PT92677A - PROCESS OF PRODUCTION OF A PLASMODIUM CIRCUMSPOROZOITE PROTEIN IN INSECT AND VACCINE CELLS CONTAINING THE REFERENCE PROTEIN - Google Patents

Description

-2- > 70 394 SKR Case 12092-1

DESCRIPTIVE MEMORY

Field of the Invention The present invention relates to the expression of the Plasmodium Circumiosporozoite protein in insect cells. More specifically, the present invention relates to the production of these proteins in Drosophila and Lepidoptera cells.

BACKGROUND OF THE INVENTION Human malaria is caused by a parasite of the genus Plasmodium. There are four species known to infect humans: P. falciparum, P. vivax, P. malariae and P. ovale. The most severe forms of human malaria are caused by P. falciparum and P. vivax; P. falciparum is the most prevalent. The malaria parasite is transmitted by mosquitoes to the liver, in the form of a sporozoite that migrates to the liver, multiplies in the hepatocytes and emerges to initiate a cyclic growth in erythrocytes. The parasite in the merozoite state, which is released at the end of each cycle, reinvents rapidly the red blood cells. The merozoite and esperazoite states are antigenically distinct, and generally the antibodies against one of the foes do not react crosswise with each other.

It has been found that mammals, including man, can be protected against challenge by Plasmodium when vaccinated with irradiated sporozoites (Clyde et al., Am 3 Trop Med Hyg, 24: 397 (1973), Nussenzweig et al., Phil Tran R Soc Lond B, 307: 117-28 (1984)). This method, although effective, is limited, due to the difficulty in culturing the sporozoites.

Sporozoites express a species-specific surface protein, the circosporozoite (CS) protein, which was first identified in P. berqhei, a rodent parasite. Monoclonal antibodies to this protein completely protected mice from challenge with infected mosquitoes (Potocnjak et al., J Exp Med, 131: 1304-13 (1980)).

Dame et al., (Science, 225: 593-9 (1984), U.S. Patent No. 4,707,357), describe the cloning of CS protein from P. falei parum in E. coli. This gene encodes a 412 amino acid protein, which contains 41 tetrapeptide repeats (37 units of invariant tetrapeptide and 4 variants). Monoclonal antibodies against the CS protein were inhibited in their binding to the protein in the presence of synthetic peptides derived from the repeat region. Enea et al., (Science, 225: 628-9 (1984)) further characterized an immunodominant top of the repeat region consisting of: (Pro-Asn-Ala-Asn) n

In addition, Arnot et al., (Science, 230: 815-18 (1985)) describe the cloning and sequence of P. vivax CS protein. The overall structure of this CS protein has been found to be similar in its overall structure to CS proteins of P. falciparum and P. Know-lesi (a species of simian malaria). That is, they consist of an amino-terminal signal sequence, a carboxy-terminal attachment domain and a large central repeat domain, flanked on both sides by conserved sequences designated regions I and II. The repeat domain is the immunodominant region of the CS protein. It has been found that the conserved regions (I and II) are highly charged.

P. falciparum sporozoite subunit vaccines have been directed against the CS protein repeat region. Young et al. (Science, 228: 958-62 (1985)) describes the cloning and expression of tandem CS repeat units in E. coli. When introduced into mice, these molecules were highly immunogenic. Antibodies raised against the mouse moiety units in mice recognize the CS protein in living sporozoites and block the sporozoite invasion of human hepatoma cells in vitro.

Additionally, Young et al. describe the very small expression of mature P. falciparum CS protein (i.e., minus the signal sequence) in E. coli.

Dame et al. , (Science, 225: 593-9 (1984)) describes the low levels of the P. falciparum CS protein at all levels in E. coli. Both references (i.e., Young et al. And Dame et al.) Do not disclose a process for the production of P. falciparum CS protein in useful amounts.

Dame et al. (U.S. Patent No. 4,707,357), Bailou et al. (Science, 228: 996-9 (1985), Zavala et al. (Science, 228, 1436-40 (1985)), Mazier et al. (Science, 231: 156-9 (1986), and Nussenzweig et al. (PCT / W086 / 05790) describe the use of synthetic peptides as subunit vaccines based on epitopes identified in the replication region More recently, Vergara et al. / W086 / 01721) and Bernardi et al. (U.S. Patent Application Serial No. 2 193 215 and 2 199 088) disclose antigenic peptides comprising regions I and / or II fused to the tetrapeptide repeats of the CS protein. vaccines, corresponding to polypeptides, synthetic or recombinant, from the requesting region, has been hampered due to their limited protective immunity. Although the subunit vaccines of P. falciparum CS protease replicates have successfully immunized mice against malaria, the vaccines so far tested in humans have have been insufficiently immunoprotective for their widespread use (Bailou et al., Lancet, I, 1277-81 (1987)).

Egan et al. (Science, 236: 453 (1987)) disclose that in the murine model of malaria (P. berqhei) the protective immunity induced by subunit and sporozoite vaccines appears to be fundamentally different. Subunit vaccines induced predominantly antibody-mediated protection whereas irradiated sporozoites induced prolonged immunity mediated by cells.

Sadoff et al. (Science, 240: 336-8 (1988)) disclose the expression of a protein comprising a full length CS protein of P. berqhei in a non-virulent strain of Salmonella ty-phimurium. This CS protein is expressed on the surface of the S. typhimurium cell and induced an antigen-specific cell mediated immunity. Sadoff et al. suggest that the CS protein contains a T cell epitope (s) which is (are) capable of inducing cell-mediated protective immunity.

Good et al., Proc Natl Acad Sci USA, 85: 1199-1203 (1988) describe T-cell domains for the P. falcipa-rum CS protein. These sites were identified by synthesizing synthetic peptides. overlapping the entire CS protein. In all these peptides three sites were located; all 3 'to the CS repeat region.

