VACCINES AGAINST HEPATITIS C

Abstract: A vaccine composition comprises QS21,3 De-O-acylated monophosphoryl lipid A (3D-MPL), an oil in water emulsion, wherein the oil in water emulsion has the following composition: a metabolisible oil, such as squalene, alpha tocopherol and tween 80, and at least one immunogen selected from the group consisting of (a) a hepatitis C virus core protein or an immunogenic derivative thereof, and (b) a hepatitis C virus envelope protein or an immunogenic derivative thereof.

Description: VACCINES AGAINST HEPATITIS C The present invention relates to novel vaccine formulations, to methods of their production and to their use in medicine. 3 De-O-acylated monophosphoryl lipid A is known from GB2220 211 (Ribi). Chemically it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in International Patent Application No. 92/116556. QS21 is a Hplc purified non toxic fraction of a saponin from the bark of the South American tree Quillaja Saponaria Molina and its method of its production is disclosed (as QA21) in US patent No. 5,057,540. Oil in water emulsions per se are known in the art, and have been suggested to be useful as adjuvant compositions (EPO 399843). Hepatitis C virus is described in EP-A-0 318 216. A particular antigenic protein of hepatitis C virus has been designated the core protein and is described by, for example, Delisse et al., J. Hepatology, 1991;13 (Suppl. 4): S20-S23 (for genotype lb). Particular envelope proteins of hepatitis C virus have been designated El and E2 and are described by, for example, Grakoui et al., 1993, J. Virology 67, 1385-1395; Spacte et al.,1992, Virology 188, 819-830; Matsumia et al., J. Virology 66, 1425- 1431, and Kohara et al., 1992, J. Gen. Virol. 73, 2313-2318. A majority of the HCV genotypes identified to date are described by Okamoto Hiroaki and Mishiro Shunji, Intervirology, 1994, 37: 68 et seq. The present invention provides a vaccine composition comprising QS21, 3 De-O-acylated monophosphoryl lipid A (3D-MPL), an oil in water emulsion, wherein the oil in water emulsion has the following composition: a metabolisible oil, such as squalene, alpha tocopherol and tween 80, and at least one immunogen selected from the group consisting of (a) a hepatitis C virus core protein or an immunogenic derivative thereof, and (b) a hepatitis C virus envelope protein or an immunogenic derivative thereof. The term "immunogenic derivative" encompasses any molecule such as a truncated or other derivative of the protein which retains the ability to induce an immune response to the protein following internal administration to a human. Such other derivatives can be prepared by the addition, deletion, substitution, or rearrangement of amino acids or by chemical modifications thereof. Immunogenic fragments of the protein, which may be useful in the preparation of subunit vaccines, may be prepared by expression of the appropriate gene fragments or by peptide synthesis, for example using the Merrifield synthesis (The Peptides, Vol 2., Academic Press, NY, page 3). The immunogenic derivative of the invention can be a hybrid, that is, a fusion polypeptide containing additional sequences which can carry one or more epitopes for other immunogens. Alternatively, the immunogenic derivative of the invention can be fused to a carrier polypeptide or to another carrier which has immunostimulating properties, as in the case of an adjuvant, or which otherwise enhances the immune response to the protein or derivative thereof, or which is useful in expressing, purifying or