WO1994012640A1 - RECOMBINANT NUCLEIC ACID, POLYPEPTIDE MATERIAL CODED THEREBY AND TRANSMISSION BLOCKING VACCINE BASED THEREON FOR CONTROLLING THE MALARIA PARASITE $i(PLASMODIUM FALCIPARUM ) - Google Patents

Abstract

A recombinant nucleic acid molecule, comprising a nucleotide sequence selected from: (a) nucleotide sequences which code for the amino acid sequence of Plasmodium falciparum protein Pfs45/48 shown in figure 2, (b) nucleotide sequences which hybridise with a nucleotide sequence complementary to a nucleotide sequence according to (a) and code for a polypeptide or protein which is recognized by an antibody capable of binding to a Plasmodium protein, and (c) fragments of a nucleotide sequence according to (a) or (b) of a length of at least 7 nucleotides. Polypeptide material comprising an amino acid sequence coded for by such nucleotide sequence, and the preparation thereof. Transmission blocking vaccine for controlling the malaria parasite Plasmodium falciparum, comprising such polypeptide material and a suitable carrier or adjuvant.

Description

Title: Recombinant nucleic acid, polypeptide material coded thereby and transmission blocking vaccine based thereon for controlling the malaria parasite Plasmodium falciparum

Field of the invention

The invention lies in the field of malaria control and concerns the preparation of a vaccine based on the identification, isolation, characterisation, manipulation and expression of a particular gene of the human malaria parasite Plasmodium falciparum. In particular, the invention concerns the use of the gene product, or parts thereof, as a vaccine component for inducing an immunological defence system which is specifically directed against the extracellular development of the parasite in the mosquito of the genus Anopheles acting as intermediate host .

Background of the invention

Since time immemorial malaria has been one of the most prevalent and dreaded tropical diseases. Approximately half of the world population is threatened by the disease and on average some 400 million people are actually ill. The annual number of fatal victims, mainly children, is estimated at some 2.5 million people. The parasitic disease malaria is caused by unicellular protozoans of the genus Plasmodium and transmitted from man to man by the female mosquito of the genus Anopheles. There are four known different species of malaria parasites for which man is an intermediate host . These species different strongly from each other with regard to the nature and severity of the clinical picture induced by them.

Both as regards infectivity, morbidity and mortality, Plasmodium falciparum is the most dangerous malaria parasite for man. In areas where malaria is endemic, it is typically the most important pathogenic organism and very often moreover resistant to drugs such as, for instance, chloroquine. Both in tropical and subtropical areas Plasmodium falciparum is fairly widespread.

The second most important and fairly widespread parasite is Plasmodium vivax- The other two species, Plasmodium ovale and Plasmodium malariae. occur only in a limited number of areas

(Plasmodium ovale only in West-Africa) . Nor do the latter parasites constitute a real threat to public health.

Human malaria parasites are highly host-specific. This means that they cannot be multiplied with the aid of animals and that therefore extremely costly in vitro cultures have to be relied on for the production/amplification of the asexual erythrocytic stages and gametocytes.

For a global control of the disease, two strategies were particularly fashionable in the 1960s en 1970s, one focussed directly on the control of the vector mosquito using larvacides and insecticides (for instance DDT) , the other focussed on the control of the unicellular parasite of the genus Plasmodium using drugs (in particular derivatives of quinine, such as for instance chloroquine) . Although these massive control campaigns initially proved to be very successful, the efficiency of these control strategies deteriorated rapidly as a consequence of a dramatic development of resistances against these control agents. The number of incident cases, which dwindled initially, was found to have increased enormously again a decade later. This may be illustrated by the following example. In 1935 the number of clinical cases in India was approximately 100 million; in 1965 this number was down to some 100 000 owing to the systematic spraying of large areas with DDT. Ten years after spraying had been stopped, this number had assumed epidemic proportions again. By the end of the 1970s, again 150 million clinical cases were reported, many of them with a fatal outcome because of the equally developed resistances to chloroquine. For this reason, and to a lesser extent for environmental reasons as well, DDT spraying was stopped by the end of the 1960s and efforts were made to find new control agents not having the last-mentioned inherent drawbacks.

Partly because of the seemingly unlimited possibilities of the then booming recombinant DNA technology, search lines were started by the end of the 1970s, which were based on a more or less "classic" approach to the problem, viz. the control of malaria parasites, Plasmodium falciparum in particular, by means of vaccines. From the very beginning the World Health Organisation vigorously espoused this alternative approach and further took upon itself the global coordination of the vaccine research. The objective of the alternative control method is to build up, by means of a focussed stimulation of the human humoral and cellular immunological defence systems, an efficient defence system which is capable of breaking through the complex life cycle of the parasite, either in the infected human or in the infected mosquito of the genus Anopheles.

Broadly, the life cycli of all malaria parasites are identical. Each parasite has its own specific host (a mosquito of the genus Anopheles) and intermediate host (for instance man) . Infection of man begins with a sting by a parasite-bearing mosquito. With the .saliva, the sporozoites or sickle germs are transferred to the intermediate host (man) . When they have entered the blood, they infect the liver parenchymal cells virtually immediately. At the expense of this cell, the parasite grows into a giant cell (schizont) with a great many nuclei (syncytium) . After exuberant membrane synthesis, this cell finally divides into some tens of thousands of minicells, the so-called merozoites. The liver cell perishes in the process and the merozoites then enter the bloodstream. The hepatocytic multiplication stage mostly goes at the expense of a few liver cells only, which is why the primary parasitic infection mostly goes completely unnoticed by the patient.

After release from the liver cell, the merozoites are dependent for their further asexual multiplication on the red blood cells (erythrocytes) which they infect virtually immediately upon release from the hepatocyte. In them the parasites initially have a ringlike structure, which grows into a freely motile trophozoite which feeds inter alia on the haemoglobin copiously present in the erythrocyte. From the trophozoite then grows an 8-16 nuclei-containing schizont which, following an extensive membrane synthesis, bursts open while releasing offspring merozoites. Virtually immediately after release, they invade new erythrocytes again and the replication process just described repeats itself. In addition to febrϋe substances, with the burst of the erythrocyte, an undigestible iron-containing polymer, the pigment or haemozoin, is also released, which is a product of the haemoglobin digestion. The malarial disease has then begun, as can be unequivocally diagnosed by demonstrating the parasite in a thick drop of blood. When the multiplication process of the merozoites in the erythrocyte proceeds synchronously (in two and three days, respectively) , the fever will also exhibit a periodicity. The number of parasites then increases logarithmically and with each erythrocytic infection as many red cells are lost. Non-infected cells, too, perish in the process, so that anaemia develops in the course of time. In addition to fits of fever and anaemia, the malarial disease is further characterised by other symptoms, such as for instance spleen enlargement or cerebral malaria. Cerebral malaria often occurs with infections with Plasmodium falciparum and is a result of the capillary blood vessels "silting up" with aggregates of infected erythrocytes.

After infection of an erythrocyte by a merozoite, it is not always merozoites that develop as a result. Through an as yet unclarified mechanism and with low frequency, the merozoite in the erythrocyte can also develop into a precursor of a female or male sex cell, the female and male gametocyte, respectively. After their development, these intracellular sexual forms continue to circulate in the blood and are cleared by the immune system when they are sufficiently "old". Only when during a blood meal they are sucked up by a mosquito, they are woken up from their rest as a result of a change in temperature and pH and in the mosquito stomach they mature virtually immediately into male microgametes or female macrogametes which fertilise each other virtually immediately after release. After the zygote formed has grown into a motile oδkinete, it penetrates the wall of the midgut to settle under the basal membrane. With simultaneous development of many sporozoites, it grows into an oδcyst. Once these are mature, they burst open while releasing the offspring sporozoites. Prompted by an as yet unclarified mechanism, the latter then migrate via the haemocoel to the salivary gland, where they settle in the lumen. When this mosquito now takes a blood meal again, a fraction of these sporozoites enter the blood of the next victim and the parasitic developmental cycle is closed.

