IE920541A1

IE920541A1 - A plasmodium falciparum blood-stage antigen, the preparation¹and use thereof

Info

Publication number: IE920541A1
Application number: IE054192A
Authority: IE
Inventors: Dagmar Nolte; Bernhard Knapp; Erika Hundt; Hans Kupper
Original assignee: Chiron Behring Gmbh & Co
Priority date: 1991-02-21
Filing date: 1992-02-20
Publication date: 1992-08-26
Also published as: DE59209544D1; UY23357A1; ATE173015T1; EP0499834B1; EP0499834A2; GR3029265T3; AU663068B2; AU1108192A; DK0499834T3; AR245782A1; MX9200711A; ES2129030T3; EP0499834A3; DE4105348A1; ZA921231B; CA2061575A1; KR920016593A; JPH06141868A

Abstract

The invention relates to a DNA sequence which is highly homologous with that of a glycophorin-binding protein, the so-called GBP 130, and has therefore been called GBP 130 h. The invention furthermore relates to the protein GBP 130 h from Plasmodium falciparum itself and to a process for the preparation thereof by recombinant DNA techniques. Finally, the invention relates to the use of the protein GBP 130 h from Plasmodium falciparum for preparing vaccines against malaria.

Description

BEHRINGWERKE AKTIENGESELLSCHAFT 9I/B 002 - Ma 862 Dr. Βΰ/Bi A Plasmodium falciparum blood-stage antigen, the preparation and use thereof The invention relates to a DNA sequence which is highly 5 homologous with that of a glycophorin-binding protein, which is called GBP 130, and has therefore been designated 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 recom10 binant DNA techniques. Finally, the invention relates to the use of the protein GBP 130 h from Plasmodium falciparum for the preparation of malaria vaccines.

The protozoon Plasmodium falciparum - a cause of malaria in humans - is a blood parasite belonging to the phylum Sporozoa. Transmission from person to person is effected exclusively by mosquitoes of the genus Anopheles (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 mosquito, the parasites are located in the red blood corpuscles. In them, they assume their ameboid forms, grow rapidly, become multinuclear and then divide into a corresponding number of daughter individuals (called merozoites) which, liberated by disintegration 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 anew, until the number of parasites (schizonts) is so large that the body of the host reacts with an attack of fever to the toxic metabolic and disintegration products of the erythrocytes and remainders of schizonts' bodies.

After a certain time, the formation of sexual forms (gamogony) starts, initially inside the red blood corpuscles, but is able to be completed only in the intes35 tine of the mosquito. Finally, after fertilization of the sexual forms forming in the mosquito they develop into - 2 what are called sporozoites which migrate from the body cavity to the salivary gland of the mosquito and there are injected, together with the anticoagulant saliva of the mosquito, into the blood circulation of the person bitten. This completes the generation cycle of Plasmodium falciparum.

The incubation period of tropical malaria, which is caused by Plasmodium falciparum, is 7 to 15 days, on average 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 forms 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 takes 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 erythrocytes may be affected. Without treatment, the clinical picture rapidly develops into a life-threatening one, with hepatomegaly, disturbances of consciousness, 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 successes but also great setbacks. The efforts of the World Health Organization make it clear how considerable are the problems associated with the occurrence of malaria for the world population. The control of malaria has for the most part followed two lines, namely control of the vector and host of Plasmodium falciparum - the Anopheles mosquito - and, on the other hand, the development of drugs for the treatment of malaria-infected people or people who have to expose themselves to an increased risk of infection. - 3 Control of the Anopheles mosquito by chemical agents 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 agents.

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 stages of Plasmodia Drugs group Asexual Tissue Gametocytes Sporozoites blood forms Quinine (P.vivax, P. malariae) 4-Aminoquinoline (+) Folic acid antagonists 8- Aminoquinoline ± Sulfonamide + 9- Quinolinemethanol ++ (mefloquine) T ari anF The prophylactic measures likewise comprise, on the one hand, control of the mosquito and, on the other hand, - 4 chemoprophylaxis. All the agents used for therapy can be employed for chemoprophylaxis.

However, over the course of time signs of resistance by the Plasmodia to all the tried and tested agents have emerged. Another disadvantage of drug therapy or prophylaxis comprises the considerable side effects borne out in the human body by the chemicals used. It is likewise disadvantageous that prophylaxis must take place for as much as several 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. The location of antigens on the surface of the merozoites 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, rosetting), 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 (MSA 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 merozoites is 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 thermostable and soluble protein which is synthesized in the trophozoite - 5 and schizont stage. It is transported into the erythrocyte 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 this connec5 tion that only a very small fraction of the GBP 130 remains weakly associated with the merozoites ((3), (5)). Instead of this, GBP 130 appears after release to bind to the erythrocyte membrane, specifically to glycophorin (2).

