CA2037151A1 - Plasmodium sporozoite antigen - Google Patents

Plasmodium sporozoite antigen

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Publication number
CA2037151A1
CA2037151A1 CA002037151A CA2037151A CA2037151A1 CA 2037151 A1 CA2037151 A1 CA 2037151A1 CA 002037151 A CA002037151 A CA 002037151A CA 2037151 A CA2037151 A CA 2037151A CA 2037151 A1 CA2037151 A1 CA 2037151A1
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CA
Canada
Prior art keywords
asn
dna
polypeptide
sequence
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002037151A
Other languages
French (fr)
Inventor
Ulrich Certa
Maria Guttinger
Hugues Matile
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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Publication of CA2037151A1 publication Critical patent/CA2037151A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • 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

Abstract

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 furthermore relates to immuno-genic 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 anti-bodies against a polypeptide according to the invention.
Processes for the preparation of the immunogenic composi-tions, of the microorganisms and of the antibodies, and the use of the polypeptides and of the immunogenic compositions for the immunisation of mammals against malaria are claimed.

Description

~3i7~

Malaria is caused in humans by four species of Plasmodium, namely by P. falciparum, P. vivax, P. ovale and P. malariae. According to a 1986 report of the World Health Organisation (~HO) 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 drug-resistant parasites and of insecticide-resistant mosquito 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 Bruce-Chwatt, 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 mosquitoes introduce sporozoites into the bloodstream of vertebrates. The-~e 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 s0veral ~multiplication cycles in the host's erythrocytes and then develop into gametocytes. The gametocytes, which represent the sexual s~age of the parasite, are picked up by feeding mosquitoes. Af~er fertilisation in the insect's intestine, the gametocytes develop in~o sporo-zoites which then migrate into the saliva~y glands of the insect. A new cycle can th~n s~art.
Thus, v~ccines against the sporozoite form of Wa/29.1.90 .~ .

: . .
. :
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Plasmodium falciparum are the first line o~ defence against malaria infection. In recent years, the so-called CS protein from sporozoites of various species of Plas-modium 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 1 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 sporo-zoite antigen with an apparent molecular weight greater than 200 kDalton (kDa) with the N-terminal amino-acid sequence (I~

- 3 ~

IS
1 .~ee Asn Lys Val Asn Ala Val .~is Lys I'e Asn Ala Val ASp Lys ,S Val Asn Ala val Asn Lys Val Asn ';er Val Asn Lys Leu Asn Val 31 Val Asn Lys T.~ Asn Val Leu Ser Lys Leu Asn Ala Val ~yr r '15 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 Ile Leu Asn r y5 Leu Asn Ala Leu Tyr Lys Met Asn Ala 91 Val ~yr Lys Met Asn Ala Leu Asn ~.ys Val Ser Ala Val Asn r ys 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 l.ys Val Asn Glu Val Asn Glu 136 Val Asn Glu Val Asn Glu Val Asn ~let Val Asn Glu Val Asn Glu 151 Leu Asn Glu Val Asn Asn Val Asn ~la Val Asn Glu Val Asn Ser 166 Val Asn Glu Val Asn G1u Met Asn Glu Val Asn Lys Val Asn G;u 181 'eu Asn Glu Val Asn G.u Val Asn Asn Val Asn Glu Val Asn Asn 196 Val Asn Val Met A5n Asn Val Asn Glu Me~ Asn A,n Met Asn G;~
211 ~et Asn Asn Val Asn Val Val Asn G;u Val Asn Asn Val Asn G'u 226 Val Asn Asn Val Asn Glu Met Asn Asn Val Asn Glu Met Asn Asn 241 ~et 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 G.u 271 Val Asn Asn Val Asn Glu Val Asn Asp Val Asn Val Val Asn Glu 286 Val Asn Asn Val Asn G1u Met Asn Asn Met Asn Glu Leu Asn Glu 301 Val Asn Gly val Asn Glu Val Asn Asn Thr Asn Glu I1e His G1u 316 Met Asn Asn Ile Asn Glu Val Asn Asn Thr Asn Glu Val Asn Asn 331 T.~r Asn G1u I'e Tyr Glu Met Asn Asn Met AS~ Asp Val Asn Asn 346 Thr Asn Glu Ile Asn Val Val Asn Ala Val ~sn Glu Val ASn Lys 361 Val Asn Asp Ser Asn Asn Ser Asn Asp Ala AS~ 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 G.n 406 Asn Thr ~hr T.~r Ser Ser Glu Asn Asn Asp Ser Leu Glu Asn Lys 471 Arg Asn Glu Glu Asp Glu Asp Glu Glu Asp ASp Gln Lys Asp Thr 436 GLn ~ys Glu Lys Asn ~sn Leu Glu Gln Glu Asp Met Ser ~ro Tyr 451 Glu Asp Arg Asn Lys Asn ASp Glu Lys Asn Ile Asn Glu Gln Asp 466 Lys Phe His Leu Ser Asn Asp Leu Gly Lys ~le Tyr ASp ~r ~yr 4a1 Asn Gln Gly Asp Glu Val Val Val Ser Lys Asn Lys Asp Lys Leu 496 Glu Lys His Leu Asn Asp Ty~ Lys Ser Ty~ ~yr Tyr ~eu Ser Lys 511 Ala T.~r Leu Met ASp rys Ile Gly Gl~ Ser G~n Asn Asn Asn Asn 526 Tyr Asn Val Cys Asn Se~ Asn Glu Leu Gly ~hr Asn Glu Ser I?e 541 Ly ~hr Asn Ser Asp Gln Asn Asp Asn Val Lys Glu Lys Asn Asp 556 Ser Asn Ile Phe Mee Lys Me~ ~le Ile Ile Ile Arg ~eu Met Ile 571 Met Ils Ile Met IlQ M~t Ile Ile Il~ Trp Tyr Leu Lys Ile Leu 586 Gln ASp Lys Ile Ile Trp Arg Asn Lys Lys Val Glu Lys Thr Ser 601 Asn ~le 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 Gl~ Ty~ Asn Tyr Gln Glu Asn Asn Ile Asn Thr Asn ;46 Tyr Asn Ile Leu ~yr ~r Pro Ser Asn CyS Gln Ile Gln Asn Asn 661 Ser Tyr Met Asn Thr Asn Glu Met Tyr Gln Pro Leu ~yr Asn ~hr 676 Tyr Pro Ser Asn Arg ~le Gln Glu Asn Ser Thr Ile Asn Asn Asn 691 :le Ile Asn Asp Ser Pro ~y~ Met Asn Asn Asp Asn Thr ~.~r Asn 706 Asn ~hr ~he I'~ Ser Gly Met ~sn , , This amino-acid sequence contains 713 amino-acid residues with the following amino-acid composition: Ala (l9), Arg (6), Asn (219), Asp (37), Cys (3), Gln (15), Glu (65), Gly (1~), His (5), Ile (34), Leu (26), Lys (48), Met (34), Phe (4), Pro (5), Ser (39), Thr (27), Trp (2), Tyr (23) and Val (92). The calculated ~lecular 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, 3~0, 387, 388, 394, ~98, 399, 406, ~14, 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 amino-acid 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 prefPrred. The sequence (NXY) is repeated about 103 times in the amino-aci~ sequence (I). Exaimples of particularly præferred sequences (NXY) are AsnGluVal, AsnLysVal and AsnGluNet. 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 (AsnGluYal)", (ValAsn&lu)4 and (GluV~lAsn)4. Comparison of the sequence of these peptides, that is to say AsnGluYal.AsnGluValAsnGluValAsnGluVal GluValAsnGluValAsnGluValAsnGluValAsn Val.~snGluValAsnGluVal~snGluYalAsnGlu , :
:

~ ~ 3 ~

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 co~es for the N-terminal c~ino-acid sequence (I):

:

, ' : ` '....... ~

2~ 30 ~0 50 50 l GAAT-CC-CG TGATCATATG AGATATAGT- --.ATGT-GA GAATATTTG, TCAAAGAAGA
i1 TGCGTCACAA GAGAAGAAAC ATAAGCuCAT -TAATAAAAT -TTATATAAG AAT.--AAAT
121 CAATCAAAAA ACAATT-AC. ATG..TCATC GTCC~AACAT GTTAAATT-A -C-TATvAT-,81 GvGATTCATT CC~AAAAGAT ATTACGuTAT TAGTAAACCA AATATTAACC -CT-ACuAAT
2~1 GGCATGGACA TvGAGCT--T GAATCATTCv --AAGTTATA TGGAGATSTA AGAAAAGATA
301 TTT-AAGTCC -ATACATC-A CTAAAAG-GT TGTGGAAAAT TTTAGA~ATA TAGAAGAAuA
361 CT-AGC.GAT .TAGATGAAG ATTCAACAGA AAATATTAAT GA~CC~AATC ATTTAGATvv 921 TCAAAATAAT AAAAACAATA GA~AAACTAA TAATGATAAT ACATTGAAAC AAAATcATGG
481 A~AATC-AG~ GGCACATCTG TACAAGGACG CAAAAATAM ATAAATCGu-u- GATCAAAAGu 541 CAAACATAAT TCTA.AAATA TTCC~AAAGA TAGAAAGACG AACATAATG. CACAAATTAA
501 TAAATTACTA TTTAATAAAA AAGATATTAA AATAAAATGT GAAGAAAGTA GTAG~GAAA

~21 -TvTAATACA AATAATAAAA ATGGAGTTTC ATTATATGAT AATTCAAAGG T--AT--vAA

a41 ATGTCGATAA AACAGA.AGA ACAATTAATA TTATATCAAA ASTC--CGG. GvTvTv~ATA
901 AGTCCAATAA CG.GAATAAT ATTAATAGTG TAAATAAGGA GAATAACATG AATAAvGT5A
961 ATGCTG.ACA TAAGA.AAAT GCTGTGGATA AGGTAAATGC TGTGAATAAG G.AAAT-C-G
1021 TCAATAAGCT AAATGT-G.G AATAAGACGA ATGTTCTGAG TAAGT-GAAT GCT5-vTA-A
1081 AGG.GAAT.C TSTACATAAG ATGAATGCTG TGAATAAGGT AAATGC--vTA AATAAGv-GA

1201 TG.ATA~GA. G~ATGC.G.G .ATAAAATGA ATGCTSTGAA TAAGG.GAGS GCTG.GAA.A
1261 AGGTGAGTGC -GTGAATAAG GTGAGTGCTG TGAATAAGAT GGGTGCTGTA AATAGGG.GA
1321 ATGGAGTAAA CAAGG.GAAT GAGGTGAATG AGGTGAATGA GGTGAATGAA GTGAATATGG
~81 TGAATGAAGT AAATGAGTTA AATGAGGTGA ATAATGTCAA TGC~GTGAAT GAAGTGAATA
1441 G.G.GAACGA GGT.AATG~A ATGAATGAGG TGAATAAGGT GAATGAGCTA AATGAGG.GA
1501 ATGAAG.GAA TAATGTCAAT GAGGTGAATA ASGTGAATGT GATGAATAAT G.GAA.GAGA
1561 TGAATAATAT GAATGAGATG AATAATG,CA ATGTAGTGAA TGAAGTG~AT AATG.CAATG
1621 AGGTGAATAA TGTGAATGAG ATGAATAATG TGAA$GAGAT GAATAATATG AATGAGA.GA