Culturing parasites in the laboratory is a particularly difficult and costly affair (human sera and blood cells are required for this) . Since the replication in vitro moreover proceeds very inefficiently, they can only be produced in very minor amounts in the laboratory. This also applies mutatis mutandis to the antigens located on the surface of these parasites. For these reasons, no attenuated or killed parasites can be used for vaccination against the malaria parasite (when killed parasites are used, not even a protective immune response is obtained) , as is often possible for the control of bacterial or viral infections, so that modern techniques from molecular biology and/or organic chemistry have to be relied on for a large-scale production of the respective antigens, or parts thereof.

Since the parasite must change host (man and mosquito, respectively) in order to complete the full course of its life cycle, three levels can in theory be designated at which the life cycle of the parasite could be broken using an activated immunological defence system. These three levels are:

1. preventing penetration and multiplication of the parasite in the human liver cell;

2. preventing penetration and multiplication of the parasite (merozoite) in the red blood cell; and

3. preventing the formation/maturation of the sex cells, their mutual fertilisation and their subsequent development into parasitic stages which are infectious to man. The antigens or parts thereof which are eligible for the development of a vaccine should be located on the surface of the parasite or infected cell. On the cellular surface of the sexual stages (gametocytes, gametes and/or zygotes) of the parasite, using transmission blocking monoclonal antibodies (TB abs) , so far three proteins, Pfs230, Pfs45/48 and Pfs25, have been demonstrated, which are not only of essential importance to the development of the parasite in the mosquito, but moreover possess the ability to evoke a vigorous "transmission blocking" immune response. Inhibition of their biological activity using TB Mabs leads to a blockade of the replication of the parasite and mutatis mutandis also to inhibition of the transmission of the parasite from mosquito to man (1, 28) .

Of these proteins, Pfs25, a protein with a molecular mass of approx. 25kDa, which is not synthesised until in the mosquito stomach, is the only protein so far whose gene has been cloned and characterised (12) . The protein components of doublet Pfs45/48 and Pfs230 are found as a stable complex on the cell surface of both gametocytes and gametes (14, 22, 28, 29) .

Studies on the biochemical and biophysical properties have demonstrated that the two protein components of the Pfs45/48 doublet are recognised by the same TB Mabs (e.g. 32F3 and 32F5) , are both highly hydrophobic glycoproteins and have the same isoelectric point (13, 22, 28, 29) . Immulogic studies have demonstrated that in these proteins at least four different epitopes occur and that the TB Mabs identified to date exclusively recognise so-called conformation- dependent epitopes, i.e. the epitopes are not recognised in proteins whose sulphur-disulphide bridges have been broken by means of reducing agents (1) .

Epidemiologic studies have demonstrated that the epitopes of Pfs45/48 in field isolates of Plasmodium falciparum are very strongly conserved (5) . All these findings, together with the discovery that even in the most natural situation (in man) the synthesis of transmission blocking antibodies can be induced (6-10, 17), suggest that Pfs45/48 could be a very important instrument for the development of a transmission blocking and self-boosting vaccine. It is for these reasons that investigations have been started into the identification and characterisation of the gene(s) that code(s) for the separate protein components of Pfs45/48 and into the possibility of expressing this gene (these genes) in heterologous prokaryotic and eukaryotic systems.

The term 'transmission blocking' as used herein means the ability of antibodies to break through the extracellular replication cycle proceeding in the mosquito stomach when they are sucked up from the blood into the mosquito stomach together with the parasite. The result of this immulogic blockade is that the same mosquito, when taking a next blood meal, will not transmit any parasites to a next victim.

Summarv of the invention

According to the invention, it has now been managed, by advanced biochemical, immunologic and molecular biological techniques, to purify extremely slight amounts of each of the proteins of the doublet Pfs45/48 (i.e. two closely related proteins with a molecular mass of 45 kDa and 48 kDa, respectively) , which are found on the surface of gametocytes as well as young ga etes/zygotes, and subsequently to characterise them by means of HPLC chromatography of their tryptic digests, followed by a determination of the amino acid sequence of some of the thus obtained peptides. As will be described herein in detail, the degenerated nucleotide sequences derived from the clarified amino acid sequences subsequently served as an instrument in the identification and isolation of the Pfs45/48 coding sequence from a cDNA bank which had been constructed from gametocyte mRNA. After various clones had been identified, the nucleotide sequence of the complete intronless Pfs45/48 gene and the corresponding amino acid sequence of the primary translation product was clarified (SEQ ID NO: 1) .

Only after it had appeared conclusively that: (a) all determined amino acid sequences of the HPLC peptides could in actual fact be traced in the 'derived' amino acid sequence of Pfs45/48, and

(b) antibodies which had been raised in a rabbit against a part of pfs45/48 protein synthesised in the gut bacterium Escherichia coli, unequivocally recognised the Pfs45/48 doublet on a Western blot, was the unequivocal conclusion drawn that the Pfs45/48 gene had in actual fact been isolated and characterised. Further, from the results obtained, the unequivocal conclusion could be drawn that the "peptide part" of the proteins Pfs45 and Pfs48 is coded by one and the same gene. The codongenic sequence is 1347 bp long and codes for a polypeptide chain of a length of 448 amino acids with a moleculair mass of 51.6 kDa. From computer-supported analyses, indications have been obtained that Pfs45/48 protein contains seven potential N- glycosylation sites and that both at the N-terminal and C-terminal end hydrophobic amino acid sequences are located. The N-terminal sequence is presumably a signal sequence, whilst the C-terminal sequence is possibly involved in a membrane anchoring of the protein chain via a glycosylphosphatidylinositol anchor. Then studies were undertaken to express the gene, or parts thereof, in heterologous prokaryotic and eukaryotic systems. With the serum which had been raised in rabbits against a Pfs45/48 peptide synthesised in Escherichia coli. the conclusion that gene Pfs45/48 had been isolated could be confirmed unequivocally. None of the recombinant Pfs45/48 peptides synthesised in Escherichia coji has so far been found capable of eliciting a transmission blocking immune response in rabbits. Since all of the transmission blocking monoclonal antibodies known to date seem to recognise exclusively conformation-dependent epitopes in the proteins of the Pfs45/48 doublet, this negative outcome had been seriously reckoned with.

For this reason, further studies were undertaken to express the Pfs45/48 gene in heterologous eukaryotic cells (COS cells and insect cells) as well. After a successful expression of Pfs45/48 protein in insect cells which had been infected with recombinant Pfs45/48 baculovirus, it could be demonstrated that in a heterologous eukaryotic expression system Pfs45/48 antigens are produced which are recognised by Pfs45/48-specific transmission blocking monoclonal antibodies. These possess the ability to induce conformation- dependent transmission blocking immunity.

In other words, these studies prove unequivocally that the • production of Pfs45/48 antigens capable of inducing transmission blocking immunity in man using heterologous eukaryotic systems is a possibility. Apparently, in the heterologous eukaryotic expression system Pfs45/48 protein is folded such that, after insertion in the surface membrane of the insect cell, it is specifically recognised by transmission blocking monoclonal antibodies of Pfs45/48. Accordingly, the production of a transmission blocking vaccine based on Pfs45/48 protein using a heterologous eukaryotic expression system has now become a real possibility.

Detailed description of the invention

The invention provides a recombinant nucleic acid molecule, comprising a nucleotide sequence selected from

(a) nucleotide sequences which code for the amino acid sequence of Plasmodium falciparum protein Pfs45/48 shown in Fig. 2;

(b) nucleotide sequences which hybridise with a nucleotide sequence complementary to a nucleotide sequence according to (a) and code for a polypeptide or protein that is recognised by an antibody capable of binding to a Plasmodium protein, and

(c) fragments of a nucleotide sequence according to (a) or (b) of a length of at least 7 nucleotides.

According to the invention, the preference is for such a recombinant nucleic acid molecule which comprises the codongenic nucleotide sequence shown in Figure 2, a nucleotide sequence deviating therefrom within the limits of the degeneration of the genetic code and coding for the amino acid sequence of Plasmodium falciparum protein Pfs45/48 shown in Figure 2, a nucleotide sequence hybridising with at least one of the nucleotide sequences ^{■ •} complementary to these nucleotide sequences and coding for a polypeptide or protein related to Plasmodium falciparum protein

Pfs45/48, or a fragment of one of these nucleotide sequences of a length of at least 8 nucleotides. Preferably, the molecule involved here is a recombinant nucleic acid molecule which codes for a polypeptide or protein that is recognised by a Plasmodium protein-binding antibody with transmission blocking properties.

