABLE 2: Nuc Leot ide sequence of the mal aria-spec if ic DNA insert of the clone 41-2, and derived amino acid sequence 30 AACATGTG-4GGAAATATTTATTTCAGCATTFCATCm. GATT TAC:'TAA.ATCTCAAGA'-TA GTA Hi sVa lTrpLysTv rLeuPheG~nHi sSe rSe rAspLe uLeuLys Se rGonAspS e r 90 12i0 TTTATGAGTATATGAT.ATGTCATAAAAkATA TTTATAAA'AATTATAAATTAC CAA I eTy r~luTy rMe t IIe CysAs pLysAsnI Ie L; u~e uAsnLys Phe IIeAs nVa 1 rcL 130 150 170 A.AGATTATGGAAATATAA.ATTC-TGCTGC CTTTGCAGCAGGT",ATTGTTGA.AGGCTTC CTCT ySAS pTy rGI yAs nlI eAs nCysAl &AIa PheAl &AI aGI yII e Va IGI uolyPh eLe uC 190
GTAGTTCTGAAT-C
ysSerSerGluPhe 21 TABLE 3: NucLeotide sequence of the malaria-specific DNA insert of the cLone 41-3, and derived amino acid sequence 30 s0 CAGTAAAAA TTTTAAAAAAAAAGAAAAATTTAAAAAAATAAAGGA-A.ACCA CTGA TGAJA Val Lys IIeLeuLys Lys LysLysAsnLeuArgLysI IeLysGl uTh rThrAspGluG 90 110 AGAAAAC TTCAGATAATGTTTC T CAAATG TATGAAAGAAAAGGTGGA CCATTAC CAC CC I uLysThrSerAspAsnVaIS e rGnetTyrG1uArgLysG1yG IyProLeuProProP 130
CCGAACTTAGAAAACA
roGluLeuArgLys -22 TABLE 4: NucLeotidle sequence of the malaria-specific DNA insert of the clone 41-4, and derived amino acid seauence 30
TGA
I IeProGluPheLeuGlyG~nTyrHi SAsnVa roxi sVa IPh eLysAspTyr~etS 90 110 GTTC CAATGATTTTATAAG TGG TATAAA TAA TA TAAATGA.ATCAGATG CTC TTTTTA..TA erSerAsr1AspPhe I IeSe rGly I IeAsn.Asn I IeAsnGluSerAspA a LeuPheAsn-.
130 150 A CATACAATATA TAXA C CA.AGC GAkATGA C CAAGAAGAAAACAA-ATT sn I IeGlnTyr I I eAsnGlniAlaAsrLAspGIn G IuGluAsn Lys to S -23- TABLE NucLeotide sequence of the malar ia-specif ic DNA insert of the clone 41-54, and derived amino acid sequence 30 TGTTTrGAT-AATAGTGATTTTATTAAA £.*CAATA-ATG3)ATTCTAATAAACAATT-AAAAkACf; PheAspAsnSerAspPhe I IeLysSe rl eMetAspSe rAsnLysGl nLeuLys Lys-' 90 110 TAAGAGAACAAAAT"TCTGATTTAA.AT CATATT.TTAATGATTCTC-AGACT7TTAAAACAA 7 euAr gGIuGl rAs nS erAspLeuAs nH isII e LeuAsn.AspS erGI nTlhrLeuLysGI nS 130 150 170 C TTTTGAAATGAT-TAAGA.ATC CAT CTTTGA TGAAAGAATTAATGAAAAATA CTGATAGAG e rPheGI uMetI11eLysAsnP roS erLeu,,tLysG uLeu~e tLysAsnTh rAspA rg."' 190 CTATTAGTAATATTGAAGCCATAC C laIJleSerAsnhleGluAlalle t o* e so~ of 0 V 24- TABLE 6 NucLeotidle sequence of the maLaria-specific DNA insert of the clone 41-6, and derived amino acid sequence 30 TCGTTGTC CTTTC CTTTGGTGATAAACGCA.ATGAGATA.AACAAAAA ATTGACACGT7T ValValLeuSerPheGlyAspLysArgAsnGlulleLysGlnLyslleAspThrPhec 90 110 GTGGTGTAAGTAATGAAGAAAAGGAGAAACTAAAGGAACAATGGAAATGCTLATGA-AGC-T
A
ysGlyValSe rAsnGluG1uLysG1uLYSLeuLYSGluG~nTrpLysCysTyrGIuA1 aL 130 150 A.ATATG TA.AAGGAGA TAATAAAA GTAAAG ysTyrValLysGluAspAsnLysSe cLys 0 p 25 TABLE 7 NucLeotidle sequence of the maLaria-specific DNA insert of the clone 41-7, and derived amino acid s~quence 30 TGAATGCCC CA:,AAAAAkTTACCAGCAGATGTr'Gc CrAAGAATTAGCAACCACCGC CCAAA AsnAla~roLysT',ysLeuProAlAspXa1AaGuG.'uLeuAIaThr'ihrA.!aC~nL 90 110