Antibodies with specificity for GBP 130 are able to inhibit invasion of merozoites in erythrocytes in vitro (6). GBP 130 has likewise been described by another group (5) as a 96 kDa antigen with thermoresistant properties. GBP 130 is recognized by antisera which have been obtained from saimiri monkeys immunized by drug-controlled infection. These sera promote protection after passive transfer from monkey to monkey (7); (θ)· Vaccination of the saimiri monkeys with a protein fraction which contains GBP 130 resulted in protective immunity.

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 GBP 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 also called Ag 78 or 96 tR, has been isolated from three different Plasmodium falciparum strains ((4), (5), (6)). It codes for a highly conserved antigen.

The amino-acid seguence derived from the DNA seguence comprises a charged 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 which codes for the possible signal seguence (10). - 6 The object which emerges from the abovementioned prior art is to find further structures or antigens which might be involved, in the widest sense, in the host-parasite interaction of Plasmodium with human cells. Antigens of this type may be able to generate a protective immunity of people against Plasmodium falciparum and thus against malaria.

The object has been achieved by finding a DNA 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 GBP 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 against Plasmodium falciparum, where this medicinal agent is preferably a vaccine.

Identification of lambda-qtll clones which code for GBP 130 h A genomic Plasmodium falciparum EcoRI* library was screened with a 32P-labelled, non-redundant 39mer oligonucleotide which was derived from a synthetic peptide which corresponds to the N-terminal protein sequence of a possible 55 kDa surface antigen. Vaccination of aotus monkeys with this synthetic peptide in combination with other proteins resulted in protective immunity against Plasmodium falciparum infection (11). The oligonucleotide which was used for testing complies with the codon usage of Plasmodium falciparum according to (12). Eight different 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 program from UWGCG (University of Wisconsin, Genetic Computer Group) showed that one of the phage clones, namely Pfa55-1, contains a 1433 bp-long DNA segment which shows 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) was 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-l/RI* repre20 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-l/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 GBP 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 falciparum 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 GBP 130 h gene. The coding regions (boxes) are depicted separated from one another by an intron sequence. The black areas correspond to the proposed signal sequence. The positions of the eight repeat units are indicated.

Tab. 1 shows a comparison of the amino-acid seguence of 5 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 lines between the corresponding amino acids and conserved amino-acid substitutions are indicated by colons.

Fig. 2 shows a Southern blot analysis of P. falciparum DNA which was digested with the restriction enzymes Rsal, Hinfl, Dral and EcoRI/Xbal 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 GBP 130- (triangles) and 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 956-1110) likewise shows a similarly high A+T content of 88%.

An intervening sequence of 179 bp which interrupts the region for the probable signal sequence has been described at the corresponding position for the GBP 130 gene (10)· Both introns start with GT and end with AG and are 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 related and that they therefore derive from a common precursor gene. - 9 The two exons of the GBP 130 h gene code for 427 amino acids with a calculated molecular weight of 48 260 Da. The ATG start codon is located at position 767 and is flanked upstream by 4 adenine residues. This is likewise consistent 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 and 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 functions as signal sequence together with the following six 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 this 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 DELE of repeats I, II, VI and VII are encoded by nucleotides which are complementary to the last twelve bases of the oligonucleotide which was used for the screening.

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

Conservation of the GBP 130 h gene DNA fragments which correspond 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 strains FCBR, FCR-3, SGE2, ItG2Gx, FVOR, FU1 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 bv different genes 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 for GBP 130. Compared with the GBP 130 sequence of the strain FCR-3 (6), this fragment shows two base-pair exchanges which result in substitution of amino acids: an A is replaced by a C in position 713 of GBP 130, and an A is 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 Pstl-XhoII DNA fragment from GBP 13Q h (compare Fig. 1) were used for the Southern blot analysis of Plasmodium falciparum DNA cut with various restriction enzymes. The two probes hybridized with different DNA fragments from a Plasmodium - 11 falciparum strain. This shows unambiguously that the genome of Plasmodium falciparum contains two different genes for GBP 130 and GBP 130 h.