When herein mention is made of nucleotide sequences that hybridise with a previously defined nucleotide sequence, the reference is to hybridisation under conventional hybridisation conditions, preferably hybridisation conditions so stringent as to avoid hybridisation with non-homologous sequences. The term 'nucleic acid molecule' is intended to comprise both DNA and RNA. Recombinant nucleic acid according to the invention can be used for many purposes, for instance for cloning purposes, for expression of the protein or polypeptide which it codes for in a heterologous host (cell) , for diagnostic purposes as a probe or primer for hybridisation or PCR analyses (also eligible for that purpose are nucleic acid molecules of a length of 7 of more, in practice 8 of more, preferably 10.or more, most preferably at least 12 of even at least 15 nucleotides), etc. However, the preference is for use of the recombinant nucleic acid for the benefit of the synthesis of Plasmodium falciparum protein Pfs45/48 in heterologous host cells. The invention further provides a polypeptide material, comprising an amino acid sequence which is coded by a nucleotide sequence as defined hereinabove. The term 'polypeptide material' is intended to comprise both oligopeptides and polypeptides and proteins (polypeptides having undergone a posttranslational modification such as processing, acylation, anchoring with glycosylphosphatidylinositol or glycosylation..

Preferably, the material involved here is a polypeptide material selected from (a) Plasmodium falciparum protein Pfs45/48 with the amino acid sequence shown in Fig. 2, (b) a polypeptide with the amino acid sequence of Plasmodium falciparum protein Pfs45/48 shown in Fig. 2,

(c) a fragment of a polypeptide according to (b) of a length of at least 7, preferably at least 8 amino acids. The invention, however, further comprises natural and artificial variants of Plasmodium falciparum protein Pfs45/48 whose amino acid sequence deviates from the sequence shown in Fig. 2, but which are recognised by an antibody binding to a Plasmodium protein, in particular an antibody binding to Plasmodium falciparum protein Pfs45/48, preferably transmission blocking antibody. The invention equally comprises polypeptides with an amino acid sequence deviating from the sequence shown in Fig. 2, which are recognised by an antibody binding to a Plasmodium protein, in particular an antibody binding to a Plasmodium falciparum protein Pfs45/48, preferably transmission blocking antibody.

According to the invention, it is preferred that the polypeptide material be recognised by an antibody capable of binding to a Plasmodium protein, preferably by a Plasmodium protein binding antibody with transmission blocking properties. Examples of antibodies with transmission blocking properties that bind to a

Plasmodium protein are the known monoclonal antibodies 32F3 and 32F5 which bind to Plasmodium falciparum protein Pfs45/48.

The polypeptide material according to the invention is a new 'synthetic' material, which is intended to exclude the material such as it occurs in nature. The invention is not limited with regard to the manner in which the material is prepared. The preparation can take place along a chemical route (conventional peptide synthesis) , but is preferably realised by expression of recombinant nucleic acid in a heterologous host cell or host. Accordingly, the invention further provides a method of preparing a polypeptide material, comprising culturing a host cell which produces the polypeptide material and recovering the polypeptide material produced, a host cell being used which has acquired the ability of synthesising the polypeptide material as a result of genetic manipulation using a recombinant nucleic acid molecule according to the invention. The genetic manipulation can be performed on a cell from which the host cell descends .

The host cell can be a prokaryotic host cell, for instance an Escherichia coli bacterium or a bacterium of a different type, but it is preferred that a eukaryotic cell be used as host cell, for instance a yeast cell, fungal cell, plant cell, insect cell (for instance Spodoptera fruσiperda) or mammalian cell (for instance Chinese hamster ovary cells) .

The invention also comprises a method of preparing a polypeptide material, comprising culturing a transgenic vegetable or animal organism which produces the polypeptide material and recovering the polypeptide material produced, a transgenic organism being used which has acquired the ability to synthesise the polypeptide material as a result of genetic manipulation using a recombinant nucleic acid molecule according to the invention. In the case of a transgenic animal, the preference is for a transgenic rodent, such as mouse, rat, guinea pig and rabbit, but also transgenic domestic amimals, such as cow, sheep and goat are eligible.

The invention further provides a method of preparing a polypeptide material, comprising carrying out such peptide synthesis steps that a polypeptide material according to the invention is obtained.

Polypeptide material according to the invention can be used for various purposes, for instance for immunisation in order to elicit an antibody response, whether or not within the framework of a method of preparing monoclonal antibodies. Also eligible are fragments of a length from 7, in practice from 8 amino acids, preferably at least 10 or even at least 12, most preferably at least 15 amino acids. The greatest preference, however, is for the use of polypeptide material according to the invention, in particular recombinant Plasmodium falciparum protein Pfs45/48, in a transmission blocking vaccine which can be deployed for the control of malaria.

Accordingly, the invention further provides a transmission blocking vaccine for control of the malaria parasite Plasmodium falciparum. comprising a polypeptide material according to the invention and a suitable support or adjuvant. Supports and adjuvants suitable for vaccine preparations are known to those skilled in the art, and so are eligible vaccination methods.

Materials and methods

Strategy Cloning Gene Pfs45/48

Using immunoaffinity chromatography, the protein doublet Pfs45 and Pfs48 was isolated from gametocyte extracts from Plasmodium falciparum. Then, from the proteins purified by means of polyacrylamide gel electrophoresis, triptic digests were made, which were separated into their constituent peptide components by means of HPLC chromatography. The amino acid sequence of seven peptides was subsequently determined using a gas phase sequenator and on the basis of these sequences three oligonucleotides with degenerated sequences were synthesised. Using two of these oligonucleotides and the polymerase chain reaction (PCR) , genomic sequences of Plasmodium falciparum were subsequently ampflified, which, after being marked with radioactive phosphate, were in turn used for fishing for complementary sequences in a cDNA library constructed from gametocyte mRNA. Using the recombinants picked up, finally the nucleotide sequence of intronless Pfs45/48 gene was determined.

Parasites

Gametocytes of Plasmodium falciparum (isolate NF54) were produced in an automated culturing system as described by Ponnudurai et al. (19) . By means of centrifugation (30 min, 6000 x g and at 24°C) through a layer of 18% Nycodenz, the gametocytes were separated from the asexual blood stages (29) .

Erythrocytes in the gametocyte fraction were lysed by incubating the cells for 5 min at 4°C in 140 mM NH₄C1. Then the mixture was centrifuged in an Eppendorf centrifuge for 10 min at 4°C.

Coupling of Pfs45/48 Specific Monoclonal Antibody to CL Sepharose For the covalent coupling of the Pfs45/48 specific monoclonal antibody IgG 32-F5 (28) to freshly prepared cyanogenic-bromide activated CL Sepharose, the conditions as described by Harlow and Lane (11) were followed.

Isolation and Purification of Pfs45/48

For the purification of each of the components of the protein doublet Pfs45/48, the starting material was a Triton X-114 extract which had been prepared by means of sonification in 40 ml Triton X- 114 buffer (2% Triton X-114 (v/v) in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl) from 3 x 10^ gametocytes. In addition to the aforementioned components, the following protease inhibitors had been added to the extraction buffer: phenylmethylsulphonyl fluoride (170 μg/ml), tosyl-phenylalanine chloromethyl ketone (100 μg/ml) , tosyl-lysine chloromethyl ketone (50 μg/ml), pepstatin (0.7 μg/ml) and leupeptin (0.5 μg/ml) . After 3 h standing on ice, the liquid phase was separated from the undissolved constituents by means of centrifugation (1 h, 100 000 x g, 4°C) .

After dilution of the supernatant with 0.1 volume lOx binding buffer (lOx binding buffer = 300 mM Tris-HCl, pH 7.5, 5 M NaCl), 5 ml CL Sepharose-coupled Pfs45/48-specific monoclonal antibody was applied with a pipette, whereafter the mixture was incubated overnight at 4°C with gentle stirring. After incubation the CL Sepharose was successively washed with twice 50 ml: 1. high-salt buffer (50 mM Tris-HCl, pH 8.2, 500 mM NaCl, 1 mM EDTA, 0.5% NP-40) ;

2. low-salt buffer (50 mM Tris-HCl, pH 8.2, 150 mM NaCl, 0.5% NP-40, 0.1% SDS) ; and

3. 150 mM NaCl. Finally, the Sepharose beads were once again washed batchwise with 50 ml distilled water. After washing the proteins were eluted from the beads using 5 ml 1% SDS and an incubation of 10 min at 70°C. After freeze-drying the eluate, 400 μl mM Tris-HCl, pH 6.8, was added to the protein, and the whole was placed in a boiling water bath until all of the protein was dissolved (approx. 10 min) . After cooling and addition of glycerol and bromphenol blue to a final concentration of 10% and 0.005%, respectively, the proteins of the doublet were separated from each other by means of SDS polyacrylamide gel electrophoresis under non-reducing circumstances (4, 18) . After electrophoresis the two protein bands were stained with Coomassie blue (18), excised from the gel and stored in the deepfreeze at -20°C.