AGCTGTTCAGCGAAAGGAATTTTAGCTGCTGATGAATCAA'ACAA~CCATTAAGA
ysLeuVa1G~nAlaGlyLysGlylleLeuAlaA)JzAspGlu~e:-ThrGrInThrxleLysL 150IS 170 AAAGATTCGAC-A.CATCAAATLAGAGAACAAATAG3AAAAC kGAGCTAGCW ACAGAGATT ysArgPheAspAsnrIe~ysLeuG'IAsnThrIeGuAsrAgAaserTy-ArgAspL S9 0 210 2130 TATTA= TGGAACTAAAGGATTAGCAAAATTCATTTCAGGAGCAATTTT,ATTTGAAGAAAk euLeuPheGlyThrLysGlyLeuGlyLys~lie 1leSe LGlyAJlalleLeuPheGluG.uT 250 0 CATTATTTCAAAAGIAATGAAGC CGGT 26 TABLE 8: Nucleotide sequence of the maLaria-specific DNA insert of the clone 41-8, and derived amino acid seqUence 30 TAACATTTTCTGTAGATACAACAAAATTTAAT~GCATC TTATCTAG-1G ACAAGA.AA, ThrPheSe rValAspTh rLySG I nAsnLeuAsniAl aSe rTyrSe rSerG IyG InG I.uAA 90 110 ATAAA CAAAATGAA T C TGATGGAkkAGAAtTG.AGAAGATGiAAGAAAATLAAGG TATATG SnLysG InA snGl uSe rAs pGl y'y sClu.A snG IuG IUAS pGl uG IuAsnLys Va ITy rA 130 150 170 ATTTAATACTGGAAAA TATAGA.AC CTAACkAAAAATACCCATCATAAAkATCGTTAAAG spLeulleLeuGl LAsn': I eGluProAsrnLys Lys I IePro I Iell eLys I IeVa ILysG 190 210 230 *stAA.ATAAAAAAAGATCTTAA TTTAAAACAAG CAAAGGATTTAG TTGATAATTTG CCACA I u I Ie Lys LysAspLeuAsnLeuLysGlraA1 a LysAspLeuValAspAsnLeuP ro 0 "O 0 *00 a 0 0 0 27 TABLE 9: NucLeotidle sequence and derived amino acid sequence of the maLaria-specific i ns e rt DNA of the :Lone 41-9 S.e S S S.
S
5*
S
S.
S S
S.
S
555 S. S S S
*S
AAAA TAAAAA TTATA CAGG TAATTCTC CAAG TCAAATAA TAAGLAAG TTA.ACGAAG C T T As nLysAsnTyrThrG IyAsrnSe rProSe rG1uAslAsn Lys Lys Va1AsnG'uA a L 90 110 TAAAATCTTACGAAAATTTTCTC CCAGMAGCAAAACTTACAACAGTTGTkACTCCAC CTC euLysSe rTyrGluAsn Ph eLeuP roGluAlaLysVa IThr~'hrVa IVa IThrProProG 130 150 170 AACC.AGATGTAACTCCATCTC CA TTATCTGTAAGGGThAGT'GGTAC,TTCAGGATCC AC; lnProAspVa IThrProSe rP roLeuSerValArgVa!Se rGlySe rSerGlySe rThrL 190 210 230 AAGAAGAAACACAAATA C CAA C".TCAGGC TCTTTA TTAACAGAATTA CAA CAA GTAG TA C ysGJluGluThrG~nrUIeProTh r SerGySerLeuLeuThrGuLeuG~nG~nVa IVa IG 250 290 55 S S
S.
S S *5
S.
55 SS S 55
S
S
*SSS
SS 55 S S
S
AATCACAAAATTATGA C GAAGA.A GATGATTCCTTAGTTG TATTAC C CATTTTTGGAGAAr I nSe rGlniAsnTyrAspG I uG I u.AspAspSe rLeuValVal LeuPro I IeP he G" yC1 US 310 330 350 CC GAAGATAATGA CGAA TATT TA GATCAAG TAG TA.AC TGGAGAAG CAATATC TG TCA CAA e rGluAspAsrLAspGluTyrLe uAspGlnValVa ITh rGlyGluA 11 leSe rVa ITh rM 370 390 TG GATAATAT C CTCT CAGGATT TGAAAATGAATATGA etAspAsn I leLeuSe rGlyPheG I uAsnGluTyr r w -28 TABLE Nucteotidle sequence and derived amino acid sequence of the maLaria-specific insert DNA of the clone 41-10 30 AATCT CATTC TGAC G,%A.ATATTGTAA CTTTA CAAGGAAA.AC T" AGAAATA CAG CTATC 7 Se rHisSerAspGIauAsn I eVa IThrLeuG~nGlyLysLeuArgAsflThrAl aI IeC 90 110 CTATAACAATGTTGATGAA-iGC:ATATTAAATAAAAGAGGTCTAACA.1TAC CTAG" GAA' ys I IeLysAsnValAspGluTrp I IeLeuAsnLysArgGlyLeuThrLeuP roSe rGluS 130 150 C.AC CTA.ATGAATCAC CTAGTGAATCAGATAGTTATCTT AA er~roAsnGluSe rProSerGluSe rAspSerTyr~eu
SC
CC 0 g. Cog C
C
*0 C S e.g.
S.
C
ggg CC 0 S S
S.