formulating the protein or derivative thereof. The invention also extends to the HCV protein or immunogenic derivative thereof when chemically conjugated to a macromolecule using a conventional linking agent such as glutaraldehyde (Geerlings et al, (1988) J, Immunol. Methods, ___, 239-244). Proteins and their immunogenic derivatives suitable for use in the present invention can be prepared by expressing DNA encoding said protein or derivative thereof in a recombinant host cell and recovering the product, and thereafter, optionally, preparing a derivative thereof. A DNA molecule comprising such coding sequence can be synthesized by standard DNA synthesis techniques, such as by enzymatic ligation as described by D.M. Roberts _i _l in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by a combination of these techniques. Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50ml or less. Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreitol, lmM spermidine, lmM ATP and 0. lmg ml bovine serum albumin, at a temperature of 4°C to ambient, generally in a volume of 50ml or less. The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982),or in other scientific publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D. Matteucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S.P. Adams et al., Journal of the American Chemical Society,1983, 105, 661; N.D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W.D. Matthes et al., EMBO Journal, 1984, 3, 801. DNA polymers which encode mutants may be prepared by site-directed mutagenesis by conventional methods such as those described by G. Winter £ al in Nature 1982, 2__, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., __, 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter ei ai in Biochem. Soc. Trans., 1984, 2, 224-225. Recombinant techniques are described in Maniatis g_. _l., Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989. In particular, a protein or immunogenic derivative for use in the present invention can be prepared using the following steps: i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said protein or an immunogenic derivative thereof; i) transforming a host cell with said vector; iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and iv) recovering said protein. The term 'transforming' is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and AJ. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term 'transformed' or 'transformant' will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest. The replicable expression vector may be prepared by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, under ligating conditions. Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired. The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses. The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis ei al cited above. The recombinant host cell is prepared by transforming a host cell with a replicable expression vector under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Maniatis ei _\ cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985. The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as E. ___ may be treated with a solution of CaCl2 (Cohen ei aL Proc. Nat. Acad. Sci., 1973, _9, 2110) or with a solution comprising a mixture of RbCl, MnC-2, potassium acetate and glycerol, and then with 3- [N-morpholino] -propane-sulphonic acid, RbCl and glycerol. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells. Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis ei al and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45°C. The product is recovered by conventional methods according to the host cell. Thus, where the host cell is bacterial, such as E. _ς_i it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian, the product may generally be isolated from the nutrient medium or from cell free extracts. Conventional protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column. Preferably, the host cell is E. coli. A particular aspect of the present invention provides a novel compound which comprises an HCV core protein, or an immunogenic derivative thereof, fused to a polypeptide containing foreign epitopes. The polypeptide is preferably an influenza protein, such as the NS1 protein, or an immunogenic derivative thereof. DNA coding for such a novel compound, vectors containing said DNA, host cells transformed with said vectors, and their use in producing said novel compound, form still further aspects of the invention claimed. The vaccines of the present invention are preferential stimulators of IgG2a production and THl cell response. This is advantageous, because of the known implication of THj response in cell mediated response. Indeed in mice induction of IgG2a is correlated with such an immune response. The vaccines of the invention enhance induction of cytolytic T lymphocyte responses. Induction of CTL is easily seen when the target antigen is synthesised intracellularly, ie during infection by the virus, because peptides generated by proteolytic breakdown of the antigen can enter the appropriate processing pathway, leading to presentation in association with class I molecules on the cell membrane. However, in general, pre-formed soluble antigen does not reach this processing and presentation pathway, and does not elicit class I restricted CTL. Therefore conventional non-living vaccines, while eliciting antibody and T helper responses, do not generally induce CTL mediated immunity. The combination of the two adjuvants QS21 and 3D-MPL together with an oil in water emulsion can overcome this serious limitation of vaccines based on recombinant proteins, and induce a wider spectrum of immune responses. In certain systems, the combination of 3D-MPL and QS21 together with an oil in water emulsion have been able to synergistically enhance interferon γ production. Additionally the oil in water emulsion may contain span 85 and/or lecithin. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in International patent application published under No. 92116556 - SmithKline Beecham Biologicals s.a. The oil in water emulsion may be utilised on its own or with other adjuvants or immuno-stimulants In a further aspect of the present invention there is provided a vaccine as herein described for use in medicine. The ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1; preferably 1 : 5 to 5 : 1 and often substantially 1 : 1. The preferred range for optimal synergy is 2.5:1 to 1:1 3D MPL: QS21. Typically for human administration QS21 and 3D MPL will be present in a vaccine in the range 1 μg - 100 μg, preferably 10 μg - 50 μg per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser. Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757. The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 2-100 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunisation adequately spaced. The formulations of the present invention may be used for both prophylatic and therapeutic purposes. Accordingly in one aspect, the invention provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient. The following examples illustrate the invention. Example 1 1.1 Construction and expression of a recombinant HCV core fusion protein Plasmid pMG81 a derivative of pMG27 (Gross et al 1985, Mol.Cell. Biol. 5: 1015) in which: (i) the 81 first codons of the NS1 coding region from influenza strain A/PR/8/34 cleaved from plasmid pASlEH/801 (Young et al. 1983, Proc. Natl. Acad. Sci. 80: 6105) have been inserted downstream of the pL promoter and ii) the ampicillin resistance gene has been replaced by the kanamycin resistance gene from transposon Tn902, was used to express the fusion protein NSl-Core. HCV genomic sequences of hepatitis C virus genotype lb (Delisse et al, 1991 J. Hepathology 13, suppl. 4:S20-23) were PCR amplified and cloned into pUC12 plasmid to give plasmid TCM 128-2. The nucleotides sequences corresponding to amino acids 2-166 of the core protein were amplified from TCM 128-2. During the polymerase chain reaction, Ncol and Xbal restriction sites have been generated at the 5' and 3' ends of the core sequences allowing insertion into the same sites of plasmid pMG81 to give pRTT 14129. pRJT 14129 contains the coding sequence for the fusion protein NS1 (flu)-core(HCV) and expresses the polypeptide described in SEQ ID NO. 1. The coding sequence for the fusion protein NS1 (flu)-core(HCV) is contained in SEQ ID NO 2. SEQ ID NO 3 shows the amino acid sequence 1-1006 of HCV genome type la (H). Plasmid pRIT14129 was introduced into E. coli AR 58 (Mott et al, 1985, Proc, Natl. Acad. Sci., 82:88) containing the thermosensitive repressor of the λpL promoter. The recombinant bacteria were grown in a 20 Litters fermentor under fed-batch conditions at 30°. The expression of the NSl-Core protein was induced by raising the temperature to 38-42°C. The cells were then harvested and mechanically disrupted. 1.2 Purification of the NSl-Core fusion protein The antigen was purified in a denatured form by preparative electrophoreses: Step 1: Bacterial cells were broken (Rannie-2 x 14,500 pi) in a 20 mM phosphate buffer pH7 containing protease inhibitors (lmM pefabloc, 0.5mg leupeptin, 0.1% aprotinin). Step 2: Lysate was centrifuged for 25 minutes, at 17,000g. At this stage the recombinant protein was insoluble and was recovered in the pellet The pellet was washed two times with lOmM phosphate pH6.8, 2M NaCl, 4M urea; three times with lOmM phosphate pH 6.8, 0.15M NaCl, and centrifuged at 17,000g for 25 minutes after each wash step. These steps were introduced in order to lower the endotoxin content of the purified product. Step 3: The washed pellets re suspended in SDS-PAGE reducing sample buffer, boiled for 5 minutes, centrifuged again at 27,000g for 25 minutes and then applied on a 12% polyacrylamide gel for separation of the remaining proteins (Prep Cell equipment Biorad). Step 4: The protein was electroluted from the gels in 25mM Tris pH8, 200mM glycine, 0.1% SDS; precipitated by 10% TCA at 0° and finally resuspended in lOmM phosphate pH 6.8, 150mM NaCl, 50mM sarcosyl. The purified antigen appears as a doublet, in the 27-30 kD range, both bands are recognised by an anti-NSl monoclonal antibody as well as by anti-core specific human monoclonal and rabbit polyclonal antibodies. 1.3 Adjuvantation of the NSl-Core Protein The two adjuvant formulations were made each comprising the following oil in water emulsion component SB26: 5% squalene 5% tocopherol 0.4% tween 80; the particle size was 500 nm size SB62: 5% Squalene 5% tocopherol 2.0% tween 80; the particle size was 180 nm 1(a) Preparation of emulsion SB 62 (2 fold concentrate) Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100 ml two fold concentrate emulsion 5g of DL alpha tocopherol and 5ml of squalene are vortexed to mix thoroughly. 