l801 AAG.GAATAA TGTA~ATGAG ATGAATAATA TGAATGAGCT AAATGAGG.G AATGGGuTAA
1861 ATGAAG.GAA TAASACG~AT GAGATACATG AGATGAATAPL TATAAATGAG GTGAATAA.A
1921 CGAATGAGGT GAATAATACG AATGAGATAT ATGAGATGAA TAASATGAAT GATG.GAATA
l9al ATACGAATGA GATAAATGTG GTGAATGCGG TTAAT~AAGT GA~TAAGGTG AATGATTCAA
2 041 ATAATTCA~A TGATGCAAAT G~AGGAAATA ACGCAAATTA TTCAAATGAT TCAAGCAATA
2101 CAAATAATAA CACATCAAGC AGCACAAATA ACTCAAAT~A TAATACATCG .GTAGT--AC

2221 ATGAAGATGA AGAAGACGAC C~AAAAGATA CACAAAAAGA AAAAAACAAT TTAGAACAGG
2281 AAGATATGAG TCCATACGAA GATAGAAATA AAAATGATGA AAAAAATA$T AATGAACAAG
2341 ATAAATTTCA MTATC~AAT GATTTGGGAA AAATATATGA TACATATAAC CAAGGAGA.G
2401 AAG..G.TGT ATC~AAGAAT AAGGACAAAT TAGA~AAGCA TTTGAATGAT TACAAGAG.T
2461 ATTATTATTT ATCTAAAGCA ACACTCATGG ACAA~ATTG~ AGAATCACAA AATAA.AACA
2521 ACTATAATG. ATG.AATTCA AASG~ACT.G GAACTAATGA ATCCAT~AAG ACAAATT-G
2591 ATCAGAATGA TAATG.AAAA GAAAAAAATG ATTCCAACAT ATTTATGAAA ATGATAATTA
2641 TAATTCGTCT TATGATAATG ATCATAATGA TAATGATAA~ AATATGGTAT TTAAAGAT-C
2 7 01 T-CAAGACAA GATAATATG~ AGAAACAAAA AAGTGGAGAA AACAAGCAAT ATTT-`AAACA
2761 A m CGATM TAATGGTAAT GATAATGATA ATGATAATGA TGATAATAAT GATAATGALA
2921 ATAATAATAA .AATAATATG AATAATCAAT ATAATTATCA AG~AAATAAT ATTAACACAA
2881 ATTATAACAT --~G. ACACT CCTTC.AATT GCCAAATCCA AAACAATTCA ~ATATGAA~A
2941 CAAATGAAAT GTACCAACCA T~ATATAATA CATATCCT~C AAATCUTATT CAAGAAAA.-3001 C~ACTATAAA TAAC~C~TT ATTAATGA.T CACCTTACAT GAATAACGAC AACACCAC-A
3061 A. AACACC__ CATXCTVG. ATGAAT~C

.

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The present invention relates to the no~el 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 Lmmunogenic determinant on a polypeptide which is produced by a specific mole-cular configuration of a part-sequence of the polypep-tide. Preferred polypeptides contain the repeating seguence NXY or its permutations YNX and XYN. Hence, particularly preferred polypeptides contain the sequence (NXY)~, (YNX)~ 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 t~pe can be represented by the general formulae A-~, 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 seguence (I). The additional peptides A and/or C can in principle be any peptides hut 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 nitrilotriace-tic acid~nickel chelate resins ~see, for ex~mple,European Patent Application, Publ. No. 253 303). Fusion polypeptides which contain such affinity peptides can be separated selectively, using nitrilotriace~ic acid/nickel chelate resins, from the other polyp~ptides (see, for example, European Patent Applications with publication numbers 282 042 and 309 746). The affinity peptide can be linked either to the C terminu~ 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 - ~ ~

8 ~ 31~
codon of the Plasmodium sporozoite antigen is also u~ed in the expres~ion 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, con-tribute to the cooperation of T and B cells in the immuneresponse 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) .
' ~, .

