CA2061575A1

CA2061575A1 - Plasmodium falciparum blood-stage antigen, the preparation and use thereof

Info

Publication number: CA2061575A1
Application number: CA002061575A
Authority: CA
Inventors: Dagmar Nolte; Bernhard Knapp; Erika Hundt; Hans Kupper
Original assignee: Individual
Current assignee: GSK Vaccines GmbH
Priority date: 1991-02-21
Filing date: 1992-02-20
Publication date: 1992-08-22
Also published as: ES2129030T3; AU1108192A; UY23357A1; ATE173015T1; DK0499834T3; ZA921231B; DE4105348A1; MX9200711A; KR920016593A; EP0499834A3; AU663068B2; EP0499834A2; GR3029265T3; EP0499834B1; IE920541A1; JPH06141868A; AR245782A1; DE59209544D1

Abstract

BEHRINGWERKE AKTIENGESELLSCHAFT 91/B 002 - Ma 862 Dr. Lp/Bi ABSTRACT OF THE DISCLOSURE

A Plasmodium falciparum blood-stage antigen, the prepara-tion and use thereof The invention relates to a DNA sequence which is highly homologous with that of a glycophorin-binding protein, which is called GBP 130, and has therefore been desig-nated GBP 130 h. The invention additionally relates to the protein GBP 130 h from Plasmodium falciparum itself and to a process for the preparation thereof by recom-binant DNA techniques. Finally, the invention relates to the use of the protein GBP 130 h from Plasmodium fal-ciparum for the preparation of malaria vaccines.

Description

BEHRINGWERKE AKTIENGBSELLSOE~T 91/B 002 - Ma 862 ~2 0 ~ 3. ~ 7 ~
A Plasmodium falciParum blood-st~ge antigen, the prepara-tion and use thereof The invention relates to a DNA set~ence which is highly homologous with that of a glycophorin-binding proiein, which is called GBP 130, and has therefore been desig-nated GBP 130 h. The invention additionally relates tt~
thP protein ~BP 130 h from Plasmodium falciparum itself and to a process for the preparation ther~of by recom-binant DNA techniques. Finally, the invention relate~i ~othe use of the protein GBP 130 h from Plasmodium fal-ciparum for the preparation of malaria vaccines.

The protozoon Plasmodium falciparum - a cause of malaria in humans - i3 a blood parasite belonging to ~he phylum Sporozoa. Transmission from person to person is effected exclusively by mosquitoes of the genus A_9Eh~l~L (in fact only the females) which on biting release or take in the parasites with the blood. For up to two weeks after the infecting bite by a most~ito, the parasites are located in the red blood corpuscles. In them, they assume their ameboid forms, grow rapidly, become multin~clear and then divide into a corresponding number of daughter indivi-duals (called merozoites) which, liberated by disintegra-tion of the blood corpuscle, immediately attack fresh blood corpuscles. This reproductive process is called ~schizogony" and takes about 24 to 48 h. It is repeated, always starting ~new, until the number of parasites (schizonts) is so large that the body of the host xeacts with an attack of fever to the toxic metabolic and disintegration products of the erythrocytes and remain-ders of schi~onts' bodies.

After a certain time~ the formation of sexual forms (gamogony) starts, initially inside the red blood cor-puscles, but is able to be completed only in the intes-tine of the mosquito. Finally, after fertilization o~ thesexual forms forming in the most~uito they develop into .

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what are called ~sporozoites~ which migrate from t~ 7 cavity ~o the salivary gland of the mosquito and ~here are injected, together with the anticoa~llant saliva of the mosquito, into the blood circulation of the person bitten. This complete~ the generation cycle of Plasmodium falciparum.

The incuba~ion period of tropit:al malaria, which i~
caused by Plasmodium falciparum, i5 7 to 15 days, on averase 12 days. Tropical malaria is the most serious and most dangerous form of malaria. In addition, it results more often than the other forms in atypical fonms of the disease which do not therefore immediately suggest malaria. The prodromal signs are more pronounced and follow one another more quickly than in tertian and quartan malaria. The increase in temperature take~ place suddenly, and the course of the fever is irregular. All the systemic symptoms are considerably more severe in tropical malaria than in the other forms. The parasitemia rapidly increases during the course of the disease. In extreme cases 20 to 30% of the erythrocyt~s may be affected. Without treatment, the clinical picture rapidly develops into a life-threatening one, with hepatomegal~, disturbances of con ciousness, hemolytic anemia and leukocytosis.

Since 1956, the World Health Organization of the United Nations has organized a world-wide malaria control campaign, which has had some great ~uccesses but also great setbacks. The efforts of the World Health Organiza-tion make i~ clear how considerable are the problems associated with the occurrence of malaria for the world population. The control of malaria has for the mos~ part followed two lines, namely control of the vector and host of Plasmodium falcipar~m - the Anopheles mosguito - and, on the other hand, the development of drugs for the treatment of malaria-infected people or people who have to expose themselve~ to an increased risk of infection.

- 3 ~ 5 Control of the Anopheles mosquito by chemical agen~ such as, for example, DDT has had only partial success because the mosquitoes have in a relatively short time developed resistance to the chemical control agen~s.

