IE910966A1 - Plasmodium Sporozoite Antigen - Google Patents

Plasmodium Sporozoite Antigen

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IE910966A1
IE910966A1 IE096691A IE96691A IE910966A1 IE 910966 A1 IE910966 A1 IE 910966A1 IE 096691 A IE096691 A IE 096691A IE 96691 A IE96691 A IE 96691A IE 910966 A1 IE910966 A1 IE 910966A1
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asn
val
glu
lys
ser
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IE096691A
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Hoffmann La Roche
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

Polypeptides which coincide in at least one specific epitope with a Plasmodium falciparum sporozoite antigen with the N-terminal amino-acid sequence shown in Fig. 1, and a process for the preparation thereof are disclosed. The invention further relates to immunogenic compositions which contain a polypeptide of this type and a suitable adjuvant, to a DNA which codes for a polypeptide of this type, to replicable microbial vectors which contain a DNA of this type, to microorganisms which contain a replicable vector of this type, and to antibodies against a polypeptide according to the invention. Processes for preparing the immunogenic compositions, the microorganisms and the antibodies, and the use of the polypeptides and of the immunogenic compositions for immunising mammals against malaria are claimed.

Description

Malaria is caused in humans by four species of 5 Plasmodium, namely by P. falciparum, P. vivax, P. ovale and P. malariae. According to a 1986 report of the World Health Organisation (WHO) there are almost 100 million cases of malaria infections throughout the world. Of these, about 1 million, usually cases of infants infected with P. falciparum, have a fatal outcome. Malaria is continuing to spread because of the appearance of drugresistant parasites and of insecticide-resistant mosguito vectors. Thus, the health authorities in India reported 100,000 cases of malaria in 1962 but already 3 million cases in 1980, mostly caused by P. vivax (compare BruceChwatt, Essential Malariology, 2nd edition, Heinemann, London [1986]). In recent years, newly developed methods have given cause to hope that it will soon be possible to produce antimalaria vaccines which are able to counteract the increasing spread of malaria (Scaife, Genetic Engineering 7, 57-90 [1988]).
The natural life cycle of P. falciparum comprises three different stages. In the first stage, feeding mosguitoes introduce sporozoites into the bloodstream of vertebrates. These sporozoites migrate in the bloodstream to the liver and penetrate into the host's hepatocytes. In the second stage, these sporozoites develop into merozoites. These merozoites pass through several multiplication cycles in the host's erythrocytes and then develop into gametocytes. The gametocytes, which represent the sexual stage of the parasite, are picked up by feeding mosguitoes. After fertilisation in the insect's intestine, the gametocytes develop into sporozoites which then migrate into the salivary glands of the insect. A new cycle can then start.
Thus, vaccines against the sporozoite form of Wa/29.1.90 IE 9196® Plasmodium falciparum are the first line of defence against malaria infection. In recent years, the so-called CS protein from sporozoites of various species of Plasmodium have been intensively investigated and its use in vaccines tested (Nussenzweig et al., Adv. Immunol. 45. 283-334 [1989]). Although vaccination experiments with the CS protein or derivatives thereof generated partial protection against subsequent malaria infection, it is not yet possible to talk of a breakthrough in the development of a generally utilisable malaria vaccine (Herrington et al., Nature 328, 257-259 [1987]). Hence the object was to find novel sporozoite antigens which can be used in native or modified form for producing novel antimalaria vaccines.
The present application discloses a novel sporozoite antigen with an apparent molecular weight greater than 200 kDalton (kDa) with the N-terminal amino-acid sequence (I) IE 91966 15 1 Met Asn Lys val Asn Ala Val His Lys He Asn Ala Val Asp Lys 16 Val Asn Ala val Asn Lys Val Asn Ser Val Asn Lys Leu Asn Val 31 Val Asn Lys Thr Asn val Leu Ser Lys Leu Asn Ala Val Tyr Lys 46 Val Asn Ser Val His Lys Met Asn Ala Val Asn Lys val Asn Ala 61 val Asn Lys val Asn Ala Val Asn Lys val Asn Val val Asn Lys 76 Lys Asp lie Leu Asn Lys Leu Asn Ala Leu Tyr Lys Met Asn Ala 91 Val Tyr Lys Met Asn Ala Leu Asn Lys Val Ser Ala Val Asn Lys 105 Val Ser Ala Val Asn Lys Val Ser Ala val Asn Lys Met Gly Ala 121 Val Asn Arg Val Asn Gly Val Asn Lys Val Asn Glu Val Asn Glu 136 Val Asn Glu Val Asn Glu Val Asn Met Val Asn Glu Val Asn Glu 151 Leu Asn Glu val Asn Asn val Asn Ala Val Asn Glu val Asn Ser 166 Val Asn Glu Val Asn Glu Met Asn Glu Val Asn Lys Val Asn Glu 181 Leu Asn Glu Val Asn Glu Val Asn Asn Val Asn Glu Val Asn Asn 196 val Asn Val Met Asn Asn val Asn Glu Met Asn Asn Met Asn Glu 211 Met Asn Asn Val Asn Val Val Asn Glu Val Asn Asn val Asn Glu 226 Val Asn Asn val Asn Glu Met Asn Asn val Asn Glu Met Asn Asn 241 Met Asn Glu Met Asn Asn val Asn val val Asn Glu val Asn Asn 256 Val Asn Glu Met Asn Asn Thr Asn Glu Leu Asn Glu val Asn Glu 271 Val Asn Asn Val Asn Glu val Asn Asp val Asn Val val Asn Glu 286 Val Asn Asn Val Asn Glu Met Asn Asn Met Asn Glu Leu Asn Glu 301 Val Asn Gly Val Asn Glu Val Asn Asn Thr Asn Glu He His Glu 316 Met Asn Asn He Asn Glu val Asn Asn Thr Asn Glu Val Asn Asn 331 Thr Asn Glu He Tyr Glu Met Asn Asn Met Asn Asp val Asn Asn 346 Thr Asn Glu He Asn Val Val Asn Ala val Asn Glu val Asn Lys 361 Val Asn Asp Ser Asn Asn Ser Asn Asp Ala Asn Glu Gly Asn Asn 376 Ala Asn Tyr Ser Asn Asp Ser Ser Asn Thr Asn Asn Asn Thr Ser 391 Ser Ser Thr Asn Asn Ser Asn Asn Asn Thr Ser Cys Ser Ser Gin 406 Asn Thr Thr Thr Ser Ser Glu Asn Asn Asp Ser Leu Glu Asn Lys 421 Arg Asn Glu Glu Asp Glu Asp Glu Glu Asp Asp Gin Lys Asp Thr 436 Gin Lys Glu Lys Asn Asn Leu Glu Gin Glu Asp Met Ser Pro Tyr 451 Glu Asp Arg Asn Lys Asn Asp Glu Lys Asn He Asn Glu Gin Asp 466 Lys Phe His Leu Ser Asn Asp Leu Gly Lys He Tyr Asp Thr Tyr 481 Asn Gin Gly Asp Glu Val Val Val Ser Lys Asn Lys Asp Lys Leu 496 Glu Lys His Leu Asn Asp Tyr Lys Ser Tyr Tyr Tyr Leu Ser Lys 511 Ala Thr Leu Met Asp Lys He Gly Glu Ser Gin Asn Asn Asn Asn 526 Tyr Asn Val Cys Asn Ser Asn Glu Leu Gly Thr Asn Glu Ser He 541 Lys Thr Asn Ser Asp Gin Asn Asp Asn Val Lys Glu Lys Asn Asp 556 Ser Asn lie Phe Met Lys Met He lie lie He Arg Leu Met He 571 Met lie lie Met He Met He He He Trp Tyr Leu Lys He Leu 586 Gin Asp tys lie He Trp Arg Asn Lys Lys Val Glu Lys Thr Ser 601 Asn He Leu Asn Asn Phe Asp Asn Asn Gly Asn Asp Asn Asp Asn 616 Asp Asn Asp Asp Asn Asn Asp Asn Asp Asn Asn Asn Asn Asn Asn 631 Met Asn Asn Gin Tyr Asn Tyr Gin Glu Asn Asn He Asn Thr Asn 546 Tyr Asn lie Leu Tyr Thr Pro Ser Asn Cys Gin He Gin Asn Asn 661 Ser Tyr Met Asn Thr Asn Glu Met Tyr Gin Pro Leu Tyr Asn Thr 676 Tyr Pro Ser Asn Arg He Gin Glu Asn Ser Thr He Asn Asn Asn 691 He He Asn Asp Ser Pro Tyr Met Asn Asn Asp Asn Thr Asn 706 Asn Phe He Ser Gly Met Asn IE 91966 •ί This amino-acid sequence contains 713 amino-acid residues with the following amino-acid composition: Ala (19), Arg (6), Asn (219), Asp (37), Cys (3), Gin (15), Glu (65), Gly (10), His (5), lie (34), Leu (26), Lys (48), Met (34), Phe (4), Pro (5), Ser (39), Thr (27), Trp (2), Tyr (23) and Val (92). The calculated molecular weight of this N-terminal part of the novel sporozoite antigen is 81,281 Da. Possible glycosylation sites are located at positions 32, 260, 308, 323, 329, 344, 362, 365, 377, 380, 387, 388, 394, 398, 399, 406, 414, 537, 554, 659, 684, 693, 702, 705 of the amino-acid sequence (I)· The essential features of the amino-acid sequence (I) are a) the frequency of the occurrence of asparagine residues in the sequence, and b) the repeated occurrence in the N-terminal half of a sequence comprising three amino-acid residues (NXY) in which N represents asparagine and X and Y represent any amino acid. The amino-acid residue X which comes directly after the asparagine residue is preferably charged, and the aminoacid residues of glutamic acid and lysine are particularly preferred. The amino-acid residue Y in the third position is preferably a hydrophobic amino-acid residue, and the amino-acid residues of valine and methionine are particularly preferred. The sequence (NXY) is repeated about 103 times in the amino-acid sequence (I). Examples of particularly preferred sequences (NXY) are AsnGluVal, AsnLysVal and AsnGluMet. It is clear to the person skilled in the art that the sequence (NXY)n also embraces the sequence permutations, that is to say also (YNX)n and (XYN)n. As the number n of repeating sequences NXY, YNX and XYN in polypeptides increases they become scarcely distinguishable immunologically. Examples which may be mentioned are the peptides (AsnGluVal )4, (ValAsnGlu)4 and (GluValAsn)4. Comparison of the sequence of these peptides, that is to say As nGluValAs nGluVa lAs nGluVa lAs nGluVal GluValAsnGluValAsnGluValAsnGluValAsn ValAsnGluValAsnGluValAsnGluValAsnGlu IE 91966 clearly shows that these peptides are identical to one another apart from the terminal amino acids.