Trypsin Digestion, Purification and Sequence Analysis of Tryptic Peptides

After excision the gel segments with, respectively, the Pfs45 and the Pfs48 protein were incubated for 1 h at 37°C in 400 μl 100 mM sodium borate, pH 9. After addition of trypsin to a final concentration of 1 μg/ml the mixture was incubated for 72 h in a water bath of 37°C. After 24 and 60 h incubation another equal amount of trypsin was added to the mixture with a pipette. Subsequent to the incubation the gel segments were separated from the incubation liquid by means of centrifugation and dithiothreitol, to a final concentration of 10 mM, was added to the incubation liquid with a pipette. After the mixture had been incubated for another 30 min at 37°C the digestion mixture was transferred to a HPLC C18 reverse phase column (Beckman, 4.7 x 250 mm) which had been preincubated in 0.063% trifluoroacetic acid (TFA) in aqua dest (= eluent A) . Elution of the peptides (elution rate: 1 ml/min) took place with gradients, of eluent B (= 0.060% TFA in acetonitrile- water (80:20)) in eluent A, of the following compositions:

1. gradient of 0-40% B in a time span of 60 min;

2. gradient of 40-75% B in 30 min; and

3. gradient of 75-100% B in 10 min. ^' The elution was monitored by determining the UV absorption at 214 nm. After elution the peak fractions were reduced by evaporation using a Speed-Vac centrifuge. Finally, the amino acid sequence of 7 peptides was determined by means of a gas phase sequenator (Applied Biosystems, Model 477A) . Oligonucleotide Synthesis and Polymerase Chain Reaction

Oligonucleotide primers for polymerase chain reactions (PCR) and sequence analyses were synthesised by means of a Cyclone Plus DNA synthesiser (Milligen/Biosearch) . A PCR reaction mixture had the following composition: 10 mM Tris-HCl, pH 9.0, 50 mM KC1, 1.5 mM MgCl₂, 0.01% gelatin, 0.1%

Triton X-100, 200 μM of each of the nucleotide triphosphates dATP, dGTP, dCTP and dTTP, 50 pmol of the 5' primer and 3' primer, 100 ng genomic Plasmodium falciparum DNA and 2.5 units Taq DNA polymerase (Promega) . DNA amplification took place in 35 cycli, each consisting of een incubation of 1 min at 96°C, 1.5 min at 50°C and 3 min at 72°C. After incubation the reaction products were analysed by means of electrophoresis on a 1% agarose gel (23) .

Screening of Gametocyte cDNA Library and Nucleotide Sequence Analysis

A gametocyte cDNA library (100,000 colonies) in pcDNA II (InVitrogen, San Diego, USA) was plated out on LB agar plates, whereafter replicas thereof were made on nitrocellulose filters according to standard procedures (23) . Then the PCR fragment of a length of approx. 750 bp was made radioactive with α^32p labelled dATP in a random-prime reaction (23) , whereafter the filters were incubated therewith overnight and at 55°C.

Composition hybridisation liquid: 6 x SSC, 5 x Denhardt's, 0.1% SDS and 100 μg/ml herring sperm DNA (20 x SSC = 3 M NaCl, 0.3 M Na citrate, pH 7.0; 50 x Denhardt's = 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin) .

After washing at 55° with 0.1 x SSC, the hybridisation signals were visualised by means of autoradiography. Positively reacting colonies were then applied pure and subsequently the nucleotide sequence of the cDNA inserts was determined using the chain- termination method developed for double-stranded DNA (2, 3, 24) .

Northern Blot Analysis From approx. 100 x 10° parasites of both the asexual stages (schizonts plus merozoites) and the sexual stages (gametocytes or . gametes) , RNA was isolated in accordance with standard techniques (23) , whereafter the RNA was fractionated on a 1% agarose/formaldehyde gel (10 μg/slot) (21) .

After blotting of the RNA on a nitrocellulose filter (23) it was incubated overnight at 5°C in the hybridisation liquid as described above and to which a PCR primer labelled at the 5' terminal with radioactive phosphate had been added with a pipette. After incubation the filter was washed several times at 40°C with 6 x SSC, whereafter the filter was inlaid for autoradiography (72 h at -80°C) .

Western Blot Analysis For the benefit of immunological analyses, proteins isolated from gametocytes were fractionated by means of electrophoresis on 10% SDS polyacrylamide gels (5 x 10⁶ gametocyte equivalents per lane) under non-reducing conditions.

After electroblotting of the proteins on a nitrocellulose filter, the filters were incubated for 3-16 h in a solution of 3% bovine serum albumin- in PBS (PBS = 10 mM phosphate buffer, pH 7.4, 150 mM NaCl) . Then the. filters were incubated with either a polyclonal antiserum (1:400 diluted with PBS) which had been raised against a recombinant peptide of Pfs45/48 synthesised in Escherichia coli (vide infra) . or with the previously mentioned Pfs45/48 specific monoclonal antibody 32F3 (final concentration 1 μg/ml) . After a second incubation with an alkaline phosphatase-conjugated antibody (goat-anti-rabbit and rabbit-anti-mouse, respectively, and 1:5000 diluted in PBS) the immune complexes were visualised with the chromogenic substrates BCIP (5-bromo-4-chloro-3-indolyl phosphate) and nitroblue-tetrazolium chloride (NBT) .

Synthesis of Pfs45/48 Specific Peptides in E. coli Using standard recombinant DNA techniques (23), a PCR fragment coding for amino acids 118 through 218 of Pfs45/48 was cloned into the fusion protein vector pGEX-3X (26) .

After induction with IPTG, the fusion protein of glutathione-S- transferase present in so-called 'inclusion bodies' was purified by means of SDS polyacrylamide gel electrophoresis (20) .^' After electrophoresis the proteins were visualised using 0.25 M KC1. Then the gel segments containing fusion proteins were excised and incubated overnight at room temperature in 100 μl elution buffer (20 mM Tris-HCl, pH 7.9 , 1 mM CaCl₂, 0.01% SDS).

After addition of KC1 to a final concentration of 50 mM, incubation took place for another 10 min at 0°C, whereafter centrifugation took place for 5 min in an Eppendorf centrifuge to remove the SDS precipitate. - Rabbits (New Zealand White) were subsequently immunised with the isolated proteins: four injections, each with 0.5 mg fusion protein; the first in Freunds adjuvants complete and the other three in Freunds adjuvants incomplete.

Expression of Gene Pfs45/48 in Insect Cells

For this purpose twee PCR primers were synthesised: a. 5 '-GCTAGCATGATGTTATATATTTCTGCG-3' (SEQ ID NO: 2) b. 5 *-GCTAGCATGAGCTAAATATATAATAATATTGC-3' (SEQ ID NO: 3) which, respectively, overlap the start and stop codon of gene Pfs45/48 and are provided at the 5' terminal with a recognition sequence of the restriction enzyme NJae_I.

After incubation of the primers with genomic Plasmodium falciparum DNA (30 cycli each consisting of an incubation of, respectively, 1 min at 96°C, 1 min at 55°C and 2.5 min at 72°C) , the amplified DNA fragment was purified by means of agarose gel electrophoresis and after incubation with Nhel inserted into the

Nhel site of the transfer vector pJVPIOZ (30) .

After the recombinant (pJV45/48P10Z) with the desired orientation of the PCR fragment had been identified and characterised, it was transfected together with the genome of the baculovirus vector Autoσrapha californica into tissue culture cells (Sf9) of the insect Spodc.ptera fruσiperda according to standard methods (27) . After positive selection of the recombinant baculoviruses with the chromogenic substrate 5-bromo-4- chlorothiogalactoside (X-gal) , they were cultured pure using the same selection techniques.