Sinigaglia et al., Nature, 336: 778-780 (1988), report that some synthetic peptides corresponding to a region of the P. falciparum protein, 3 'to the repeating region, can be recognized by class molecules II of different MHC T cells. The production of an effective vaccine, however, depends on the ability to produce large amounts of antigen. Barr et al. (J Exp Med, 165: 1160 (1987)) describe the expression of a P. vivax recombinant CS protein in yeast. This contains the entire repeat domain, plus 15 amino acids preceding the repeats, and 48 amino acids following the repeats, on the carboxyl-terminal side.

Valenzuela et al. (U.S. Patent 2 4 722 840) and Rutgers et al. (Bio / Technoloqy, 6: 1065-70 (1988)) disclose multi-length P. falciparum CS replicates fused to hepatitis surface antigen B. These fusions are expressed as viral particles in Saccharomyces cerevisiae.

De Wilde et al., PCT / W088 / 05817, describe the expression of the mature P. falciparum CS protein (i.e., minus the sialyl sequence) in S. cervisiae.

Smith et al. (Science, 224: 397-9 (1984)) describe the expression of P. knowlesi CS protein in a recombinant virus. Mammalian cells infected with the recombinant virus expressed a CS polypeptide that was recognized by a monoclonal antibody against the CS protein of P. knowlesi. 4,745 Q51) and Matsuura et al. (3 Gen Virol, 68: 1233-50 (1987))

In the baculovirus expression system, a strong, temporally regulated promoter can be used to express high levels of heterologous genes. Smith the al. (U.S. Patent No. 6,704,394 SKR Case 12092-1 disclose baculovirus vectors containing the polyhedrin gene promoter to express prokaryotic and eukaryotic genes.

Miller et al. (PCT / W088 / 02030) describe a method for producing heterologous genes using a mixture of at least two genetically distinct baculoviruses.

Cochran et al. (European Patent Application No. 228 036) show expression of the growth factor of the vaccine (VGF) in a recombinant baculovirus. In addition, Cochran et al. Presents a hypothetical list of proteins that can be expressed on a recombinant virus, including the hepatitis B surface antigen and Plasmodium polypeptides.

In addition to the baculovirus expression system, it has also been reported that Drosophila cells express foreign bacterial genes. H. Johansen et al., 28th Annual Drosophila Conference, p. 41 (1987) is a summary published by the inventors of the present application which briefly states that E. Coli galk genes, regulated by a Dro sophila metallothionein promoter, were expressed in Drosophila cell lines.

In a further publication, Johansen et al., Genes & Dev, 3: 882-889 (1989) describe the expression of human H-ras ocongene in Drosophila host cells. A. Vanderstraten et al., "Proceedings of the 7th International Conference on Invertebrate and Fish Tissue Culture," Abstract, University of Tokyo Press, Japan, (1987) and A. Vanderstraten et al., &Quot; Invertebrate and Fish Tissue Culture ", Eds. Y. Kuroda et al. , Japan Scientific Societies Press, Tokyo, pp. 131-134, (1988) are also publications of the present inventors, which disclose the use of a hygromycin B selection system for expression of foreign genes in cultured D. melanogaster cells. The same discloses that the system was used to co-introduce and over-express the E. coli galk gene and other genes of mammalian origin. B. J. Bond et al., Mol Cell Biol., 6 (6): 2080 (1986) describes the structure of the actin 5C gene of Drosophila melanogaster. This publication discloses the two transcription initiation sites of actin g and e 5C and fusions between the promoter sequences and the bacterial chloramphenicol acetyltransferase gene -7- 70 394 SKR Case 12092-1, inserted into host cells of D melanoqaster It is therefore an object of the present invention to express proteins in insect cells and to use these recombinant products as vaccine and diagnostic agents.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to the process of preparing a Plasmodium circunosporo-zoito gene expression unit which includes a DNA coding sequence for the desired protein and the regulatory sequences necessary for the transcription of the sequence of encoding the protein and substituting the translation into an insect cell.

In related aspects, the present invention relates to the process of preparing a recombinant DNA or baculovirus vector comprising the expression unit of the gene of the present invention.

In yet another related aspect, the present invention describes an insect cell transfected with the recombinant DNA or recombinant baculovirus of the present invention.

In further related aspects, the present invention relates to the process for the preparation of a Plasmodium circrosphozoite protein, or a derivative thereof, comprising its production by the insect cells transfected herein. The derivative encompasses any Plasmodium circosporozoite protein such as those resulting from deletions, additions, substitutions or rearrangements of the amino acids or their chemical modifications, which retain the ability to be recognized by antibodies raised against wild-type Plasmodium circosporozoite protein.

In a further aspect the present invention relates to the process for the preparation of a vaccine to provide protection against malaria infection comprising an immunoreactive and non-toxic amount of Plasmodium circosporozoite protein prepared according to invention. The present invention therefore relates to the process for the preparation of a porcine porous protein from P1 to spore. This process comprises culturing a host cell transfected with the expression unit of the present invention in a suitable culture medium.

Detailed Description of the Invention The present invention relates to the expression of Plas-modium proteins, and their derivatives, in insect cell cultures. The insect cells are transfected using conventional techniques to introduce foreign DNA into an insect host cell without adversely affecting the host cell. The thus-constructed combining host host cells produce PIasmodium proteins which are completely free of contaminating materials. These can be expressed, intracellularly, or bound to the membrane, or secreted into the culture medium of the cells. Upon secretion, the protein is available to be purified by conventional techniques. The intracellularly expressed protein can be extracted from the host cells also using standard techniques. The DNA coding sequence for the Plasmodium falciparum circunosporozoite protein is described by Dame et al. ,