90ml of PBS Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and finally microfluidised by using an Ml 10S microfluidics machine. The resulting oil droplets have a size of approximately 180 nm. 1(b) Preparation of emulsion SB26 This emulsion was prepared in an analogous manner utilising 0.4% tween 80. 1(c) Other emulsions as depicted in the Table were made in an analogous manner. 1(d) Preparation of fusion protein/QS21/3D MPL/ oil in water formulation. To the emulsion of 1 a) or b) or c) an equal volume of twice concentrated fusion protein(either 20μg or lOOμg) was added and mixed. This was combined with 50μg/ml of 3D-MPL and 20μg/ml of QS21 to give the final formulation. Buffer was set according to salt content and pH. Example 2 2.1 Preparation of a recombinant E1E2 oligomeric protein Oligomeric forms of E1-E2 HCV envelope proteins can be prepared form mammalian cells infected with recombinant vaccinia virus expressing HCV envelope sequences as a polyprotein. The coding sequences for a polyprotein covering the amino acids 167- 1006 of HCV genome of type la (H) can be inserted in vaccinia virus vectors using procedures known in the art and the resulting plasmid used to prepared vaccinia recombinant virus that will lead to expression of the polyprotein in infected cells. The expressed polyprotein is processed and retained intracellularly. E1-E2 oligomeric form can be purified from cell extracts in which the E1/E2 protein complex has been solubilized using specific detergent (Ralston et al, 1993, J. Virology 67:6753) (Dubuisson et al 1994, J. Virology 68:6147). 2.2 Preparation of vaccine formulations Formulations of oligomeric E1E2 are prepared analagously to the formulations of Example 1. Example 3 Formulations containing both the fusion protein of Example 1 and the E1E2 oligomer of Example 2 are prepared analagously to the formulations of Example 1, each formulation containing between 50 and lOOμg of each protein. Table 1 Vehicles two fold concentrated Emulsions SB Tocopherol % Squalene % Tween 80 % Span 85 % Lecithin Ψo Size 26 5 5 0.4 0 0 500 nm 90-100% 800 nm 10-0% 26.1 5 5 0.4 0 0.1 500 nm 63 5 5 0.6 0 0 500 nm 64 5 5 0.8 0 0 500 nm 61 5 5 1 0 0 250-300 nm 62 5 5 2 0 0 180 nm 40 5 5 0.4 1 0 500 nm 80-100% 800 nm 20-0% 40.1 5 5 0.4 1 0.1 500 nm 60 5 5 1 1 0 300 nm 65 5 5 0.4 1.5 0 500 nm 66 5 5 0.4 2 0 500 nm SEQ ID NO 1 1 MDPNTVSSFQ VDCF WHVR RVADQELGDA PFLDRLRRDQ KSLRGRGSTL 51 GLDIETATRA GKQIVERILK EESDEALKMT MSTNPKPQRK TKRNTNRRPQ 101 DVKFPGGGQI VGGVYLLPRR GPRLGVRATR KTSERSQPRG RRQPIPKARQ 151 PEGRAWAQPG YP PLYGNEG MGWAGWLLSP RGSRPS GPT DPRRRSRNLG 201 KVIDTLTCGF ADLMGYIPLV GAPPGGAARA LAHGVRVLED GVNYAT SEQ ID NO 2 1 GAATTCGTAC CTAGATCTCT CACCTACCAA ACAATGCCCC CCTGCAAAAA 51 ATAAATTCAT A AAAAAACA TACAGATAAC CATCTGCGGT GATAAATTAT 101 CTCTGGCGGT GTTGACATAA A CCACTGG CGGTGATACT GAGCACATCA 151 GCAGGACGCA CTGACCACCA TGAAGGTGAC GCTCTTAAAA AT AAGCCCT 201 GAAGAAGGGC AGCATTCAAA GCAGAAGGCT TTGGGGTGTG TGATACGAAA 251 CGAAGCATTG GCCGTAAGTG CGATTCCGGA TTAGCTGCCA ATGTGCCAAT 301 CGCGGGGGGT TTTCGTTCAG GACTAC ACT GCCACACACC ACCAAAGCTA 351 ACTGACAGGA GAATCCAGAT GGATGCACAA ACACGCCGCC GCGAACGTCG 401 CGCAGAGAAA CAGGCTCAAT GGAAAGCAGC AAATCCCCTG TTGGTTGGGG 451 TAAGCGCAAA ACCAGTTCCG AAAGATTTTT TTAAC A AA ACGCTGATGG 501 AAGCGTTTAT GCGGAAGAGG TAAAGCCCTT CCCGAGTAAC AAAAAAACAA 551 CAGCATAAAT AACCCCGCTC TTACACATTC CAGCCCTGAA AAAGGGCATC 601 AAATTAAACC ACACCTTAAG GAGGATATAA CATATGGATC CAAACACTGT 651 