For immunofluorescence experiments (IFA) Sf9 cells were cultured on cover slips and subsequently infected with the recombinant baculovirus (approx. 1 plaque-forming unit (pfu) per cell) . Three days after infection it was examined with the IFA assay whether

Pfs45/48 protein had been synthesised and whether it was exposed on the external surface of the cell membrane of the infected cells, analogously to the authentic protein.

Immunofluorescence Assay

Infected cells were immobilised with ethanol/acetone (1:1) and subsequently incubated at room temperature for 30 min with either polyclonal antiserum (1:100 diluted in PBS), or monoclonal antiserum 32F5 (10 μg/ml in PBS) . After washing with PBS, a fluorescein isothiocyanate (FITC)-labelled second antibody (goat-anti-rabbit IgG, 1:100 diluted in Evans Blue or rabbit-anti-mouse IgG, 1:200 diluted in Evans Blue; Evans Blue = 0.5% glucose, 0.05% Evans Blue in PBS) was applied to the cells with a pipette and after incubation in the dark for 30 min the cells were washed with PBS and after drying analysed using a fluorescence microscope.

Results and Discussion

Isolation and Purification of Pfs45/48 Since Pfs45/48 is a highly hydrophobic membrane protein (22, 28) , the dissolution thereof requires that the solvent contain a detergent (Triton X-114 or SDS) . With the procedure described, Pfs45/48 could efficiently be extracted from the gametocyte membranes and subsequently be efficiently bound to the CL Sepharose- coupled monoclonal antibody 32F5. The extreme affinity of 32F5 ^'for Pfs45/48 made it possible for stringent conditions to be employed both in the binding and in the washing of the affinity column. This prevented any other antigens from binding aspecifically to the affinity column. Conversely, such a high affinity necessitated the use of "strong" elution agents.

After column chromatography the separate protein components of the doublet Pfs45/48 were separated from each other under non- reducing conditions by means of SDS polyacrylamide gel electrophoresis (4, 18) . With this second purification step, it was further accomplished that IgG, too, which always leaks to a greater or lesser extent from an immunoaffinity column, was separated from the respective protein components. On the basis of the intensity of the Coomassie blue stained protein bands, it could be concluded that each of the components had a purity exceeding 90% and that the estimated yield of Pfs45 was approx. 20 μg and that of Pfs48 was approx. 16 μg. After staining the protein bands were excised from the gel and, to verify that Pfs45 and Pfs48 had in actual fact been isolated, a part of the protein fractions was again fractionated under non-reducing conditions on a polyacrylamide gel (18) . After electrophoresis the proteins were blotted on a nitrocellulose filter and incubated with t-he monoclonal antibody 32F5. After visualisation of the immune complexes with a second conjugated antibody and a chromogenic substrate (Materials and Methods) it could indeed be unequivocally concluded that the two components of the doublet had been isolated free of IgG or other protein components.

Trypsin Digestion

To obtain as complete a digest as possible, the proteins were incubated for a prolonged time in the presence of as low a trypsin concentration as possible. After digestion the tryptic peptides were separated from each other using reverse phase HPSC chromatography. The huge similarities between the elution patterns of the peptides which had been released by trypsin from Pfs45 and Pfs48 confirmed our previous suspicions that these two proteins could only differ from each other to a slight extent. To further verify this, some corresponding peptide fractions of the HPLC eluates of Pfs45 and Pfs48 were put together and then fractioned again using the HPLC column. In all cases only one peak could be eluted, which proves again that the peptide fractions were pure and that the corresponding fractions of the column eluates of Pfs45 and Pfs48 contained peptides with an identical amino acid sequence.

After fractionation on the HPLC column the amino acid sequences of five Pfs45 specific peptides and of four 'mixed' peptides of Pfs45 and Pfs48 were determined. These analyses showed that the trypsin digestions were still incomplete and that on the basis of sequence overlap between the peptides, seven, for the greater part unequivocal, amino acid sequences (Table 1) could be derived from the results obtained. It is clear that peptide 2, and possibly also peptide 5, is the product of an incomplete proteolytic digestion.

Combination of the amino acid sequences of the peptides 1, 2 and 3 yields the (most likely) amino acid sequence: V Y T D Y E N R V E T D I S E L G L I E Y E I E (SEQ ID NO: 4) .

The sequences underscored in Table 1 were used for designing the degenerated oligonucleotides for the benefit of the PCR ampflication (vide supra) .

PCR and Northern Blot Analysis

In the design of degenerated oligonucleotide primers for the benefit of the PRC ampflication, use was made inter alia of the codon use of Plasmodium falciparum_r published previously (25) . On the basis of the amino acid sequence of peptide 1 (Table 1) , both a 'sense' primer and an 'antisense' primer were synthesised; on the basis of the amino acid sequence of peptide 5 only a (degenerated) antisense primer was synthesised. For the sense and antisense primers of peptide 1 the nucleotide sequences were, respectively: 1. 5 '-ATT TCA GAA TTA GGT TTA ATT GAA TAT GAA ATT GAA-3'

A A A A

(SEQ ID NO: 5) and 2. 5*-TTC AAT TTC ATA TTC AAT TAA ACC TAA TTC TGA AAT-3'

T T T T

(SEQ ID NO: 6)

The antisense primer derived from peptide 5 had the nucleotide sequence:

5*-ATC TGG TAT TAT ATC TCC TGG TCT ATT TAA TCC TAC-3'

A A A A A

(SEQ ID NO: 7)

After synthesis the antisense primers were screened for their specificity by means of hybridisation with Northern blots of RNA fractions which had been isolated from asexual and sexual stages of the parasite (Figure 1) . Apart from the fact that specific hybridisation signals were obtained, these experiments confirmed that the expression of Pfs45/48 is sexual stage-specific and the Pfs45/48 gene is expressed by means of two mRNAs of lengths of 2.3 kb and 2.8 kb (Figure 1) .

The antisense probe which had been derived from peptide 1 reacted considerably more strongly with the RNA blots than did the antisense probe derived from peptide 5. As appeared afterwards, this was a consequence of the fact that the amino acid sequence of peptide 5 proved not to have been determined entirely unequivocally. In particular, it appeared from the nucleotide sequence analyses that the amino acid arginine (Table 1) does not occur in the peptide (vide infra) . Despite this base mismatch, PCR amplification of genomic DNA with this primer, and the sense primer of peptide 1, resulted nonetheless in a product of a length of 750 base pairs. Sequence analysis of this product resulted in an 'open reading frame' (ORF), spanning the entire sequence, in which, surprisingly enough, the clarified amino acid sequences of three other peptides (Table 1) occurred as well.

Isolation and Sequence of Gene Pfs45/48 The PCR fragment of a length of 750 base pairs was used for screening a gametocyte-specific cDNA library. From it 12 clones were picked up with inserts varying in size from 1000 to approx. 2500 bp. After sequence analysis a unique nucleotide sequence could be unequivocally assembled from this, with only one long ORF of 1347 bp and coding for a protein of a length of 448 amino acids and a molecular mass of 51.6 kDa (Figure 2) .

As could be expected, the amino acid sequences of all tryptic peptides whose sequence had been determined could be traced in the ORF. On the basis of these unequivocal results the conclusion seems justified that the clarified nucleotide sequence codes for the peptide part of each of the two protein components of the Pfs45/48 doublet.

This conclusion is further corroborated by the fact that the derived amino acid sequence exhibits all characteristics of a membrane protein, i.e. a hydrophobic signal sequence at the N- terminal end and a highly hydrophobic amino acid sequence adjacent the C-terminal end. The fact that the C-terminal hydrophobic sequence is not followed by any other sequence is presumably an indication of the fact that, like many other membrane proteins of

Plasmodium falciparum, the proteins Pfs45 and Pfs48 are anchored to the external surface of the gametocyte/gamete via a glycosyl- phosphatidylinositol anchor (15) .

Computer-supported sequence analyses further taught that seven potential N-glycosylation sites occur in the amino acid sequence of Pfs45/48 (Figure 2) . All of these characteristics agree excellently with the known biochemical and physicochemical properties of Pfs45/48. On the basis of Southern blot analyses and size comparisons of PCR fragments which had been obtained from amplifications of genomic DNAs and cDNAs, the further conclusion could be drawn that the codongenic sequence .of the Pfs45/48 gene contains no introns and that only one copy of this gene occurs in the Plasmodium falciparum genome.