A similar resistance problem arose when various types of drugs had been developed for the ]prophylaxis and control of Plasmodium falciparum and other Plasmodium species.
Not all the development stages of Plasmodium which occur in the human body respond to the same drugs. It is therefore necessary to divide the latter into various groups based on their mechanism action:

Action of antimalaria agents on various development stag~s of Plasmodia Drugs Ase~ Tissue G~x~tes Sporozoi~s group blood forms stages QL~ne +~ - +
(P.vivax, P.m~lariae) 4-km~inoline +~ - (+

Folic acid + + +
antagDnists 8-~mlox~n~line 1 ++ +

Sul~onamide + ? ~ -9~nol~ + ? +
(meflo~ne) ~r~

The prophylactic measures likewise compri~e, on the one hand, control of the mosquito and, on the other handr `

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~ 4 ~ 2~157~
chemoprophylaxis. All the agents used for therapy can be employed for chemoprophyla~is.

However, over the course of time signs of resistance by the Plasmodia to all ~he tried ,and tested agents have emerged. Another disadvan~age of drug therapy or prophy-laxis compris~s the considerable side effects borne out in the human body by the chemicals used. It is likewise disadvanta~eous that prophylaxis must take place for as much as æeveral weeks before and after possible contact with Plasmodium-infected mosquitoes in order to ensure reasonably successful protection from malaria.

This is why there has recently been world-wide discussion of another possibility for controlling malaria. This is the idea of vaccination against the malaria pathogen.
Considerable research effort has therefore been directed at the identification of antigens which are suitable for the development of a vaccine against the asexual blood stage of Plasmodium falciparum. ~he location of antigens on the surface of the mervzoites or of the infected erythrocytes is regarded as one possibility. No genetic information for parasite-encoded antigens, which might function as carrier or receptor ("cytoadherence", "roset-ting~), located on the surface of the infected host cell has been found to date. In contrast to this, genes for the antigens located on the merozoite surface tMSA I, MSA
II) have already been isolated and described in detail (1) .

Another antigen located on the merozoite surface binds to glycophorin (2). Glycophorin is a sialoglycoprotein on the surface of erythrocytes. Glycophorin-binding protein (GBP 130) which is located on the surface of the mero-zoites i6 probably partly responsible for the recognition of the erythrocytes by the merozoites and controls, in a manner which is still unknown, the invasion of merozoites into the erythrocytes (2). GBP 130 is a ~hermostable and soluble protein which is synthesized in the trophozoite -~. , .

_ 5 _ 20~
and schizont staqe. It is transported into the erythro-cyte cytoplasm (3); (4); (5). GBP 130 is released into the culture supernatant in vitro at the time the schizonts are released. It has been shown in th.is connec-tion that only a very small fraction of the GBP 130remains weakly associated with the merozoites ((3), (5)).
Instead of this, GBP 130 appears after release to bind to the erythrocyte membrane, speciiically to glycophorin (2).

Antibodies with specificity for GBP 130 are able ~o inhibit invasion of merozoites in erythrocytes in vitro (6). GBP 130 has likewise been described by anothex group (5) as a 96 kDa antigen with thermoresistant properti0s.
GBP 130 is recognized by antisera which have been obtained from saimiri monkeys immunized by drug-con-trolled infection. These sera promote protection after passive transfer from monkey to monkey (7); (8). Vac-cination of the saimiri monkeys with a protein fraction which contains GBP 130 resulted in protective Lmmunity.
The sera from the monkeys protected in this way moreover showed a strong reaction with the 96 kDa band (9~. It was additionally possible to show that antibodies against G~P
130 occurred exclusively in the sera from immune adults and not in the sera from children or adults who had already lost their immunity ~9).

The gene coding for GBP 130, which is al50 called Ag 78 or 96 tR, has been isolated from three different Plasmodium falc parum strains ((4~, (5), (6)). It codes for a highly conserved antigen.

The amino-acid sequence derived from the DNA sequence comprises a chargsd N-terminal region of 225 amino acids followed by 11 highly conserved repeats of 50 amino acids. The gene contains a small intron which interrupts the sequence wh.ich codes for the possible signal sequence (10).

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The object which emerges from the abovementioned prior art is ~o find further structures or antigens which might be involved, in the widest sense, in the host-parasite interaction of Plasmodium with h~uman cells. Antigens of this type may be able to generate a protective immunity of people against Plasmodium falcipar~m and ~hus against malaria.

The object has been achieved by finding a DN~ with a sequence which is called No. 1 in Seq. ID, which codes for the protein GBP 130 h which is homologous with GBP
130.

The antigen called GBP 130 h has large homologous regions with the already known GBP 130. Both antigens have been investigated by the inventors with regard to the gene structure, the gene localization and for conserved structures in various parasite strains.

The invention embraces all DNA sequences which hybridize with the DNA sequence shown in Seq. ID No. 1 and, at the same time, code for the protein GBP 130 h.

The present invention likewise relates to the protein &BP
130 h and to a process for the preparation thereof by recombinant DNA techniques.

Finally, the invention embraces the use of the protein GBP 130 h for the preparation of a medicinal agent agains~ Plasmodium falciparum, where this medicinal agent is preferably a vaccine.

Identification of lambda-qtll clones which code for GB?
130 h A genomic Plasmodium falciparum EcoRI library was screened with a 32P-labelled, non-redundant 39mer oligo-nucleotide which was derived from a synthetic peptide which corresponds to the N-terminal protein ~equence of ... .