Science, 225: 593-1 9 (1984) as the "5 'ATG follows: T AGCTATTTT ATGAG AAAAT 24 ATCTGTTTCT TCCTTTTTAT TTGTTGAGGC CTTATTCCAG GAATACCAGT 74 GCTATGGAAG TTCGTCAAAC ACAAGGGTTC TAAATGAATT AAATT ATGAT 124 AATGCAGGCA CTAATTTATA TAATGAATTA GAAATGAATT ATT 174 ATGGGAA ACAGGAAAAT TGGTAT AGTC TTAAAAAAAA T AGTAGATCA CTTGGAGAAA 224 ATGATGATGG AAATAATAAT AATGGAGAT The ATGGTCGTGA AGGTAAAGAT 274 GAAGATAAAA GAGATGG AAA T T AACGAAGAC AACGAGAAAT AAGGAAACC 324 AAAACAT AAA AAATT AAAGC AACCAGGGGA TGGTAATCCT GATCCAAATG -9 70 394 Case SKR 12092-1374 CAAACCCAAA TGTAGATCCC AATGCCAACC CAAATGTAGA TCCAAATGCA 424 AACCCAAATG T AGATCCAAA TGCAAACCCA AATGCAAACC 474 CAAATGCAAA CCCAAATGCA AACCCAAATG CAAACCCAAA TGCAAACCCA AATGCAAACC 524 CAAATGCAAA CCCAAATGCA AACCCCAATG CAAATCCT AA TGCAAATCCT 574 AATGCAAACC CAAATGCAAA TCCTAATGCA AACCCAAATG CAAACCCAAA 624 CGT AGATCCT AATGCAAATC CAAATGCAAA CCCAAATGCA AACCCAAACG 674 CAAACCCCAA TGCAAATCCT AATGCAAACC CCAATGCAAA TCCTAATGCA 724 AATCCT AATG CCAATCCAAA TGCAAATCCA AATGCAAACC CAAACGCAAA 774 CCCCAATGCA AATCCT AATG CCAATCCAAA TGCAAATCCA AATGCAAACC 824 CAAATGCAAA CCCAAATGCA AACCCCAATG CAAATCCT AA T AAAAACAAT 874 CAAGGT AATG GACAAGGTCA CAATATGCCA AATGACCCAA ACCGAAATGT 924 AGATGAAAAT GCT AATGCCA ACAATGCTGT AAAAAAT AAT AATAACGAAG 974 AACCAAGTGA TAAGCACAT The GAACAAT ATT T AAAGAAAAT AAAAAATTCT 1024 ATTTCAACTG AATGGTCCCC ATGT AGTGTA ACTTGTGGAA ATGGT ATTCA 1074 AGTTAGAATA AAGCCTGGCT CTGCTAATAA ACCTAAAGAC GAATTAGATT 1124 ATGAAAATGA TATTGAAAAA AAAATTTGT A AAATGGAAAA ATGTTCCAGT 1174 GTGTTT AATG TCGTAAATAG TTCAAT AGGA TTAATAATGG T ATT ATCCTT 1124 Where CTTGTTCCTT the first AATTAG 3 'ATG encodes an N-terminal methionine and the β1 codon, TAG, is an end signal of translation (ie, stop).

DNA molecules comprising the coding sequence of the present invention can be obtained from P. falciparum mRNA using known techniques, e.g. e.g., by making complementary DNAs or cDNAs from an mDNA template or by the polymerase chain reaction (see, Mullis et al., U.S. Patent 1040394 SKR Case 12092-1 4-800-159) . Alternatively, this coding sequence may be synthesized by conventional DNA synthesis techniques. In addition, there are numerous recombinant host cells containing the P. falciparum circumsporozoite DNA molecule, which are commonly available. The present invention is not limited to the sequence specifically described, but includes all the CS protein DNA coding sequences as described below. As used herein, the terms " CS protein ", " circosporozoite protein " or " CS polypeptide " include the full-length P. falciparum circosporozoite protein, substantially as described above, and all derivatives thereof. The term " derivative " encompasses any CS DNA coding sequence, such as a truncated CS coding sequence or other derivatives encoding a protein retaining the ability to induce an immune response to the CS protein of wild type after internal administration in man. These other derivatives may be prepared by the addition, deletion, substitution, or rearrangement of amino acids or their chemical modifications. The CS DNA coding sequence of the invention comprises the full-length CS protein, or a CS protein derivative which retains substantially the entire conserved region I (bars 319-363) and the conserved region II (bases 1030-1068 ), and at least one tetrapeptide unit Asn-Ala-Asn-Pro, as hereinafter described in detail.

As an example, the CS DNA coding sequence used in the present invention is substantially the same (ie, differs by no more than about 10 amino acids) than the DNA encoding sequence encoded by the sequence shown above, or lacks all or part of the carboxy-tej region of attachment? (approximately, amino acids 392-412), or lacking the signal peptide (approximately, amino acids 1-18), or lacking a signal peptide and an attachment region. Examples of preferred embodiments are set forth in Example 1. The derivative of the invention may be a hybrid, i.e. a fusion polypeptide containing the α-dideoxy-encoding sequences which may comprise one or more epitopes of other immo-genes of sporozoite, other PIasmodium immunogens, or other non-Plasmodium immunogens. Alternatively, the derivative of the invention may be fused to a carrier polypeptide which has immunostimulatory properties, as in the case of adjuvants, or which otherwise improves the immuno-response to the CS polypeptide, or which is useful in the expression, purification or formulation of the CS polypeptide.

As an example, a desirable DNA coding sequence for the CS protein of the present invention can be constructed by fusing the DNA coding sequence of the mature CS protein with a heterologous signal sequence, eg, the tissue plasminogen activator (tPA) sequence, . This signal sequence functions to direct the secretion of the host cell protein. The signal sequence may also be derived from other available signal sequences, e.g., from the HSV-I gD gene of the Herpes Simplex virus (Lasky et al., Science, supra.). The CS DNA coding sequence may also be followed by a polyadenylation region (poly A), such as an initial poly A region SV40. The poly A region, which functions in the polyadenylation of RNA transcripts, appears to play a role in the stabilization of transcription. A poly A region may be derived from a variety of genes in which it is naturally present. This region may also be modified to alter its sequence as long as the polyadenylation and transcription stabilization functions are not adversely affected. The recombinant DNA coding sequence of the present invention may also comprise a genetic selection marker. The selection marker may be any gene or genes that cause an easily detectable phenotypic change in a transfected host cell. This phenotypic change may be, for example, resistance to a drug, such as the hygromycin B resistance gene.