The GBP 130 gene specifies a gene family of three dif5 ferent genes 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 Rsal, Hinfl, Dral and EcoRI/Xbal. Three different genes can be detected under mild washing conditions (55°C, 2XSSC, 0.1% SDS). It was possible on the basis of the known restriction maps of the GBP 130 gene (4, 5, 6) and of the GBP 130 h gene (Pig. 1), and with the aid of a Southern blot analysis with GBP 130 and GBP 130 h specific DNA fragments (351 bp Xbal-Spel fragment for GBP 130, 108 bp Pstl-XhoII fragment for GBP 130 h), which was carried out under stringent conditions, to assign unambiguously the GBP 130 and GBP 130 h specific hybridization fragments (Fig. 2A). In addition, it was possible to detect a third gene, the GBP 130 h probe cross-hybridizing with an approximately 22kb EcoRI/Xbal fragment, a 1.7 kb Dral fragment, a 0.8 kb Rsal fragment and a 2.2 kb Hinfl fragment. Washing of the filter under more stringent conditions (65’C; 0.5XSSC, 0.1% SDS) results in no detectable hybridization of the GBP 130 h probe with the GBP 130 gene, but the DNA 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 gene is only very weakly expressed in blood stages of P. falciparum Starting from poly(A)+ RNA from schizonts, a Northern blot analysis was carried out with a 108 bp Pstl-XhoII fragment (specific GBP 130 h gene fragment) and with a 351 bp Xbal-Spel fragment (specific GBP 130 gene fragment). The - 12 two fragments used for the hybridization have approximately 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 time of 8 days. In this case, one of the mRNAs can be assigned to the GBP 130 h gene; the second band might represent 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 GBP 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 similar frequency rates, it must be assumed that GBP 130 occurs very frequently in P. falciparum schizonts, and GBP 130 h tends to be underrepresented. This different distribution of these 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 diverting the immune system, in which case the underrepresented GBP 130 h might simultaneously exert its essential function.

Table 2 shows the sequences of oligonucleotides which have been constructed for the polymerase chain reaction (PCR). - 13 Examples Preparation of DNA and mRNA Standard methods (17) were used for the cultivation of the Plasmodium falciparum strains FCBR (Colombia), FCR-3 (Colombia), FVOR (Vietnam), SGE2 (Zaire), 11636! (Brazil), FU1 (Uganda) and #13 (Senegal), for the enrichment of schizonts and for the preparation of DNA and poly (A)+ RNA. The analysis of DNA and mRNA of the strain FCBR by Southern and Northern blot technology is likewise des10 cribed in the prior art (18).

Construction of a genomic EcoRI* library pg of DNA of the Plasmodium falciparum strain FCBR were incubated with 14 units of the restriction enzyme EcoRI in 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithio15 threitol and 40% (v/v) glycerol at 37®C overnight. Under these conditions, EcoRI shows star activity. This means that the DNA is digested at its tetranucleotides AATT. The DNA fragments with a size up to 10 kb resulting from this were fractionated using a 0.8% agarose gel. Frag20 ments between 500 bp and 7 kb were electroeluted and then inserted 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).

Screening of the EcoRI* library 1.5 x 105 phage clones from this library were screened by standard methods (20) using a 5'-32P-labelled oligonucleotide 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 TTC TGC TTC TAA TTC ATC-3'. - 14 The oligonucleotide was constructed on the basis of the codons most commonly used by Plasmodium falciparum (16). Eight phage clones were isolated and one of these, which was called Pfa55-1, was used for the subsequent inves5 tigations. The DNA of the phage clone Pfa55-1 was digested with the restriction enzymes EcoRI and Kpnl. This resulted in a 2.4 kb fragment which, besides the malariaspecific fragment, carried a 1 kb of lambda gtll region, which was deleted by means of partial PvuII restriction.

The 1.4 kb fragment obtained in this way was cloned into the pKS(+) Bluescript vector, which was digestible with EcoRI and Smal, resulting in the plasmid p55-l/RI*.

Isolation of a 5'-overlapping gene fragment by inverse PCR In order to isolate the complete gene, a fragment 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 kb Sau3AI fragment which extends the 5' region of the inserted DNA of the phage clone Pfa55-1 by 985 bp was identified by Southern blot analysis (20), using a 3ZP-labelled 108 bp fragment (Pstl/XhoII digestion of p55-l/RI*) as probe. 90 pg of Sau3AI-digested P. falciparum DNA were fractionated on a 0.8% agarose gel, and the DNA fragments with a size between 1.2 and 1.3 kb were electroeluted. The Sau3AI cleavage sites were selfligated and, after restriction with the enzyme Pstl, the known DNA sequences were converted to the 5' and 3' end. 50 ng of this genomic DNA and 500 ng of the oligonucleotides pi and p2 (Tab. 2) were used for the PCR, which was carried out under standard conditions using the Gene-AmpR kit from Perkin Elmer Cetus. The 1.25 kb fragment obtained after this was phosphorylated at the 5 * ends. This was followed by a fill-in reaction with the Klenow enzyme (20). This DNA fragment was then inserted into the pKS vector which had been digested with Smal.