The gene which codes for the novel sporozoite antigen contains the following nucleotide sequence (1) which codes for the N-terminal amino-acid sequence (I): 20 30 I 40 50 60 1 GAATTCCTCG 7GA7CA7ATG I AGA7A7AG77 x x χ Λ i U X-GA GAATATTTGT TCAAAGAAGA 51 TGCGTCACAA GAGAAGAAAC A7AAGCGCA7 77AATAAAA7 777ATATAAG AAT777AAAT 121 CAATCAAAAA ACAATTTACT A X u χ χ * x GTCCAAACAT GTTAAATTTA mm x 1» - -λ x wn x - 181 GGGATTCATT CCAAAAAGAT A77ACGG7A7 7AG7AAACCA AATATTAACC 7C77ACGAAT 241 GGCATGGACA TGGAGCTTTT GAATCATTCG 77AAGT7A7A TGGAGATTTA AGAAAAGATA 301 TTTTAAGTCC TATACATCTA C7AAAAG7G7 TGTGGAAAAT TTTAGAAATA TAGAAGAAGA 261 CTTAGCTGAT 77AGATGAAG ATTCAACAGA AAATAT7AAT GAACCTAATC ATTTAGATGG 421 TCAAAATAAT AAAAACAA7A GAAAAAC7AA 7AATGATAAT ACATTGAAAC AAAATCATCG 481 AAAATCTAGG GGCACATCTG TACAAGGACG CAAAAA7AAA ATAAATCGGG GATCAAAAGG 541 CAAACATAAT 7C7A7AAATA TTCCAAAAGA 7AGAAAGACG AACATAATGT CACAAAT7AA 601 TAAATTACTA 777AATAAAA AAGA7A77AA AATAAAA7G7 GAAGAAAGTA GTAGTTCAAA 661 TTATGAAGAG GGAAA7AG77 CGAG7AATGA AGAAAA7AAT ATATCAACTG ATAAAAATAT 721 TTGTAATACA AATAATAAAA ATGGAGTTTC ATTATATGAT AATTCAAAGG χ--λ x- -jAA 781 AGGAGATTAT AAAAT77AGC AAACAGTTTC ATAAG7ACAT ACACAAGAAC C7777AGATA 341 ATGTCGATAA AACAGA7AGA ACAAT7AA7A 77ATATCAAA ATTC7TCGGT GGTGTGAATA 901 AGTCCAATAA CG7GAA7AA7 A77AA7AG7G TAAATAAGGA GAATAACATG AATAAGGTGA 961 ATGCTGTACA 7AAGA7AAAT GCTGTGGATA AGG7AAATGC TGTGAATAAG GTAAATTCTG 1021 TCAATAAGCT AAATGT7G7G AATAAGACGA ATGTTCTGAG TAAGTTGAAT mmmmr* m « m « xuxu-λ-Α 1081 AGGTGAATTC 7G7ACATAAG ATGAATGC7G TGAATAAGGT AAATGCTGTA AATAAGGTGA 1141 ATGCTGTAAA 7AAGG7AAAT GTCGTGAATA AGAAGGATAT TCTGAATAAG 77GAA7GC77 1201 7G7A7AAGA7 GAATGC7G7G 7ATAAAA7GA ATGCTTTGAA TAAGGTGAGT GCTGTGAATA 1261 AGGTGAG7GC 7G7GAATAAG GTGAGTGCTG TGAATAAGAT GGGTGCTGTA AATAGGG7GA 1321 ATGGAGTAAA CAAGG7GAAT GAGGTGAATG AGGTGAATGA GGTGAATGAA GTGAATA7GG 1381 7GAA7GAAGT AAATGAGTTA AATGAGGTGA ATAATG7CAA TGCAGTGAAT GAAGTGAATA 1 441 G7G7GAACGA GGTTAATGAA ATGAATGAGG TGAATAAGGT GAATGAGCTA AATGAGGTGA 1501 A7GAAG7GAA TAATGTCAAT GAGGTGAATA ATGTGAATGT GATGAATAAT G7GAA7GAGA 1561 7GAA7AA7AT GAA7GAGA7G AATAATGTCA ATGTAGTGAA TGAAGTGAAT AATGTCAATG 1621 AGG7GAA7AA 7GTGAATGAG ATGAATAATG 7GAATGAGAT GAATAATATG AATGAGA7GA 1681 ATAA7G7CAA TGTAGTGAAT GAAGTGAATA ATGTAAATGA GATGAATAAT ACGAATGAGC 1741 7AAA7GAGG7 GAATGAAGTG AATAATGTGA ATGAGGTGAA TGATGTCAAT GTAGTGAACG 1801 AAGTGAATAA TGTAAATGAG ATGAATAATA TGAATGAGCT AAATGAGGTG AATGGGG7AA 1861 ATGAAG7GAA TAATACGAAT GAGATACATG AGATGAATAA TATAAATGAG GTGAATAATA 1921 CGAATGAGGT GAATAATACG AATGAGATAT ATGAGATGAA TAATATGAAT GATG7GAATA 1981 ATACGAA7GA GATAAATGTG GTGAATGCGG TTAATGAAGT GAATAAGGTG AATGATTCAA 2041 ATAATTCAAA TGATGCAAAT GAAGGAAATA ACGCAAAT7A TTCAAATGAT 7CAAGCAATA 2101 CAAATAATAA CACATCAAGC AGCACAAATA ACTCAAATAA TAATACATCG 7GTAG77CAC 2161 AGAACACCAC AACTAGCAGC GAAAATAATG ATTCATTAGA AAATAAAAGA AATGAAGAAG 2221 A7GAAGA7GA AGAAGACGAC CAAAAAGATA CACAAAAAGA AAAAAACAAT T7AGAACAGG 2281 AAGATATGAG TCCATACGAA GATAGAAATA AAAATGATGA AAAAAATATT AATGAACAAG 2341 A7AAAT77CA TTTATCAAAT GATTTGGGAA AAATATATGA TACATATAAC CAAGGAGA7G 2401 AAGTTGTTGT ATCTAAGAAT AAGGACAAAT 7AGAAAAGCA TTTGAATGAT TACAAGAGTT 2461 A7TA77AT77 A7C7AAAGCA ACACTCATGG ACAAAATTGG AGAATCACAA AATAATAACA 2521 AC7ATAA7G7 ATCTAATTCA AATGAACTTG GAACTAATGA ATCCATAAAG ACAAATTC7G 2581 ATCAGAA7GA 7AA7G7AAAA GAAAAAAATG ATTCCAACAT ATTTATGAAA ATGATAAT7A 2641 7AAT7CG7CT 7ATGA7AATG ATCATAATGA 7AATGATAAT AATATGGTAT T7AAAGA77C 2701 T7CAAGACAA GATAATATGG AGAAACAAAA AAGTGGAGAA AACAAGCAAT ATT77AAACA 2761 atttcgataa TAATGGTAAT GATAATGATA ATCATAATGA TGATAATAAT GATAATGATA 2821 ΑΤΑΑ7ΑΑ7ΑΑ 7AATAATATG AATAATCAAT ATAATTATCA AGAAAATAAT ATTAACACAA 2881 ATTATAACAT TTTGTACACT CCTTCTAATT GCCAAATCCA AAACAATTCA TATATGAATA 2941 CAAATGAAAT G7ACCAACCA 7TATATAATA CATATCCTTC AAATCGTATT CAAGAAAATT 3001 CTACTATAAA TAACAACATT ATTAATGATT CACCTTACAT GAATAACGAC AACACCACTA 3061 A7AACACC77 CATTTCTGGT ATGAATTC IE 91966 The present invention relates to the novel Plasmodium sporozoite antigen per se and derivatives thereof, especially polypeptides which coincide in at least one specific epitope with the Plasmodium sporozoite antigen with the N-terminal amino-acid sequence (I). A specific epitope is defined as an immunogenic determinant on a polypeptide which is produced by a specific molecular configuration of a part-sequence of the polypeptide. Preferred polypeptides contain the repeating sequence NXY or its permutations YNX and XYN. Hence, particularly preferred polypeptides contain the sequence (NXY)n, (YNX)n or (XYN)n in which n is a number between 3 and 120. The invention also relates to polypeptides as defined above and which additionally are covalently linked to another peptide at the N terminus and/or at the C terminus. Fusion polypeptides of this type can be represented by the general formulae A-B, B-C or A-B-C in which B is a polypeptide which coincides in at least one specific epitope with the Plasmodium sporozoite antigen with amino-acid sequence (I). The additional peptides A and/or C can in principle be any peptides but are preferably affinity peptides or T-cell epitope peptides. By an affinity peptide are meant those peptides which contain an amino-acid sequence which preferentially binds to an affinity chromatography support material. Examples of such affinity peptides are peptides which contain at least two, preferably six, histidine residues. Such affinity peptides bind selectively to nitrilotriace30 tic acid/nickel chelate resins (see, for example, European Patent Application, Publ. No. 253 303). Fusion polypeptides which contain such affinity peptides can be separated selectively, using nitrilotriacetic acid/nickel chelate resins, from the other polypeptides (see, for example, European Patent Applications with publication numbers 282 042 and 309 746). The affinity peptide can be linked either to the C terminus or to the N terminus of the polypeptide B defined above, but linkage to the N terminus is preferred, for example when the natural stop IE 91966 *-----/ I codon of the Plasmodium sporozoite antigen is also used in the expression of the polypeptide according to the invention. By a T-cell epitope peptide are meant those peptides which act on T cells and, in this way, con5 tribute to the cooperation of T and B cells in the immune response to the malaria antigen. Particularly preferred are universal T-cell epitopes as described, for example, in European Patent Application with the publication number 343 460.
