MXPA97010523A - Vaccines against hepatitis - Google Patents

Abstract

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

Description

VACCINES AGAINST HEPATITIS C The present invention relates to new formulations of vaccines, to methods for their production and to their use in medicine. The lipid A monophosphoryl 3 De-O-acylated is known from GB2 220 21 1 (Ribi) Chemically it is a mixture of lipid A monophosphoryl 3 De-O-acylated with 4,5, or 6 chain adidas and is produced by Ribi Immunochem Montana. A preferred form of monophosphoryl 3 De-O-acylated lipid A is described in International Patent Application No. 92/1 16556. QS21 is a non-toxic fraction purified by Hplc from a saponin from the bark of the South American tree Quillaja Saponaria Molina and its production method is described (as QA21) in U.S. Patent No. 5,057,540. Oil-in-water emulsions per se are known in the art and have been suggested to be useful as auxiliary 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 1 b). The particular envelope proteins of hepatitis C virus have been designated E1 and E2 and are described by, for example, Grakoui et al. , 1 993, 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, De-O-acylated monophosphoryl lipid A, an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: a metabolizable oil, such as squalene, alpha tocopherol and tween 80, and at least one immunogen selected from the group consisting of (a) a core protein of hepatitis C virus or an immunogenic derivative thereof, and (b) a hepatitis C virus envelope protein or a 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 followed by internal administration to a human. These 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 expressing 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, i.e., 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 immunostimulatory properties, as in the case of an auxiliary, or which otherwise enhances the immune response to the protein or derivative of the invention. same, 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 binding agent such as a glutaraldehyde (Geerlings et al, (1988) J, Immunol. Methods, 106, 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 subsequently, optionally, preparing a derivative of the same A DNA molecule comprising such a ciphering sequence can be synthesized by standard DNA synthesis techniques, such as by enzymatic ligation as described by D.M. Roberts et al in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by a combination of these techniques. Enzymatic DNA polymerization can be performed in vitro using a DNA polymerase such as DNA I polymerase (fragment Klenow) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP, dTTP as required at a temperature of 10 ° -37 ° C, generally in a volume of 50 ml or less.

Enzymatic ligation of DNA fragments can be performed using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05 M Tris (pH 7.4), 0.01 M MgCl 2, 0.01 M dithiothreitol, 1 mM spermidine, ATP 1 mM and 0.1 mg / ml of bovine serum albumin, at a temperature of 4 ° C to ambient, generally in a volume of 50 ml or less. The chemical synthesis of the DNA fragments or polymer can be carried out by conventional phosphotriester, phosphite or phosphorite chemistry, using solid phase techniques such as those described in "Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual" (ed. .G Gassen and A. Lang), Verlag chemie, Weinheim (1982), or in other scientific publications, for example MJ 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. Bann arth, 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, 19084, 12, 4539; and H .W. D. Matthes et al. , EMBO Journal, 1984, 3, 801. DNA polymers which encode mutants can be prepared by site-directed mutagenesis by conventional methods such as those described by G. Winter et al in Nature 1 982, 299, 756-758 or by Zoller and Smith 1982; Nucí Acids Res., 10, 6487-6500, or by deletion mutagenesis as described by Chan and Smith in Nuci.

Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans. , 1984, 12, 224-225. Recombinant techniques are described in Maniatis et. to the. , Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989. In particular, an immunogenic protein or derivative for use in the present invention can be prepared using the following steps. i) preparing an integrating or replicable expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence encoding said protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector; iii) culturing said transformed host cell under conditions that allow the expression of said DNA polymer to produce said protein; and iv) recovering said protein.

The term "transform" is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate viral or plasmid vector using for example conventional techniques as described in Genetic Engineeering; Eds. YE. Kingsman and A .J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term "transformed" or "transformant" will be applied subsequently to the resulting host cell containing and expressing the foreign gene of interest.

The replicable expression vector can be prepared by cutting a vector compatible with the host cell to provide a segment of Linear DNA 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 ligation conditions. In this way, the DNA polymer can 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. Such suitable vectors include plasmids, bacteriophages, cosmid and recombinant viruses. The preparation of the replicable expression vector can be carried out conventionally with enzymes suitable for the restriction, polymerization and ligation of DNA, by methods described in, for example, Maniatis et al mentioned above. The recombinant host cell is prepared by transforming a host cell with a replicable expression vector under transformation conditions. Suitable transformation conditions are conventional and are described in, for example, Maniatis et al cited above, or "DNA Cloning" Vol. II, D.M. Glover ed. , IRL Press Ltd, 1985. The choice of transformation conditions is determined by the host cell. In this way, a bacterial host such as E. coli can be treated with a CaCl 2 solution (Cohen et al, Proc. Nat. Acad.