. ~ ~; , - 7 - ~0~'~ 3 7 a possible 55 kDa surface antigen. Vaccination of aotus monkeys with this synthetic peptide in combination with other proteins resulted in protective ~mmunity against Plasmodium falci~arum infection tll). The oligonucleotide which was used for testing complies with the codon usagç
of Plasmodium falciparum according to (12). Eigh~ dif-ferent phage clones were isolated. Sequencing of their integrated DNA showed that none coded for the N-terminal sequence of the 55 kDa antigen determined in (11).
However, computer analysis using the best fit prosram from UWGCG tuniversity of wisconsin, Genetic Computer Group) showed that one of ~he phage clones~ namely Pfa55-1, contains a 1433 bp long DNA segment which ~hows homology with a sequence which codes for the C-terminal region of glycophorin-binding protein GBP 130 (6). The protein coded by this segment (insert) wa~ therefore called GBP 130 homologous protein, namely GBP 130 h.

Isolation of the complete_GBP 130 h gene The inserted DNA fragment of the plasmid p55-1/RI repre-sents parts of an intron followed by an exon with a TAA
stop codon and 261 bp of 3' non-coding region ~Seq. ID
No. 1). The inverse polymerase chain reaction method (13) was used to isolate a 5'-overlapping subclone. Starting from a genomic 1.25 kb Sau3AI fragment specific for GBP
130 h, a DNA sequence which extends the 5'-region of the DNA fragment of plasmid p55-1/RI by 985 bp was amplified (Seq. ID No. 1). The two DNA fragments represent the complete coding region and 5' and 3' non-coding sequences of the ~BP 130 h gene.

The sequence listed in Seq. ID No. 1 shows, besides the nucleotide sequence of the complete GBP 130 h gene of the Plasmodium fal iParum strain FCBR, the amino-acid sequence of GBP 130 h derived from the DNA sequence.

Fig. 1 shows a restriction map and the structure of the ~BP 130 h gene. The coding regions (boxes) are depicted . .

2 ~ 7 ~
separated from one another by an intron sequence. The black areas correspond to the proposed si.gnal 0equence.
The positions of the eight repeat units are indicated.

Tab. 1 shows a comparison of the amino-acid ~equence of GBP 130 h with the amino-acid sequence of GBP 130. This comparison was carried out using the GAP program from UWGCG. Identity is indicated by lin s between the corres~
ponding amino acids and conserved ~mino-acid substitu tions are indicated by colons.

Fig. 2 shows a Southern blot analysis of P. falciparum DNA which was digested with the restriction enzymes RsaI, HinfI, DraI and EcoRX/XbaI and was hybridized with a 32P-labelled XhoII-TaqI fragment which contains the repetitive region of the GBP 130 h gene. The filter was washed under mild (A) and stringent (B) conditions. In this way GsP 130- (triangles) an~ GBP 130 h-specific DNA
fragments, and DNA fragments of a third gene (arrows) which shows greater homology with GBP 130 h than with GBP
130, were detected.

The 5' (nucleotides 1-766) and the 3' (nucleotides 2202-2418) non-coding regions of the GBP 130 h gene are extremely A~T rich (89% and 80.5% respectively). This has already been described for non-coding regions of other Plasmodium falciparum genes (14). The 155 bp-long intron (nucleotide 9S6-1110) likewise shows a similaxly high A+T
content of 88%.

An intervening sequence of 179 bp which interrupts the region for the probable signal seguence has been des-cribed at the corresponding positisn for the GBP 130 gene (10). Both introns start with GT and end with AG and axe thus consistent with introns of other eukaryotes ~15).
The nucleotide sequences of the two introns show a homology of 81%. This shows that the two genes are very closely relatecl and that they therefore derive from a common precursor gene.

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2 ~ ~1 i r~5 The two exons of the GBP 130 h ~ene code ~or 427 amino acids with a calculated molecular weight of 48 260 Da.
The ATG start codon is located at position 767 and i~
flanked upstream by 4 adenine residues. This iB likewise consisten~ with the start consensus sequences of other Plasmodium falciparum genes (16). The N-terminus of GBP
130 h starts with a very hydrophilic region of 50 amino acids in which lysine, serine ancl asparagine occur very frequently. This structure has likewise been found in GBP
130 (5), (6). This region is followed by a hydrophobic sequence of 13 amino acids which are encoded by the 3' end of the first exon. This region, which is highly conserved between GBP 130 and GBP 130 h, probably func-tions as signal sequence together with the following 8iX
amino acids which are encoded by the second exon. The predicted signal peptidase cleavage site of GBP 130 is glycine 69 (6). An alanine residue was found at the position in GBP 130 h corresponding to this site.

The C terminus of GBP 130 h comprises an extended repeat region which represents 74.5% of the entire protein. This region contains eight repeat units with 40 amino acids, and ~his structure is very characteristic of GBP 130 h.
The repeats show only slight variations, and two of these repeats, namely IV and V, can boast of only 39 amino acids. The last four amino acids DE~E of repeats I, II, VI and VII are encoded by nucleotides which are com-plementary to the last twelve bases of the oligo-nucleotide which was used for the screening.