Alternatively, a selection system using the drug methotrexate prokaryotic dihydrofolate to-reductase (DHFR) may be used with Drosophila cells -12- 70 394 SKR Case 12092-1. The endogenous eukaryotic DHFR of cells is inhibited by methotrexate. Thus, by transfecting cells with a prokaryotic DHFR-containing plasmid, which is insensitive to methotrexate, and then selecting with methotrexate, it will be seen that only the cells transfected with and expressing the prokaryotic DHFR will survive.

In addition, in contrast to methotrexate selection of transformed mem- brane and bacterial cells, methotrexate can be used in the Drosophila system to initially have a high copy number of transfectants. In addition, the cells having the prokaryotic prokaryotic DHFR gene will survive. Concurrently, these cells have the desired expression level of the gene.

Also included in the recombinant molecule are the necessary or desirable regional regions for transcription of the CS protein coding sequence and for subsequent translation and ex post pressure in the host cell. The regulatory region typically contained a promoter region that functions at the polymyrase RNA binding and at the start of RNA transcription. The promoter region is typically upstream of the CS protein coding sequence.

Promoters known to be useful in Drosophila cells include mammalian cell promoters as well as Drosophila promoters, the latter being preferred. Examples of useful Drosophila promoters include the Drosophila metallothionein promoter. (Lastowski-Perry et al., J. Biol. Chem., 260: 1527 (1985)). This inducible promoter directs the transcription with levels and carried of the gene in the presence of metals, e.g., CuSOâ, ". The use of the Drosophila metallothionein promoter results in the expression system of the invention maintaining complete regulation even for high copy number. This is in direct contrast to the use of the mammalian metallothionein promoter in mammalian cells in which the regulatory effect of the metal decreases as the number of copies increases. In the Proso-13- 70 394 SKR Case 12092-1 phila expression system, this retained inducibility effect increases expression of the gene product in the Drosophila cell for a number of copies and is carried out. The 5 C promoter of Drosophila actin (B.J. Bond et al., Mol. Cell Biol., 2080 (1986)) is also a desirable promoter sequence. The actin 5 C promoter is a constitutive promoter and does not require the addition of metal. Thus, it is better adapted for use in a large-scale production system, such as an infusion system, than the Drosophila metallothionein promoter. A further advantage is that the absence of a high concentration of copper in the medium keeps the cells in a healthier state for longer periods of time.

Examples of other known Drosophila promoters are, e.g., heat shock inducible promoters (Hsp 70) and COPIA LTR. The SV40 early promoter results in lower expression levels than the Drosophila metallothionein promoter. Promoters which are commonly used in cell expression vectors including, for example, Rousse's virus LTR / avian sarcoma and simian virus (SV40 early promoter), demonstrate poor function and expression in the Drosophila system .

Promoters for use in Lepidoptera cells include promoters of a baculovirus genome. The promoter for the po-1-hedrin gene is preferred because the polyhedrin protein is naturally overexpressed relative to other baculovirus proteins. The preferred polyhedrin gene is AcMNPV baculovirus. See, Summers et al., U.S. Patent 4,745,051; Smith et al. , Proc Natl Acad Sci USA, 82: 8404 (1985); and Cochran, EP-A-228,036.

Insect cells that may be used in the invention include Drosophila SI, S2, S3, KC-0 cells and D. hydei cells. See, for example, Schneider et al., J Embryol Exp Morph, 27: 353 (1972); Scultz et al., Proc Natl Acad Sci USA, 183: 9428 (1986); Sinclair et al., Mol Cell Biol., 5: 3208 (1985).

A preferred Drosophila cell line for use in the practice of the present invention is line S2. S-cells (Schneider, J. Embryol. Exp. Morph., 27, 353 (1972)) are spleen cell cultures 70 394 SKR Case 12092-1 polypeptides of Drosophila embryonic cells. The use of the Drosophila S7 cell has many advantages which includes, but is not limited to, the ability to grow to a high density at room temperature. The stable integration of the selection system produced up to 1000 copies of the transfected gene expression unit on the chromosomes of the cell.

Other systems for culturing Drosophila cells may also be useful in the present invention. Some possibly useful cells are, for example, the Drosophila Me-lanoqaster KC-0 cell line which is a serum-free cell line (Schulz et al., Proc Natl Acad, Sci., USA, 83: 9428 (1986)). Preliminary studies using the KC-0 line have suggested that transfection is more difficult than with cells " Another cell line that may be useful is a Drosophila hydei cell line. The expression of the protein can be monitored using the hydei cell line; however, transfection in this cell line can result in the expression of transfected DNA with very low efficiency (Sinclair et al., Mol. Cell Biol., 3208 (1985)). Other available Drosophila cell lines, which may be used in the present invention, include the lines and S2.

Drosophila cells selected for use in the present invention may be cultured in a variety of suitable media, including, e.g., M meio medium. The M meio medium consists of a formulation of balanced salts and essential amino acids at a pH of 6.6. The preparation of the medium is substantially done as described in Lindquist, PIS (" Drosophila Information Services "), 58: 163 (1982). Other conventional means for culturing Drosophila cells may also be used. Useful Lepidoptera cells include cells from Trichoplu-sia ni, Spodoptera fruqiperda, Heliothis zea, Autoqraphica californica, Rachiplusia or Galleria melonella, Manduca sexta or other cells that can be infected with baculovirus. These include polyhedrosis virus (NPV), single nucleocapsid virus (SNPV) and multiple nucleocapsid virus (MNPV). Preferred baculoviruses are NPV or MNPV baculoviruses because they contain the polyhedrin gene promoter which is expressed at high levels in infectious cells. In particular, the MNPV virus of Autoqraphica california (AcMNPV) is exemplified hereinafter. However, other MNPV and NPV viruses such as the host bug, Bom-byx mori, may also be used. Lepidoptera cells are transfected with a recombinant bacteriophage of the invention according to conventional transfection techniques. These include, but are not limited to, calcium phosphate precipitation, electroporation, and liposome mediated transfer. The cells are cultured according to standard culture techniques in a variety of nutrient media including, for example, TC100 (Gibco Europe; Gardiner et al., J Invert Path, J25: 363 (1975)) supplemented with 10% fetal calf (FCS), and at a temperature of 27 ° C to 28 ° C for a period of 18 to 24 hours. Excellent yields of virus-encoded products are obtained by infecting actively growing cultures at densities of 1 to 1.2 x 10 cells / ml. See, Miller et al., In Setlow and Hollander (eds.), Genetic Enzymes: Principles and Methods, Vol. 8, p. 277-98 Plenum Pu-blishing Co. New York, 1986.