GTCAAGCTTT CAGGTAGATT GCTTTCTTTG GCATGTCCGC AAACGAGTTG 701 CAGACCAAGA ACTAGGTGAT GCCCCATTCC TTGATCGGCT TCGCCGAGAT 751 CAGAAATCCC TAAGAGGAAG GGGCAGCACT CTTGGTCTGG ACATCGAGAC 801 AGCCACACGT GCTGGAAAGC AGATAGTGGA GCGGATTCTG AAAGAAGAAT 851 CCGATGAGGC ACTTAAAATG AcCATGAGCA CAAATCCTAA ACCCCAAAGA 901 AAAACCAAAC GTAACACCAA CCGTCGCCCA CAGGACGTTA AGTTCCCGGG 951 CGGTGGTCAG ATCGTtGGTG GAGTTTACcT GTTGCCGCGC AGGGGCCCCA 1001 GGTTGGGTGT GCGcGCGACT AGGAAGACTT CCGAGCGGTC GCAACCTCGT 1051 GGAAGGCGAC AgCCTATCCC CAAGGCTCGC CaGCCCGAGG GtAGGgCCTG 1101 GGCaCAGCCc GGGTATCCTT GGCCCCTCTA TGGCAATGAG GGCaTGGGGT 1151 GGGCAGGATG GCTCCTGTCA CCCCGCGGCT CcCGGCCTAG TTGGGGCCCC 1201 AcgGACCCCC GGCGTAGGTC GCGTAATTTG GGTAAGGTCA TCGATACCCT 1251 cACgTGCGGC TTCGCCGACC TCATGGGGTA CATTCCGCTC GTCGGCGCCC 1301 CCccAGGGGG CGCTGCCAGG GCCtTGGCAC ATGGTGTCCG GGTTCTGGAG 1351 GACGGCGTGA ACTATGCAAC AtaaTCTAGA ATCGATAAGC TTCGACCGAT 1401 GCCCTTGAGA GCCTTCAACC CAGTCAGCTC CTTCCGGTGG GCGCGGGGCA 1451 TGACTATCGT CGCCGCACTT ATGACTGTCT TCTTTATCAT GCAACTCGTA 1501 GGACAGGTGC CGGCAGCGCT CTGGGTCATT TTCGGCGAGG ACCGCTTTCG 1551 CTGGAGCGCG ACGATGATCG GCCTGTCGCT TGCGGTATTC GGAATCTTGC 1601 ACGCCCTCGC TCAAGCCTTC GTCACTGGTC CCGCCACCAA ACGTTTCGGC 1651 GAGAAGCAGG CC TTATCGC CGGCATGGCG GCCGACGCGC TGGGCTACGT 1701 CTTGCTGGCG TTCGTCCAGT AATGACCTCA GAACTCCATC TGGATTTGTT 1751 CAGAACGCTC GGTTGCCGCC GGGCGTTTTT TATTGGTGAG AATCGCAGCA 1801 ACTTGTCGCG CCAATCGAGC CATGTCGTCG TCAACGACCC CCCATTCAAG 1851 AACAGC AGC AGCATTGAGA ACTTTGGAAT CCAGTCCCTC TTCCACCTGC 1901 TGACGACGCG AGGCTGGATG GCCTTCCCCA TTATGATTCT TCTCGCTTCC 1951 GGCGGCATCG GGATGCCCGC GTTGCAGGCC ATGCTGTCCA GGCAGGTAGA 2001 TGACGACCAT CAGGGACAGC TTCAAGGATC GCTCGCGGCT CTTACCAGCC 2051 TAACTTCGAT CACTGGACCG CTGATCGTCA CGGCGATTTA TGCCGCCTCG 2101 GCGAGCACAT GGAACGGGTT GGCATGGATT GTAGGCGCCG CCCTATACCT 2151 TGTCTGCCTC CCCGCGTTGC GTCGCGGTGC ATGGAGCCGG GCCACCTCGA 2201 CCTGAATGGA AGCCGGCGGC ACCTCGCTAA CGGATTCACC ACTCCAAGAA 2251 TTGGAGCCAA TCAATTCTTG CGGAGAACTG TGAATGCGCA AACCAACCCT 2301 TGGCAGAACA TATCCATCGC GTCCGCCATC TCCAGCAGCC GCACGCGGCG 2351 CATCTCGGGC AGCGTTGGGT CCTGGCCACG GGTGCGC TG ATCGTGCTCC 2401 TGTCGTTGAG GACCCGGCTA GGCTGGCGGG GTTGCCTTAC TGGTTAGCAG 2451 AATGAATCAC CGATACGCGA GCGAACGTGA AGCGACTGCT GCTGCAAAAC 2501 GTCTGCGACC TGAGCAACAA CATGAATGGT CTTCGGTTTC CGTGTTTCGT 2551 AAAGTCTGGA AACGCGGAAG TCAGCGCCCT GCACCATTAT GTTCCGGATC 2601 TGCATCGCAG GATGCTGCTG GCTACCCTGT GGAACACCTA CATCTGTATT 2651 AACGAAGCGC TGGCATTGAC CCTGAGTGAT TTTTCTCTGG TCCCGCCGCA 2701 TCCATACCGC CAGTTGTTTA CCCTCACAAC GTTCCAGTAA CCGGGCATGT 2751 TCATCATCAG TAACCCGTAT CGTGAGCATC CTCTCTCGTT TCATCGGTAT 2801 CATTACCCCC ATGAACAGAA ATTCCCCCTT ACACGGAGGC ATCAAGTGAC 2851 CAAACAGGAA AAAACCGCCC TTAACATGGC CCGCTTTATC AGAAGCCAGA 2901 CATTAACGCT TCTGGAGAAA CTCAACGAGC TGGACGCGGA TGAACAGGCA 2951 GACATCTGTG AATCGCTTCA CGACCACGCT GATGAGCTTT ACCGCAGCTG 3001 CCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC 3051 CGGAGACGGT CACAGCTTGT CTG AAGCGG ATGCCGGGAG CAG CAAGCC 3101 CGTCAGGGCG CGTCAGCGGG TGTTGGCGGG TGTCGGGGCG CAGCCATGAC 3151 CCAGTCACGT AGCGATAGCG GAGTGTA AC TGGCTTAACT ATGCGGCATC 3201 AGAGCAGATT GTACTGAGAG TGCACCATAT ATGCGGTGTG AAATACCGCA 3251 CAGATGCGT AGGAGAAAAT ACCGCATCAG GCGCTCTTCC GCTTCCTCGC 3301 TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT 3351 CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG 3401 AAAGAACATG TGAGCAAAAG GCC GCAAAA GGCCAGGAAC CGTAAAAAGG 3451 CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC 3501 AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG 3551 ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA 3601 CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG 3651 GCGCTTTCTC AATGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT 3701 TCGCTCCAAG CTGGGCTGTG TGC CGAACC CCCCGTTCAG CCCGACCGCT 3751 GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC 3801 TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGG A 3851 TGTAGGCGGT GC ACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA 3901 CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC 3951 GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG 4001 CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT 4051 CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC 4101 GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT 4151 CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA 4201 TATATG GTA AACTTGGTCT GAC GTTACC AATGCTTAAT CAGTGAGGCA 4251 CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC 4301 CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG 4351 CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA 4401 ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT 4451 ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA 4501 GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TGCAGGTCGA 4551 CGGATCAGCC TCGAGGTGAG GTCTGCCTCG TGAAGAAGGT GTTGCTGACT 4601 CATACCAGGC CTGAATCGCC CCATCATCC GCCAGAAAGT GAGGGAGCCA 4651 CGGTTGATGA GAGCTTTGTT GTAGGTGGAC CAGTTGGTG TTTTGAACTT 4701 TTGCTTTGCC ACGGAACGGT CTGCGTTGTC GGGAAGATGC GTGATCTGAT 4751 CCTTCAACTC AGCAAAAGTT CGATTTATTC AACAAAGCCA CGTTGTGTCT 4801 CAAAATCTCT GATGTTACAT TGC CAAGAT AAAAATATAT CATC TGAAC 4851 AATAAAACTG TCTGCTTACA TAAACAGTAA TACAAGGGGT GTTATGAGCC 4901 ATATTCAACG GGAAACGTCT TGCTCGAGGC CGCGATTAAA TTCCAACATG 4951 GATGCTGATT TA ATGGGTA TAAATGGGCT CGCGATAATG TCGGGCAATC 5001 AGGTGCGACA ATCTATCGAT TGTATGGGAA GCCCGATGCG CCAGAGTTGT 5051 TTCTGAAACA TGGCAAAGGT AGCGTTGCCA ATGATGTTAC AGATGAGATG 5101 GTCAGACTAA ACTGGCTGAC GGAATTTATG CCTCTTCCGA CCATCAAGCA 5151 TTTTATCCGT ACTCCTGATG ATGCATGGTT ACTCACCACT GCGATCCCCG 5201 GGAAAACAGC ATTCCAGGTA TTAGAAGAAT ATCCTGATTC AGGTGAAAAT 5251 ATTGTTGATG CGCTGGCAGT GTTCCTGCGC CGGTTGCATT CGATTCCTGT 5301 TTGTAATTGT CCTTTTAACA GCGATCGCGT ATTTCGTCTC GCTCAGGCGC 5351 AATCACGAAT GAATAACGGT TTGGTTGATG CGAGTGATTT TGATGACGAG 5401 CGTAATGGCT GGCCTGTTGA ACAAGTCTGG AAAGAAATGC ATAAGCTTTT 5451 GCCATTCTCA CCGGATTCAG TCGTCACTCA TGGTGATTTC TCACTTGATA 5501 ACCTTATTTT TGACGAGGGG AAATTAATAG GTTGTATTGA TGTTGGACGA 5551 GTCGGAATCG CAGACCGATA CCAGGATCTT GCCATCC AT GGAACTGCCT 5601 CGGTGAGTTT TCTCCTTCAT TACAGAAACG GCTTTTTCAA AAATATGGTA 5651 TTGATAATCC TGATATGAAT AAATTGCAGT TTCATTTGAT GCTCGATGAG 5701 TTTTTCTAAT CAGAATTGGT TAATTGGTTG TAACACTGGC AGAGCATTAC 5751 GCTGACTTGA CGGGACGGCG GCTTTGTTGA ATAAATCGAA CTTTTGCTGA 5801 GTTGAAGGAT CAGATCACGC ATCTTCCCGA CAACGCAGAC CGTTCCGTGG 5851 CAAAGCAAAA GTTCAAAATC ACCAACTGGT CCACCTACAA CAAAGCTCTC 5901 ATCAACCGTG GCTCCCTC C TTTCTGGCTG GATGATGGGG CGATTCAGGC 5951 CTGGTATGAG TCAGC ACAC CTTCTTC CG AGGCAGACCT CACCTCGAGG 6001 CTGATCCCCG SEQ ID NO 3 1 MSTNPKPQRK TKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR GPRLGVRATR 51 KTSERSQPRG RRQPIPKARR PEGRT AQPG YPWPLYGNEG CG AGWL SP 101 RGSRPSWGPT DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GAPLGGAARA 151 LAHGVRVLED GVNYATGNLP GCSFSIF LA L SC TVPAS AYQVRNSSGL 201 YHVTNDCPNS SIVYEAADAI HTPGCVPCV REGNASRC V AVTPTVATRD 251 GK PTTQ RR HIDLLVGSAT LCSA YVGDL CGSVFLVGQL FTFSPRRHWT 301 TQDCNCSIYP GHITGHRMAW DMMMNWSPTA ALWAQLLRI PQAIMDMIAG 351 AHWGVLAGIA YFSMVGNWAK VLWLLLFAG VDAETHVTGG NAGRTTAGLV 401 GLLTPGAKQN IQLINTNGSW HINSTALNCN ESLNTGWLAG LFYQHKFNSS 451 GCPERLASCR RLTDFAQGWG PISYANGSGH DERPYCWHYP PRPCGIVPAK 501 SVCGPVYCFT