~33 7~i7 g 3 1O `~
1 Ue~ A-g Gly Ser ~s ~is ~s His ~s ~lS G;y Ser Val Asn Ser ~5 Val Asn rys Glu Asn Asn ~et Asn T yS Val Asn Ala Val :s ;y5 i1 .le Asn Ala Val As? L s Val Asn Ala Val ~sn 'ys Val Asn Ser ~6 Val Asn r yS Leu Asn Val Val Asn ' y5 ~ Asn Val :eu Ser rjs 5 1 t eu Asn Ala Val ~y_ Lys Val Asn Ser Val Y~s Lys ~et Asn Ala ,6 Val Asn r yS Val Asn Ala V21 Asn Lys Val Asn Ala Val Asn _ys 91 Val Asn Val Val Asn Lys Lys Asp I.e Leu Asn ys _eu Asn Aia 106 reu ~yr ~ys Met Asn Ala Val Tyr ys Met Asn Ala Leu Asn 'ys 121 Val Ser Ala Val Asn Lys val Ser Ala Val Asn Lys Val Ser Ala 135 Val Asn ~ys Met Gly Ala Val Asn Ar~ Val Asn G;y Val Asn T j.5 ,51 Val ~sn Glu Val Asn Glu Val Asn Glu Val Asn G;~ Val ;s- Uet '66 Val Asn Glu Val Asn G u 'eu Asn G;u Val Asn Asn Val ;_~ Ala 81 Val ~sn Glu Val Asn Ser Val Asn G ~ Val Asn G'~ ~et Asn G.u ~6 Val Asn 'ys Val Asn G.u T eu Asn Glu Val Asn G;u Val Asn Asn ,1 Val ~sn G'u Val Asn Asn Val ~sn Val Met Asn Asn Val Asn G:u 226 Met ~sn Asn ~et ~sn Glu Met Asn Asn Val Asn Val Val Asn G.u 2~1 Val ~sn ~sn Val Asn G'u Val Asn Asn Val Asn G~u Met Asn ~sn 2_5 Val Asn Glu Uet Asn Asn ~et Asn Glu ~et Asn Asn Val Asn Val 271 Val Asn G u Val Asn Asn Val Asn Glu Met Asn Asn ~~- Asn G.u 235 t eu ~sn G.u Val Asn G;u Val Asn Asn Val Asn Glu Val Asn As~
_01 Val ~sn Val 'al ~sn G.u Val Asn Asn val Asn Gl~1 ~et Asn Asn i16 ~et ~sn Glu _eu Asn Gl~ Val Asn Gly Val Asn Glu Val Asn Asn _31 .~ sn G;u le riS G-u ~et Asn Asn ~'e Asn Glu Val Asn Asn `~6 T.~_ Asn Glu Val Asn Asn ~ sn G`u ;le Tyr G;u Met Asn Asn 61 ~et Asn As? Val Asn Asn .~_ Asn Glu Ile Asn Val Val Asn Ala ;/6 Val Asn G;u Val Asn Lys Val Asn Asp Ser Asn Asn Ser Asn As~
_31 Ala Asn Gl1 Gly Asn Asn Ala Asn .yr Ser Asn ~s? Ser Ser Asn ~06 ~.~_ Asn Asn Asn ~ Ser Ser Ser ~s Asn Asn Ser Asn Asn Asn ~21 ~.'_ Ser Cys Ser Ser Gln Asn .~r ~r ..~5 Ser Ser Glu Asn Asn :i5 Asp âer 'eu Glu Asn Lys Ary Asn Glu Glu Asp Glu Asp Glu G.~
;51 Asp Asp G.n Lys Asp ..~s Gln 'ys Glu Lys Asn Asn eu Glu Gln ~65 Glu As~ ~et Ser ~:o ~yz Glu Asp Arg Asn 'ys Asn Asp Gl~ 'ys ~61 Asn .le Asn Gly .!e Arg Arg Pro Ala Ala Lys L2u Asn ,. . .. .

~ J.

This polypeptide contains 493 amino~acid resi-dues, where part A comprises amino-acid residues 1 to 21, part B comprises ~mino-acid residues 22 to 483 and part C comprises amino-acid residue; 484 to 493. The poly-peptide has a calculated molecular weight of55,239 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, pre-ferably against the sporozoite antigen with the N-ter-minal amino-acid sequence (I) of P. falciparum. The invention also relates to DNAs which code for a poly-peptide according to the in~ention, and to replicable microbial vectors which contain a DNA of this type, especially expression vectors, that is to say replicable micro~ial vectors in which a DNA which codes for a polypeptide according to the invention is joined to an expxession-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 proces~es for ~he 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 agains~
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 :

~ ~ , . .

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, heu/Val, Ala/Glu and vice versa (compare Doolittle, in "The Proteins", ed. Neurath, H. ancl ~ill, R. L., Academic Press, New York [1979]).
The polypeptides according to the Lnvention can be covalently bonded to a carr:ier material or adsorbed thereon. Suitable carrier materials are natural or synthe~ic 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 haemo-cyanin), serum proteins (for example gamma globulin, serum albumin) and ~oxoids (for example diphtheria or tetanus toxoid). Other suitable c~rrier materials are known to the person skilled in the art.
The covalent bonding of the polypeptides accor-ding 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 correspon~ing groups on the carrier material, or indirectly by using conventional bifunctional reagents such as, for example, m-maleimidobenzoyl-N-hydroxy-succinimide esters (MBS) or succinimidyl 4-(p-maleimido-phenyl)butyrate (SMPB). The e and other bifunctional reagents can be obtained commercially, for example from Pierce Chemical Company, Rockford, Illinois, USA. It is also possible to use C27-dialkanals such as, for example, glutaraldehyde (Avrameas, Immunochem. 6, 43-52 tl969]).
The carrier material with the polypeptides bound thereto can be separated from unbound polypeptides and, where appropriate, from excess reagen~s using known metho~s (for example dialysis or column chromatography).
The polypeptid~s of the present invention, ~J) ~'i.i~ 3 ~

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 synthesiséd, which is coupled in protected ~orm to an appropriate ~0 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 hydroxy-methylated support material or by forming an amide linkage to a ben~hydrylamine (BHA)-, methylbenzhydryl-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 xemoving 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-chlorobenzylo~ycarbonyl (2Cl-Z), the dichlorobenzyl (Dcb) and the 3,4-dimethylbenzyl (Dmb) group.
After removal of the ~-amino protective group of the C-terminal amino acid linked to the support, the protected amino acids are coupled on stepwise in thP
required sequence. It is possible to synthesise a com-plete 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 a.rt, with dicyclohexylcarbodiimide (DCC) being particularly suit-able.
Every protected amino acid or peptide is placed in excess in the- solid-phase synthesis reaction ~essel, :, !L

and the coupling reaction can be carried out in dimethyl-formamide (DMF) or methylene chloride (CH2Clz) or a mixture of the two. In cases of incomplete coupling, the coupling reaction is repeated before the N-terminal ~-amino protective group is removed for the coupling of the next amino acid. ~he 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 ou~ automatically.
The peptide can be clea~ed 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 0C for 1 hour, possibly followed by a second reaction lS with HF in the presence of p-cresol at 0C for 2 hours.
Peptides cleaved off chloromethylated or hydro~ymethy-lated 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. Enz~mol. 68, 90-108 [1979] or using the phosphodiester method (Brown et al., Meth. Enzymol. 68, 109-151 [1979]). Both methods entail initial synthe~is of relati~ely 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 polypeptidP 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 , :. , :..... . -. :