CC
S S
CC
CC
C 0
SC
S. C C
CS
S S Cggg C 55CC *S 50 CS C C C -29- TABLE 11: NucLeotide sequence of the maLaria-specific DNA insert of the clone 41-12, and derived amino acid sequence 30 AT GAGGAGkAGA TTATA T TAATGA TGA T AAAT~ATTA CA TAT TGA TA CAT T TG GluGlyGluGluLeulleLeu.AsnAspAspGlnLAsnLysLeuHislleAspThrPheG 90 110 2 AAAAATACAAATATCTC-ATTTGTGAAA.ATATTAATAATGACAAATTTGTTATAAAAAA:A luLysTyrLysTyrLeulleCysGlu.AsnlleAsnLAsnLAspLysPheValIleLysAsaA 130 150 170 AT CAAATTA CAA CAT TTGAAAA CTTTTTGAAAA.AC CAA CAAAATTTTGAAA ATA.A snG~nIleThrThrPheGluAsrPheLeuLysAsnG~nG~n.AsnPheGlulle 5* 0 S S0 .00 0 00 0 S 0 *0060 TABLE 12: Nuc Leot ide sequence and der ived am ino ac id sequence of the malaria-specific insert of clone 41-13.
40 AsnAsnGI uAsnMe tAspLysGI rAsnVa1As n 11eGlnAs nGIuGI yAsflGIyPh eA 100 120 ATA.ATAATAAAAA TAATAATGAT CTTTTAAATGTTTATA TATCAC CTA.ATATGA TTAAT C Sri.AsnAsnLysAsrnAsrnAsnAspLeuLeuAsnVaI TyrI I e Se rP roAsnMe tlI1 eAs-H 140 160 180 ATT C TTTATC TT CA.ACTT TGAAAAAAAAATAAAGAAGATAA CAAAA TGA.ATGA CAA TA i s Se rLe u Se r Se r Th r C ys G IuLy sLysA s nLys G 1uAspA s nLy sMe tAs rAspA s nL *200 220 240 AATTTCTTAATAG CAG TAG TAAAATGAAAATTC CAGAGATAAG TA CGAACAACTCAAATG :900: y s Ph eLe uAs n Se rS er Se, rL ys M e tLy sI Ie P roG Iu II e Se r Th rA sAs nS erAsn G *260 280 300 AAAAGATTGTTAATGTGTCAAATGATGAAATGTTAGTATATCATAATTTAACCGTATT1,A luLysileValAsnValSerAsrAspGlu~etLeuValTyrHi sAQ~nLeuThrVa1LeuA :320 340 360
KATGTAAAGGAACAAGGAGGTGTA.ACAGAAGAATCCGAGCTGTATAAAACGCACALTATTTTG
snValLysGluGlnGlyGlyvalThrGluGluSerSerCyslleLysArgThrTyrPheV *:380 400 420 TGGCATCAATTTTATGATTCATATAATATGAAATGAAAAAkTAACAGATGATAATATGC alAspGlnPheTyrAspSerTyrAsnietArgAsnGlULysIleThrAspAspAsn etG *0*440 460 480
AAGTAGAAGATATATATA.ATGTAAAGGAAAATATAAAAAGAACTCTAAAAGGTGATGGTC
InValGluAsplI eTyrAsriValLysGluAsnI leLysArgThrLeuLysGlyAspGlyP 500 520 540 fee* CTGA TGATGT CAAAA CGA.ATATG CTGAG TGAAGATAA TAG TTATG CAAG TGG TTTA TGG G roAspAspValLysThrAsnle tLeuSe rGluAspAsnSe rTyrAl aSe rGIyLeuTrpG 560 580 600 GTAACGAAATAAACTTTATTAGTAATAATCAAAATTGTTTAAATAG CTATG.ATATATCAT IyAsnGlul IeAsnPhell eSe rAsnAsnGluAsnCySLeuAsnSe rTyrAspl Ie Se rC 620 640 GTGATGAGAAATATATC CCAAATGAAGAGGAACAGGA ysAspGluLysTyrIleProAsnGluGluGluCln -31 TABLE 13: Nucleotidle sequence and derived amino acid sequence of the malaria-specific insert of clone 41-14.
30 ACAA CAA TATGAA CA GAATAAA TAG TTTAA-A CAA TAAAAA TAA TA TTA.A C C C-TA TAAA 7 As nAsn~le t.AsnArg I IeAsnSe rLeuAsnAsnLysAsrnAsn I eAsnPro I I eAsnG 90 110 kATACAATGATGAAAAACAAAACTTACTTAACGNCCATCTTCAGTYCAATCAAGTAA.ALT In TyrAsn~spG uLysG InAsnLeuLeuAsnXxxRi sLeuG IanXxAsnGIn Va1As~nT 130 150 170 ATNATXATA.AC CTTG TGAATG G C YTTCATANAANNAAATTTTTAAGCAATAATAA-TTA TA yrXxxAsnAsnLeuValAsnG IyXxxHi sXxxXxxLys Ph eLeuSe rAsrnAsnLAsnTyr I 190 210 230 0 0 0 TTA.ATACTACAGATATTAATGGAAATAATATGATTAGTCATAATGATCATATGAATA.ATA 0 009 1eAsnThrThrAsp I IeAsnG IyAsnAsnMe t IIeSe rHi sAsriAsp~i s~1etAsnAsnL :.250 270 290 *AATTATACAG TAATA TAAA CAk TAATTA TTATTATA.ATAGGG CTAAGAATGAAATTC CTA :ysLeuTyrSe rAsn I IeAsrnAsnAsrnTyrTyrTyrAsn.ArgAl aAsn.AsnGl u 11eP roA 310 330 350 ATAATAATAGTAA CkATCATAATAATXATTT CAATATA TATGAATC CAAATAC CAAAC CA SO srLAsnAsriSe rAsrnAsnHisAsrnAsrisnPheAsnleTyrGuSerLysTyrG~nThrM 55*.*370 390 410 TGATT CATAACAACAACATAGGACA.GAT CTAAAACAACAAATAAATAATTATA.ATGAAA et~leHa sAsnAsaAsnIleGlyGlnls LeuLysG~nG~nIleAsnAsnTyrAsnGluA S430 450 470
ATACATCTTCTAATAATAATTTAAGTATATCTCAATTACTTGAGGGAAATACAAATTTTA
snThrSerSerAsnAsnAsnLeuSerlleSerGnLeuLeuGluiGlyAsnThrAsnPheI *490 510 530 0*55TAAATATTTCTAATACATTTATTAATACGAATTATTCTAATGATTTTCATCA 0 leAsnhleSerAsnl-hrPhelleAsflThrAsflTyrSerAsnAspPheHis 32 TABLE 14: NucLeotidle sequence and derived amino acid sequence of the macaria-specific insert of clone 41-15.