The plasmid formed in this way was called p55-l/PCR. - 15 DNA sequencing Both strands of the inserted DNA fragments of the plasmids p55-l/RI* and p55-l/PCR were sequenced by the dideoxy method using the sequenase system from USB (Cleveland, OH) . Suitable subfragments were obtained by subcloning at available restriction sites into the Bluescript vector pKS. The sequencing data were analyzed using the UWGCG program (21).

Amplification and sequencing of specific gene regions of 10 various P. falciparum isolates 0.5 pg of DNA from the P. falciparum strains FCBR, FCR-3, SGE2, ItG2Glz FVOR, FU1 and #13 were used in combination with, in each case, 300 ng of the oligonucleotides p3 and p4 (Tab. 2) to amplify a genomic fragment. The Gene-AmpR kit from Perkin Elmer Cetus was used for this. The genomic fragments of the seven different parasite isolates were phosphorylated, then subjected to a fill-in reaction using the Klenow enzyme by standard methods (20) and subsequently inserted into an Smal cleavage site of the vector pKS for 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 specific 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 from this was digested with Xbal and Spel and then ligated into the vector pKS, resulting in the plasmid pKS/GBP. The identity of the GBP 130 specific fragment was checked by DNA sequencing. - 16 Southern blot analysis of the GBP 130 and GBP 130 h gene The GBP 130 specific fragment was isolated from the plasmid pKS/GBP using the restriction enzymes Xbal and Spel, and labelled with 32P by nick translation. A 108 bp5 long GBP 130 h specific DNA fragment was isolated analogously from the plasmid p55-l/RI* using the restriction enzymes Pstl and XhoII and was labelled with 32P by nick translation. Both probes were used for the Southern blot analysis by standard methods (20) of P. falciparum DNA which was digested with the restriction enzymes Ddel, TaqI, Alul, Sau3AI, Rsal, Hinfl, Dral and EcoRI/Xbal.

Southern blot analysis of genomic P. falciparum DNA using the repetitive GBP 130 h probe The plasmid p55-l/RI* was digested with the restriction 15 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 32P by nick translation (20) and used for the Southern blot analysis of P. falciparum DNA which was digested with the restriction enzymes Rsal, Hinfl, Dral and EcoRI/Xbal. 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), 0.1% SDS (sodium 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 65"C twice for 15 minutes each time and subsequently exposed over30 night.

Northern blot analysis pg samples of a poly(A)+ RNA which was isolated from schizonts of the P. falciparum strain FCBR were fractionated using a 0.8% agarose/formaldehyde gel, then transferred to a Gene-Screen membrane (Du Pont) using the supplier's protocol, and hybridized with a nick-translated 108 bp-long Pstl-XhoII fragment of the GBP 130 h gene which was obtained from the plasmid p55-l/RI*, and with a 351 bp Xbal-Spel fragment of the GBP 130 gene which was obtained from the plasmid pKS/GBP. The filter was washed twice for 15 min. in 0.5XSSC, 0.1% SDS at 55°C, and autoradiographed.

Expression of a partial GBP 130 h sequence The vector pSEM (Biotechniques 8, pages 280-281 (22)) was used to express a part-sequence of the plasmid p55-l/RI*, the fusion protein carrying the 375 N-terminal amino acids of 0-galactosidase. The oligonucleotides p7 and p8 (Table) and 100 ng of the plasmid DNA from p55-l/RI* were used to amplify a 680 bp fragment by PCR. The amplified fragment was digested with Sacl and Pstl and then ligated into the pSEMl vector which had been linearized with the same restriction enzymes. The E, coli strain DH5alpha was transformed with the ligated plasmid. After this, colo20 nies which contained the inserted DNA fragments of the correct size were isolated. Single colonies were cultivated overnight and then induced with 1 mM IPTG (isopropyl thiogalactoside) for 2 h. The expression products were analyzed by SDS polyacrylamide gel electrophoresis.