One example of a fusion polypeptide according to the invention, of the general formula A-B-C is the polypeptide with the amino-acid sequence (II) IE 91966 1 Met Arg «□.γ Ser His His His His His His Gly Ser Val Asn Ser 15 Val Asn Lvs Gx u Asn Asn Met Asn Lys Val Asn Ala val His Lys 31 lie Asn Ala val Asp Lys val Asn Ala Val Asn Lys val Asn Ser 45 Val Asn Lys Leu Asn val val Asn Lys •U v Asn Val Leu Ser Lys 51 Leu Asn Ala val ^y«r Lys val Asn Ser Val His Lys Met Asn Ala 75 val Asn Lys Val Asn Ala Val Asn Lys val Asn Ala val Asn Lvs 91 val Asn Val Val Asn Lys Lys Asp lie Leu Asn Lys Leu Asn Ala ICS Leu Tyxr Lys Met Asn Ala val Tyr Lys Met Asn Ala Leu Asn Lys 121 val Ser Ala val Asn Lys val Ser Ala Val Asn Lys val Ser Ala 126 Val Asn Lys Met Gly Ala Val Asn Arg Val Asn Gly Val Asn Lys 151 val Asn Glu Val Asn Gx u Val Asn Glu Val Asn Glu Val AS*. Met 165 Val Asn Glu val Asn Glu Leu Asn Glu Val Asn Asn Val Ac.. Ala 1S1 Val Asn Glu val Asn Ser Val Asn Glu Val Asn Glu Met Asn Glu 196 Val Asn Lys val Asn Glu Leu Asn Glu Val Asn Glu Val Asn Asn 211 Val Asn Glu val AST* Asn val Asn Val Met Asn Asn val Asn 226 Met Asn Asn Met Asn Glu Met Asn Asn val Asn val Val Asn Glu 241 Val Asn Asn val Asn GxU val Asn Asn val Asn Glu Met Asn Asn 255 Val Asn Glu Met Asn Asn Met Asn Glu Met Asn Asn Val Asn Val 271 Val Asn Glu val Asn Asn val Asn Glu Met Asn Asn Asn Glu 255 Leu Asn GxU val Asn Glu val Asn Asn val Asn Glu val Asn Asp 201 Val Asn Val Val Asn Glu Val Asn Asn Val Asn Glu Met Asn Asn 315 Met Asn Glu Leu Asn Glu Val Asn Gly val Asn Glu Val Asn Asn 221 mU v Asn Glu lie His Glu Met Asn Asn lie Asn Glu val Asn Asn 246 Thr Asn Glu Val Asn Asn ff'k φ·’ Asn Glu lie Tyr Glu Met Asn Asn 251 Met Asn Asp Val Asn Asn mUy Asn Glu lie Asn val val Asn Ala 37S Val Asn Glu Val Asn Lys val Asn Asp Ser Asn Asn Ser Asn Asp 291 Ala Asn Glu Gly Asn Asn Ala Asn Tyr Ser Asn Asp Ser Ser Asn 406 ~»u Asn Asn Asn wk * Ser Ser Ser mU * Asn Asn Ser Asn Asn Asn 421 Ser Cys Ser Ser Gin Asn Thr mU mu Ser Ser Glu Asn Asn 435 Asp Ser Leu Glu Asn Lys Arg Asn Glu Glu Asp Glu Asp Glu Gx- 451 Asp Asp Gin Lys Asp mu Gin Lys Glu Lys Asn Asn Leu Glu Gin 465 Glu Asp Met Ser Pro Tyr Glu Asp Arg Asn Lys Asn Asp Glu Lys 4S1 Asn lie Asn Gly lie Arg Arg Pro Ala Al 3 Lys Leu Asn IE 9196® --— This polypeptide contains 493 amino-acid residues, where part A comprises amino-acid residues 1 to 21, part B comprises amino-acid residues 22 to 483 and part C comprises amino-acid residues 484 to 493. The poly5 peptide has a calculated molecular weight of 55,239 Dalton. Part A is an affinity peptide with six histidine residues. Part B corresponds to the N-terminal part of the sporozoite antigen of the present invention, and contains amino-acid residues 1 to 462 of amino-acid sequence (I). Part C is any peptide encoded by a vector sequence.
The invention also relates to polypeptides and fusion polypeptides which have been derived from the amino-acid sequences shown above by additions, deletions or insertions, with the proviso that these polypeptides are still able to elicit an immune response to the circumsporozoite stage of the malaria parasites, preferably against the sporozoite antigen with the N-terminal amino-acid sequence (I) of P. falciparum. The invention also relates to DNAs which code for a polypeptide according to the invention, and to replicable microbial vectors which contain a DNA of this type, especially expression vectors, that is to say replicable microbial vectors in which a DNA which codes for a polypeptide according to the invention is joined to an expression-control sequence in such a way that the polypeptide encoded by the DNA can be expressed in microorganisms. In addition, the present invention relates to microorganisms which contain a replicable vector of this type or an expression vector, and to processes for the preparation of these vectors and microorganisms. Furthermore, the present invention relates to processes for preparing the polypeptides and to the use thereof for immunisation of mammals against malaria.
Since certain substitutions in the amino-acid sequence of a polypeptide have no effect on the spatial structure or the biological activity of the polypeptide, it is possible' for the amino-acid sequence of the IE 91966 polypeptides according to the invention to differ from the amino-acid sequences shown above. Examples of such amino-acid substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and vice versa (compare Doolittle, in The Proteins, ed. Neurath, H. and Hill, R. L., Academic Press, New York [1979]).
The polypeptides according to the invention can 10 be covalently bonded to a carrier material or adsorbed thereon. Suitable carrier materials are natural or synthetic polymeric compounds such as, for example, copolymers of one or more amino acids (for example polylysine) or sugars (for example polysaccharide). Other suitable carrier materials are natural polypeptides such as haemocyanins (for example KLH = keyhold limpet haemocyanin), serum proteins (for example gamma globulin, serum albumin) and toxoids (for example diphtheria or tetanus toxoid). Other suitable carrier materials are known to the person skilled in the art.
The covalent bonding of the polypeptides according to the invention to the carrier materials can be carried out in a known manner, for example directly by forming a peptide or ester linkage between free carboxyl, amino or hydroxyl groups of the polypeptides and the corresponding groups on the carrier material, or indirectly by using conventional bifunctional reagents such as, for example, m-maleimidobenzoyl-N-hydroxysuccinimide esters (MBS) or succinimidyl 4-(p-maleimido30 phenyl)butyrate (SMPB). These and other bifunctional reagents can be obtained commercially, for example from Pierce Chemical Company, Rockford, Illinois, USA. It is also possible to use C2.7-dialkanals such as, for example, glutaraldehyde (Avrameas, Immunochem. 6, 43-52 [1969]).
The carrier material with the polypeptides bound thereto can be separated from unbound polypeptides and, where appropriate, from excess reagents using known methods (for example dialysis or column chromatography).
The polypeptides of the present invention, IE 91966 especially those with fewer than 50 amino-acid residues, can be prepared by conventional methods of peptide synthesis, in liquid or, preferably, on solid phase by the method of Merrifield (J. Am. Chem. Soc. 85, 2149-2154 [1963]) or using other equivalent methods of the prior art.
The solid-phase synthesis starts with the C-terminal amino acid of the peptide to be synthesised, which is coupled in protected form to an appropriate support material. The starting material can be prepared by linking an amino acid with protected amino group via a benzyl ester bridge to a chloromethylated or hydroxymethylated support material or by forming an amide linkage to a benzhydrylamine (BHA)-, methylbenzhydryl15 amine (MBHA)- or benzyloxybenzyl alcohol-support material. These support materials are commercially available and their preparation and use are well known.
General methods for protecting and removing protective groups from amino acids which can be used in this invention are described in The Peptides, Vol. 2 (edited by E. Gross and J. Meienhofer, Academic Press, New York, 1-284 [1979]). Protective groups comprise, for example, the 9-fluorenylmethoxycarbonyl (Fmoc), the tertiary butyloxycarbonyl (Boc), the benzyl (Bzl), the t-butyl (But), the 2-chlorobenzyloxycarbonyl (2C1-Z), the dichlorobenzyl (Deb) and the 3,4-dimethylbenzyl (Dmb) group.
After removal of the α-amino protective group of the C-terminal amino acid linked to the support, the protected amino acids are coupled on stepwise in the required sequence. It is possible to synthesise a complete polypeptide in this way. As an alternative to this, it is possible to construct small peptides which are then joined together to give the required polypeptide.
Suitable coupling reagents belong to the prior art, with dicyclohexylcarbodiimide (DCC) being particularly suitable .
Every protected amino acid or peptide is placed in excess in the* solid-phase synthesis reaction vessel, 91966 and the coupling reaction can be carried out in dimethylformamide (DMF) or methylene chloride (CH2C12) or a mixture of the two. In cases of incomplete coupling, the coupling reaction is repeated before the N-terminal a-amino protective group is removed for the coupling of the next amino acid. The yield of each coupling step can be determined, specifically and preferably by the ninhydrin method. The coupling reactions and the washing steps can be carried out automatically.
The peptide can be cleaved off the support material by methods which are well known in peptide chemistry, for example by reaction with hydrogen fluoride (HF) in the presence of p-cresol and dimethyl sulphide at 0°C for 1 hour, possibly followed by a second reaction with HF in the presence of p-cresol at 0°C for 2 hours.