5 ~L
_ 14 --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 18, 199-209 [1982]). Elowever, 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 expr~ssion of the polypeptide.
The DNA fragment which codes for the Plasmodium sporozoite antigen according to the invention or for the part-se~uence B of the fusion polypeptide according to the invention can also be obtained by cleaving genomic DNA o~ 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 ~ phage vector gtll. The vector gtll is described by Young et al., Proc. Na~l. Acad~ Sci. USA 80, 1194-1198 [1983] and can be obtained from the American Type Culture Collection (~TCC), 12301 Parklawn Drive, Rockville, ~aryland, 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 lO0,000 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 poly-peptide according to the invention~ of the genomic DNA/
or antibodies which recognise the sporozoite antigen produced by ~-gtll phages. The manner in which these prohes are selected and used is known to the person skilled in the art. Phages which contain the required DNA
fragment are replicated, and the DN~ is isolated. The DNA
fragment can then be incorporated into a suitable replic-able microbial vector, preferably into an expression :

h,~ J~ b;, ~ 15 -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 ~ector is the vector pDS56/RBSII,62His, 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, l9B6. 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 poly-peptide 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 RRl and Eo coli W3110, all of which can be obtained from ATCC, DSM or other institutions.
The manner in which ~he polypeptides according ~o the invention are expressed depends on the expres ion vector used and on the host organism. The host organisms which contain the expression ~ector are normally propaga-ted under conditions which are optimal for growth of thehost 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 ~emperature. ~he expression in the f`~ L
- 16 ~
expression vector used in the present invention is controlled by the lac repressor which binds to a control sequence. ~he repressor is removed by adding isopropyl-b-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 cor-responds 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 ter-minus. In certain expression systems this N-terminal methionine residue is cleavecl 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 i5 inter~
ference 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 polypep~ides produced in the host organisms may be secreted out of th~ 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., ~feth. Enzymol. 221 476-556 [1971]), enz~matically (lysozyme treatment) or chemically (deter-gent treatment, treatment with urea or guanidine HCL
etc.), or by a combîn~tion of the~e.
The polypeptides according to the invention can then be purified by known methods such as, for examplet by centrifugation at different speeds, by precipitation with ammonium sulphate, by dialysis (under atmospheric pressL:e or reduced pressure), by preparative isoelectric focusing, by preparative gel electrophoresis or by various chromatographic methods such as gel filtra~ion, .~ , . , ' Jri ~ L

high performance liquid chromatogxaph~ (HPLC), ion exchange chromatography, reverse phase chromatography and affinity chromatography (for e~ample on Blue Sepharose CL-6B on monoclonal antibodies which are directed against 5 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. Multi-mers can also result when polypeptides are produced inprokaryotic host organisms, especially owing to the formation of disulphide linkages between cysteine resi-dues.
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. 4, 369-388 [1986]; Morein, Nature 332, 287-~88 ~0 [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 Lmmunogenic compositions according to the in~ention into mammals acti~ates their imimune system and raises antibodies against the Plasmodi~m sporozoite antigen according to the invention. These antibodies can be isolated from the serum. The present invention also relates to antibodies of this t~pe. The antibodies according to the invention react with malaria parasites ~nd 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 requi~ed. The antibodies can be purified in a `: :

: : :
.. ~

,p~ tf~

known manner, for ex~mple by precipitat:ion with ammoni~n 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 5 et al. (Nature, 256, 495-497 [1975]). Polyclonal or monoclonal antibodies can also be used for the purifica-tion by affinity chromatography of the polypeptides of the present invention or their natural equivalents.
Th~ polypeptides according to the invention and the immunogenic compositions can be used to immunise mammals against malaria. The modle of administration, the dosage and the number of injections can be optimised by the person skilled in the art in a known manner. Typi-cally, several injections are administered over a lengthy period in order to obtain a high titre of antibodies against the malaria paxasites, 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 Fiqures Fig. 1 Amino-acid sequence of the N-terminal part of the P. falciparum sporozoite antigen = amino-acid sequence (I) Fig. ~ Nucleotide sequence of the P. falciparu~ gene which codes for the sporozoite antigen = nucleo~
tide sequence (1) Fig. 3 ~mino-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, Ml 3-NXY and pDS-NXY. The vector NXY contains nucleotides 1-3088 of nucleo-tide 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 E:c~RI fragment (nucleotides 1-1084) and a . .