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. Bonnefoy, S. and Mercereau-Puijalon, 0. (1989) Plasmodium falciparum: An Intervening Sequence in the GBP 130/96 tR Gene. Exp. Parasitol. 69., 37-43 . 11. Patarroyo, Μ. E., Romero, P., Torres, M. L., Clavijo, P., Moreno, A., Martinez, A., Rodriguez, R., Guzman, F. and Cabezas, E. (1987) Induction of protective immunity against experimental infection with malaria using synthetic peptides. Nature 328, 629-632. 12. Hyde, J. E., Kelly, S. L., Holloway, S. P., Snewin, V.A. and Sims, P. F. G. (1939) A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms. Mol. Biochem. Parasitol. 82., 247-262. 13. Triglia, T., Peterson, M. G. and Kemp, D. J. (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucl. Acids Res. 16 . 818 6. 14. Weber, J. L. (1987) Analysis of sequences from the extremely A+T-rich genome of Plasmodium falciparum. Gene 52, 103-109.

. Mount, S. M. (1982) A catalogue of splice junction sequences. Nucl. Acids. Res. 10, 459-472. 16. Saul, A. and Battistutta, D. (1990) Analysis of the sequences flanking the translational start sites of Plasmodium falciparum. Mol. Biochem. Parasitol. 42. 55-62. 17. Knapp, B., Shaw, A., Hundt, E., Enders, B. and Kupper, H. A. (1988) A histidine-alanine-rich recombinant antigen protects Aotus monkeys from Plasmodium falciparum infection. Behring Res. Commun. .82., 349-359. 18. Knapp, B., Hundt, E., Nau, U. and Kupper, H. A. (1989) Molecular cloning, .genomic structure and localization of a blood stage antigen of Plasmodium falciparum characterized by a serine stretch. Mol. Biochem. Parasitol. 32., 73-84. 19. Huynh, Τ. V., Young, R. A. and Davis, R. W. (1985) Constructing and screening cDNA libraries in lambdagtlO and lambdagtll (in: DNA-cloning Vol. 1 pp. 49-78 ed. by Glover, D. M.).

. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 21. Devereux, J.; Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12, 387-395 22. Knapp, S., Broker, M. and Amann, E. (1990) pSEM Vectors: High Level Expression of Antigenic Determinants and Protein Domains. Biotechniques 8, 280-281. IE 920541 - 22 Table 1 * · · · · 1 MRISKASNIESTGVSNCXNFNSXNCSXYSLMEVQNXNEXKRSLTSFEAKN l|:||.|:|.||||l|:|||||||:|||||||| .11111.II·.II.I. 1 MRLSXVSOIKSTGVSNYXNFNSXNSSXYSLMEVSXKNEKKNSLGAFHSXK ITLIFGIIYVALLGVYICASQYKQAADYSFRESRVLAEGXSTSKKNAXTA I I II I I I I 1. 11 ·. 111:.·I.. I·f 1: 11 111:11 I I.. I: :..11.

ILLIFGIIYWLLNAYICGDKYEXAVDYGFRESRILAEGEDTCARKEKTT 101 LRKTXQTTL.........................................