Peptides cleaved off chloromethylated or hydroxymethylated support materials are peptides with a free C terminus; peptides cleaved off benzhydrylamine- or methylbenzhydrylamine-supports are peptides with amidated C terminus.
On the other hand, the polypeptides of the present invention can also be prepared using the methods of recombinant DNA technology (Maniatis et al. in Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory [1982]). For example, a piece of DNA, that is to say DNA fragment which codes for a polypeptide of this type, can be synthesised by conventional chemical methods, for example using the phosphotriester method as described by Narang et al. in Meth. Enzymol. 68, 90-108 [1979] or using the phosphodiester method (Brown et al., Meth. Enzymol. 68., 109-151 [1979]). Both methods entail initial synthesis of relatively long oligonucleotides, which are attached together in a predetermined manner. The nucleotide sequence of the DNA fragment can be identical to that nucleotide sequence which encodes the natural polypeptide in the Plasmodium parasite. Since the genetic code is degenerate, there is, on the other hand, the possibility that a partly or completely different nucleotide sequence encodes the same polypeptide. It is 91966 possible, where appropriate, to choose for the nucleotide sequence those codons which are also preferentially used by the host organism which is used for the expression of the polypeptide (Grosjean et al., Gene 1(3, 199-209 [1982]). However, it is necessary to ensure in this connection that the DNA fragment obtained in this way contains no part-sequences which impede the construction of the expression vector, for example by introducing an undesired restriction enzyme cleavage site, or which prevent the expression of the polypeptide.
The DNA fragment which codes for the Plasmodium sporozoite antigen according to the invention or for the part-sequence B of the fusion polypeptide according to the invention can also be obtained by cleaving genomic DNA of a Plasmodium strain with one or more suitable restriction endonucleases, for example EcoRI. Fragments with a length of 1.5 to 6 * 103 base pairs are isolated and incorporated in a suitable vector, for example into the X phage vector gtll. The vector gtll is described by Young et al., Proc. Natl. Acad. Sci. USA 80, 1194-1198 [1983] and can be obtained from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, USA or from other institutions . The recombinant phage DNA can be packaged in vitro in phage protein coats. The infectious phages obtained in this way are introduced into suitable host cells, for example into E. coli Y1088 containing the plasmid pMC9 (obtainable from ATCC). About 100,000 recombinant phages are screened to find those phages which react with a suitable probe.
Suitable probes of this type are oligonucleotides which correspond to a part-sequence, which codes for a polypeptide according to the invention, of the genomic DNA, or antibodies which recognise the sporozoite antigen produced by )\-gtll phages. The manner in which these probes are selected and used is known to the person skilled in the art. Phages which contain the required DNA fragment are replicated, and the DNA is isolated. The DNA fragment can then be incorporated into a suitable replicable microbial vector, preferably into an expression IE 91966 vector which provides the required expression signals and which, where appropriate, codes for part-sequences A and/or C of the abovementioned fusion polypeptides. A preferred expression vector is the vector pDS56/RBSII,6xHis, which is described in the examples.
The polypeptides of the present invention can, after appropriate adaptation of the nucleotide sequence, also be prepared in other suitable expression vectors. Examples of expression vectors of this type are described in the European Patent Application, Publication No. 186 069, which was published on July 2, 1986. Other expression vectors are known to the person skilled in the art.
The expression vector which contains a DNA fragment with the DNA sequence which codes for a polypeptide according to the invention is then introduced into a suitable host organism. Suitable host organisms are microorganisms, for example yeast cells or bacterial cells which are able to express the polypeptide encoded by expression vectors. The preferred host organism is E. coli SG13009. Other suitable host organisms are E. coli M15 (described as DZ291 by Villarejo et al. in J. Bacteriol. 120, 466-474 [1974]), E. coli 294, E. coli RR1 and E. coli W3110, all of which can be obtained from ATCC, DSM or other institutions.
The manner in which the polypeptides according to the invention are expressed depends on the expression vector used and on the host organism. The host organisms which contain the expression vector are normally propaga30 ted under conditions which are optimal for growth of the host organisms. Towards the end of exponential growth, when the increase in the cell count per unit time decreases, expression of the polypeptide of the present invention is induced, that is to say the DNA encoding the polypeptide is transcribed, and the transcribed mRNA is translated into protein. The induction can be brought about by adding an inducer or a derepressor to the growth medium or by altering a physical parameter, for example altering the temperature. The expression in the IE 91θ66 - — - 16 expression vector used in the present invention is controlled by the lac repressor which binds to a control sequence. The repressor is removed by adding isopropylb-D-thiogalactopyranoside (IPTG) and this induces the synthesis of the polypeptide. Other induction systems are known to the person skilled in the art.
The translation start signal AUG, which corresponds to the ATG codon at the DNA level, has the effect that all polypeptides synthesised in a prokaryotic host organism have a methionine residue at the N terminus. In certain expression systems this N-terminal methionine residue is cleaved off. However, it has emerged that the presence or absence of the N-terminal methionine has scarcely any effect on the biological activity of a polypeptide (compare Winnacker, in Gene und Klone (Genes and Clones), page 255, Verlag Chemie, Weinheim, FRG [1985]). In cases where there is interference from the N-terminal methionine, it can be cleaved off using a peptidase specific for N-terminal methionine.
Miller et al. (Proc. Natl. Acad. Sci. USA 84, 2718-2722 [1987]) have described the isolation of a peptidase of this type from Salmonella typhimurium. The present invention therefore relates to polypeptides with or without N-terminal methionine residue.
The polypeptides produced in the host organisms may be secreted out of the cell by specific transport mechanisms, or isolated by disruption of the cell. The disruption of the cell can be brought about mechanically (Charm et al., Meth. Enzymol. 22, 476-556 [1971]), enzymatically (lysozyme treatment) or chemically (detergent treatment, treatment with urea or guanidine HCL etc.), or by a combination of these.
The polypeptides according to the invention can then be purified by known methods such as, for example, by centrifugation at different speeds, by precipitation with ammonium sulphate, by dialysis (under atmospheric pressure or reduced pressure) , by preparative isoelectric focusing, by preparative gel electrophoresis or by various chromatographic methods such as gel filtration, 91966 high performance liquid chromatography (HPLC), ion exchange chromatography, reverse phase chromatography and affinity chromatography (for example on Blue Sepharose CL-6B on monoclonal antibodies which are directed against the polypeptide and are bound to a carrier, or on metal chelate resins).
The polypeptides of the present invention can be in the form of multimers, in particular in the form of dimers, as depicted diagrammatically in Fig. 8A. Multi10 mers can also result when polypeptides are produced in prokaryotic host organisms, especially owing to the formation of disulphide linkages between cysteine residues .
The present invention also relates to immunogenic compositions which contain a polypeptide according to the present invention, and a suitable adjuvant. Suitable adjuvants for use in humans and animals are known to the person skilled in the art (Warren et al., Ann. Rev. Immunol. £, 369-388 [1986]; Morein, Nature 332. 287-288 [1988]; Klausner, BIO/TECHNOLOGY 6, 773-777 [1988] and Bromford, Parasitology Today 5, 41-46 [1989]). The polypeptides and immunogenic compositions according to the invention can be in the form of lyophilisates for reconstitution with sterile water or with a saline solution, preferably a sodium chloride solution.
Introduction of the polypeptides and immunogenic compositions according to the invention into mammals activates their immune system and raises antibodies against the Plasmodium sporozoite antigen according to the invention. These antibodies can be isolated from the serum. The present invention also relates to antibodies of this type. The antibodies according to the invention react with malaria parasites and can therefore be used for passive immunisation or for diagnostic purposes.
Antibodies against the polypeptides according to the invention can be produced in monkeys, rabbits, horses, goats, guinea pigs, rats, mice, cows, sheep etc. but also in humans. The antiserum or the purified antibodies can be used as requited. The antibodies can be purified in a IE 91966 known manner, for example by precipitation with ammonium sulphate. It is also possible to produce monoclonal antibodies which are directed against the polypeptide of the present invention, by the method developed by Kohler et al. (Nature, 256, 495-497 [1975]). Polyclonal or monoclonal antibodies can also be used for the purification by affinity chromatography of the polypeptides of the present invention or their natural equivalents.
The polypeptides according to the invention and the immunogenic compositions can be used to immunise mammals against malaria. The mode of administration, the dosage and the number of injections can be optimised by the person skilled in the art in a known manner. Typically, several injections are administered over a lengthy period in order to obtain a high titre of antibodies against the malaria parasites, that is to say against the Plasmodium sporozoite antigen of the present invention.
The figures and the detailed example which follows contribute to explaining the present invention. However, it is not the intention to give the impression that the invention is restricted to the subject-matter of the example or of the figures.
Key to the Figures Fig. 1 Amino-acid sequence of the N-terminal part of the P. falciparum sporozoite antigen = amino-acid sequence (I) Fig. 2 Nucleotide sequence of the P. falciparum gene which codes for the sporozoite antigen = nucleotide sequence (1) Fig. 3 Amino-acid sequence of the fusion protein with amino-acid sequence (II) Fig. 4 Partial restriction enzyme map of the vectors NXY, NXY-L, NXY-H, Ml3-NXY and pDS-NXY. The vector NXY contains nucleotides 1-3088 of nucleotide sequence (1) in the lambda phage gtll. The black bar defines the coding region, which presumably starts with the ATG start codon at position 948-950 of nucleotide sequence (1). A 1 kb EcoRI fragment (nucleotides 1-1084) and a 91966 -- 2 kb EcoRI fragment (nucleotides 1085-3088) were isolated from the vector NXY and integrated into the vector M13 mpl8. This resulted in the vectors NXY-L containing the 1 kb EcoRI fragment and NXY-H containing the 2 kb EcoRI fragment from the vector NXY. The vector M13-NXY corresponds to the vector M13 mpl8 containing nucleotides 1-3088 of nucleotide sequence (1). This vector thus contains the same DNA as NXY. The vector pDS-NXY contains the 1413 bp Asel fragment (nucleotides 922-2332 of nucleotide sequence (1)) from M13-NXY which, after the protruding ends had been filled in with Klenow polymerase, was provided with BamHI linkers (10-mer) and cloned into pDS56/RBSII,6xHis. The correct orientation to the promoter was established by restriction analysis. E. coli cells which have been transformed with pDS-NXY produce, after induction, the recombinant Plasmodium sporozoite antigen of the general formula A-B-C with the sequence shown in Fig. 3.