In a preferred embodiment of the present invention, a CS protein is expressed in Lepidoptera cells to produce immunogenic polypeptides. For the expression of the CS protein in Lepidoptera cells, the use of a baculovirus expression system is preferred. In this system, a " cassette " expression sequence comprising the CS-coding sequence of the CS protein operably linked to a baculovivus promoter in a shuttle vector. This vector contains sufficient amount of bacterial DNA to propagate the shuttle vector in E. coli or any other suitable prokaryotic host. This shuttle vector also contains a sufficient amount of bacteriophage DNA flanking the coding sequence of the CS protein in order to allow recombination between a wild-type baculovirus and the heterologous gene. The recombinant vector is then co-transfected into Lepidoptera cells with DNA from a wild-type baculovirus. Recombinant baculoviruses resulting from homologous recombination are then selected and plaque purified by standard techniques. See Summers et al. TAES Buli-16-70 394 SKR Case 12092-1 (Texas Agricultural Experimental Station Bulletin) NR 1535, May 1987. Production in insect cells can also be performed by infecting insect larvae. For example, a CS protein can be produced in Heliothis virescens caterpillars by feeding the bovine virus of the invention together with traces of the wild type baculovirus and then extracting the CS protein from the hemolymph after about two days. See, for example, Miller et al., PCT / W088 / 02030.

In another preferred embodiment, a CS protein is expressed in Drosophila host cells. For expression in Drosophila cells it is preferred to use a two-carrier system. In such a system, a unit of expression of the CS protein gene is typically in an expression vector. The expression vector comprises a regulatory region which functions in Drosophila, e.g., a metallothionein or actin 5C promoter, a gene expression unit and a polyadenylation region, e.g., the SV40 poly A site. The second vector of the two-vector system comprises a selection marker gene expression unit, e.g., hi-Gromycin B or DHFR. The pCOHYGRO vector, as further described in Example 1, encodes the hygromycin B phosphotransferase gene which, when expressed, imparts resistance to the hygromycin to the transfected host cells which are expressing said selectable marker gene. Another example is the dihydrofolate reductase (DHFR) gene which, when expressed, is useful as a selectable marker in the presence of methotrexate. These selectable markers, in conjunction with co-transfection of Drosophila cells are further described by Johansen et al. , U.S. Patent Application Serial No. 07 / 477,736, filed May 8, 1987 (equivalent to Eur. Application Ser. No. 290,261) and is hereby incorporated by reference.

Drosophila S células cells are especially suitable for the production of high level protein in the process of the present invention. Cells can be maintained in suspension cultures at room temperature (24 ° C). The culture medium is medium supplemented with 5 to 10% (v / v) of heat-deactivated fetal bovine serum (FBS). In the preferred embodiment of the invention the culture medium contains 5% FBS. After induction, the cells are cultured in serum-free medium. When the pCOHYGRO vector site is used, the medium is also supplemented with 300 pg / ml hygromycin B. In this medium, the S células cells can be cultured as suspension cultures, for example in shaken flasks of 250 ml to 2000 ml, with stirring at 50-60 rpm. Cell densities are typically maintained at between 10 and 10 cells per ml. In one embodiment of the present invention, the cells are cultured before induction in shaken flasks of 1500 ml in media containing 5% of serum. Transcription and expression of the CS protein coding sequence in the above-referenced systems can be controlled. For example, Southern blot analysis can be used to determine the number of CS gene copies. Northern blot analysis provides information regarding the size of the transcribed genetic sequence (see, e.g., Maniatis et al., Molecular Clopidogenesis Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)). The level of transcription can also be quantified. Expression of the selected CS protein in the recombinant cells can be further verified by Western blot analysis. Purification of the CS polypeptide from the cell culture is accomplished by standard techniques of protein isolation, eg, selective precipitation, absorption chromatography, and affinity chromatography, including a monoclonal antibody affinity column, as described in Example 3 .

Proteins produced by Drosophila cells according to the present invention are completely free from mammalian, bacterial and protozoan contaminant materials, and, more importantly, from other Plasmium dium materials. The vaccine of the present invention comprises a P. falciparum CS protein immunoprotective amount, prepared according to the method of the present invention. The term " immunoprotective " refers to the amount needed to induce an immuno-response against a subsequent challenge so that the disease is prevented or minimized. In a preferred embodiment, the vaccine of the present invention comprises an aqueous solution of the insect-derived CS protein that can be used directly. Alternatively, the CS protein, with or without prior lyophilization, may be admixed or absorbed with any of the various known adjuvants. These adjuvants will include, but are not limited to, aluminum hydroxide, muramyl dipeptide, and saponins such as Quil A. As a further alternative example the CS protein can be encapsulated within micropertiums such as liposomes. In yet another exemplary alternative, the CS protein may be conjugated to an immunostimulatory macromolecule, such as a dead Bordetella or a tetanus toxoid. The preparation of the vaccine is generically described in New Trends and Developments in Vaccines, Voller et al. (Eds.), University Park Press, Baltimore, Maryland, 1978. Liposome encapsulation is described by Fullerton, U.S. Patent No. 4 235 877. Conjugation of proteins with macromolecules is described, for example, by Likhite, U.S. Patent 4,372,945, and Armor et al., U.S. Patent No. 4,474,757. The use of Quil A is referred to in Dalsgaard et al. , Acta Vet Scand, 18: 349 (1987).