30 A CAG CAACAACAATAATAATAATAATAATAATA TTAG TAA TATA TTA GTA ATAA Se rAsnAs nAs nAS rAS rAS rlAS As rAsnI11e Se rAs riAs n IIe Se rA 5n.As nLysA 90 110 ATTGTGATCGAATACGATTATCAGGAAGAATATTTGAAAGATAAAG CATTATACGATT1CAG spCysAspGl uTy rAs p'y rGl nGl uG UTy rLeuLysAs pLysAl aLeuTy rAs pS e rA 130 1S0 170 ATATGGAC GAAAATACAAAT CAAC TT CACAATAATGAACATCATA CAAATCAkACATCAC G so* sple tAspGl As nTh rAsnGin Le uHi SASnrAsnGl uHi s~is Th rAsnG InH 1s'i sA 00190 210 230 of CAAATTGG CAT CAT CACAAACAT CAAAAG CAA CATTT CAAA CAAC TTA TTGAT CA TAA CA 8:46 1 &AsnTrpHi sHi sH is LysHi s GInLy s GnHi sPh eLysGI nLeu I eAspH i SAS nA :0.
0 0 so 250 270 ATATGATAAATAATAATGATAATAATATTAT
CA.ATAA
a o sn~e t I IeAsnAs rAs rAs pAsnAs n Ie I IeAsn see* a a~ I I 33 TABLE NucLeoticle sequence and derived amino acid sequence of the ma aria-specific insert of cLone 41-17 40 GCACTG C CAC C CTTG TTG CGGAAGAATTG CAC CAG C TCGG CTATTC-AC TG GCGCTGGSTC ThrAlaThrLeuValAlaGluGluLeuHisGInLeuGlyTyrSerLeuAlaLeuGlyA 100
GCGAAGTAGTTAATGAAAGTAGCCGGATGGGATTACCTGATGAATTC
rgGluVa I Va !As nGluSe rSe rArgMetGlyLeuProAspGluPhe off 0 .::Oq 00 *fee f34 TABLE 16: sqec- Nuc~otile equnceand derived amino acid sequence of the 51 end of the P.faLciparum (isoLate FCB3R) gene specified by the insert of the c~one 41-2.' 30 TTTAAAAATTTTAT AAATAATTTTTCTCTTTTATATTTAATACATCTATAAGTATATAT3, 90 110 TAATA.ATTTGATACACAAGAA.ATGTGTATTLTTTAATATATATATATATATATATATATAtT 130 150 170
ATATATATATATATATATATATATATATATATGATATATATAAATATATACATTTATT"TA
190 210 230 TTC CATAATTTATTAAAkAATAAATTTATATATTTTATTTTATTTTTTATTTATTTATTTG 0 00 250 270 290 so* TATATATTAAATCTTTTCAATGGAATAAAATTCA.ATCGGATCGTTATATAAACTTTATTA 06 96 *0310 330 350
*~.TATCAAATAAA.ACACTTTTTATAATAATACGAAAA.ATATATTTCCTTATTTTTATGTT-T
sees 370 390 410 CAAAATTTTAGTAGACTTATA.ATATTATTATGGATAACATLTAACAAATAAAATATTAT-i 430 450 470
AGTATA.ATATGTAAATTATTTTTTTTTTTTTACAGTTTATATJTTTATGACATATAATG
49 51053 Ste TGATA.AATAAAATTGATTAATTATTATTATATATAATTACTCTTGTAATTTAITTAAAAk
TG
00S 550 570 590
GTATATTATATATATATATATAATTTTTTTTATATTATTTGAATAAAAATATTAAATAXA
610 630 650
AATTTTGTGTTTGGGTAAATCATAATAAGTGCTAACGTTCATAATTTATCTCATTAAAAA
04670 690 710 *00 ATAGAA.ATGAAATATAATATTTACGACAGTACATATATATATATGTATATTATTAAAAA :S050 0 730 750 770
AA.ATAAAAATAACACATATATATATATATATATATATATATTGATAATATATATGTTTTA
790 810 830 AGTATGGATAAATCAAAAAGTTCCATAGAGAAAGAkATTAAkATAGGATAAAAr-AG GATGTG Me tAspLysS e rLysSe rSer IlIeGluLysGluLeu; snA rgII e LysGl nAspVa 1 850 870 890 AGCTTAAGCGCATTTAGTATCCTCTTTAGTGAAATGGTAC ,TATTGTTTATATAAXAGT Se rLeuS erAl aPhe Se r 11eLeuPhe Se rGl uMe tva IGInTy rCysLeuTy rLys Se r 910 930 950
AAAAGAGGATATCGAATAGAAGATTGTTTACATGAAATGGGTTTACGTGTAGGTTATAA