As was shown by comparison of the amino-acid sequences of GBP 130 h and ~BP 130, there is 69~ identity between the two sequences. This corresponds to a very high degree of homology. The main difference relates to a hi~hly charged segment of 116 amino acids from position 110 to 225 in GBP 130, this segment not occurring in ~BP 130 h. The first 46 amino acids, which are encoded by the ~econd exon, show only 54% identity between the two proteins, which means that this segment is the most divergent :
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10 -- 2 0 ~
region be~ween GBP 130 and GBP 130 h. In addition, khe proteins differ in the number and ~he length of the repeats. GBP 130 contains eleven repeats of 50 amino acids, whereas ~BP 130 h shows only eight repeats with 40 amino-acid residues which correspond to amino acids 2 to 41 in the GBP 130 repea~s.

Conservation of the GBP 130 h genle DNA fragments which corxespond to nucleotide positions 767 to 1232, which contain exon 1 and exon 2 upstream from the repeat regions and the intervening sequence of the GBP 130 h gene, were amplified and then sequenced.
The DNA in this case was from the Plasmodium falciparum ~;trains FCBR, FCR-3, SGE2, Il;~:2Gl, FVOR, FUl and #13. This 465 bp region of the GBP 130 h gene is identical in its sequence for all parasite isolates which have been analyzed to date. This shows that the GBP 130 h gene is highly conserved.

GBP 130 h and GBP 130 are encoded by different qenes It was possible by PCR on a genomic DNA of the Plasmodium falciparum strain FCBR, using oligonucleotides p5 and p6 (Table), to isolate a 360 bp-long fragment which codes for the highly charged region specific fox GBP 130.
Compared with the GBP 130 sequence of the str2in FCR-3 (6), ~his fragment shows two base-pair exchange~ which result in substitution of amino acids: an A is replaced by a C in position 713 of GBP 130, and an A i6 replaced by a G in position 758. This base exchange in position 713 of GBP 130 has also been reported for the Palo Alto isolate (10).

This GBP 130-specific probe and a 108 bp PstI-XhoII DNA
fragment from GBP 130 h (compare Fig. 1) were used for the Southern blot analysis of Plasmodium falciparum DNA
cllt with various restriction enzymes. ~he two probes hybridized with different DNA fragments from a Plasmodium , ' , :

2 0 ~ ~ ~ 7~
falciparum strain. This shows unambiguously that the genome of Plasmodium falciparum contains two different genes for &BP 130 and GBP 130 h.

The _GBP_130 qene specifies a ~ene family of three dif-ferent qenes The repetitive region of the GBP 130 h gene was isolated and used as probe for the Southern blot analysis of genomic P. falciparum DNA digested with the restriction enzymes RsaI, HinfI, DraI and ~coRI/XbaI. Three different genes can be detected under mild washing conditions t55~C, 2XSSC, 0.1% SDS). It was possible on the ba~is of the known restriction maps of the GBP 130 gene (4, 5, 6) and of the GBP 130 h gene (Fig. 1), and with the aid of a Southern blot analysis with GBP 130 and GBP 130 h specific DNA fragments (351 bp XbaI-SpeI fragment for GBP
130, 108 bp PstI-XhoII fragment for GBP 130 h), which was carried out under stringent conditions, to assign unambi-guously the GBP 130 and ~BP 130 h specific hybridization fragments (Fig. 2A). In addition, it was pos~ible to detect a third gene, the GBP 130 h probe cross-hybridi-zing with an approximately 22kb EcoRI/XbaI fragment, a 1.7 kb DraI fragment, a 0.8 kb RsaI fragment and a 2.2 kb HinfI fragment. Washing of the filter under more strin-gent conditions (65C; 0.5XSSC, 0.1% SDS) results in no detectable hybridization of the GBP 130 h probe with the GBP 130 gene, but the DN~ fragments which are assigned to the third as yet unknown gene are detected (Fig. 2B).
This shows that this gene is more homologous with the GBP
130 h gene than with the GBP 130 gene.

The GBP 130 h aene is only very weaklY xpressed in blood staqes of_P. falciparum Starting from poly(A)+ RNA from schi20nts, a Northern blot analysis was carried out with a 108 bp Ps~I-XhoII frag-ment (specific GBP 130 h gene fragment) and with a 351 bp XbaI-SpeI fragment (specific GBP 130 gene fragment). ~he : ;' 12 - 2~ 7~
two fragments used for the hybridization have appxoxi-mately the same specific radioactivity. The GBP 130 probe detected a unique mRNA of about 6.5 kb as dominant band after overnight exposure. Similar results have already been described in the literature for GBP 130 (4, 5, 6).
In contrast with this, the GBP 130 h probe hybridized with two mRNA bands of about 2.5 kb and about 2.8 kb which were detectable only after a very long exposure tLme of 8 days. In this case, one of the mRNAs can be assigned to the GBP 130 h gene; the second band might repr~sent the mRNA of the third gene of the GBP gene family, which evidently shows high homology with the GBP
130 h gene (Fig. 2). There is a noticeable discrepancy between the expression rate of the G~P 130 gene and that of the gene which codes for the protein GBP 130 h: the GBP 130 gene is transcribed with very high efficiency, whereas the transcription rate of the GBP 130 h gene, and evidently of the third gene of this gene family, is very weak. Supposing that the proteins translated by these mRNAs have approximately simil~r frequency rates, it must be assumed that GBP 130 occurs very frequently in P.
falciparum schizonts, and GBP 130 h tends to be und~r-represented. This different distribution of the~e homologous proteins might be utilized for the so-called smokescreen effect: this entails GBP 130 being released in large quantity after the merozoites have been released from the schizonts, and it might have the task of divert-ing the Lmmune system, in which case the under-represented GBP 130 h might simultaneously exert its essential function.