The amino-acid sequence of this protein is encoded by nucleotides 922 to 2332 of nucleotide sequence (1).
Fig. 5 Graphical representation of the distribution of positively (ordinate upper half) and negatively (ordinate lower half) charged amino acids in the amino-acid sequence (I). The numbering of the amino acids (abscissa) corresponds to that in Fig. 1. The graph was constructed by the PC/gene program NOVOTNY (Genofit SA, Geneva, Switzerland).
Fig. 6 Graphical representation of the hydrophobic protein domains in amino-acid sequence (I) calculated by the method of Kyte et al., J. Mol.
Biol. 157, 105-132 (1982). Values above the number -5 (ordinate) indicate hydrophobic domains. The numbering of the amino acids (abscissa) corresponds to that in Fig. 1.
Fig. 8 Fig. 9 Fig. 10 Potential B-cell epitopes in amino-acid sequence (I). Values above the number zero (ordinate) signify the presence of possible B-cell epitopes. Calculation was by the method of Hopp et al., Proc. Natl. Acad. Sci. 78, 3824-3828 (1981). The numbering of the amino acids (abscissa) corresponds to that in Fig. 1.
(A) Model of the possible dimer formation by two molecules of the sporozoite antigen according to the invention by means of intermolecular interaction between the positively and negatively charged regions in the N-terminal amino-acid sequence of the Plasmodium sporozoite antigen according to the invention (see also Fig. 5). (B) shows the regions with different properties in amino-acid sequence (I) according to the computer calculations (see Fig. 5 to 7). + = positively charged domain; - = negatively charged domain; glyc = potential N-glycosylation region; ag = antigenic region (that is to say increased probability of the presence of B-cell epitopes; tm = possible transmembrane sequence.
A) Analysis of Sspl-digested genomic DNA from 9 different P. falciparum isolates (Gentz et al., EMBO J. 7, 225-230 [1988]). The following isolates were tested: lane 1 = T9/96.2; lane 2 = #13; lane 3 = CPG-1; lane 4 = 542; lane 5 = Kl; lane 6 = MAD20; lane 7 = R053; lane 8 = T9/94; lane 9 = RO-59.
B) Analysis of Dral- (lanes 1 and 3) and Hindlll(lanes 2 and 3) digested genomic DNA from two different Plasmodium species (P. falciparum lanes 1 and 2; P. berghei lanes 3 and 4).
Nucleotide sequence of the plasmid PDS56/RBSII,6xHis.
The abbreviations, buffers and media mentioned in the present application, and methods 1 to 15 used in the example correspond to those described in European Patent Application, publication number 309 746, pages 13 to 20.
IE 9196® Example Isolation of a P. falciparum sporozoite gene from a genomic expression gene bank using antibodies Preparation of an immune serum against P. falciparum sporozoites Sporozoites of the P. falciparum isolate NF54 were isolated by conventional methods from infected Anopheles stephensis mosquitoes. A rabbit was immunised in 2-week intervals on each occasion with 106 of these sporozoites in complete Freund's adjuvant. The immune serum was obtained in a customary manner.
Construction of the P. falciparum expression gene bank P. falciparum cells (KI isolate) were cultured by conventional methods (Trager et al., Science 193, 673-675 [1976]) in 10 culture dishes and then washed in culture medium containing 0.1% saponin. The washed parasites were resuspended in 2 ml of 10 mM EDTA [pH 8.0] 0.5% (w/v) SDS. After addition of 50 mg of proteinase K (Merck), the mixture was incubated at 65°C for 10 minutes and then 2 ml of phenol (saturated with 1 M tris/HCl [pH 8.0]) were added. The phases were mixed by shaking and separated again by centrifugation (10 minutes at 6,000 RPM, 20 °C). The phenol extraction was repeated twice (an interphase ought no longer to be visible) . The DNA in the aqueous phase was precipitated as in method 1, washed with ethanol and dried. The DNA was dissolved in 2 ml of water and mechanically sheared, that is to say forced 80 times through a syringe with a 0.5 x 16 mm needle. Then 0.2 volume 5 x EcoRI methylase buffer (50 mM tris/HCl [pH 7.5], 0.25 M NaCl, 50 mM EDTA, 25 mM ^-mercaptoethanol, 0.4 mM S-adenosylmethionine) was added. 10 μg of DNA were methylated with 50 units of EcoRI methylase (New England Biolabs Beverly, Massachusetts, USA) at 37 °C for 30 minutes. The DNA was extracted once with phenol as described above and precipitated as in method 1. The DNA was dissolved in 200 μΐ of T4 polymerase buffer and, after addition of 5 μΐ of 5mM dATP, dCTP, dGTP and dTTP, as well as 10 units of T4 polymerase, was incubated at 91966 37°C for 30 minutes. The DNA was again extracted with phenol and precipitated as in method 1. The DNA was dissolved in 50 pl of T4 DNA ligase buffer and, after addition of 0.01 OD260 units of phosphorylated EcoRI oligonucleotide adaptors (New England Biolabs) and 2 μΐ of T4 DNA ligase (12 Weiss units), ligated at 14 °C overnight. The DNA was precipitated as in method 1, dissolved in 20 pl of lx DNA gel-loading buffer and fractionated on a 0.8% (w/v) agarose gel (method 2). DNA fragments with a length of 2 to 6 kb (1 kb = 1,000 nucleotides) were isolated as in method 3. The resulting DNA was dissolved in 50 pl of water and, after addition of 6 pl of 10 x ligase buffer, 2 pi of dephosphorylated lambda arms (Promega Biotech., Madison Wl, USA) and 6 Weiss units of T4 DNA ligase, ligated at 14°C overnight.
The DNA was precipitated (method 1) and dissolved in 5 pl of water. After addition of 20 pl of packaging extract (Genofit S.A., Geneva, Switzerland), the DNA was packaged in lambda phage particles at 20°C for 2 hours in accor20 dance with the supplier's instructions. After addition of 500 pl of SM buffer and 50 pl of chloroform, the gene bank was ready for the antibody assay.
Gene bank antibody assav E. coli Y1090 was incubated in 3 ml of LB medium containing 40 pg/ml ampicillin in a shaker bath at 37°C overnight. The next morning the cells were sedimented (10 minutes at 7,000 x g, 20°C) and resuspended in 1 ml of SM buffer. To this cell suspension were added 106 infectious phage particles from the gene bank and incu30 bated at room temperature for 30 minutes. Then 60 ml of 0.8% (w/v) agar solution in LB medium which had been equilibrated at 42°C were added and thoroughly mixed. The soft agar with the infected cells was distributed over 6 LB-agar plates (diameter 135 mm) containing 40 pg/ml ampicillin and incubated at 42°C for 5 hours. A nitrocellulose filter dipped in 100 mM IPTG solution and dried was placed on each dish and incubated at 37°C overnight. The next day the position of the filter relative to the dish was marked * and the marked filter was stored in x TBS. A new nitrocellulose filter treated in 100 mM IPTG solution was placed on the plate, marked and incubated on the plates at 37°C for 4 hours. Both sets of filters were shaken in 1 x TBS for 10 minutes and then 5 incubated in 1 x TBS, 20% FCS (fetal calf serum) for 20 minutes. The sporozoite-specific rabbit antiserum was diluted 1:1000 with 1 x TBS/20% FCS and both sets of filters were incubated in a shaker bath at room temperature for 1 hour. The filters were now washed three times 10 in 1 x TBS, 0.1% Triton® X-100 for 10 minutes each time in a shaker bath, followed by incubation with 5 pCi [125I]protein A (Amersham, Aylesbury, GB; Catalogue No. 1M.144) in 1 x TBS, 0.1% protease-free bovine serum albumin for 1 hour. The filters were again washed as above and then 15 dried at room temperature. The filters were exposed to Kodak XAR x-ray film overnight. Plaques which had a positive reaction on both plates were identified with the aid of the marks on the film and picked off the Petri dishes on the basis of the marking. The phage solution 20 was again plated out in various dilutions in soft agar as in method 4, and individual positive plaques were again identified as described above. One positive plaque, called NXY hereinafter, was picked out, the lambda phages were grown as in method 5, and the DNA was isolated. pg of NXY DNA were dissolved in 490 pi of T4 polymerase buffer and digested with 50 units of EcoRI at 37°C for 1 hour. The DNA was precipitated (method 1) and fractionated on a 0.8% (w/v) agarose gel (method 2). As a control, 10 pg of gtll DNA were digested with EcoRI and 30 analysed. Two EcoRI fragments (2.0 and 1.0 kb) were present only in the lane with the NXY DNA. The EcoRI fragments were isolated (method 3) and dissolved in 50 pi of water. For the cloning, 50 ng of EcoRI-cut, dephosphorylated Ml3 mp!8 DNA (Pharmacia Uppsala, SE; method 35 6) were mixed with 10 pi of each of the dissolved EcoRI fragments from the NXY DNA, and 2 pi of 10 x ligase buffer, 6 pi of water and 6 Weiss units of T4 DNA ligase were added, and the DNAs were ligated at room temperature for 1 hour. Competent TG-1 E. coli cells (Amersham) were IE 9196® transformed with the ligated DNA (method 7) . Two plaques from each mixture were isolated and amplified, and sufficient DNA for determining the sequence was isolated (method 8). The DNA sequence was determined by method 9.
The M13 mpl8 clones which contained the EcoRI fragments and were used were called NXY-L (1 kb fragment) and NXY-H (2.0 kb fragment).