LysArgGlyTyrArglleGluAspCysLeuHi sGluMetGlyLeuArgValGlyTyrLys 970 990 1010 TTAA.ATGAATATTTAACATATAAGA.ATAAAGTGAAAkAGAAGCATAAATATTATTAkATATT LiuAsnGl uTyr LeuTh rTy rLysAsflLysVal LysA r g~er 11eAsnI Ie I IeAsn I Ie 35 TABLE 16 1030 1 O5' 1070 TTAACATTCATATCTAAAC-ATGTGTGGAAPATAT TTATTTCAGCATTC-ATCTGATTTACTT- Le UmhrPh e 1e Se rLysi sVa T rpL'ySTy rLeuPheGl2Hi sSeS e rAspLe uLe u 1090 1110 1130 AATCCAAGA TAG TATTTA TGAG TATA TGATATG TGATAAAAATATTTTA TTAAA.APJ LysS e rGIaAs pS e r IIe Ty rG uTy rMet I Ie CySASpLysAsn I Ie Le uLe UASnLys 1150 1170 1190 TTTATAA.ATGTAC CAAAAGATTATGGAAATATAAATTGTGCTGCCTTTGCAGCAGGTATT ?he I IeAs nVa IP roLysAs pTy rGIyAsnl eAsnCysA aAaPheA aA aG'y I e 1210 1230 GTTGAAGG C TrCC TC T0TA OTT CTGkATT C Va IGI uG yPhe Le uCys Se r S erG.IuPhe e.g.
C
C. S S 0*O* 0e .09 SS C Ce
CS
C C S
SC
S.
CO
S
OS CS S S
CC..
00 *e
C
0
A*
Nwfis -36 TABLE 17: Nucteotidle sequence and derived amino acid sequence of the 3' end of the P.faLciparum (isoLate FCBR) gene specified by the insert of the cLone 41-2.
1240 1260 1280 GAATTCCAkAGCAGATGTTACAGCGCACACTATTCATGAAGGCGATGATAkATTATAACACT GluPheGlnAlaAspvalThrAlaHisThrIeHisGluGyAspAspAsflTyrAslThr 1300 1320 1340 ACTATT TTTATTAAAT TTTAT CCG GAAG TA GTGGAAAGA GAAAAAAC CAC TAGA TA TTC ThrIlePheleLysPheTyrProGluValValGluArgGluLysAsiHi s 1360 1380 1400 ATATAAGGGTC-ACACAkATAAkATATATATATATAATACATGTTGTATAAG TTGTCAAAAAA." 1420 1440 1460 000 TTTATATGGAAAAAAATAAATTAAATATGTAAATATATATATATATATATATATATATAT 1480 1500 1520
::TCTTTCTTTCTTTCTTTTTTTTTTTTTTTTGTTATTATTAATGTATTATTTATCCTTATG
gos1540 1560 1580 CATG GGATTATTTAA CAAA TTTATTGA TAAA.A TAAA TGTA CCC CTTT TTTTTTTTTC TTT 1600 1620 1640 ofTTTTTC TTTTTT TTT TT TTTG TATA.AA CA TAT TTA TATA TA TTTATATTTAA TTAAA CCT 1660 1680 1700 go TTTTTAACATTTTAAATCTATATGAAATA-ATAAATGAAGAC-ATGACTATTTTAATACAAG "sees, 1720 1740 1760 50 ATTAATGGTTCCTTAAATTTCACATAAAL.ATAAAACATAT-ATAATATAT 0*0 ~1780 1800 1820 toATA TA TATATAAAA CA CTTGG TTC TA.ATTT TTTTTTTTTT TTTTTTTTTTTTTTTTTTAA 1840 1860 1880 sof TTTGTATGGAGATATTATAATATTTAAACAATATATATGAC-ATATATAGAGGACATA
CTG
1900 1920 1940 go TTACCJATATTTTCAATACATTGTTGGAATTTTTTATTTTTTCATATATCA,'TACATAAGA 1960 1980 2000 C CT C TGGAAAAGAAAAAAG TAATAAAGTGTC TTATATA CTATTAATTTTGAATATAGAT 2020 2040 2060
TTTTTTTCTTTTCTTTCAAAATTAAAAGTATTCTATCAATGTATGTAAAATATATAATT
2080 2100 2120 TTA CTTTTTTTTTG TT CTTTTTTC TATTTT TAA.ATA CG TATG TC CTCG TTTTTTTTTTTT 2140 2160 2180 rTTTATAACATTATTTTGCATATTCCAAATTTTTTCTATGTGTCCA&1KhAAALA A 2200 2220 2240
AAAA.AAATAAAGTGTTAATAAAAAAATTAAATAATATATGTGAAGATACTTTTTTTAATA
2260 TGCATATGTATATATATTTATATATAT~hGATC TABLE 18: NucLeotidle sequence and derived amino acid sequence of the 41 kD protein of P. faLciparum 30 AATTTTTTTTTTGAA TATTCTTTTTAG CATTTGATATAATATTA TTTTGAAAATGG TPJAG 90 110 AATATAAAACATTTAAGAAATAAATAAAAG TACAGTGTTTATATATAC CG'rATAAAT7;A.A 130 150 170 TA.AGTG TATATATATATATATA TATTAAA TACATTTATATTA TTAATTTA TACCAATGCA 210 230 TAGTTATA', ATATATATACTATTATATATGTATTCATTTTATTCTGCTCACATTATTTAT 250 270 290 GCATATG CTTC C TTTATAATAAkATATATTC GTATTAACATTCAAGAAATGAGGACGAAA.