Table 2 shows the ~equences of oligonucleotides which have been constructed for the polymerase chain reaction (PCR).

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Examples Pre~earation of DNA and mRNA

Standard methods (17) were used for the cultivation of the Plasmodium falc_parum strains FCBR tColombia), FCR-3 (Colombia), FVOR (Vietnam), SGE2 (Zaire), ItG2Gl (srazil)~
FUl (Uganda) and #13 (Senegal), for the enrichment of schizonts and for the preparation of DNA and poly(A)+ RNA.
ThP analysis of DNA and mRNA of the strain FCBR by Southern and Northern blot technology is likewise des-cribed in the prior art (18).

Construction of a qenomic EcoRI :LibrarY

2 ~g of DNA of the Plasmodium falciparum strain FCBR wereincubated with 14 units of the restriction enz~me EcoRI
in 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 ~M dithio-threitol and 40~ (v/v) glycerol at 37C overnight. Underthese conditions, EcoRI shows star activity. This means that the DNA is digested at its tetranucleotides AATT.
The DNA fragments with a ~ize up to 10 kb resulting from this were fractionated using a 0.8% agarose gel. Fra~y-ments between 500 bp and 7 kb were electroeluted and theninserted into the vector lambda gtll by the method described in ~19). A genomic EcoRI library of 5 x 105 recombinant phage clones was obtained in this way, and these phages were then amplified by standard methods (20).

Screeninq of the EcoRI library 1.5 x 105 phage clones from thi~ library were screened by standard methods (20) using a 5'-32P-labelled oligo-nucleotide which was derived from the N-terminal protein sequence of a possible 55 kDa Plasmodium falciparum surface antigen (11). The oligonucleotide had the base sequence~ 5'-TGC TGC ATA TAC ATT TTG TGT ~TC TGC TTC TAA
TT(: ATC-3 ' .

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The oligonucleo~ide was constructed on the basis of the codon~ most commonly used by Plasmodium falciparum (16).
Eight phage clones were i~olated and one of these, which was called Pfa55-1, was used for the subsequent invez-tigations. The DNA of the phage clone Pfa55-1 was diges-ted with the restriction enzymes EcoRI and KpnI. This resulted in a 2.4 kb fra~ment which, besides the malaria-specific fragment, carried a 1 kb of lambda gtll region, which was deleted by means of par~ial PvuII restriction.
The 1.4 kb fragment obtained in this way was cloned into the pKS(+) Bluescript vector, which was digestible with EcoRI and SmaI, resulting in the plasmid pS5-1/RI .

Isolation of a 5'-overlap~ina qene fraqment by inverse PCR

In order to isolate the complete gene, a fragmenk of which is contained in the phage clone Pfa55-1, the 5' region of the gene was extended by means of inverse PCR
(13). A genomic 1.25 ~b Sau3AI fragment which extends ~he 5~ region of the inserted DNA of the phage clone Pfa55-1 by 985 bp was identified by Southern blot analysis (20~, using a 32P-labelled 108 bp fragment (PstI/XhoII digPstion of p55-1/RI ) as proba. 90 ~g of Sau3AI-digested P.
falciparum DNA were fractionated on a 0.8% agarose gel, and the DNA fragments with a si~e between 1.2 and 1.3 kb were electroeluted. The Sau3AI cleavage sites were self-ligated and, after restriction with the enzyme PstI, the known DNA sequences were converted to the 5' and 3' end.
50 ng of this genomic DNA and 500 ng of the oligo nucleotides pl and p2 (Tab. 2) were u~ed for the PCR, which was carried out under standard conditions u~ing the Gene-AmpR kit from Perkin Elmer Cetus. The 1.25 kb frag-ment obtained ater this was phospho~ylated at the 5' ends. This was followed by a fill-in reaction with the Klenow en~yme (20). This DNA fragment was then inserted into the pKS vector which had been digested with SmaI.
The plasmid formed in this way was called pS5-1/PCR.

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DNA sequencing Both strands of the inserted DNA fragments of the plas-mids p55-ltRI~ and p55-1/PCR were sequenced by the dideoxy method using the sequenase system from ~SB (Cleveland, OH). Suitable subfragments were obtained by subcloning at available restriction sites in~o the Bluescript vector pRS. The sequencing data were analy~ed using the ~WGCG
program (21).

Amplification and sequencin~ of specific ~ene reqions of various P. falciparum isolates 0.5 ~g of DNA from the P. falciparum strains FCBR, FCR-3, SGE2, ItG2Gl, FVOR, FU1 and #13 were used in combination with, in each case, 300 ng of the oligonucleotides p3 and p4 (Tab. 2) to ~mplify a genomic fragment. ~he Gene-AmpR
kit from Perkin Elmer Cetus was u~ed for this. The genomic fragments of the seven different parasite iso-lates were phosphorylated, then sub~ected to a fill-in reaction using the Klenow enzyme by standard methods (20) and subsequently inserted into an SmaI cleavage site of the vector pKS or sequencing.