The following examples are illustrative but not limitative of the present invention. Restriction enzymes and the other reagents were used substantially in accordance with the instructions of the vendors.

Examples

Example 1. Constructions of vectors A) BACULOVIRUS VECTORS: i) Full-length P. falciparum CS protein The WR201 plasmid is obtained from the Walter Reed Army Institute of Research. It contains a 2.3 kilobase EcoRI fragment of the phage > mPfl, which encodes the full-length 412-amino acid, including the signal sequence, of the full-length P. falcipa-95-94 SKR Case 12092-1. See Dame et al., Science, 225: 593-9 (1984). From the WR201, a StuI-AsuII fragment encoding the amino acids 18-412 of the CS protein plus the untransfected 3 'DNA is isolated. To this fragment of the CS gene is ligated a synthetic HindiII-StuI oligomer: 5'-3 '

AGCTTACCATGATGAGAAAATTAGCTATTTTATCTGTTTCTTCCTTTTTATTTGTTGAGG ATGGTACTACTCTTT AATCGAT AAAAT AGACAAAGAAGGAAAAAT AAACAACTCC 3 '5' to recreate amino acids 1-17, but without the 5 'untranslated region of DNA. The new fragment (ie, HindIII-StuI-AsuII) is treated with E. coli DNA polymerase I, Large fragment (ie Klenow fragment) and ligated at a cloning site into a vector capable of replicating in E. coli . For example, the HindIII-StuI-AsuII fragment is inserted into blunt-ended Mlul-Bell sites of a typical shuttle vector, herein called TigND, to create a plasmid designated pNlV2103. The TigND plasmid is a derivative of the TND plasmid (Connors et al., DNA, 7: 651-661 (1988) or U.S. Patent Application Serial No. 07 / 137,792, filed Dec. 28, 1987). The TigND plasmid differs from the TND plasmid because Rous's LTR is replaced by the mouse, gamma 2B heavy chain immunoglobulin improved sequence. Alternatively, the HindIII-StuI-AsuII CS gene fragment may be inserted into the HindIII-Accl sites of pUC19 or pBR322, or other conventional cloning vectors. Plasmid pAcYM1 is a shuttle vector of baculovirus containing the sequences of the AcMNPV genome which includes the promoter of the polyhedrin gene, but not the polyhedrin gene, and the sequences of a high number of bacterial plasmids, pUC8. See Matsuura et al., 3 Gen Virol, 68: 1233-50 (1987). From pNIV2103, hybridize to E. coli DNA polymerase I and isolate a 1261-pair HindIII-Ddel fragment, encoding the full-length CS protein, including P. falciparum DNA, 20-70 394 SKR Case 12092-1 3 'untranslated from WR201, the blunt-ended fragment is then ligated to a BamHI blunt-ended site of plasmid pAcYM1 to create a plasmid designated herein as pNIV2112. ii) CS Protein " no fixation "

To create a CS gene lacking the last 21 amino acids (ie, at the carboxyl terminus), the HindIII-StuI-AsuII gene fragment of Example (i) is first cloned into the HindIII-AccI sites of M13mp18, a standard vector used in subcloning, sequencing and a lot of genesis. Then, using standard site-directed mutagenesis techniques, the CS gene was fused to the 5 'ACACTGGAGCACTTTCCAT 3' primer to introduce a HgiAI site at amino acid codon 390. The vector was digested with 5 'in relative to the HindIII site) and with HgiAI obtaining a CS gene fragment encoding amino acids 1-390. This fragment is then ligated and a synthetic adapter, encoding amino acid 391, a stop codon and a Bell site , 5 'CCAGTTGATGATCATCTAGAGG 3' 3 'ACGAGGTCAACTACTAGTAGATCTCC 3' to the MluI-Bell sites of the plasmid TigND to generate plasmid pNIV2104. From pNIV2104, a 1180 base pair blunt-ended HindIII-Bell fragment, encoding amino acids 1-391, is then ligated to a bamHI blunt-ended site of pAcYM1 to create plasmid pNIV2105. This plasmid is essentially the same as pNIV2112 with the expectation of encoding a CS protein lacking carboxyl terminus. iii) CS protein without signal sequence and without fixation From the plasmid pNIV21Q5, a subsequent vector, pNIV2111, is constructed, which lacks the first 17 codons (ie the signal sequence) beyond the last 21 codons of the gene of the CS protein. This vector is constructed (1) by removing the Xhol-StuI fragment from pNIV2105, which comprises the polyhedrin gene promoter in the CS signal sequence (amino acids 1-17), 70 394 SKR Case 12092-1 and (2) by replacing this fragment with a synthetic ligand: 5 'GATCCACCATGG 3' 3 'GTGGTACC 5' and with an XhoI-BamHI fragment isolated from pAcYM1. The resulting vector, pNIV2111, encodes a CS protein from amino acids 18 to 391 , which lacks a signal sequence and an attachment sequence.