III.II-1 101 LRKSKQKTSTRTVATQTKXDEENKSWTEEQKVESDSEKQKRTKXWKKQ 109 ........ . ..........’.........’.........’.........ί 151 INIGDTENQKEGKNVKKVIKKEXXXEESGXPEENXHANEASKXXEPXASX 109 .........*.........’..........’.........’.........’ 201 VSQKPSTSTRSNNEVKIRAASNQETLTSADPEGQIMREYAADPEYRXHLE 109 .........*.........'.........’__________’.........’ 51 IFYXILTNTDPNDEVERRNADNXEDLTSADPEGQIMREYASDPEYRKELE 109 .................................................. 301 IFYXILTNTDPNDDVERRNADNXEDLTSADPEGQIMREYAADPEYRKELE 110 ..........................TSADPEGQIMKAWAADPEYRKBLN 1111 I I I I I I:.: I I I I I I I I I I ·· 51 VFEKILTNTDPNDEVERENADNXEDLTSADPEGQIMREYAADPEYRXELE 4 VLYQILNNTDPNDELZ..........TSADPEGQIMKAYAADPEYRKELN ::.. I I . I I I I I I I : I I 11 I I I I I 11: -1 I I I I I I I I I I ·· 401 IFEKILTNTDPNDEVERRNADNXEDLTSADPEGQIMREYAADPEYRKELE 174 VLYQILNNTDPNDEVE.........SSADPEGQIMXAYAADPEYRKEVNV I :.. I I . II I I I I I I I · I -I I I ! I I I : · I I I I I I I I I I :: : .4 51 VFHXILTNTDFNDEVERRNADNXELTSSDPEGQIMREYAADPEYRXELEI 215 LYQILNNTDPNDELE..........TSADPEGQIMXAYAADPEYRKEVNV :·· I I · II I I I I I : I I I I I I I I I I I :. I I I I I I I II ; ::: 501 FEKIL TN TDPND EVERRNADNXED LTSAD PEGQI MRS YAAD P ZYRXELEI LYQILNETDSS . EVE..........TSADPEGQIMXAYAADPEYRKEVNV : I · I I . : I I · · III I I . I I ·| I I I I : · I I I I I I I I I I : : : 551 FYXILTNTDPNDEVERRNADNKEELTSSDPEGQIMREYAADPEYRKHLEI 294 LYQILNETDSS.EVE..........TSADPEGQIMXAYAADPEYRKEVNV : . . I I - : I I .. Ill I I I I I I I I I I :· I I I I I I I I I I 601 FKKILTNTDPNDEVERRNADNXZDLTSADPEGQIMREYAADPEYRKELZI 33 LYQILNNTDPNDELE..........TSADPEGQIMXAYAADPEYRKEVNV : I - I I . I I I I I I I : I I I I I I I I I I I : · I I · I I I I I I I : : : 651 FYKILTNTDPNDEVERRNADNKEDLTSADPEGQIMREYASDPEYRKELEI 3 LYQILNNTDPNDELE..........TSADPEGQIMXAYAADPEYRKEVNV : I -I I . I I I I I I ::1 I I I I I I ! I 11: · I I I I I ! I I I I = = ·· 701 FYXILTNTDPNDDVERRNADNXEpLTSADPEC-QIMREYAADPEYRKELEI 413 LYQILNNTDPNDESS......... 427 :. - I I · I I 11 I I I . 751 FHXILTNTDPNDEVERQNADNNEA 774 100 100 109 150 109 200 109 250 109 300 109 350 133 400 173 450 214 500 254 550 293 600 332 650 372 700 412 750 - 23 CN Table •H s a H H Μ l-l on CN « « r* If) cn VO r-~ σι -S' CN co r-4 r-l CO r-i Ύ 00 1 r4 1 r-l 1 co If) 00 CO If) o If) co CN CN ** t r4 00 r-l r-l r-l - m m ι a a, § a (H U Λ P O M-4 Ό Φ 4-) υ +J ra υ to φ TJ •r| +J O Φ 4J υ CP •H (0 Φ 4-) •id to O •r| 4-> U •rl M 4-) to Φ M Φ P H Φ Φ tP 4-) Ό Φ to g a s z g g η s n § a ι o HOHO noon g m g m g n g § h inmmminmmm ua'ai'a’ai.u to Φ Ό 4-> vo o Φ Φ O Φ P Φ to Φ M CU Φ M tQ Φ Ό •r| 4-) O Φ Ό Φ •H & Φ (0 corresponding to the GBP 130 o o + ♦ SEQ ID NO: 1 TYPE OF SEQUENCE: nucleotide with corresponding protein STRANDEDNESS: single strand TOPOLOGY: linear TYPE OF MOLECULE: genome DNA ORIGINAL SOURCE ORGANISM: Plasmodium falciparum IMMEDIATE EXPERIMENTAL SOURCE NAME OF THE STRAIN: P. falciparum FCBR FEATURES: from 767 to 955 BP exonl from 1111 to 2202 BP exon2 from 1249 to 2202 BP repetitive region PROPERTIES: gene which codes for an antigen homologous 15 