Expression of (subfragments) of Gene Pfs45/48 1. Expression in Escherichia coli

A PCR fragment coding for the amino acids 118 through 218 of Pfs45/48 (Figure 2) was recombined with the 3' end of the glutathione-S-transferase gene of the fusion vector pGEX-3X and subsequently expressed in Escherichia coli. The result of this construct was that the peptide after induction was synthesised in large amounts, the vast majority thereof being deposited in the cell in the form of so-called 'inclusion bodies'.

After harvesting of the 'inclusion bodies' the protein was purified by means of SDS polyacrylamide gel electrophoresis and subsequently used for raising Pfs45/48 specific antisera (Materials and Methods) .

Subsequently immunological analyses were carried out with the obtained antisera on Western blots which had been made from proteins isolated from the asexual (schizont, merozoite) stages and sexual (gametocytes/gametes) stages. From the results it could be unequivocally concluded that the antisera reacted specifically with the Pfs45/48 proteins in the sexual stages but not with the proteins which had been isolated from the asexual blood stages (Figure 3) . In an immunofluorescence assay, too, the antisera appeared to react exclusively with antigens occurring on the surface of the sexual stages. The antisera, however, did not possess any transmission blocking activity. Since all transmission blocking monoclonal antibodies of Pfs45/48 isolated to date are directed against conformation-dependent epitopes (1) , this outcome was seriously reckoned with. The specific recognition of Pfs45 and Pfs48 by the peptide antiserum, however, confirms the conclusion drawn previously that the characterised gene contains the codongenic information for the protein doublet Pfs45/48.

2. Expression of Gene Pfs45/48 in Insect Cells

The fact that all characterised transmission blocking monoclonal antibodies of Pfs45/48 recognise conformation-dependent epitopes (1) suggests that in order to obtain a transmission blocking immune response an authentic folding of the protein is indispensable. For this reason, studies were undertaken to express the entire codongenic sequence of Pfs45/48 in insect cells using recombinant baculovirus.

Analysis of the expression of the Pfs45/48 gene using immunofluorescence assays showed that the synthesis of Pfs45/48 specific proteins could be demonstrated unequivocally both with the polyclonal antiserum (vide supra) and with the transmission blocking antibody 32F5 (Figure 4) . Although, on account of the as yet low expression, we have not yet succeeded in demonstrating the synthesis of Pfs45/48 specific proteins with Western blots as well, these studies nevertheless prove that the induction of transmission blocking immunity by Pfs45/48 specific proteins which are synthesised in a heterologous eukaryotic expression system is a real possibility. The experiments accordingly show that it is possible to produce a transmission blocking vaccine with heterologous eukaryotic expression systems.

Discussion of the Figures

Figure 1: Northen blot analyses of asexual, gametocyte and gamete RNA

Total RNA (10 μg per lane) of asexual blood stage parasites (lane 1), gametocytes (lane 2), or gametes (lane 3) of isolate NF54 of Plasmodium falciparum was blotted on a membrane of nitrocellulose after electrophoresis. For the detection of Pfs45/48 specific mRNAs the membrane was incubated with a PCR primer rendered radioactive at the 5' terminal (specific activity 2 x 10° cpm/μg) and subsequently washed as described in Materials and Methods. Autoradiography lasted for 72 hours. The lengths of the RNA markers (BRL) are specified in kilobase pairs (kb) .

Figure 2: Nucleotide sequence and derived amino acid sequence of gene Pfs45/48

The codongenic sequence (1347 nucleotides) is indicated by capitals. Solid lines: peptide sequences which were clarified using microsequence analysis of purified tryptic peptides of Pfs45 and Pfs48. Dotted lines: undetermined or wrongly assigned amino acids after amino acid sequence analyses. Double solid lines: tryptic peptide sequences from which nucleotide sequences of degenerated PCR primers are derived. Open circles: potential N-glycosylation sites. Broken lines : hydrophobic amino acid sequences .

Figure 3: Western blot analysis of gametocyte proteins

Protein extracts of gametocytes (5 x 10° gametocyte equivalents per lane) were electrophoresed through a SDS polyacrylamide gel and then blotted on nitrocellulose. Subsequently the respective lanes were incubated with:

(a) rabbit preimmune serum (lane 1) ;

(b) immune serum raised against the recombinant fusion protein (lane 2); and

(c) monoclonal antibody 32F3 (lane 3) .

After a second incubation with an alkaline phosphatase- conjugated goat-anti-rabbit and rabbit-anti-mouse antibody, respectively, the immune complexes were visualised as described in Materials and Methods. The molecular masses of the marker proteins are specified in kDa.

Figure 4: Immunofluorescence analysis (IFA) of insect cells infected with recombinant baculovirus Sf9 cells were cultured on cover slips and infected with recombinant baculovirus. Three days after infection the cells were immobilised and incubated with, respectively:

(A) antibodies which had been raised against the recombinant fusion protein; (B) the monoclonal antibody 32F5.

After a second incubation with a FITC-labelled goat-anti-rabbit and rabbit-anti-mouse antibody, respectively, the cells were examined under a fluorescense microscope . Table 1: Amino acid sequences of tryptic peptides of protein Pfs45/48 of Plasmodium falciparum

1: V E T D I S E L G L I E Y E I E P W C (SEQ ID NO: 8)

2: V Y T (I) Y E N ? (V) E T (D) I S E L S

(SEQ ID NO: 9)

3: V Y T D Y E N R (SEQ ID NO: 10)

4: A P F Y V T S K (SEQ ID NO: 11)

5: Y N H L V G L N (R) P G D I I P D (SEQ ID NO: 12)

6: T I T I (S P F S P)

(SEQ ID NO: 13)

-7: N L T I F K (SEQ ID NO: 14)

The identity of the residues in parentheses has not been unequivocally established. The same applies to the places where several amino acids are specified in one position.

REFERENCES

1. Carter, R., Graves, P.M., Keister, D.B. & Quakyi, I.A.: Parasite Immunol. 12, 587-603 (1990) .

2. Casanova, J.L., Pannetier, C, Jaulin, C. & Kourilsky, P.: Nucl . Acids Res. 18, 4028 (1990) . 3.DelSal , G., Manfioletti , G. and Schneider, C: Nucl. Acids Res. 16, 9878 (1988) . 4. Doucet, J.P. & Trifaro, J.M.: Anal. 3iochem. 168, 265-271 (1988) .

5. Foo, A., Carter, R., Lambros, C, Graves, P., Quakyi, I., Targett, G.A.T., Ponnudurai, T. and Lewis, G.E.: Am. J. Trp. Hyg. 44, 623-631 (1991) . 6. Graves, P.M., Carter, R., Burkot, T.R., Quakyi, I.A. & Kumar, N. K.: Parasite Immunol. 10, 209-218 (1988) .

7. Graves, P.M., Bhatia, K., Burkot, T.R., Prasad, M., Wirtz, R.A. ^~ Beckers, P.: Clin. Exp. .Immunol. 78, 418-423 (1989) .

8. Graves, P.M., Doubrovsky; A., Carter, R., Eida, S. & Beckers, P.: Am. J. Trop. Med. Hyg. 42, 515-520 (1990) .

9. Graves, P.M., Doubrovsky, A. & Beckers, P.: Parasite Immunol. 13, 291-299 (1991) .

10. Graves, P.M., Doubrovsky, A., Sattabongkot, J. ^~ Battistutta, D.: Am. J. Trop. Med. Hyg. 46, 711-719 (1992) . 11. Harlow, Ξ. & Lane, D.: Antibodies. A Laboratory Manual, (CSH, New York, 1988) .

12. Kaslow, D.C, Quakyi, I.A., Syin, C, Raum, M.G., Keister, D.B., Coligan, J.E., McCutchan, T.F. & Miller, L.H.: Nature 333, 74-76

(1988) . 13. Kumar, N. & Carter, R. : Mol. Biochem. Parasitol. 13, 333-342 (1984) .