Construction of a GBP 130 specific probe Comparison of the coding sequences of GBP 130 h and GBP
130 (6) resulted in the discovery of a 351 bp-long fragment which is present only in the GBP 130 gene. This GBP 130 ~pecific fragment was amplified by a genomic DNA
of the P. falciparum strain FCBR, specifically using the oligonucleotides p5 and p6 ~Tab. 2~. The 360 bp fragment resulting ~rom this was digested with XbaI and SpeI and then ligated into the ~ector pRS, resulting in the plasmid pKS/GBP. The identity of the GBP 130 ~pecific fragment was checked by DNA sequencing.

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- 16 - 2~ 7 Southern blot analysis of ~he GBP 130 and ~BP 130 h qene The GBP 130 specific fragment was isolated from the plasmid pKS/G~P using the xestriction en~ymes XbaI and SpeI, and labelled with 32p by nick translation. A 108 bp-long GBP 130 h specific DNA fra~ment was isolated analo-gously from the plasmid pS5-l/RI~ using the restriction enzymes Pstl and XhoII and was labelled with 32p by nick translation. soth probes were usecl for the Southern blot analysis by standard methods (20) of P. falciparum DNA
which was digested with the r~s1,riction enzymes DdeI, TaqI, AluI, Sau3AI, RsaI, HinfI, DraI and EcoRI/XbaI.

Southern blot analysis of genomic P. falci~arum DNA usinq the repetitive GBP 130 h probe The plasmid p55-l/RI* was digested with the restriction enzymes XhoII and TaqI, and it was possible to isolate a 965 bp DNA fragment which contains the 8 repeat units of the GBP 130 h gene. This DNA fragment was radioactively labelled with 3ZP by nick translation (20) and used for ~he Southern blot analysis of P. falciparum DNA which was digested with the restriction enzymes RsaI, HinfI, Dra~
and EcoRI/XbaI. After the hybridization, which was carried out using standard conditions (20), the membrane was first washed in 2XSSC (2XSSC is 300 mM NaCl, 30 mM
sodium citrate), O.l~ SDS (sodlum dodecyl sulfate) at 55~C twice for 15 minutes each time and subsequently autoradiographed for 3 h. After development of the autoradiograph, the membrane was washed under more stringent conditions in 0.5XSSC, 0.1% SDS at 65C twic~
for 15 minutes each time and subsequently expo~ed over-night.

Northern blot analysis 10 ~g samples of a poly(A)+ RN~ which was isolated fromschizonts of the P. falciparum strain ~CBR were frac-tionated using a 0.8% agarose/formaldehyde gel, then ~ .

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- 17 ~ 75 transferred to a Gene-Screen membrane tDu Pont) using the supplier's protocol r and hybridized with a nick-trans-lated 108 bp-long PstI-XhoII fragment of the GBP 130 h gene which was obtained from the plasmid p55-1/RI~, and with a 351 bp XbaI-SpeI fragment of the GBP 130 gene which was obtaine~ from the pla3mid pRS/GBP. The filter was washed twice for 15 min. in 0.5XSSC, 0.1% SDS at 55C/ and autoradiographed.

Expression of a part al BP 130 h se~lence The vector pSEM tsiotechni~ue~ 8,]pages 280-281 (22)) was used to express a part-sequence of the plasmid p55-1/RI~, the fusion protein carrying the 375 N-terminal amino acids of ~-yalactosidase. The oligonucleotides p7 and p8 (Table) and 100 n~ of the plasmid DNA from p55-1/RI were used to amplify a 680 bp fragment by PCR. The amplified fragment was digested with SacI and PstI and then ligated into the pSEM1 vector which had been linearized with the same restriction enzymes. The E. coli strain DHSalpha was transformed with the ligated plasmid. After this, colo-nies which contained the inserted DN~ fragments of the correct size were isolated. Single colonies were cul-tivated overnight and then induced with 1 mM IPTG (iso propyl thiogalactoside) for 2 h. The expression products were analyzed by SDS polyacrylamide gel electrophore~is.
In this case i was possible for a fusion protein of 70 kDa to be expressed in high yield.

, - 22 - 2~6~3~
Tahle 1 .
1 MRIS-~SNIESTGVS~1C:~NFNSXNCSXYSLMEVQN~NE~X~SLTSF~KN 50 11:11-1:1-111111:1111111:11111111 IlllI.li..ll.l-1 MRLSXVSDIKSTGVSNYXMFNS~NSSXYSL-'qEVSX~NEKgNSLG~F35~X 50 51 ITLIFG~ IYVAL~GVYIC~SQYRQAADYS~RES~VLAEG~STSXRNA~TA 100 51 ILLI~GIIYWLLNAYICG3RYE~AVDYG~ESRII,AEGEDTC~RREgTT 100 .
101 LRKTRQTTL................... ...........................109 I I I I I I
lGl LRKSXQXTST~TV~TQTRXDE~NRS W TEcQKVESDSEXQXRTX~W RKQ 150 109 .'.......................... ...........................109 151 INIG~TENQKEGXNVX;;VIXKrXXXEESGXPEENXaANEASRXXEP.YASX 200 109 ............................ ...........................109 201 VSQ~?STST~SNNEVRIR~ASNQETLTS~DPEGQI~REY~DP~YRK~LE 250 ~ 09 ........................... ...........................109 25i IFYXLLTNTDPNDEVERRNADNXEDLTSADP''GQIMREYAS3P~YRXcL~ 300 109 ............................ ...........................109 301 IFV~ILTNTDPNDDVcRRNADNXEDLTSADPEGQI~qREY~ADPEYRXHLE 350 710 ............................ .TS~DPEGQIMKAWAADPEYRK~LN 133 1111111111:.:1111111111:
351 V~-~Rl~TNTDP~lD~VERRNADNX_3LTSADP~G~I~REY~ADPEYRX:'L_ 400 134 VLYQILNNTDPND~L~............ .TS~DP-5QIMRAVAAD?EYRR;;LN 173 ::- 11-1111111:1 11111l1111:-11111111111:
~01 lEE~ILTNTDpNDEvrRRNADN~EDLTs~Dp-GQIMREvA~Dpry~R~L~ 450 77q VLYQIL.~NTDPNDEVE........... SSADPrC-QI~q.YAY.~DPrY~X~-VNV 214 451 VF3~ILTNTD~NDEVLRRNADNX_LTSSDPEGQI:qRE~V`~ADP~Y~XuLrI 50O