with the GBP130 protein GATCTATATT AAAAAAAATA TACAAGGAAA AGATGTGTTA AACAATATAC ATTTATATAA 60 TATATAATTA ATATAATATA ATATAATGTA GTATTTCATG AAATATATTA TGTAGGTTTG 120 ATTTAAATTT AATCATTATA- TAAATATTAC ACATATGAAT AAATAAATAA ATATATATAT 180 ATATATATAA ATATATTTAC TTATATTTAG TAATTTTTAA CATTGTATAT TTAAAAAGAA 240 AATATTTTTT ATTTCATATT TTTTAATATT TTATAATAGA AAAAGATAGA TACAAAAGAA 300 CATGTGGAAA AAATATATAT TATAAAATAT ATTCTAAATT CTTTTATTAA TTTTAAGATT 360 CAATAAAAAT AATATATCAA TGAAATAATT AATTATTTTA TAATCAATAG TCGTAAGTGT 420 AATACATATA TTATTTCTAG CTTCTTGTAA CTTACATAAT TATAAATATA TCATATATTC 480 TTTCAAGGAT ATGATAATTT ATATCATTGA AAAAATATAT ATATAGTATT TATCTTTTAT 540 GAAAAAAAAC ATTGAAATGT AATTTATGTA AAAAAAAAAA AAATTAAAAT AAAATAATAA 600 AAAAATATTT ATGTATTGTT TTTTTTTTTT ATTTTTATTT TATTATTTTA AAATATATAT 660 TTAAAAAAAA AAAAATATAT ATATATATAA TATTTATATT TATAATTATT TTAGAAACAC 720 ACAAATTAGA AAAAACATAT ATATTCTTAT TTTCTTCTAA GTAAAA ATG CGT ATT 775 Met Arg Ile TCA Se r AAA Lys 5 GCA Ala AGT Ser AAT Asn ATT lie GAA TCT ACA Thr GGA GTT Gly Val TCG Se r 15 AAT Asn TGT Cys AAA Lys AAT Asn 823 Glu 10 Ser TTC AAT TCG AAA AAT TGC TCT AAA TAT TCT TTG ATG GAA GTA CAA AAT 871 Phe Asn Ser Lys Asn Cys Ser Lys Tyr Ser Leu Met Glu Val Gin Asn 20 25 30 35 AAA AAT GAA AAG AAA CGT TCC TTA ACT TCC TTC CAT GCC AAA AAC ATC 919 Lys Asn Glu Lys Lys Arg Ser Leu Thr Ser Phe His Ala Lys Asn Ile 40 45 50 ACA TTG ATT TTT GGA ATA ATA TAC GTA GCC TTA TTG GTATGA 1 TAATATAAAT 971 Thr Leu Ile Phe Gly Ile Ile Tyr val Ala Leu Leu 55 60 AAAATATATA ATTGAATTTT TTTCTTTTTT AATTAATGAA TTTAAATATC CTTATTAGAA 1031 ATATATAGAT ACATAGATAC ATACATAAAT ATATATTTAT ATATATATAT TTTTTTCTGT 1091 TTGCATCATT TATTTTTAG GGT GTT TAT ATA TGT GCA AGC CAA TAC AAA CAA 114 3 Gly Val Tyr lie Cys Ala Ser Gin Tyr Lys Gin 70 GCT Ala 75 GCA GAT TAT Tyr AGT Ser TTT AGA GAA AGC AGA GTT TTA GCT GAA GGT AAA Lys 90 1191 Ala Asp Phe Arg 80 Glu Ser Arg Val 85 Leu Ala Glu Gly AGT ACC AGT AAA AAA AAC GCA AAA ACC GCA TTA AGA AAA ACT AAG CAA 1239 Se r Thr Se r Lys Lys Asn Ala Lys Thr Ala Leu Arg Lys Thr Lys Gin 95 100 105 ACA ACC TTA ACT AGC GCA GAT CCA GAA GGA CAA ATA ATG AAA GCC TGG 1287 Thr Thr Leu Thr Ser Ala Asp Pro Glu Gly Gin Ile Met Lys Ala Trp 110 115 120 GCT GCT GAT CCA GAA TAT CGT AAA CAC CTA AAT GTT CTT TAC CAA ATA 1335 Ala Ala Asd Pro Glu Tyr Arg Lys His Leu Asn Val Leu Tyr Gin Ile 125 130 135 TTA AAT AAC ACT GAT CCA AAT GAT GAA TTA GAA ACT AGC GCT GAC CCA 1383 Leu Asn Asn Thr Asp Pro Asn Asp Glu Leu Glu Thr Se r Ala Asp Pro 140 145 150 GAA GGA CAA ATA ATG AAA GCT TAT GCT GCT GAT CCA GAA TAT CGT AAA 1431 Glu Gly Gin Ile Met Lys Ala Tyr Ala Ala Asp Pro Glu Tyr Arg Lys 155 160 165 170 CAC CTA AAT GTT CTT TAC CAA ATA TTA AAT AAT ACC GAC CCA AAT GAT 1479 His Leu Asn Val Leu Tyr Gin lie Leu Asn Asn Thr Asp Pro Asn Asp 175 180 185 GAA GTA GAA TCT AGC GCT GAC CCA GAA GGA CAA ATA ATG AAA GCT TAT 1527 Glu Val Glu Se r Ser Ala Asp Pro Glu Gly Gin Ile Met Lys Ala Tyr 190 195 200 GCT GCT GAT CCA GAA TAT CGT AAA CAC GTA AAT GTC CTT TAC CAA ATA 1575 Ala Ala Asd Pro Glu Tyr Arg Lys His Val Asn Val Leu Tyr Gin Ile 205 210 215 TTA AAT AAC