L
- 19 -- ' 2 kb EcoRI fragment (nucleotides 1085-308~) 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 Ml3-NXY corresponds to the vector M13 mpl8 containiLng nucleotides 1-3088 oP
nucleotide sequence ~1). This vector thus con-tains ~he same DNA as NXY. The vector pDS~NXY
contains the 1413 bp AseI 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 b~ restriction analysis.
E. coli cells which have been transformed with pDS~NXY produce, after induction, the recombinant Plasmodium sporozoite antigen of the general fvrmula 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 t1).
Fig. 5 Graphical representation of the distribution of positively (ordinate upper half) and negativeIy (ordinatP lower half) charged amino acids in the amino-acid ~equence (I). The numbering of the amino acids (abscissa) corresponds to that in Fig. 1. The graph was constructed by the PC/gene program "NOVOTNY" (Geno~it 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. 7 Potential s-cell epitopes in am:ino-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) cor-responds to that in Fig. 1.
Fig. 8 (A) ~odel of the possib:Le dimer formation by two molecules of the sporozoite antigen according to the invention by means of intermolecular inter-action between the positiv~ly 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 char~ed domain; - = negatively charged domain;
glyc = potential N-~lycosylation region; ag =
antigenic region (that is to say increased probability of the presence of B-cell epitopes;
tm = possible transmembrane sequence.
Fig. 9 A) Analysis of SspI-digested genomic DN~ 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-l; lane 4 = 547; lane 5 = ~1; lane 6 = NAD20; lane 7 = R053 lane & =
T9/94; lane 9 = R0-59.
B) Analysis of DraI- (lanes 1 and 3) and HindIII~
(lanes 2 and 3) digested genomic DNA from two different Plasmodium species (P. falciparum lanas 1 and 2; P. ber~hei lanes 3 and 4).
Fig. 10 Nucleotide sequence of the plasmid pDS56/RBSII~6xHis.
The abbreviations, bufferq and media mentioned in the present application, and methods 1 to 15 u-qed in the example correspond ~o those described in European Patent Application, publication number 309 746, pages 13 to 20.

. . . .
:
:

3 ::
~ 21 ~
Example Isolation of a P. falciparum s~oroz~ite ~ from a qenomic expression qene bank using an~ibodies Preparation of an immune serum aqainst P. falci~arum 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 occ:asion with 106 of these sporozoi~es in complete Freund~s adjuvant. The immune serum was obtained in a customa~ manner.
Construction of -the P. falciparum expression qene bank P. falciparum cells (Kl 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 incu~ted at 65C for 10 minutes and then ~ ml of phenol (saturated with 1 M tris/HCl [pH 8.0]) were added. The phases were mixed by shaking and separa-ted again by centrifugation (10 minutes at 6,000 RPM, 20C). The phenol ex~raction was repeated twice (an interphase ought no longer to be visible). The DNA in the aqueous phase was precipitated as in method 1I 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 tpH
7.5], 0.25 M NaCl, 50 mM EDTA, 25 mM ~-mercaptoethanol, 0.4 mM s-adenosylmethionine) was added. 10 ~g of DNA were methylated with 50 units of EcoRI methylase (New England Biolabs Beverly, Massachusetts, USAj at 37C for 30 minutes. The DNA was extracted once with phenol as described ahove and precipita~ed as in m~thod 1. The DNA
was dissolved in 200 ~l of T4 polymerase buffer and, after addition of 5 ~l of 5mM dATP, dCTP, dGTP and dTTP, as well as lO units of T4 polymerase, was incubated at ~
i ~, . .

~ 3 7 :~ .3 ~

37C for 30 minutes. The DNA was again extracted with phenol and precipitated as in method 1. The DNA was dissolved in 50 ~1 of T4 DNA ligase buffer and, after addition of 0.01 OD260 units of phosphorylated EcoRI
5 oligonucleotide adaptors (New England Biolabs) and 2 ~1 of T4 DN~ ligase (12 Weiss units), ligated at 14C
overnight. The DNA was precipitated as in method 1, dissolved in 20 ~1 of 1 x 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 = l,C00 nucleotides) were isolated as in method 3. The resulting DNA was dissolved in 50 ~1 of water and, after addition of 6 ~1 of 10 x ligase buffer, 2 ~1 of dephosphorylated lambda arms (Promega Biotech., Madison WI, USA) and 6 Weiss uni~s of T4 DNA ligase, ligated at 14C overnight.
The DNA was precipitated (method 1) and dissolved in 5 ~1 of water. After addition of 20 ~1 of packaging extract (Genofit S.A., Geneva, Switzerland), the DNA was packaged in lambda phage particles at 20C for 2 hours in accor-dance with the supplier~s instructions. After addition of500 ~1 of SM buffer and 50 ~1 of chloroform, the gene bank was ready for the anti~ody assay.
Gene bank antibody assay E. coli Y1090 was incubated in 3 ml of LB medium containing 40 ~g/ml ampicillin in a shaker bath at 37C
overnight. The next morning the cells were sedimented (10 minutes at 7,000 x g~ 20C) and resuspended in 1 ml of SM buffer. To this cell suspension were added 106 infectious phage particles from the gene bank and incu-bated at room temperature for 30 minutes. Then 60 ml ofO.8~ (w/v~ agar solution in LB medium which had been equilibrated at 42C were added and thoroughly mixed. The soft agar with the infec~ed cells was distributed over 6 LB-agar plates (diameter 135 mm~ containing 40 ~g/ml ampicillin and incuba~ed at 42C for 5 hours. A nitro-cellulose filter dipped in 100 mM IPTG solution and dried was placed on each dish and incubated at 37C overnight.
The next day the position of the filter relative to the dish was marked~and the marked filter was stored in :.
`;;
.~