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275 LYQILN~TDPNDELr............. TSADP~rQ_~g~VAADP~'~Rr~v~v 254 : I I I I I 1'1 1 1: 1 1 1 1 1 1 1 1 1 1 1: I I I I I I I I I 1:::
501 -.XILTNTD5NDEVER~NADNXEDLTSADaEGQ_~RrY~A3P~YRKr.LE 550 25~ LYQILNr.TDSS._VE............ TSAD2_C-QIiYRAYA~D2~YRR~NV 293 :1-11-:11-- 111 II-IIlilll:-llllllllll:::
~1 Fy~ILT~TDpNDEvER~NADNxrELTssDprGQI;qREyAADpEy~Rr3r; 600 2S4 LYQIL~2TDSS.~VE............. TSADP--C-~IM~VAADP~YRXEV~ 332 601 FEKILT~TDPNDEVERRNADNgEDLT5ADPEGQIMREY~ADPEYRXr3 _I 6;0 .
333 LYQIL~NTDPNDEL_............. TSADP_r-QI;~XAYAADP~YRXF.~NV 372 :1-11-1111111:1 Illlllilll:-ll-lllllll:::
6~1 FYXILT~TDP~DEVE~RNADNR~DLTSADP~GQIMREVA53PrYRgr.LE~ 700 373 LYQIL~NTDPNDELE............. TSrDP~5QI~qKAY.~ADPrYRXn~NV 412 701 EYXILT~TDP~DDVEP~RNADNXrDLTS.;DP--C-2Il~REV~ADP~YRK~LEl /;0 . _ . .
413 LYQIL~NTDPNDESS............. 427 : I I I I I I I I I
751 FUXIL~TDP~DEVERQNADN~'EA 774 ,~

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- ~4 -SEQ ID NO: 1 2 0 6 1 ^ r7 ~
TYPE OF SEQUENCE: nucleotide with corresponding pro~.ein STRANDEDNE5S: single strand TOPOLOGY: linear TYPE OF MOLECU~E: genome DNA

ORGANISM: Plasmodium falciparum IMMEDIATE EXPERIMEN~AL SOURCE
NAME OF THE STRAIN: P. falciparum FCBR
FEATURES:
from 767 to 955 BP exonl from 1111 to 2202 ~P exon~
from 1249 to 2202 BP repetitive rlegion PROPERTIES: gene which code~ for an antigen homologous with the GBP130 protein GA$CTATATT AAAAAAAATA TACAAGGAAA AGATGTG~TA AACAATATAC ATTSATATAA 60 CAATAAAAAT AATATA$C.;A TG~AATAATT AATTATTTTA ,AATCAATAG TCGTAAGTGT 420 TTTCAAGGAT ATGATAA.TT ATATCATTGA AAAAATATA- ATATAGTATT TATCTTTTAT 540 GAA~U~AAAC ATTGAAA.GT AATTTATGTA AAu~vuuU~A AAATTAAAAT AAAATAATAA 600 AAAAATATTT ATGTATTG.T TTTTTTTTTT ATTTTTATTT TATTATTTTA AAATATATAT 660 ACAAATTAGA AAAAACAT.iT ATATTCTTAT TTTCTTCT~; GTAAAA ATG CGT ATT 775 Met Arg Ile Ser Lys Ala Ser Asn Ile Glu Ser Thr Gly V21 Sec Asn Cys Lys Asn ; 10 15 TTC AAT TCG AAA AAT TGC TCT AAA TAT TCT T-S ATG GAA GTA CAA AAT a7 Phe Asn Ser Lys Asn Cy5 Ser Lys Tyr Ser Le~a Met Glu Val Gln Asn ZS ;o 35 AAA AAT GAA AAG AAA CGT TCC TTA ACT TCC T-_ CAT GCC AAA AAC ATC 919 Lys Asn Glu Lys Lys Arg Ser Leu Thr Ser Phe ~is Ala Lys Asn Ile Thr Leu Ile Phe Glv Ile Ile Tyr Val Ala Leu Leu 5; 60 TTGCATCATT TATTTTTAG GGT GTT TAT ATA TGT G~ AGC CAA TAC AAA CAA 1143 Gly Val Tyr Ile Cys Ala Ser Gln Tyr Lys Gln - 25 - 20~ ~7~
GCT GCA GAT TAT AG~ TTT AGA G~A AGC AGA GTT T~A GCT GAA GG~ AAA 1191 Ala Ala Asp Tyr S~r Phe Arg Glu Ser Arg Val Leu Ala Glu Gly Lys Ser Thr Ser Lys Ly~ Asn Ala Lys Thr Ala Leu Arg Lys Thr Lys Gln Thr Thr Leu Thr Ser Ala Asp Pro Glu Gly Gln Ile Met 1ys Ala Trp Ala Ala As? Pro Glu Tyr Arg Lys Hls Leu Asn Val Leu Tyr Gln Ile Leu Asn Asn Thr ASD Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro Glu Gly Gln }le Met Lys Ala Tyr Ala Ala ~sp Pro Glu Tyr Arg Lys His Leu Asn Val Le~ Tyr Gln Ile Leu Asn Asn Thr Asp Pro Asn Asp 175 180 laS