B) VECT0RE5 DE DR0S0PHILA

iv) pMTtPA

As the basic vector for expression of the Drosophila gene, the tPA expression vector, pMTtPA (also referred to as pDMtPA) was used. This vector is a derivative of vector pML1, a small pBR322 vector containing the beta-lactamase gene which eliminated the venom sequelae [Mellon et al, Cell, 27: 297 (1982)]. These sequences are inhibitors of vector amplification. This vector was digested with SalI and Aat2 which removed a small piece of pBR322, and the digested ends were filled. The missing part of pBR322 is then replaced with a " cassette " containing the Drosophila metallothionein promoter on an EcoR1-Stul fragment of filled ends followed by a filled HindIII-SacI fragment from pDSPI [d.S. Pfarr et al. , DNA, .4 (6): 461 (1985)] containing a signal sequence of tPA, opre-peptide and the entire coding region of tPA. The tPA gene in this fragment is followed by an SV40 initial polyadenylation site.

v) pCOHYGRO

A commercially available plasmid, pUC18 [Bethesda Research Laboratories, Gaithersburg, MD, U.S.A.] containing a BamHI and Smal site was used. The 5 'LTR of an integrated COPIA element (357 base pairs) was cloned into the BamHI site of vector pUC18, resulting in the vector designated pUCOPIA. COPY is a representative member of the dispersed intermediate repeat sequences, which are dispersed in the Drosophila genome [Rubin et al., In Cold Spinq Harbor Symp. Quant. Biol., 45: 619 (1980)]. The pUCOPIA vector was cut at the SmaI site and the E. coli C gene was cloned to hygromycin B (" cassett " cassett " cassett " 70394 SKR Case 12092-1 miclin B) phosphotransferase in pUCOPIA using conventional cloning techniques. A " cassette " of hygromycin B was isolated on a 1481 bp HindIII-BamHI fragment of the DSP-Higro vector [Gertz et al., Gene, 25: 179 (1983)]. A " cassette " of hygromycin B contains the coding sequence of the hygromycin B phosphotransferase gene and the initial poly A region SV40. The HindIII and BamHI sites were filled using DNA polymerase and the " cassette " of the hygromycin B was ligated to the SmaI site of the PUCOPIA vector to produce the pCOHYGRO vector. vi) pMTcsp391

To prepare a coding sequence for mature P. falciparum CS without the codons of the last 21 amino acids, an EcoRI-AsuII fragment of plasmid WR201 (see Example (i)) was cloned into the EcoRI-AccI sites of M13mp18, which was also quoted in Example (i). Using conventional site-directed mutagenesis techniques, the CS gene was subjected to mutagenesis as primer of Example (ii) to introduce a HgiAI site at the amino acid codon 390. The vector was then digested with EcoRI and HgiAI obtaining a fragment of the CS gene encoding amino acids 1-390. This fragment and a synthetic adapter encoding amino acid 391, a stop codon, a Bell site and an Xba I sc site were then ligated. 5 'CCAGTTGATGATCAT 3' 3 'ACGAGGTCAACTACT.AGTAGATC 5' at the EcoRI-XbaI sites of the plasmid pULB1221 (see, Pierard et al., DNA, 8: 321-328 (1989)) to generate the plasmid pULB1221CSPdel. From plasmid pULB1221CSPdel a BstXI-XbaI fragment encoding amino acids 26-391 was isolated and a BqIII-BstXI synthetic adapter encoding serine and amino acids 19-25 of CS: 5'GATCTTTATTCCAGGAATACCAGTGCT 3 3 'AAATAAGGTCCTTATGGTC 5' at the BglII-XbaI sites of a vector with the Drosophila metallothionein promoter, a tPA signal sequence, and an SV40 initial polyadenylation site, such as the pMTtPA vector derived from 394 SKR Case 12092-1 from vector pgpl60A32 (Johansen et al., U.S. Patent Application Serial No. 07 / 278,386, filed December 1, 1988). The resulting vector, pMTcsp391, encodes a mature CS protein lacking a carboxy-terminal attachment sequence (ie, without amino acids 392-412) and with an additional serine residue at the amino terminal end, ie, 391 (vii) pMTcsp398

Another vector was constructed, containing a modified P. falciparum CS coding sequence, digesting the vector pMTcsp391 with the restriction endonucleases HgiAl and XbaI, and subsequently substituting the HgiAl-XbaI fragment for a synthetic oligonucleotide adapter having the sequence:

5 'CCAGTGTGTTTAATGTCGTGAATTCTTGAT 3' ACGAGGTCACACAAATTACAGCACTTAAGAACTAGATC 5 'The adapter encodes amino acids. From CS, 390-398 plus a stop codon. This resulting vector, designated pMTcsp398 (ct), cleaves a mature CS protein with an additional serine at the amino terminus and which also lacks the carboxyl terminal fixing codons 399-412, ie, CS-398 * viii) pMTcsp412

A 1492 bp BstX1-EcoRV fragment of the P. falciparum CS protein (amino acids 26-412) was isolated from the plasmid WR201 (supra). This BstX1-EcoRV fragment and the same synthetic adapter that was used to construct the vector (vi) at the BqIIII-Saci (from blunt-end) sites of the vector, pMTtPA (see construct (iv)) were ligated. The final construct, pMTcsp412, encodes a mature CS protein (amino acids 19 - 412) plus an additional serine at the amino terminus, i.e., - CS 19-412

Example 2. Expression in insect cells

A) SPODOPTERA FRUGIPERDA CELLS

Spodoptera frucciperid 9 (Sf9) cells are available from the ATCC (Rockville, MD, U.S.A.). Sf9 cells were co-transfected with one of the following recombinant vectors, plasmids pNTV2112, pNIV2105 or pNIV2111, and wild-type AcMNPV DNA, 50 μg SKR Case 12092-1 with 50 μg and 1 μg, respectively, substantially as described in Summers et al. , TAES Buli, NR 1535, May 1987, supra.

The resulting virus particles were collected by collecting the supernatants. The virus-containing medium was then used to infect Sf9 cells in a plaque assay. Subsequent infection of the Sf9 cells with a plaque-purified recombinant baculovirus resulted in cells expressing the CS protein instead of the polyhedrin protein.

Combined baculovirus-infected cells from pNIV2112 were found to express intracellularly a CS protein, with approximately 3 Âμg / ml infected culture medium supernatant. Western blot analysis essentially showed a band at about 60 Kilodalton.

Recombinant baculovirus infected cells derived from pNIV2105 express a " unfixed CS " protein, i.e., without a carboxy-terminal fixation, at a level of approximately 1 psg / ml supernatant from a 7 day culture. Western blot analysis of the proteins in the supernatant and in the cell extract showed two immuno-reactive doublet bands at about 50 and 60 kilodaltons.