~ 3 1 x TBS. A new nitrocellulose filter treated in 100 mM
IPTG solution was placed on the plate, marked and incu-bated on the plates at 37C for 4 hours. Both sets of filters wer~ shaken in 1 x TBS for 10 minutes and then 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 sha};er bath at room tempera-ture for 1 hour. The filters were now washed three times in 1 x TBS, 0.1% Triton~ X-100 for 10 minutes each time in a shaker bath, followed by incubation with 5 ~Ci [125I]-protein A (Amersham, Aylesbury, GB; Catalogue No. lM.144) in 1 x TBS, 0.1% protease-free bovine serum albumin for 1 hour. The filters were again washed as above and then dried at room temperature. The filters were exposed to Kodak XAR x-ray film overnight. Plaque~ 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 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.
10 ~g of NXY DNA were dissolved in 490 ~1 of T4 polymerase buffer and digested with 50 units of EcoRI at 37C 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 ~g of gtll DNA were digested with EcoRI and 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 ~1 of water. ~or the cloning, 50 ng of EcoRI-cut, dephos-phorylated M13 mpl8 DNA (Pharmacia Uppsala, SE; method 6) were mixed with 10 yl of each of the dissol~ed EcoRI
fragments from the NXY DNA, and 2 ~1 of 10 x ligase buffer, 6 ~1 of water and 6 Weiss uni s of T4 DNA ligase were added, analthe D~s were ligated at roo~ temperature for 1 hour. Competent TG-1 E. coli cells (Amersham) were ..

transformed with the ligated DNA (method 7). ~wo plaques from each mixture were isolated and amplified, and sufficient DNA for determining the sequence was isolated tmethod 8). The DNA sequence was determined by method 9.
S 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 determinatlon 1 ~g each of NXY-L and NXY-H DNA were completely digested with BamHI and PstI. After one hour, the DN~s wexe precipitated (method 1) and the pellet was dissolved in 100 ~1 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 37C. 30 ~1 samples were taken after 1, 3 and 6 minutes and 3 ~1 of 0.5 M EDT~ were added. The samples were precipitated by method 1. The DNAs were dissolved in 50 ~1 of Sl nuclease buffer (0.3 M potassium acetate, 10 mM zinc sulphate, 5~ (v/v) glycerol). 10 UllitS of Sl nuclease were added and then the mixture wa~
incubated at room temperature for 30 minutes. The samples were e~tracted 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 37C for 2 minutes. 1 ~1 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 ~1 of 10 x ligase buffer and 400 units of ~4 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. ~) was analysed for the charge distribution ~Fig. 5), hydrophobicity (Fig. 6) and the presence of possible B-cell epitopes (Fig. 7). This showed that ~he amino-acid seguence (I~ hasl starting at the N terminus, a cluster of positive charges followed by a cluster of negative charges. These are followed by amino~acid resi.duas which can be N glycosylated (Fig. 8~) -, I''J ~r.i` C~

followed by a region which contains potential immunogenic B cell epitopes. A typical transmembrane sequence (Eig.
6 and 8B) is located in the C-terminal part of ~he amino-acid sequencP (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 accor-ding to the invention can form dimers by means of the cha.rged 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 qene from the malaria parasite P.
berghei 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 x 105 phage par~icles on two Petri dishes of diameter 135 mm). After five hours, when plaques bec2me visible, the Petri dishes were removed from the 37C incuhator 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 lifte~ off the plate and placed, with the side , ~: . ..
~ ~ ' ~ 3 ~ ~L ~3,l- ~6 -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 Lmpregnated with the alkaline solution. The filters were then briefly dried on a 3MM filt;er 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/HCl [p~ 8.0]. The filters were then dried in the air and baked at 80~C in vacuo for 90 minutes. A P. falci-parum probe was prepared by i,solating 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 sequ~nced (methods 6, 7, 8 and 9). The first 47 amino acids after the ATG start codon ~hich are encoded by the PB-B DNA are identical to the corresponding amino acids of the P.
falciparum sporozoite antigen with the N-terminal amino-acid sequence (I)~ This sequence homology is exceptional.
Thu~, for example, there is little sequence homoloqy in the case of the repatitive sequences of the circum-sporozoite antigen CS in the various Plasmodium species.
Preparation of a polypeptide accordinq to the invention in E. coli 5 ~g of NXY ~NA were partially cleaved with 10 units of ~coRI a~ 37C for 2 minutes. The resulting 3 kb fragment was isolated by methods 2 and 3 and introduced into the EcoRI cleavage site of ~13 mpl8 (method 6). The subclone was called M13-NXY.
An AseI fr~gmen~ specific for P. falciparum was isolated from the M13-NXY clone by methods 1 to 3 as follows. 6 ~g of M13-NXY DNA were digested with 30 units of AseI in lO0 ~l of 1 x T4 polymerase buffer at 37C for one hour. The ~NA was precipitated (method l) and ' :

' :

~'J ~ 3 fractionated on a 0.8~ (w/v) agarose gel (method 2), and a 1400 bp fra~nent was isolated (method 3). The ends were filled in with Xlenow polymerase as described above. The fragment was resuspended in 20 ~l of water and, after addition of 10 pmol of a phosphorylated BamHI oligo-nucleotide adaptor (10-mer: CCGGATCCGG; New England Biolabs), 2.5 ~1 of lO 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 ~l of 1 x T4 polymerase buffer and, after addition of 40 units of BamXI, digested at 37C for 1 hour. The DNA was precipi-tated (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 ~.1 of water. To prepare the vector (see method 6), 1 ~g of pDS56~RBSII,6xHis vector DNA was digested with 10 units of BamHI in T4 polymerase buffer at 37C 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,5xHis 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 con-taining the plasmids pDS56/RBSII,6xHis and pDMI,1 since April 6, 1989, a~ the Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 16 in Braunschweig, Garmany, under DSM No. 5298, specifically in connection with European Patent Application, publication no. 393 502, in accordance with ~he Budapest treaty. The vector DNA was dephosphorylated (method 4), extracted once with phenol (see above), purified on a U.8% (w/v) agarose gel and subsequently isolated by method 3. The isolated DNA
was dissolved in 50 ~l of water.

: ~ :
.~ .