ACC GAT CCA AAT GAT GAA TTA GAA ACT AGC GCA GAC CCA 1623 Leu Asn Asn Thr Asp Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro 220 225 230 GAA GGA CAA ATA ATG AAA GCC TAC GCA GCT GAT CCA GAA TAT CGT AAA 1671 Glu Gly Gin Ile Met Lys Ala Tyr Ala Ala Asa Pro Glu Tyr Arg Lys 235 240 245 250 CAT GTA AAT GTC CTT TAC CAA ATA TTA AAT CAC ACC GAC TCA AGT GAA 1719 His Val Asn Val Leu Tyr Gin Ile Leu Asn His Thr Asp Ser Ser Glu 255 260 265 GTA GAA ACT AGC GCA GAC CCA GAA GGA CAA ATA ATG AAA GCT TAT GCT 1767 val Glu Thr Ser Ala Asp Pro Glu Gly Gin lie Met Lys Ala Tyr Ala 270 275 280 GCT GAT CCA GAA TAT CGT AAA CAC GTA AAT GTC CTT TAC CAA ATA TTA 1815 Ala Asp Pro Glu Tyr Arg Lys HiS val Asn Val Leu Tyr Gin Ile Leu 285 290 295 AAT CAC ACC GAC TCA AGT GAA GTA GAA ACT AGC GCA GAC CCA GAA GGA 1863 Asn His Thr Asp Ser Ser Glu Val Glu Thr Se r Ala Asp Pro Glu Gly 300 305 310 CAA ATA ATG AAA GCC TAC GCA GCT GAT CCA GAA TAT CGT AAA CAC GTA 1911 Gin lie Met Lys Ala Tyr Ala Ala Asp Pro Glu Tyr Arg Lys His Val 315 320 325 330 AAT GTC CTT TAT CAA ATA TTA AAT AAC ACT GAT CCA AAT GAT GAA TTA 1959 Asn Val Leu Tyr Gin Ile Leu Asn Asn Thr Asp Pro Asn Asp Glu Leu 335 340 345 GAA ACC AGC GCA GAC CCA GAA GGA CAA ATA ATG AAA GCT TAT GCA GCT 2007 Glu Thr Ser Ala Asp Pro Glu Gly Gin Ile Met Lys Ala Tyr Ala Aia 350 355 360 GAT CCA GAA TAT Tyr CGT Arg AAA CAC GTA AAT GTC CTT TAT CAA ATA TTA Leu AAT Asn 2055 Asp Pro Glu 365 Lys His Val 370 Asn Val Leu Tyr Gin 375 Ile AAC ACT GAT CCA AAT GAT GAA TTA GAA ACT AGT GCT GAC CCA GAA GGA 2103 Asn Thr Asp Pro Asn Asp Glu Leu Glu Thr Ser Ala Asp Pro Glu Gly 380 385 390 CAA ATA ATG AAA GCT TAT GCT GCT GAT CCA GAA TAT CGT AAA CAC GTT 2151 Gin Ile Met Lys Ala Tyr Ala Ala Asp Pro Glu Tyr Arg Lys His Val 395 400 405 410 AAT GTC CTT TAC CAA ATA TTA AAT AAC ACT GAT CCA AAT GAT GAA AGT 2199 Asn Val Leu Tyr Gin Ile Leu Asn Asn Thr Asp Pro Asn Asp Glu Ser 415 420 425 TCC TAAGAA Ser TGTATCTCCC TTCGAAAAAT AAGAGAAAAC AAAATTTGCA AATGAATTAG 2258 AAAGTACGAT TATGATAATT AAGAGATGTA TGAATTTGAA TGTAAAAATG ACATTTTTTA 2318 TAATAACGTA CAATATTTTA ATAATTAATT ATCAAAAATG AAATATATAA TACTATTTAT 2378 GGTATTTGAT ATTATTTAGA TGAGGAAGAA AAAAGGAATT 2418

Claims

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

2. A DNA sequence as claimed in claim 1, with the 5 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 10 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 15 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 20 than with GBP 130.

9. A process for the preparation of GBP 130 h, which comprises transforming 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 25 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 16. . 16A vaccine which contains GBP 130 h as claimed in any of claims 5 to 7 or proteins as claimed in claim 8The 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 falciparumA process as claimed in claim 9, substantially as hereinbefore described and exemplifiedThe protein GBP 130 h, whenever prepared by a process claimed in a preceding claimA vaccine as claimed in claim 12, substantially as hereinbefore described.