310 330 350 AT TCC TTA.AT TTA CATATG TAT TTTTTTAT TAA TTAAAAAAAAAAAA TAG TAAAA..
*370 390 410 *TAAGTATAGGCATATATTGAATk.ATGTGCTGTTGAATTGATTTATATATATATATATATA :**430 450 470 :TATATGTATATT TATTTATATTTATACATATGGGAATATTATATATTT TC CTTTTTTC TT 490 510 530
ATTTTTATATTTTTATATTTTTTTTTAGGCTCATTGCACTGATATATGATGCCCCAAAA
MetAsnJlaProLys *'550 570 590 0****AA.ATTAC CAGCAGATG TTG CCGAAGAATTAG CAAC CAC CG CC CAAAAG CTTGTTCAAGC T Lys LeuP roAl aAspValAl aGluGl uLeuAl aTh rTh rAl aGl nLys LeuValGlnAl a .*:610 630 650 GGAALAGGGAATTTTAGCTGCTGATGAATCAA CACAAAC CATTALAGAAAAGATTCGACAAC Gl yLy sGlI ie L euAl &AIaA spG;Iu Se r Thr G InTh r I leLy sLysA r gPh eA spAs n *670 690 710 ATCAAATTAGAGAACAC.A.ATAGAAAACAGA GCTAG CTACAGAGATTTATTATTTGGA.AC T I Ie Ly sLeuG 1uA s nTh r 1 e G IuAs nA rgAIa S e r Ty rA rgASpLe ULeu Ph eGlIyTh r 730 750 770 Amw AAAGGATTAGGAAAATTCATTTCAGGA.A-ATTTTATTTGAAGAAACATTATTTCAAAG Lys GlIyLe uG, IyLys P he 1 leS e r G IyAlaI Ie Le uPh eGIu G IU Th rLeuPh e GIn Ly s 790 810. 830 AATGAAGCCGGTGTAC CAATGGTTAATTTATTACACA)P.TAAA.ATA TAATTCCAGGTATT AsnG Iu.Ala G 1yVaIP r oM etVa 1A s nLeu L eUH isAs nGuA s n I Ie I Ie ProG IyII e 850 870 890 AAGGTTGATAAkAGGTTTGGTTAACATTCCATGCACAGATGMGAAAATCAACTCAAGGT Ly sValAs pLy sG Iy L euVa lAs nIIe P r oCy sTh rAs pG 1 UG Iu y sS e rTh r G1nGly 910 930 950 TTAGATGGA'TAGCAGAkAGATGCAAAGAGTATTATAAAGCTG2GCAAGGTTTGCTAA.A 38 TABLE 18: LeuspGlyLeu8 GuAr gCysLysG uTy rTy rLysAiGI yAlAr PheA a Lys 970 990 1010 TGGAGA.ACAGTTTTAGTTATTGACACAGCCAAAGGAAAACCAACTGli
TTATCAATT'CAC
TrpArgThrValLeuValleAspThrAaLyslvyys?:L~rrrAcpLeuSerIlel44 s 1030 1050 1070
GAAACTGCATGGGGATTGGCTAGATATGCATC'LATTTGTCAACAAAATAGATTAGTTCCA
t ,luThrAaTrpGlyLeuA1 a.ArgTyrAlaSr~IeCysGJ~nG~rnAsfLArgLeuValPro 1090 1110 1130 A TTGTTGAAC CTGAAAlTTTTAGCTGATGGACCACACTCAATTGAAGTTTG
TGCAGT~TTGTA
'UevalGluProGlulleLeuAlaAspGlyProHisSerIeG~iValCysA aValVa 1150 1170 1190
ACTCAAAAAGTTTTATCATGTGTATTTAAAGCTTTACAAGAAA---TGGTGTJ'-.TTATTAGAA
Th rGJnLysValLeuSe rCysVa1PheLy-sAlaLeuGlnGluAsnGlyValLeuLeuG1 u 1210 1230 1250 GGTGrCATTGTTAAkACCAAAC.ATGGTTACTGCTGGTTATGAATGTACTGCTA.PAACACT GlyAlaLeuLeuLysProAsnMetVaIThrAlaGlyTyrG1uCysThrAlaLysThrTh r 1270 1290 1310 a a ACTCALAGATGTTGGTTTCTTAACTGTCAGAACCTTA,'AGGAGAACTGTACCACCAGCCTTA a. ThrGlnAspVa1GlyPheLeuThrVaArgThrLeuArgArgThrVa1ProProAlaLeu 1330 13b0 1370 CCAGGTGTTGTATTTTTATCTGGAGGACAATCAGA.AGAkAGAGGCTTCTGTTAATTTAAAT ProGlyValValPh eLeuSe rGlyGlyGlInSe rGluGluGluAl aSe rValAsnLeuAsn 1390 1410 1430