~ ~ r3J ~ ~j 3 5 ~1 of the linearised pDS56/RBSII,6xHis vector DNA, which had been digested with BamHI and dephosphory-lated (metnod 6), were incubated with 5 ~,1 of the 1400 bp fragment, 1.2 ~1 of 10 x ligase buffer and 6 Weiss units of T4 DNA ligase at room temperature for one hour. 10 ~1 of DNA were then transformed into competent E. coli SG13009 (pUHA1) cells (method 7) and plated on LB plates containing 100 ~g/ml ampicillin and 25 ~g/ml kanamycin.
Individual colonies were picked out with a toothpick and transferred into 3 ml of LB medium containing 100 ~g/ml ampicillin and 25 ~g/ml kanamycin. The cultures were incubated in a shaker bath at 37C until the optical density at 600 nm (OD~oo) with pure medium as reference was 0.6. An aliquot of 50Q ~1 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 ~1 of the induced culture were removed and centrifuged together with the non induced sample (3 minutes at 12,000 RPM, 20C). The supernatant was aspirated off, and the cell ~edLment was resuspended in 100 ~1 of SDS sample buffer. The samples were incubated in boiling wa~er 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 wi~h 0.1~ (w/v) Coomassie blue in 30~ (v/v) acetic acid and 10% (~/v) methanol on the shaker for 30 minutes. The gel was destained in 10% (v/v) methanol and 10% (v/v) acetic acid a~ 65DC 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.
58~6 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 repre~sor. It is a derivative of the plasmid pDMI,l which is described in detail in European Patent Application, publication no. 309 746. The plasmid pU~l differs from the plasmid pDMI,1 by replacement of the lacIq allele by the lacI a:LlPle which contains the wild-t~pe promoter. This replacement ensures that an optimum amount of lac reprPssor is produced in the strain SG13009 (pDS-NXY; pUHAl). This makes possible ~ parti-cularly efficient expression of a recombinant protein.
The strain SG13009 (pUHAl) can be obtained in a known manner starting from the strain 'iG13009 ~pDS-NXY; pU~1).
Western blot analysis of the expression products of vDS-NXY
A lysate of E. coli SG13009 (pDS-NXY; pUHA1) 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 para~ites 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 ~lasmodium 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 kDa ~rotein with sporo20ites and blood staqes of P.
falciparum E. coli S~13009 (pDS-NXY; pUHA1) was culti~ated 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 nitrilo~riacetic acid/nickel chelate r~sin. The pu~ified 69 kDa protein was used to immunise rabbits. After two booster in~ections, the serum was tested for unfixed Plasmodium sporozoites and mero-zoites by immunofluorescence. The serum recognised both stages of the parasite. The rabbit serum (dilution 1:500) was therefore tested on ~estern 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 ~een described by Wahlgren et al., Proc. Natl. Acad. Sci. USA
83, 2677-2681 [1986J. Compar:ison of the amino-acid sequence of these 50-53 kDa proteins with the amino-acid sequence of the sporozoite antigen of the present inven-tion shows that they must be different proteins. It wasalso found that, when the MXY-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.
Recoqnition of the NXY qene in 9 P. falciparum isolates and in P. berqhei Genomic DNA from 9 different P. falciparum isolates was in each case digested with Sspl, and the fragments were separated on a 1.2% (w/v) a~arose 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) (60C, 2xSSC; methods 2, 3, 12, 15). Fig. 9 part A shows clearly that two bands (abou~ 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 ~ene 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 DraI (lanes 1 and 3) or HindIII (lanes 2 and 4) and tested as above with the radioactive probe from the M13-NXY DNA. ~igure 9 part B shows clearly that an NXY-specific band can be `:

.

detected in all 4 lanes. Although the detected NXY-specific DNA has a different size in each case, there iis not, however, a non-specific cross-reaction because, as was shown above, the nucleotide sequence coding for the firist 47 amino acids in the P. falciparum DNA is identi-cal to the corresponding nucleotide sequence in the P.
berghei DNA.

' ; ~ ' ', ;~
~ , .: .

Claims (23)

1. Polypeptides which coincide in at least one specific epitope with the Plasmodium falciparum sporozo-ite antigen which contains the N-terminal amino-acid sequence (I)
2. Polypeptides according to Claim 1, which are covalently linked to an affinity peptide, for example the polypeptide with the amino-acid sequence (II)
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, 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 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.
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 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 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 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 poly-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:

(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 expres-sion 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 posi-tion 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 posi-tion 3088 in Figure 2 or a part-sequence thereof, is integrated in a vector;
(b) the transformed vector is replicated in a micro-organism; and (c) the replicated microbial vectors are isolated from the microorganism.
13. A process fox the preparation of a transformed microorganism which is able to produce a polypeptide according to Claim l, 2 or 3, characterised in that:
(a) a microorganism which contains a replicable micro-bial vector, where the vector contains a DNA which codes for a polypeptide according to Claim l, 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 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.
17. Use of 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.
18. A polypeptide prepared by a process according to claim 10 or 11.
19. A replicable microbial vector prepared by a process according to claim 12.
20. A transformed microorganism prepared by a process according to claim 13.
21. Antibodies prepared by a process according to claim 14.
22. The invention as made and hereinbefore described.
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.
CA002037151A 1990-03-23 1991-02-26 Plasmodium sporozoite antigen Abandoned CA2037151A1 (en)

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EP0278941A1 (en) * 1987-01-30 1988-08-17 Smithkline Biologicals S.A. Expression of the P. falciparum circumsporozoite protein by yeast
AU625713B2 (en) * 1988-02-19 1992-07-16 Microgenesys, Inc. Method for producing recombinant protein derived from the circumsporozoite gene of plasmodium falciparum
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