Introduction of deletions for sequence determination pg each of NXY-L and NXY-H DNA were completely 10 digested with BamHI and Pstl. After one hour, the DNAs were precipitated (method 1) and the pellet was dissolved in 100 pi of 66 mM tris/HCl [pH 8.0], 6.6 mM MgCl2. 10 units of exonuclease III were added and then the mixture was incubated at 37°C. 30 pi samples were taken after 1, 3 and 6 minutes and 3 pi of 0.5 M EDTA were added. The samples were precipitated by method 1. The DNAs were dissolved in 50 pi of SI nuclease buffer (0.3 M potassium acetate, 10 mM zinc sulphate, 5% (v/v) glycerol). 10 units of SI nuclease were added and then the mixture was incubated at room temperature for 30 minutes. The samples were extracted once each with phenol and ether and then precipitated by method 1, The DNAs were dissolved in HIN buffer and incubated with 5 units of Klenow polymerase at 37eC for 2 minutes. 1 pi of 0.25 mM dATP, dCTP, dTTP and dGTP was added and then the mixture was incubated for a further 2 minutes. Addition of 5 pi of 10 x ligase buffer and 400 units of T4 DNA ligase was followed by ligation at room temperature overnight. Then E. coli cells were transformed with the DNA by method 7. The DNA sequence was analysed by method 8 and 9.
Analysis of the NXY protein sequence The protein sequence (I; Fig. 1) derived from the nucleic acid sequence (1; Fig. 2) was analysed for the charge distribution (Fig. 5), hydrophobicity (Fig. 6) and the presence of possible B-cell epitopes (Fig. 7). This showed that the amino-acid sequence (I) has, starting at the N terminus, a cluster of positive charges followed by a cluster of negative charges. These are followed by amino-acid residues which can be N-glycosylated (Fig. 8B) 91966 followed by a region which contains potential immunogenic B-cell epitopes. A typical transmembrane sequence (Fig. 6 and 8B) is located in the C-terminal part of the aminoacid sequence (I) and might serve for anchoring of the protein in the sporozoite membrane. It appears possible on the basis of this analysis of amino-acid sequence (I) that the charged regions, including the immunogenic domain, are exposed on the sporozoite surface and thus to the immune system. This hypothesis is supported by the recognition of the sporozoite antigen according to the invention by human sera from malaria-exposed individuals (see below). Figure 8A shows how two polypeptides according to the invention can form dimers by means of the charged N termini. In the case of an immunisation with NXY, this dimer formation might also lead to direct binding of polypeptides according to the invention which contain the N-terminal part of amino-acid sequence (I) to the natural Plasmodium sporozoite antigen of the present invention and, owing to this reaction, to impeding or even inhibition of hepatocyte invasion.
Analysis of the NXY gene from the malaria parasite P, berqhei Mice were infected intravenously with P. berghei (Anka isolate). Blood was taken from the mice at 40% parasitaemia. The parasites were isolated from the blood by the method of Trager et al., Science 193, 673-675 [1976].
A gene bank of the Plasmodium berghei genome was prepared as described above for the Kl isolate of P. falciparum. This P. berghei lambda gene bank was then plated as described above (2 χ 105 phage particles on two Petri dishes of diameter 135 mm). After five hours, when plaques became visible, the Petri dishes were removed from the 37*C incubator and stored in a refrigerator overnight. PALL nylon filters (PALL, Basle, Switzerland) were then placed on the cold dishes, and the relative position of the filters on the Petri dishes was marked with a felt pen. After 5 minutes, the filters were cautiously lifted- off the plate and placed, with the side ΙΕ 9ΐθ66 with the plaques upwards, on Whatmann 3MM paper which had previously been impregnated with alkaline solution (0.5 N sodium hydroxide and 0.5 M tris) . After a few minutes, the filters were placed on a new Whatmann 3MM paper impregnated with the alkaline solution. The filters were then briefly dried on a 3MM filter paper and then placed twice for five minutes on Whatmann 3MM paper which had previously been impregnated with 1.5 M NaCl, 0.5 M tris/HCI [pH 8.0]. The filters were then dried in the air and baked at 80°C in vacuo for 90 minutes. A P. falciparum probe was prepared by isolating the 2 kb EcoRI fragment from clone NXY-H (methods 1, 2 and 3) and radioactively labelling it (method 11). A positive clone (PB-B) was obtained using this P. falciparum probe and was isolated (methods 4 and 12) and analysed. The clone PB-B was propagated by method 5, and the DNA was isolated and digested with EcoRI (method 2). A 400 bp fragment which was not present in the control DNA (lambda gtll) was isolated and cloned in M13 mpl8 and sequenced (methods 6, 7, 8 and 9). The first 47 amino acids after the ATG start codon which are encoded by the PB-B DNA are identical to the corresponding amino acids of the P. falciparum sporozoite antigen with the N-terminal aminoacid sequence (I). This sequence homology is exceptional.
Thus, for example, there is little sequence homology in the case of the repetitive sequences of the circumsporozoite antigen CS in the various Plasmodium species. Preparation of a polypeptide according to the invention in E. coli pg of NXY DNA were partially cleaved with 10 units of EcoRI at 37°C for 2 minutes. The resulting 3 kb fragment was isolated by methods 2 and 3 and introduced into the EcoRI cleavage site of M13 mpl8 (method 6). The subclone was called M13-NXY.
An Asel fragment specific for P. falciparum was isolated from the M13-NXY clone by methods 1 to 3 as follows. 6 μζ of M13-NXY DNA were digested with 30 units of Asel in 100 μΐ of 1 χ T4 polymerase buffer at 37°C for one hour. The DNA was precipitated (method 1) and IE 91966 a ' fractionated on a 0.8% (w/v) agarose gel (method 2), and a 1400 bp fragment was isolated (method 3). The ends were filled in with Klenow polymerase as described above. The fragment was resuspended in 20 μΐ of water and, after addition of 10 pmol of a phosphorylated BamHI oligonucleotide adaptor (10-raers CCGGATCCGG; New England Biolabs), 2.5 μΐ of 10 x ligase buffer and 6 Weiss units of T4 DNA ligase, ligated at 14°C overnight. The DNA was precipitated (method 1), dissolved in 50 μΐ of lx T4 polymerase buffer and, after addition of 40 units of BamHI, digested at 37°C for 1 hour. The DNA was precipitated (method 1) and fractionated on a 1.0% (w/v) agarose gel (method 2). A 1400 bp fragment was isolated (method 3) and dissolved in 10 μΐ of water. To prepare the vector (see method 6), 1 pg of pDS56/RBSII,6xHis vector DNA was digested with 10 units of BamHI in T4 polymerase buffer at 37°C for 1 hour. The vector pDS56/RBSII,6xHis is a derivative of the vector pDS56/RBSII which is described in detail in European Patent Application, publication no. 282 042. The nucleotide sequence of the vector pDS56/RBSII,6xHis is shown in Fig. 10. The vector pDS56/RBSII,6xHis differs from the vector pDS56/RBSII by having an additional DNA fragment which codes for 6 histidine residues. The vector pDS56/RBSII,6xHis can be prepared from the vector pDS56/RBSII by conventional methods of recombinant DNA technology, for example by incorporating a suitable synthetic DNA fragment into the vector pDS56/RBSII. The vector pDS56/RBSII,6xHis has been deposited in the form of a culture of E. coli M15 con30 taining the plasmids pDS56/RBSII,6xHis and pDMI,l since April 6, 1989, at the Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 16 in Braunschweig, Germany, under DSM No. 5298, specifically in connection with European Patent Application, publication no. 393 502, in accordance with the Budapest treaty. The vector DNA was dephosphorylated (method 4), extracted once with phenol (see above), purified on a 0.8% (w/v) agarose gel and subsequently isolated by method 3. The isolated DNA was dissolved in *50 μΐ of water. ΙΕ 9ΐθ66 ' 28 _ ——5 μΐ of the linearised pDS56/RBSII, 6xHis vector DNA, which had been digested with BamHI and dephosphorylated (method 6), were incubated with 5 pl of the 1400 bp fragment, 1.2 pl of 10 x ligase buffer and 6 Weiss units of T4 DNA ligase at room temperature for one hour. 10 pl of DNA were then transformed into competent E. coli SG13009 (pUHAl) cells (method 7) and plated on LB plates containing 100 pg/ml ampicillin and 25 pg/ml kanamycin. Individual colonies were picked out with a toothpick and transferred into 3 ml of LB medium containing 100 pg/ml ampicillin and 25 pg/ml kanamycin. The cultures were incubated in a shaker bath at 37 °C until the optical density at 600 nm (OD600) with pure medium as reference was 0.6. An aliquot of 500 pl of the culture was taken as non-induced control. IPTG (1 mM final concentration) was added to the remainder of the culture, and the induced culture was incubated for a further 3 hours. Then 500 pl of the induced culture were removed and centrifuged together with the non-induced sample (3 minutes at 12,000 RPM, 20°C). The supernatant was aspirated off, and the cell sediment was resuspended in 100 pl of SDS sample buffer. The samples were incubated in boiling water for 7 minutes and then the proteins were fractionated on a 12% SDS polyacrylamide gel (method 13) by electrophoresis (three hours at 50 mA constant current). The gel was stained with 0.1% (w/v) Coomassie blue in 30% (v/v) acetic acid and 10% (v/v) methanol on the shaker for 30 minutes. The gel was destained in 10% (v/v) methanol and 10% (v/v) acetic acid at 65°C for 2 hours. Clones which showed additional bands, compared with the uninduced sample, with the apparent molecular weight of 69 kDa (= 69,000 Dalton) were called E. coli SG13009 (pDS-NXY; pUHAl). The strain E. coli SG13009 (pDS-NXY? pUHAl) was deposited on March 16, 1990, at the Deutsche Sammlung von Mikroorganismen in Braunschweig, Germany under DSM No. 5846 in accordance with the Budapest treaty. The strain E. coli SG13009 is described by Gottesman et al. in J. Bacteriol. 148, 265-273 (1981). The plasmid pUHAl codes for the lac repressor. It is a derivative of the ,E 91966 plasmid pDMI,1 which is described in detail in European '/ Patent Application, publication no. 309 746. The plasmid pUHAl differs from the plasmid pDMI,l by replacement of the lacl*1 allele by the lacl allele which contains the wild-type promoter. This replacement ensures that an optimum amount of lac repressor is produced in the strain SG13009 (pDS-NXY; pUHAl). This makes possible a particularly efficient expression of a recombinant protein. The strain SG13009 (pUHAl) can be obtained in a known manner starting from the strain SG13009 (pDS-NXY; pUHAl).