Cells infected with pNIV2111 express intracellularly a CS protein, at a level of approximately 3æg / 1.5x10 4 cells, 3 days post infection. Western blot analysis of the proteins in the cell extract showed essentially 2 bands with approximately 50 kilodaltons. B) DR0S0PHILA CELLS:

Each of the MTcsp vectors of Example 1 were co-transfected with pCOHYGRO into Drosophila 5 'cells, with a ratio of MTcsp vector to pCOHYGRO of 20: 1. Transfection was performed by conventional techniques using CaPO 3 precipitation. Polyclonal cell lines were generated after selection with hygromycin B for 2 weeks. Simple clonal cell lines were generated by serially diluting the polyclonal cell line and placing in one well, one cell per well in 96-well plates. The cell lines were seeded and CS protein expression was followed after 6 days of CuSO4 induction. of a culture with a cell density of 5x10 4 cells / ml. The production of the CS proteins was measured by ELISA or conventional Western blot techniques using antibodies raised against the CS repeat regions, substantially as described by Gross et al in U.S. Patent Application Serial No. 07 / 256,181, deposited October 11, 1988. The highest production levels achieved for a single clonal cell line were 9 mg / l in 100 ml shaken flasks with a 6-day induction period with a cell density of 5 x 10 cells / ml.

One skilled in the art can express other Plasmodium circum sporozoite proteins and their fragments, described by the present invention, using substantially the same systems and procedures as exemplified above as well as using similar systems and materials which are publicly available.

Example 3. Purification of recombinant proteins expressed in insect cells

The recombinant CS proteins expressed in 5.fruqiperda were purified as follows: Sf9 infected cells were lysed for 3 cycles of freeze-thawing in RIPA buffer (50 mM tris-HCl pH 7.2, 0.15M NaCl, of triton X-100, 0.1% of SDS, 1% of Na-deoxycholate). The crude extract was centrifuged for 30 minutes at 1300 rpm. The supernatant was pelleted with DNAse for 30 minutes at 37 ° C, pH 7.5. The pH was adjusted to 5.5 and RNAse and MgSO4 were added. After an additional 30 minute incubation at 37 DEG C., the 1 M MgCl2 and 1 M NaCl cell extract became; then the pH was adjusted to 1.6 by the addition of concentrated HCl and the sample was incubated for 1 hour at 0 ° C and centrifuged for 30 minutes at 13,000 rpm. The supernatant was dialyzed against 20 mM ammonium carbonate, pH 7.5, and applied to an affinity column prepared from a monoclonal antibody recognizing the CS protein repeats (See Nardin et al., J Exp Med, 156 : 20 (19 8 2)). Elution was performed with 100 mM Na-acetate at pH 4.0 in the presence of 500 mM NaCl. 70 394 SKR Case 12092-1

This purification technique is also effective with recombinant CS proteins expressed in a Drosophila host.

Example 4. Vaccine containing CS protein

An exemplary vaccine of the present invention is prepared as follows: the insect-derived recombinant CS protein of the invention is added with agitation to a final concentration of 10-2000 yLtg / ml, preferably 600-1600 μg / ml, to a buffered saline solution (150 mM NaCl, 10 mM sodium phosphate, pH 6.8, and sterilized by filtration) containing 1.0 mg of Al 2+ (as aluminum hydroxide gel) per ml . The pH is maintained at pH 6.8 and the mixture is allowed to stand overnight at about 4 ° C. Thimerosal is added to a final concentration of 0.0005%. The pH is checked and adjusted, if necessary, to pH 6.8.

Each dose of vaccine contains 0.5 ml to 3.0 ml of the CS vaccine formulation, prepared as described above. As an example, the amount of polypeptide per dose is from about 1 to about 2000æg. Preferably, each dose will contain about 800æg of polypeptide / 0.5ml.

Preferably, the vaccine is administered parenterally, e.g., intramuscularly (im) or subcutaneously (sc), although other routes of administration may be used to induce a protective response. The vaccine is administered in a single dose or in multiple doses, e.g., with 2 to 4 supplementary doses. Preferably, multiple doses are administered over a period of 1-6 weeks. Vaccines may then be readministered as needed, e.g., annually. The foregoing description and examples fully describe the invention and its preferred embodiments. The present invention is not limited by the embodiments specifically described but encompasses all of its variations and modifications falling within the scope of the following claims.

Claims (10)

A process for producing a Plasmo-dium CS protein in insect cells, characterized in that it comprises culturing in suitable medium insect cells transfected with a gene expression unit (SEQ ID NO: of the Plasmodium CS protein and cells capable of expressing said protein are seen. 2. A method according to claim 1, characterized in that the insect cells are Drosophila cells. 3. A method according to claim 2, wherein said cells are transfected with a first vector containing the coding sequence of the hygomicin B transfecase and with a second vector containing the coding sequence of a gene expression unit of the Plasmodium CS protein. A process according to claim 3, characterized by: wherein said first vector is pCOHYGRO. 3. The method of claim 3, wherein said second vector comprises the CS DNA coding sequence of pMTcsp391, pMTcsp398 or pMTcsp412. A method according to claim 2, characterized in that the Drosophila cells are cells of D. melanoqaster S2 * A method according to claim 6, wherein said S células cells are co-transfected with the pCOHYGRO vector and with a vector comprising the CS gene expression unit as present in pMJcsp391, pMTcsp398 or pMT csp412 . 8. The process of claim 1, wherein the insect cells are Lepidoptera cells. A method according to claim 8, wherein said cells are co-transfected with a first vector comprising a plasmid DNA, which contains the coding sequence of a Plasm-dium CS gene expression unit. 70 394 k SKR Case 12092-1 * -28- 10. A method according to claim 8, characterized in that the Lepidoptera cells are from S. fructusi. 11. A process for the preparation of a vaccine for protection against malaria infection characterized in that it comprises the combination of an immunoprotective and non-toxic amount of the CS protein produced according to claim 1. 15 Lisboa, By SMITHKLINE BIOLOGICALS