TCAATCAATGCTTTGGGTCCACACCCATGGGCTTTAACCTTCTCTTACGGTAGAGCTT-A
SerIleAsnAlaLeuGlyProHisPrOTPA~aLeuThrPheSerTyrGlyArgAlaLeu *1450 1470 1490 CAAGCTTCAGTATTGAACACATGGCAAGGAAAGAAAGAAAATGTTGCAAkAGGCAAGAGA U GlrLAaSerValLeuAsnThrTrpGlnGlyLysLysGluAsflValA1aLysAlaArgGlu 1510 1530 1550
GTTTTATTACAAAGAGCTGAAGCCACTCCTTAGCAACTTATGGTAAATACAA~AGGAGGT
Go ValLeuLeuGlnArgAlaGlu~kl&AsnSerLeuAlaThrTyrGlyLysTyrLysGlyGly 1570 1590 1610
GCAGGTGGTGAAAATGCAGGTGCTTCATTATATGA~AGAAATATGTCTATTAAAAACTT
AlaGlyGlyGlu.AsrxA~aGyAaSe rLeuTyrG1IJLysLysTyrVa1 Tyr 1630 1650, 1670 CACCAACCAAAAATGAkATAATAATAATAATAAATAAATTACTAAATGAATGGTACTATAT 1690 1710 1730
TTTTAAAATAAGGGTAATATATTTCTGTATGTATATATATATATATATATATACAAATA
1750 1770 1790 TG TGATATAAAAAAAAAAAATG TAATATATATC GAT CAATG TATAT CTACGA TAT 1810 1830 1850 .4 39 T AB E 18: ATAA.A TA TATA TTTATT CATA TC TC CT TTTTTTAGA rCA TA TA T Tk. TAA TA C C TAAJ..A 1870 1890 19)10 AT. T-r.TATTTATT.TATTATTATTTTATTTATTTAATAkATTTTTTTTTATTAGTAAATJGA: 1930 1950 1970 AATAAkATTTTTTAAACGTTTTTTCAACGTTTTATTAAATGTG TAAATAT AAATLA2AAATA 1990 2010 2030 TTAT ATATA TA TATATATATA TATG TATG TAT.TTATTTATTTATTTA TATA, CATA CAT 2050 2070 2090 ACCTGTTGACATTCATGTAATATAATAAAGGkACACATGC rATTTTG7ATTATATATCT 2110 2130 2150 T..V CTCTACT4TTTTAATAAAAAATGTCAAAZGCAGGAAATAAAAACTTTT:AATTTAACC 2170 2190 2210 4 4 p A.AAP.'AATATAATTIAATGATGTACACT4TATAGATATTGATACAAGAAAAACATTATA
A
2230 2250 2270 TT-TTTTTTTTrC'TTTCTTTTTTTTTTTTTTTTTTTTAAT-A-'AACAAAAAA ATTTATTA 2290 2310 2330 T~eTAATAATTTTAAATGAATGATG CAAATTTAATGAGC CATTTTATTTATATT TTAAA 2350 2370 ***TAATTATAATAA,,TAACGTACATATATAAAATGGTGATr'GAAIT 14 .4 a of
L
Claims (3)
1. Individual proteins of Plasmodium falciparum each having an amino acid sequence as shown respectively in tables 1 to 1 8, and partial sequences thereof having an antigenic activity. 2* 9* DBM/KJS/CH (DOC.1 3) AU2761 988.WPC I- i I I -~*ypaa_ S- MM WX^XXXXMXX TE SEFINING TH4E- N -1-VENlL ARE-AS- OTLLO WS 1. Proteins of odium falciparum with amino acid sequences shown in Tab to 18, and partial sequences thIp rpn-f h i nn- en I act4v^5?-- I *0 I I* I I..