Western blot analysis of the expression products of PDS-NXY A lysate of E. coli SG13009 (pDS-NXY; pUHAl) was fractionated on a mini SDS gel, and the proteins were transferred to nitrocellulose filters (methods 13 and 14) . The filter was then analysed by the Western blot technique (Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350-4354 [1979]) using anti-sporozoite rabbit serum or human sera from malaria-exposed individuals. It was possible to show in this way that these sera recognise antigens specific for the malaria parasites in the lysate. This means that the synthetic antigen with the amino-acid sequence (II) is recognised by antibodies against the natural antigen, that is to say that it coincides in at least one specific epitope with the Plasmodium falciparum sporozoite antigen with the N-terminal amino-acid sequence (I). It is also possible to conclude indirectly that the natural antigen can be recognised in vivo by the immune system of infected individuals.
Reaction of antibodies against the recombinant 69 kPa protein with sporozoites and blood stages of P. falciparum E. coli SG13009 (pDS-NXY; pUHAl) was cultivated by conventional methods and induced to express the recombinant polypeptide with the amino-acid sequence (II) (= 69 kDa protein). The recombinant 69 kDa protein was then isolated from the E. coli lysate and purified by affinity chromatography on a nitrilotriacetic acid/nickel IE 91966 - 30 chelate resin. The purified 69 kDa protein was used to immunise rabbits. After two booster injections, the serum was tested for unfixed Plasmodium sporozoites and merozoites by immunofluorescence. The serum recognised both stages of the parasite. The rabbit serum (dilution 1:500) was therefore tested on Western blots of sporozoite and blood-stage extracts . The serum recognised a band of over 200 kDa in the sporozoite extract, while three bands of about 50-55 kDa were recognised in the blood-stage extract. 50-53 kDa proteins from blood stages have been described by Wahlgren et al., Proc. Natl. Acad. Sci. USA 83, 2677-2681 [1986]. Comparison of the amino-acid sequence of these 50-53 kDa proteins with the amino-acid sequence of the sporozoite antigen of the present inven15 tion shows that they must be different proteins. It was also found that, when the NXY-DNA is used as probe, no NXY transcripts are detectable in blood-stage RNA. Thus the Plasmodium antigen according to the invention is specific for the sporozoite stage.
Recognition of the NXY gene in 9 P. falciparum isolates and in P. berqhei Genomic DNA from 9 different P. falciparum isolates was in each case digested with SspI, and the fragments were separated on a 1.2% (w/v) agarose gel. The DNAs were transferred to a nitrocellulose membrane and hybridised with a radioactive Plasmodium sporozoite antigen gene-specific probe from M13-NXY DNA (2 kb EcoRI fragment) (60°C, 2xSSC; methods 2, 3, 12, 15). Fig. 9 part A shows clearly that two bands (about 1.7 kb and about 15 kb relative to the markers) can be detected with all 9 isolates which were analysed. Differences in the intensity are caused by different amounts of DNA.
In order to test whether the NXY gene can also be detected in the mouse malaria parasite P. berghei, in each case P. falciparum DNA (lanes 1 and 2) and P. berghei DNA (lanes 3 and 4) were cut with Dral (lanes 1 and 3) or Hindlll (lanes 2 and 4) and tested as above with the radioactive probe from the M13-NXY DNA. Figure 9 part B shows clearly that an NXY-specific band can be \E 9A96® detected in all 4 lanes. Although the detected NXYspecific DNA has a different size in each case, there is not, however, a non-specific cross-reaction because, as was shown above, the nucleotide sequence coding for the first 47 amino acids in the P. falciparum DNA is identical to the corresponding nucleotide sequence in the P. berghei DNA.

Claims (36)

1. Patent claims
1. Polypeptides which coincide in at least one specific epitope with the Plasmodium falciparum sporozoite antigen which contains the N-terminal amino—acid 5 sequence (I) 5 10 15 1 Met Asn Lys Val Asn Ala Val His Lys He Asn Ala Val Asp Lys 16 Val Asn Ala Val Asn Lys Val Asn Ser Val Asn Lys Leu Asn Val 31 Val Asn Lys Thr Asn Val Leu Ser Lys Leu Asn Ala Val Tyr Lys 46 Val Asn Ser Val His Lys Met Asn Ala Val Asn Lys Val Asn Ala 61 val Asn Lys Val Asn Ala Val Asn Lys Val Asn Val Val Asn Lys 76 Lys Asp lie Leu Asn Lys Leu Asn Ala Leu Tyr Lys Met Asn Ala 91 Val Tyr Lys Met Asn Ala Leu Asn Lys Val Ser Ala Val Asn Lys 106 Val Ser Ala Val Asn Lys Val Ser Ala Val Asn Lys Met Gly Ala 121 Val Asn Arg Val Asn Gly Val Asn Lys val Asn Glu Val Asn Glu 136 Val Asn Glu Val Asn Glu Val Asn Met Val Asn Glu Val Asn Glu 151 Leu Asn Glu Val Asn Asn Val Asn Ala Val Asn Glu Val Asn Ser 166 val Asn Glu Val Asn Glu Met Asn Glu Val Asn Lys val Asn Glu 181 Leu Asn Glu Val Asn Glu Val Asn Asn Val Asn Glu val Asn Asn 196 Val Asn Val Met Asn Asn Val Asn Glu Met Asn Asn Met Asn Glu 211 Met Asn Asn Val Asn Val Val Asn Glu Val Asn Asn Val Asn Glu 226 Val Asn Asn Val Asn Glu Met Asn Asn Val Asn Glu Met Asn Asn 241 Met Asn Glu Met Asn Asn Val Asn Val val Asn Glu val Asn Asn 256 Val Asn Glu Met Asn Asn Thr Asn Glu Leu Asn Glu Val Asn Glu 271 Val Asn Asn Val Asn Glu Val Asn Asp Val Asn Val Val Asn Glu 286 Val Asn Asn Val Asn Glu Met Asn Asn Met Asn Glu Leu Asn Glu 301 Val Asn Gly Val Asn Glu Val Asn Asn Thr Asn Glu lie His Glu 316 Met Asn Asn lie Asn Glu Val Asn Asn Thr Asn Glu Val Asn Asn 331 Thr Asn Glu He Tyr Glu Met Asn Asn Met Asn Asp Val Asn Asn 346 Thr Asn Glu He Asn Val Val Asn Ala Val Asn Glu Val Asn Lys 361 Val Asn Asp Ser Asn Asn Ser Asn Asp Ala Asn Glu Gly Asn Asn 376 Ala Asn Tyr Ser Asn Asp Ser Ser Asn Thr Asn Asn Asn Thr Ser 391 Ser Ser Thr Asn Asn Ser Asn Asn Asn Thr Ser Cys Ser Ser Gin 406 Asn Thr Thr Thr Ser Ser Glu Asn Asn Asp Ser Leu Glu Asn Lys 421 Arg Asn Glu Glu Asp Glu Asp Glu Glu Asp Asp Gin Lys Asp Thr 436 Gin Lys Glu Lys Asn Asn Leu Glu Gin Glu Asp Met Ser Pro Tyr 451 Glu Asp Arg Asn Lys Asn Asp Glu Lys Asn He Asn Glu Gin Asp 466 Lys Phe His Leu Ser Asn Asp Leu Gly Lys lie Tyr Asp Thr Tyr 481 Asn Gin Gly Asp Glu Val Val Val Ser Lys Asn Lys Asp Lys Leu 496 Glu Lys His Leu Asn Asp Tyr Lys Ser Tyr Tyr Tyr Leu Ser Lys 511 Ala Thr Leu Met Asp Lys He Gly Glu Ser Gin Asn Asn Asn Asn 526 Tyr Asn val Cys Asn Ser Asn Glu Leu Gly Thr Asn Glu Ser He 541 Lys Thr Asn Ser Asp Gin Asn Asp Asn Val Lys Glu Lys Asn Asp 556 Ser Asn lie Phe Met Lys Met lie He He He Arg Leu Met lie 571 Met He lie Met He Met He He He Trp Tyr Leu Lys He Leu 586 Gin Asp Lys He He Trp Arg Asn Lys Lys val Glu Lys Thr Ser 601 Asn lie Leu Asn Asn Phe Asp Asn Asn Gly Asn Asp Asn Asp Asn 616 Asp Asn Asp Asp Asn Asn Asp Asn Asp Asn Asn Asn Asn Asn Asn 631 Met Asn Asn Gin Tyr Asn Tyr Gin Glu Asn Asn He Asn Thr Asn 646 Tyr Asn lie Leu Tyr Thr Pro Ser Asn Cys Gin He Gin Asn Asn 661 Ser Tyr Met Asn Thr Asn Glu Met Tyr Gin Pro Leu Tyr Asn Thr 676 Tyr Fro Ser Asn Arg lie Gin Glu Asn Ser Thr He Asn Asn Asn 691 lie lie Asn Asp Ser Pro Tyr Met Asn Asn Asp Asn Thr Asn 706 Asn Phe He Ser Gly Met Asn 91966
2. Polypeptides according to Claim 1, which are covalently linked to an affinity peptide, for example the polypeptide with the amino-acid sequence (II) 1 Met Arg Gly Ser Kis His His His His His C-ly Ser Val Asn Ser 16 Val Asn Lys Glu Asn Asn Met Asn Lys Val Asn Ala Val His Lys 31 lie Asn Ala Val Asp Lys Val Asn Ala Val Asn Lys Val Asn Ser 46 val Asn Lys Leu Asn Val Val Asn Lys Thr Asn Val Leu Ser Lys 61 Leu Asn Ala val Tyr Lys val Asn Ser Val His Lys Met Asn Ala 75 Val Asn Lys Val Asn Ala Val Asn Lys Val Asn Ala Val Asn Lys 91 Val Asn Val Val Asn Lys Lys Asp lie Leu Asn Lys Leu Asn Ala 106 Leu Tyr Lys Met Asn Ala Val Tyr Lys Met Asn Ala Leu Asn Lys 121 Val Ser Ala Val Asn Lys Val Ser Ala Val Asn Lys val Ser Ala 136 Val Asn Lys Met Gly Ala Val Asn Arg Val Asn Gly val Asn Lys 151 Val Asn Glu Val Asn Glu Val Asn C-lu Val Asn C-lu Val Asn Met 166 Val Asn Glu Val Asn Glu Leu Asn Glu Val Asn Asn Val Asn Ala 181 Val Asn Glu Val Asn Ser Val Asn Glu Val Asn Glu Met Asn Glu 196 Val Asn Lys Val Asn Glu Leu Asn Glu Val Asn Glu Val Asn Asn 211 Val Asn Glu Val Asn Asn Val Asn Val Met Asn Asn val Asn Glu 226 Met Asn Asn Met Asn Glu Met Asn Asn Val Asn Val val Asn Glu 241 Val Asn Asn Val Asn Glu Val Asn Asn Val Asn Glu Met Asn Asn 256 Val Asn Glu Met Asn Asn Met Asn Glu Met Asn Asn val Asn Val 271 Val Asn Glu val Asn Asn Val Asn Glu Met Asn Asn Thr Asn Glu 285 Leu Asn Glu val Asn Glu Val Asn Asn Val Asn Glu Val Asn Asp 301 •Val Asn Val Val Asn Glu Val Asn Asn Val Asn Glu Met Asn Asn 316 Met Asn Glu Leu Asn Glu Val Asn Gly Val Asn Glu Val Asn Asn 331 Thr Asn Glu He Kis Glu Met Asn Asn He Asn Glu Val Asn Asn 346 Thr Asn Glu Val Asn Asn Thr Asn Glu He Tyr Glu Met Asn Asn 361 Met Asn Asp Val Asn Asn Thr Asn Glu He Asn Val Val Asn Ala 376 Val Asn Glu Val Asn Lys Val Asn Asp Ser Asn Asn Ser Asn Asp 391 Ala Asn Glu Gly Asn Asn Ala Asn Tyr Ser Asn Asp Ser Ser Asn 406 Thr Asn Asn Asn Thr Ser Ser Ser Thr Asn Asn Ser Asn Asn Asn 421 Thr Ser Cys Ser Ser Gin Asn Thr Thr Ser Ser Glu Asn Asn 436 Asp Ser Leu Glu Asn Lys Arg Asn Glu Glu Asp Glu Asp Glu Glu 451 Asp Asp Gin Lys Asp Thr Gin Lys Glu Lys Asn Asn Leu Glu Gin 466 Glu Asp Met Ser Pro Tyr Glu Asp Arg Asn Lys Asn Asp C-lu Lys 481 Asn lie Asn Gly He Arg Arg Pro Ala Ala Lys Leu Asn IE 9196®