14. Kumar, N. and Wizel, B.: Mol. Biochem. Parasitol. 53, 113-120 (1992) .

15. Low, M.G. and Saltiel, A.R.: Science 239, 268-275 (1988) . 16. Minotti, A.M., Loeb, L.M., Cook, R., Boggs, B.A. & Cabral, F.:

Anal. Biochem. 184, 28-34 (1990) .

17. Ong, C.S.L., Zhang, K.Y., Eida, S.J., Graves, P.M., Dow, C,

Looker, M., Rogers, N.C., Chiodini, P.L. & Targett, G.A.T.: Parasite

Immunol. 121, 447-456 (1990) . 18 .Plaxton, W.C. & Moorhead, G.B.C. : Anal. Biochem. 178, 391-393

(1989) .

19. Ponnudurai, T., Lensen, A.H.W. & Meuwissen, J.H.E.Th.: Parasitol. 87, 439-445 (1983) .

20. Prussak, C.E., Almazan, M.T. & Tseng, B.Y.: Anal. Biochem. 178, 233-238 (1989) . 21. Rave, N., Crkvenjakov,_. R. & Boedtker, H.: Nucl. Acids Res. 6, 3559-3567 (1979) .

22. Rener, J., Graves, P.M., Carter, R., Williams, J.L. & Burkot, T.R.: J. Exp. Med. 158, 976-981 (1983) . 23. Sambrook, J., Fritsch, E.F. & Maniatis, T.: Molecular Cloning, 2nd ed. (CSH, New York, 1989) .

24. Sanger F., Nicklen, S. & Coulsen, R. : Proc. Natl. Acad. Sci. USA 74, 5463-5468 (1977) .

25. Saul, A. & Battistutta, D.: Mol. Biochem. Parasitol. 27, 35-42 (1988) .

26. Smith, D.B. & Johnson, K.S.: Gene 67, 31-40 (1988) .

27. Summers, M.D. & Smith, G.E.: Texas Agricult. Exp. Stat. Bullet. 155 (1987) .

28. Vermeulen, A.N., Ponnudurai, T., Beckers, P.J.A., Verhave, J.P., Smits, M.A. & Meuwissen, J.H.E.Th.: J. Exp. Med. 162, 1460-1476

(1985) .

29. Vermeulen, A.N., van Deursen, J., Brakenhoff, R.H., Lensen, A.H.W., Ponnudurai, T. & Meuwissen, J.H.E.Th.: Mol. Biochem. Parasitol. 20, 155-163 (1986) . 30. Vialard, J., Lalumiere, M., Vernet, T., Briedis, D., Alkhatib, G., Henning, D., Levin, D. and Richardson, C: J. Virol. 64, 37-50 (1990) .

SEQUENCE LIST

SEQ ID NO: 1

SEQUENCE TYPE: nucleotide with corresponding protein SEQUENCE LENGTH: 1593 base pairs and 448 amino acids STRANDEDNESS: single

acat atatatatat atatatatat atatataatt atacataatt tattttcttt acatattcac ttaaaatttt ttaaaatttt tacaattttt tcttttatac

MET Met Leu Tyr lie Ser Ala Lys Lys Ala Gin Val Ala Phe lie Leu

1 5 10 15

ATG ATG TTA TAT ATT TCT GCG AAA AAG GCT CAA GTT GCT TTT ATC TTA 48

Tyr lie Val Leu Val Leu Arg lie lie Ser Gly Asn Asn Asp Phe Cys

20 25 30

TAT ATA GTA TTA GTA TTA AGA ATA ATA AGT GGA AAC AAT GAT TTT TGT 96

Lys Pro Ser Ser Leu Asn Ser Glu lie Ser Gly Phe lie Gly Tyr Lys 35 40 45

AAG CCT AGC TCT TTG ^'AAT AGT GAA ATA TCT GGA TTC ATA GGA TAT AAG 144

Cys Asn Phe Ser Asn Glu Gly Val His Asn Leu Lys Pro Asp Met Arg 50 55 60 TGT AAT TTT TCA AAT GAA GGT GTT CAT AAT TTA AAG CCA GAT ATG CGT 192

Glu Arg Arg Ser lie Phe Cys Thr lie His Ser Tyr Phe lie Tyr Asp 65 70 75 80

GAA CGT AGG TCT ATT TTT TGC ACC ATC CAT TCG TAT TTT ATA TAT GAT 240

Lys lie Arg Leu lie lie Pro Lys Lys Ser Ser Ser Pro Glu Phe Lys

85 90 95

AAG ATA AGA TTA ATA ATA CCT AAA AAA AGT TCG TCT CCT GAG TTT AAA 288 lie Leu Pro Glu Lys Cys Phe Gin Lys Val Tyr Thr Asp Tyr Glu Asn

100 105 110

ATA TTA CCA GAA AAA TGT TTT CAA AAA GTA TAT ACT GAT TAT GAG AAT 336

Arg Val Glu Thr Asp lie Ser Glu Leu Gly Leu lie Glu Tyr Glu lie

115 120 125

AGA GTT GAA ACT GAT ATA TCG GAA TTA GGT TTA ATT GAA TAT GAA ATA 384

Glu Glu Asn Asp Thr Asn Pro Asn Tyr Asn Glu Arg Thr lie Thr lie 130 135 140

GAA GAA AAT GAT ACA AAC CCT AAT TAT AAT GAA AGG ACA ATA ACT ATA 432

Ser Pro Phe Ser Pro Lys Asp lie Glu Phe Phe Cys Phe Cys Asp Asn

145 150 155 160 TCT CCA TTT AGT CCA AAA GAC ATT GAA TTT TTT TGT TTT TGT GAT AAT 480

Thr Glu Lys Val He Ser Ser He Glu Gly Arg Ser Ala Met Val His

165 170 175

ACT GAA AAG GTT ATA TCA AGT ATA GAA GGG AGA AGT GCT ATG GTA CAT 528

Val Arg Val Leu Lys^•Tyr Pro His Asn He Leu Phe Thr Asn Leu Thr

180 185 190

GTA CGT GTA TTA AAA TAT CCA CAT AAT ATT TTA TTT ACT AAT TTA ACA 576

Asn Asp Leu Phe Thr Tyr Leu Pro Lys Thr Tyr Asn Glu Ser Asn Phe

195 200 205

AAT GAT CTT TTT ACA TAT TTG CCG AAA ACA TAT AAT GAA TCT AAT TTT 624

Val Ser Asn Val Leu Glu Val Glu Leu Asn Asp Gly Glu Leu Phe Val 210 215 220

GTA AGT AAT GTA TTA GAA GTA GAA TTG AAT GAT GGA GAA TTA TTT GTT 672

Leu Ala Cys Glu Leu He Asn Lys Lys Cys Phe Gin Glu Gly Lys Glu

225 230 235 240 TTA GCT TGT GAA CTA ATT AAT AAA AAA TGT TTT CAA GAA GGA AAA GAA 720 Lys Ala Leu Tyr Lys Ser Asn Lys He He Tyr His Lys Asn Leu Thr

245 250 255

AAA GCC TTA TAT AAA AGT AAT AAA ATA ATT TAT CAT AAA AAC TTA ACT 768

He Phe Lys Ala Pro Phe Tyr Val Thr Ser Lys Asp Val Asn Thr Glu

260 265 270

ATC TTT AAA GCT CCA TTT TAT GTT ACA TCA AAA GAT GTT AAT ACA GAA 816

Cys Thr Cys Lys Phe Lys Asn Asn Asn Tyr Lys He Val Leu Lys Pro 275 280 285

TGT ACA TGC AAA TTT AAA AAT AAT AAT TAT AAA ATA GTT TTA AAA CCA 864

Lys Tyr Glu Lys Lys Val He His Gly Cys Asn Phe Ser Ser Asn Val 290 295 300

AAA TAT GAA AAA AAA GTC ATA CAC GGA TGT AAC TTC TCT TCA AAT GTT 912

Ser Ser Lys His Thr Phe Thr Asp Ser Leu Asp He Ser Leu Val Asp

305 310 315 320 AGT TCT AAA CAT ACT TTT ACA GAT AGT TTA GAT ATT TCT TTA GTT GAT 960

Asp Ser Ala His He Ser Cys Asn Val His Leu Ser Glu Pro Lys Tyr

325 330 335

GAT AGT GCA CAT ATT TCA TGT AAC GTA CAT TTG TCT GAA CCA AAA TAT 1008

Asn His Leu Val Gly Leu Asn Cys Pro Gly Asp He He Pro Asp Cys

340 345 350

AAT CAT TTG GTA GGT TTA AAT TGT CCT GGT GAT ATT ATA CCA GAT TGC 1056

Phe Phe Gin Val Tyr Gin Pro Glu Ser Glu Glu Leu Glu Pro Ser Asn 355 360 365

TTT TTT CAA GTA TAT CAA CCT GAA TCA GAA GAA CTT GAA CCA TCC AAC 1104

He Val Tyr Leu Asp Ser Gin He Asn He Gly. Asp He Glu Tyr Tyr 370 375 380 ATT GTT TAT TTA GAT TCA CAA ATA AAT ATA GGA GAT ATT GAA TAT TAT 1152