Glu Val Glu Ser Ser Ala Asp Pro Glu Gly Gln Ile Met Lys Ala Tyr GCT GCT GA. CCA GAA TAT CGT AAA CAC GTA AAT GTC CTT TAC CAA ATA 1575 Ala Ala As~ Pro Glu Tyr Arg Lys His Val Asn Val Leu Tyr Gln ~le Le~ ~sn Asn ~hr Aso Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro Glu Gly Gln Ile Met Lys Ala Tyr Ala Ala A5? 2ro Glu Tyr Arg Lys 235 240 2~5 250 8is Val As~ Val Leu Tyr Gln Ile Leu Asn 9is Thr Asp Ser Ser GLu ~5, 260 26;
GTA GAA AC- AGC GCA GAC CCA GAA GGA CAA ATA ATG AAA GCT TAT GCT 176, Val Glu Thr Ser Ala Asp Pro Glu Gly Gln Ile .~et Lys Ala Tyr Ala 270 . 275 2eo GCT GAT CCA GAA TA. CGT AAA CAC GTA AAT GTC CTT TAC CAA ATA TTA 1815 Ala Asp Pro Glu Tyr Arq Lys His Val Asn Val Leu Tyr Gln Ile Leu 285 ~90 295 Asn 8is Thr Asp Ser Ser Glu Val Glu Thr Ser Ala Asp Pro Glu Gly CAA ATA ATG AAA GCC TAC GCA GCT GAT CCA GAA TAT CGT AAA CAC GT~ 1911 Gln Ile Met Lys Ala Tyr Ala Ala Asp Pro Glu Tyr Arg Lys His Val AAT GTC CTT TAT CAA ATA TTA AAT AAC ACT GAT CCA AAT GAT G~A TTA 1959 Asn Val Leu Tyr Gln Ile Leu Asn Asn Thr Asp ~ro Asn Asp Glu Leu Glu Thr Ser Ala Asr 2ro Glu Gly Gln Ile Met Lyr Ala Tyr Ala Ala 350 ' 355 360 , . ~ . , ~ :
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GAT CCA GAA TAT CGT AAA CAC GTA AAT GTC CTT TAT CAA ATA TTA AAT ~ 5 Asp Pro Glu Tyr Arg Lys Hls V~l Asn Val Leu Tyr ~ln Ile Leu Asn Asn Thr ASp Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro Glu Gly Gln Ile Met Lys Ala Tyr Ala Ala Asp Pro Glu Tyr Arg Lys ~is V~l Asn Val Leu Tyr Gln Ile Leu Asn Asn Thr Asp Pro Asn Asp Glu Ser TCC TAAGAA TGTATCTCCC TTCG~AAAAT AAGAG-~AAAC AAAATTTGCA AATGAATTAG 2258Ser AAAGTACGAT TATGATAATT AAGAGATGTA TGAAT'rTGAA TGTAAAAATG ACATTTTTTA 2318 TAATAACGTA CAATATTTTA ATAATTAATT ATCAA~ATG AAATATATAA TACTATTTAT 2378 GGTATTTGAT ATTATTTAGA TGAGGAAGAA AAAAGGAATT 2ql8 .

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Claims

1. DNA which codes for the protein GBP 130 h shown in Seq. ID No. 1.

2. A DNA sequence ad dlaimed in claim 1, with the sequence shown in Seq. ID No. 1 or parts thereof.

3. A DNA sequence which hybridizes with any DNA
sequence of claims 1 or 2 and codes for the protein GBP 130 h.

4. A DNA sequence which cross-hybridizes with any DNA
sequence of claims 1 or 2 and does not code for the proteins GBP 130 or GBP 130 h.

5. The protein GBP 130 h with an amino-acid sequence indicated in Seq. ID No. 1.

6. The protein GBP 130 h as claimed in claim 5, which has been prepared with the aid of recombinant DNA
techniques.

7. The protein GBP 130 h, which is encoded by a DNA
sequence as claimed in claims 1 to 3.

8. A protein which is more homologous with GBP 130 h than with GBP 130.

9. A process for the preparation of GBP 130 h, which comprises trandforming a host organism with a DNA
sequence as claimed in any of claims 1 to 3, and isolating the protein after cultivation of the host organism and expression.

10. GBP 130 h as claimed in any of claims 5 to 7 as medicinal agents.

11. A protein as claimed in claim 8 as medicinal agent.

12. A vaccine which contains GBP 130 h as claimed in any of claims 5 to 7 or proteins as claimed in claim 8.

13. The use of GBP 130 h as claimed in any of claims 5 to 7 or of protein as claimed in claim 8 for the production of a medicinal agent against Plasmodium falciparum.