3. Polypeptides according to Claim 1 or 2, with a part-sequence (NXY) n , (YNX) n or (XYN) n in which N is an asparagine residue, 5 X is a charged amino-acid residue, for example an amino-acid residue of glutamic acid or lysine, Y is a hydrophobic amino-acid residue, for example an amino-acid residue of valine or 10 methionine and n is a number between 3 and 120.
4. Polypeptides according to Claim 1, 2 or 3, which are adsorbed onto a carrier material or covalently bonded thereto. 15
5. A DNA which codes for a polypeptide according to Claim 1, 2 or 3, in particular a DNA which contains the nucleotide sequence from position 948 to position 3088 in Figure 2. or a part-sequence thereof.
6. A replicable microbial vector which contains a 20 DNA which codes for a polypeptide according to Claim 1, 2 or 3, in particular a DNA which contains the nucleotide sequence from position 948 to position 3088 in Figure 2. or a part-sequence thereof, where the DNA is preferably linked to an expression-control sequence so that the 25 polypeptide encoded by the DNA can be expressed.
7. A transformed microorganism which contains a replicable microbial vector, where the vector contains a DNA which codes for a polypeptide according to Claim 1, 2 or 3, in particular a DNA which contains the nucleotide 30 sequence from position 948 to position 3088 in Figure 2 or a part-sequence thereof, where the DNA is preferably linked to an expression-control sequence so that the polypeptide encoded by the DNA can be expressed.
8. Antibodies which are directed against a poly35 peptide according to Claim 1, 2 or 3.
9. Polypeptides according to one of Claims 1 to 4 for the immunisation of mammals against malaria.
10. A process for the preparation of polypeptides according to Claim 1, 2 or 3, characterised in that: IE 91966 (a) a microorganism which has been transformed with a replicable microbial vector which contains a DNA which codes for a polypeptide according to Claim 1, 2 or 3, in particular a DNA which contains the nucleotide sequence from position 948 to position 3088 in Figure 2 or a part-sequence thereof, is cultivated under conditions which permit the expression of the polypeptide encoded by the DNA; and (b) the polypeptide is isolated from the culture.
11. A process for the preparation of polypeptides according to Claim 1, 2 or 3, characterised in that: (a) peptides are prepared by conventional methods of peptide synthesis; and (b) several peptides are linked together in the required sequence.
12. A process for the preparation of a replicable microbial vector which contains a DNA which codes for a polypeptide according to Claim 1, 2 or 3, in particular a DNA which contains the nucleotide sequence from position 948 to position 3088 in Figure 2 or a part-sequence thereof, characterised in that: (a) a DNA which codes for a polypeptide according to Claim 1, 2 or 3, in particular a DNA which contains the nucleotide sequence from position 948 to position 3088 in Figure 2 or a part-sequence thereof, is integrated in a vector; (b) the transformed vector is replicated in a microorganism; and (c) the replicated microbial vectors are isolated from the microorganism.
13. A process for the preparation of a transformed microorganism which is able to produce a polypeptide according to Claim 1, 2 or 3, characterised in that: (a) a microorganism which contains a replicable microbial vector, where the vector contains a DNA which codes for a polypeptide according to Claim 1, 2 or 3, in particular a DNA which contains the nucleotide sequence from position 948 to position 3088 in Figure 2 or a part-sequence thereof, where the DNA IE 91966 3 6is linked to an expression-control sequence so that the polypeptide encoded by the DNA can be expressed, is transformed; and (b) the transformed microorganism is grown in a culture medium.
14. A process for the preparation of antibodies which are directed against a polypeptide according to Claim 1, 2 or 3, characterised in that (a) a polypeptide according to one of Claims 1, 2, 3 or 4 is injected into a suitable host organism which is capable of an immunological reaction against the polypeptide; and (b) the antibodies which are produced and are directed against the polypeptide are isolated in a known manner.
15. Immunogenic compositions which contain a polypeptide according to one of Claims 1 to 4 and a suitable adjuvant.
16. Immunogenic compositions according to Claim 15, as a vaccine. 37 ,E 91966
17. Useof a polypeptide according to one of Claims 1 to 4 or of an immunogenic composition according to one of Claims 15 or 16 for the immunisation of mammals against malaria. IE 91966 - 38 - 18. or 11. 19. A polypeptide prepared by a process according to claim 10 A replicable microbial vector prepared by a process according to claim 12.
18. 20. A transformed microorganism prepared by a process according to claim 13.
19. 21. Antibodies prepared by a process according to claim 14. IE 91966 - 39
20. 22. The invention as made and hereinbefore described. IE 91966
21. 23. A method for the immunisation of mammals against malaria, characterised in that the immune system of these mammals is stimulated with an immunising amount of a polypeptide according to one of Claims 1 to 4 or of an immunogenic composition according to one of Claims 15 or 16.
22. 24. A polypeptide according to Claim 1, substantially as hereinbefore described with particular reference to the accompanying drawings.
23. 25. A DNA according to Claim 3, substantially as hereinbefore described.
24. 26. A replicable microbial vector according to Claim 6, substantially as hereinbefore described with particular reference to the accompanying drawings.
25. 27. A transformed microorganism according to Claim 7, substantially as hereinbefore described.
26. 28. An antibody according to Claim 8, substantially as hereinbefore described.
27. 29. A process for the preparation of a polypeptide according to Claim 1, substantially as hereinbefore described and exemplified.
28. 30. A polypeptide according to Claim 1, whenever prepared by a process claimed in any one of Claims 10, 11 or 29.
29. 31. A process for the preparation of a replicable microbial vector according to Claim 6, substantially as hereinbefore described and exemplified.
30. 32. A replicable microbial vector according to Claim 6, whenever prepared by a process claimed in Claim 12 or 31. IE 91966 - 41
31. 33. A process for the preparation of a transformed microorganism according to Claim 7, substantially as hereinbefore described and exemplified.
32. 34. A transformed microorganism according to Claim 7, whenever prepared by a process claimed in Claim 13 or 33.
33. 35. A process for the preparation of an antibody according to Claim 8, substantially as hereinbefore described and exemplified.
34. 36. An antibody according to Claim 8, whenever prepared by a process claimed in Claim 14 or 35.
35. 37. An immunogenic composition according to Claim 15, substantially as hereinbefore described.
36. 38. Use according to Claim 17, substantially as hereinbefore described.
IE096691A 1990-03-23 1991-03-22 Plasmodium Sporozoite Antigen IE910966A1 (en)

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