Glu Asp Ala Glu Gly Asp Asp Lys He Lys Leu Phe Gly He Val Gly 385 390 395 400 GAA GAT GCT GAA GGA GAT GAT AAA ATT AAA TTA TTT GGT ATA GTT GGA 1200

Ser He Pro Lys Thr Thr Ser Phe Thr Cys He Cys Lys Lys Asp Lys

405 410 415

AGT ATA CCA AAA ACG ACA TCT TTT ACT TGT ATA TGT AAG AAG GAT AAA 1248

Lys Ser Ala Tyr Met Thr Val Thr He Asp Ser Ala Tyr Tyr Gly Phe

420 425 430

AAA AGT GCT TAT ATG ACA GTT ACT ATA GAT TCA GCA TAT TAT GGA TTT 1296

Leu Ala Lys Thr Phe He Phe Leu He Val Ala He Leu Leu Tyr He 435 440 445

TTG GCT AAA ACA TTT ATA TTC CTA ATT GTA GCA ATA TTA TTA TAT ATT 1344

TAGctcatga tatgttatta aaaataaatt aaagttaaaa ttaaaattaa aattaaaaga aatgaataca caaattatta aagattcaag attatttata atgtatatta attatacttt tatggtatat atatctatat atata

SEQ ID NO: 2 SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 27 nucleotides STRANDEDNESS: single

GCTAGCATGA TGTTATATAT TTCTGCG 27

SEQ ID NO: 3

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 32 nucleotides STRANDEDNESS: single GCTAGCATGA GCTAAATATA TAATAATATT GC 32

SEQ ID NO: 4 SEQUENCE TYPE: amino acid

SEQUENCE LENGTH: 24 amino acids STRANDEDNESS: single

Val Tyr Thr Asp Tyr Glu Asn Arg Val Glu Thr Asp He Ser Glu Leu 1 5 10 15

Gly Leu He Glu Tyr Glu He Glu 20

SEQ ID NO: 5

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 36 nucleotides STRANDEDNESS: single

ATTTCAGAAT TAGGTTTAAT TGAATATGAA ATTGAA 36

A A A A

SEQ ID NO: 6 SEQUENCE TYPE: nucleotide

SEQUENCE LENGTH: 36 nucleotides STRANDEDNESS: single

TTCAATTTCA TATTCAATTA AACCTAATTC TGAAAT-3 ' 36 T T T T ^'

SEQ ID NO: 7

SEQUENCE TYPE: nucleotide SEQUENCE LENGTH: 36 nucleotides STRANDEDNESS: single ATCTGGTATT ATATCTCCTG GTCTATTTAA TCCTAC-3' 36

A A A A A

SEQ ID NO: 8

SEQUENCE TYPE : amino acid SEQUENCE LENGTH : 16 amino acids STRANDEDNESS : single

Val Glu Thr Asp He Ser Glu Leu Gly Leu He Glu Tyr Glu He Glu Pro Trp

Cys 1 5 10 15

SEQ ID NO: 9

SEQUENCE TYPE: amino acid SEQUENCE LENGTH: 16 amino acids STRANDEDNESS: single

Val Tyr Thr He Tyr Glu Asn Xaa Val Glu Thr Asp He Ser Glu Leu Ser

1 5 ^• 10 15

SEQ ID NO: 10 SEQUENCE TYPE: amino acid SEQUENCE LENGTH: 8 amino acids STRANDEDNESS: single

Val Tyr Thr Asp Tyr Glu Asn Arg 1 5

SEQ ID NO: 11

SEQUENCE TYPE: amino acid SEQUENCE LENGTH: 8 amino acids STRANDEDNESS: single

Ala Pro Phe Tyr Val Thr Ser Lys 1 5

SEQ ID NO: 12 SEQUENCE TYPE: amino acid SEQUENCE LENGTH: 16 amino acids STRANDEDNESS: single

Tyr Asn His Leu Val Gly Leu Asn Arg Pro Gly Asp He He Pro Asp 1 5 10 15

SEQ ID NO: 13

SEQUENCE TYPE: amino acid SEQUENCE LENGTH: 9 amino acids STRANDEDNESS: single

Thr He Thr He Ser Pro Phe Ser Pro 1 5

SEQ ID NO: 14 SEQUENCE TYPE: amino acid

SEQUENCE LENGTH: 6 amino acids STRANDEDNESS: single

Asn Leu Thr He Phe Lys 1 5

Claims

1. A recombinant nucleic acid molecule, comprising a nucleotide sequence selected from

(a) nucleotide sequences which code for the amino acid sequence of Plasmodium falciparum protein Pfs45/48 shown in Figure 2 , (b) nucleotide sequences which hybridise with a nucleotide sequence complementary to a nucleotide sequence according to (a) and code for a polypeptide or protein which is recognised by an antibody capable of binding to a Plasmodium protein, and

(c) fragments of a nucleotide sequence according to (a) or (b) of a length of at least 7 nucleotides.

2. A recombinant nucleic acid molecule according to claim 1, comprising the codongenic nucleotide sequence shown in Figure 2, a nucleotide sequence which deviates therefrom within the limits of the degeneration of the genetic code and codes for the amino acid sequence of Plasmodium falciparum protein Pfs45/48 shown in Figure

2, a nucleotide sequence which hybridises with at least one of the nucleotide sequences complementary to these nucleotide sequences and codes for a polypeptide or protein related to Plasmodium falciparum protein Pfs45/48, or a fragment of one of these nucleotide sequences of a length of at least 8 nucleotides.

3. A recombinant nucleic acid molecule according to claim 1 or 2, which codes for a polypeptide or protein which is recognised by an antibody, binding to a Plasmodium protein, with transmission blocking properties.

4. A polypeptide material comprising an amino acid sequence coded by a nucleotide sequence as defined in claim 1 or 2.

5. A polypeptide material according to claim 4, selected from (a) Plasmodium falciparum protein Pfs45/48 with the amino acid sequence shown in Figure 2, (b) a polypeptide with the amino acid sequence of Plasmodium falciparum protein Pfs45/48 shown in Figure 2, (c) a fragment of a polypeptide according to (b) of a length of at least 7 amino acids.

6. A polypeptide material according to claim 4 or 5, which is recognised by an antibody capable of binding to a Plasmodium protein.

7. A polypeptide material according to claim 6, which is recognised by an antibody, binding to a Plasmodium protein, with transmission blocking properties.

8. A method of preparing a polypeptide material, comprising culturing a host cell which produces the polypeptide material and recovering the polypeptide .material produced, a host cell being used which has acquired the ability to synthesise the polypeptide material as a result of genetic manipulation using a recombinant nucleic acid molecule according to any one of claims 1-3.

9. A method according to claim 8, wherein a eukaryotic cell is used as host cell.

10. A method according to claim 9, wherein an insect cell or mammalian cell is used as host cell.

11. A method of preparing a polypeptide material, comprising culturing a transgenic vegetable or animal organism which produces the polypeptide material and recovering the polypeptide material produced, a transgenic organism being used which has acquired the ability to synthesise the polypeptide material as a result of genetic manipulation using a recombinant nucleic acid molecule according to any one of claims 1-3.

12. A method of preparing a polypeptide material, comprising carrying out such peptide synthesis steps that a polypeptide material according to any one of claims 4-7 is obtained.

13. A transmission blocking vaccine for controlling the malaria parasite Plasmodium falciparum, comprising a polypeptide material according to claim 7 and a suitable carrier or adjuvant.