PSPVWGTTD RSGAPTYSWG ANDTDVFVLN NTRPPLGNWF 551 GCTWMNSTGF TKVCGAPPCV IGGVGNNTLL CPTDCFRKHP EATYSRCGSG 601 PWITPRCMVD YPYRLWHYPC TINYTIFKVR MYVGGVEHRL EAACNWTRGE 651 RCDLEDRDRS ELSPLLLSTT QWQVLPCSFT TLPALSTGLI HLHQNIVDVQ 701 YLYGVGSSIA SWAIKWEYW LLFLLLADAR VCSCLWMMLL ISQAEAALEN 751 LVILNAASLA GTHGLVSFLV FFCFAWYLKG RWVPGAVYAL YGMWPLLLLL 801 LALPQRAYAL DTEVAASCGG WLVGLMALT LSPYYKRYIS WCMWWLQYFL 851 TRVEAQLHVW VPPLNVRGGR DAVILLMCW HPILVFDITK LLLAIFGPLW 901 ILQASLLKVP YFVRVQGLLR ICALARKIAG GHYVQMAIIK LGALTGTYVY 951 NHLTPLRDWA HNGLRDLAVA VEPWFSRME TKLITWGADT AACGDIINGL 1001 PVSARR

Claims: Claims l. A vaccine composition comprising: QS21; 3 De-O-acylated monophosphoryl lipid A (3D-MPL); an oil in water emulsion, wherein the oil in water emulsion has the following composition: a metabolisible oil, alpha tocopherol and tween 80; and at least one immunogen selected from the group consisting of (a) a hepatitis C virus core protein or an immunogenic derivative thereof, and (b) a hepatitis C virus envelope protein or an immunogenic derivative thereof. 2. A vaccine composition according to claim 1 wherein the HCV protein or immunogenic derivative thereof is chemically conjugated to a carrier molecule. 3. A vaccine composition according to claim 1 or 2 wherein the immunogenic derivative is a fusion polypeptide. 4. A vaccine composition according to claim 3 wherein the fusion polypeptide comprises an HCV core protein or an immunogenic derivative thereof fused to an influenza protein or an immunogenic derivative thereof. 5. A vaccine composition according to claim 4 wherein the influenza protein is the NS1 protein. 6. A compound which comprises an HCV core protein, or an immunogenic derivative thereof, fused to a polypeptide containing foreign epitopes. 7. A compound according to claim 6 wherein the polypeptide containing foreign epitopes is an influenza protein or an immunogenic derivative thereof. 8. A compound according to claim 7 wherein the influenza protein is the NS1 protein. 9. A method of treating or preventing HCV infection, which comprises administering to a patient in need thereof an effective amount of a composition according to any one of claims 1 to 5 or a compound according to any one of claims 6 to 8. 10. Use of a composition according to any one of claims 1 to 5 or a compound according to any one of claims 6 to 8 in the manufacture of a medicament for use in the prevention or treatment of HCV infection. 11. A process for the preparation of a composition according to any one of claims 1 to 5, which process comprises mixing the constituents thereof in the required proportions. 12. A process for the preparation of a compound according to any one of claims 6 to 8, which process comprises expressing DNA encoding said compound in a recombinant host cell and recovering the product. 13. A DNA molecule encoding a compound according to any one of claims 6 to 8. 14. A recombinant vector comprising the DNA of claim 13. 15. A host cell transformed with the recombinant vector of claim 14.

Inventors: CABEZON SILVA TERESA, GARCON NATHALIE MARIE-JOSEPHE, MOMIN PATRICIA MARIE, CABEZON SILVA, TERESA, GARCON, NATHALIE, MARIE-JOSEPHE, CLAUDE, MOMIN, PATRICIA, MARIE
Applicants: CABEZON SILVA TERESA, GARCON NATHALIE MARIE JOSEPHE, MOMIN PATRICIA MARIE, SMITHKLINE BEECHAM BIOLOG, CABEZON SILVA, TERESA, GARCON, NATHALIE, MARIE-JOSEPHE, CLAUDE, MOMIN, PATRICIA, MARIE, SMITHKLINE BEECHAM BIOLOGICALS S.A.
Application year: 1996
Main classification: C12N15
IPC codes: A61K39, A61P1, A61P31, C07K14, C07K19, C12N1, C12N15, C12P21, C12R1