2. Proteins as claimed in claim 1, obtained by expression of the sequences of the clones 41-1, 41-2, 41-4,
41-5, 41-6, 41-7, 41-8, 41-9, 41-10, 41-12, 41-13, 41- 14, 41-15, 41-17, 41-2gen and 41-7gen, 3. Fusion proteins which contain proteins as claimed in claim 1. 4. DNA and RNA coding for proteins as claimed in claim 1. 5. Purified and isolated DNA sequences shown in Table 1 to Table 18, coding for proteins as cla:med in claim 1, as well as sequences complementary and hybridizing therewith. 6. Vectors and DNA structures which code 'or proteins as claimed in claim 1 or 2 or fusion proteins as claimed in claim 3. 7. Vectors or DNA structures which contain DNA sequences as claimed in claim 8. Host cells containing DNA as claimed in claim 4 or or a vector as claimed in claim 6 or 7. 9. Proteins expressed by a hos elL as claimed in claim 8. A process for the preparation of proteins as claimed in claim 1 or 2, which comprises expression, in pro- or eukaryotic host systems, of DNA sequences coding for the proteins shown in Table 1 to Table 18. I Si a *r r,. I' 11. Polyclonal or monoclonal antibodies against proteins as claimed in claim 1, 2 or 3. 1 2. A vaccine containing one or more of the antigens as claimed in claim 1, 2 or 3. 13. A vaccine containing proteins or parts thereof, which are obtained by expression of the clones 41-1, 41-2 and 41-3. 14. An agent for immunoprophylaxis, containing antibodies as claimed in claim 11. A composition comprising DNA or RNA as claimed in claim 4, 5, 6 or 7. S 16. A composition comprising antibodies as claimed in claim 11. 17. A composition comprising proteins as claimed in claim 1, 2 or 3. 18. Proteins as claimed in any of claims 1-3 isolated using antibodies as claimed in claim 11. DATED this 21st day of January 1992 BEHRINGWERKE AKTIENGESELLSCHAFT I ee WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3744495 | 1987-12-30 | ||
DE3744495 | 1987-12-30 | ||
DE3831351 | 1988-09-15 | ||
DE3831351A DE3831351A1 (en) | 1987-12-30 | 1988-09-15 | MALARIA-SPECIFIC DNA SEQUENCES, THEIR EXPRESSION PRODUCTS AND THEIR USE |
Publications (2)
Publication Number | Publication Date |
---|---|
AU2761988A AU2761988A (en) | 1989-07-06 |
AU625315B2 true AU625315B2 (en) | 1992-07-09 |
Family
ID=25863316
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU27619/88A Ceased AU625315B2 (en) | 1987-12-30 | 1988-12-30 | Malaria-specific DNA sequences, expression products thereof, and the use thereof |
Country Status (11)
Country | Link |
---|---|
EP (1) | EP0322712B1 (en) |
JP (2) | JPH022356A (en) |
AU (1) | AU625315B2 (en) |
DE (2) | DE3831351A1 (en) |
DK (1) | DK727688A (en) |
ES (1) | ES2045075T3 (en) |
FI (1) | FI99144C (en) |
GR (1) | GR3007126T3 (en) |
IL (1) | IL88818A (en) |
PT (1) | PT89363B (en) |
YU (1) | YU48126B (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE4041836A1 (en) * | 1990-12-24 | 1992-06-25 | Behringwerke Ag | PROTECTIVE PLASMODIUM FALCIPARUM HYBRID PROTEINS COMPRISING PARTIAL SEQUENCES OF MALARIA ANTIGENE HRPII AND SERP, THEIR PREPARATION AND USE |
MX9304767A (en) * | 1992-08-07 | 1994-02-28 | Schering Corp | MALARIA PFEMP3 ANTIGEN, ANALOGS, ANTIBODIES AND USES THEREOF. |
EP0969714A4 (en) * | 1997-01-10 | 2004-10-06 | Univ California | Rg nucleic acids for conferring disease resistance to plants |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE64957T1 (en) * | 1984-02-22 | 1991-07-15 | Wellcome Found | CLONING DNA FOR PROTOZOA ANTIGENS. |
EP0309746A1 (en) * | 1987-09-08 | 1989-04-05 | F. Hoffmann-La Roche Ag | Antimalaria vaccines |
-
1988
- 1988-09-15 DE DE3831351A patent/DE3831351A1/en not_active Withdrawn
- 1988-12-20 ES ES88121299T patent/ES2045075T3/en not_active Expired - Lifetime
- 1988-12-20 EP EP88121299A patent/EP0322712B1/en not_active Expired - Lifetime
- 1988-12-20 DE DE8888121299T patent/DE3876227D1/en not_active Expired - Fee Related
- 1988-12-28 FI FI886001A patent/FI99144C/en not_active IP Right Cessation
- 1988-12-28 YU YU237588A patent/YU48126B/en unknown
- 1988-12-28 JP JP63335713A patent/JPH022356A/en active Pending
- 1988-12-28 IL IL8881888A patent/IL88818A/en not_active IP Right Cessation
- 1988-12-29 PT PT89363A patent/PT89363B/en not_active IP Right Cessation
- 1988-12-29 DK DK727688A patent/DK727688A/en not_active Application Discontinuation
- 1988-12-30 AU AU27619/88A patent/AU625315B2/en not_active Ceased
-
1993
- 1993-02-23 GR GR930400202T patent/GR3007126T3/el unknown
-
1999
- 1999-03-17 JP JP11072817A patent/JPH11318475A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
DK727688D0 (en) | 1988-12-29 |
EP0322712B1 (en) | 1992-11-25 |
EP0322712A3 (en) | 1990-05-02 |
YU48126B (en) | 1997-05-28 |
IL88818A0 (en) | 1989-07-31 |
EP0322712A2 (en) | 1989-07-05 |
DE3831351A1 (en) | 1989-07-13 |
ES2045075T3 (en) | 1994-01-16 |
GR3007126T3 (en) | 1993-07-30 |
FI99144B (en) | 1997-06-30 |
PT89363B (en) | 1993-08-31 |
JPH022356A (en) | 1990-01-08 |
IL88818A (en) | 1997-02-18 |
YU237588A (en) | 1991-06-30 |
FI886001A (en) | 1989-07-01 |
AU2761988A (en) | 1989-07-06 |
FI99144C (en) | 1997-10-10 |
DE3876227D1 (en) | 1993-01-07 |
JPH11318475A (en) | 1999-11-24 |
PT89363A (en) | 1989-12-29 |
DK727688A (en) | 1989-07-01 |
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