Sci., 1973, 69, 21 1 0) or with a solution comprising a mixture of RbCI, MnCl2, potassium acetate and glycerol, and then with 3- [N-morpholino] -propane-sulphonic acid, RbCI and glycerol. The mammalian cells in culture can be transformed by calcium co-precipitation of the vector DNA into the cells. The culture of the transformed host cell under conditions that allow the expression of the DNA polymer is conventionally performed, as described in, for example, Maniatis et al.

"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. In this manner, the host cell is bacterial, such as E. coli, can be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian, the product may be generally 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 NS 1 protein, or an immunogenic derivative thereof. The ciphering of DNA for such a novel compound, vectors containing said DNA, host cells transformed with said vectors, and their use to produce said compound, still form additional aspects of the claimed invention. The vaccines of the present invention are preferential stimulators of the production of IgG2a and cellular response TH1. This is advantageous, due to the known involvement of the TH1 response in the cell-mediated response. Indeed in the induction in mice of IgG2a is correlated with such immune response. The vaccines of the invention enhance the induction of cytolytic T lymphocyte responses. The induction of CTL is easily seen when the target antigen is synthesized intracellularly, ie during virus infection, because the peptides generated by proteolytic cleavage of the antigen can enter the appropriate processing path, leading to presentation in association with the molecules of class I in the cell membrane. However, in general, the pre-formed soluble antigen does not reach this path of presentation and processing, and does not produce restricted class I CTL. Consequently, conventional non-living vaccines, although they produce T helper and antibody responses, generally do not induce CTL-mediated immunity. The combination of the two auxiliaries 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 broader 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 intensify interferon production.

Additionally, the oil-in-water emulsion may contain span 85 and / or lecithin. A preferred form of De-O-acylated monophosphoryl lipid A is described in the international patent application published under No. 921 16556 - SmithKine Beecham Biologicals s.a. The oil-in-water emulsion can be used alone or with other auxiliaries or immunostimulants. In a further aspect of the present invention there is provided a vaccine as described herein 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. Normally 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 oil in water will comprise 2 to 10% squalene, 2 to 10% alpha tocopherol and 0.3 to 3% tween 80. Preferably the squalene: alpha tocopherol ratio is equal to or less than 1 since this provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it may be advantageous if the vaccines of the present invention also contain a stabilizer. The preparation of the vaccine is described generally in New Trends and Developments in Vaccines, edited by Voller et al, University Park Press, Baltimore, Maryland, EU 1978. Encapsulation within liposomes is described, for example, by Fullerton, patent US 4,235,877. The conjugation of proteins to macromolecules is described, 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 vaccinates. Generally, it is expected that each dose will comprise 1 -100 μg of protein, preferably 2-100 μg. An optimal amount for a particular vaccination can be ascertained by standard studies involving the observation of appropriate immune responses in the subjects. Following the initial vaccination, subjects can receive one or several booster immunizations spaced appropriately. The formulations of the present invention can be used for both prophylactic and therapeutic purposes. According to 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 first 81 codons of the NS1 coding region of the influenza species A / PR / 8 / 34 cut from plasmid pAAS1 EH / 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 gene of kanamycin resistance of the Tn902 transposon, was used to express the NS1-nucleus fusion protein. The HCV genomic sequences of the hepatitis C virus genotype 1 b (Delisse et al, 1991 J. Hepathology 13, suppl.4: s20-23) were amplified by PCR and cloned into the pUCM plasmid to give plasmid TCM 128-2. The nucleotide sequences corresponding to amino acids 2-166 of the core protein were amplified from TCM128-2. During the polymerase chain reaction, the Ncol and Xbal restriction sites have been generated at the 5 'and 3' ends of the core sequences allowing the insertion in the same sites of the plasmid pMG81 to give pRIT 14129. pRIT 14129 contains the ciphering sequence for the fusion protein NS1 (flu) -neucleus (HVC) and expressing the polypeptide described in SEQ ID NO.1. The coding sequence for the fusion protein NS1 (flu) -nucleus (HVC) is contained in SEQ ID NO 2. SEQ ID NO 3 shows the sequence of amino acids 1-1006 of the HCV genome type 1 to (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? PL promoter.

The recombinant bacteria were grown in a 20 liter fermenter under 30 ° batch feed conditions. The expression of the NS1-core protein was induced by raising the temperature to 38-42 ° C. The cells were then harvested and mechanically dissolved. 1. 2 Purification of the NS1 fusion protein - Nucleus The antigen was purified in a denatured form by preparative electrophoresis: Step 1: Bacterial cells were broken (Rannie-2 x 14,500 pi) in 20 mM phosphate buffer 20 mM pH7 containing protease inhibitors (1 mM pephabloc, 0.5 mg / leupeptin, 0.1% aprotinin).

Step 2: The lysate was centrifuged for 25 minutes at 17,000 g. At this stage the recombinant protein was insoluble and was recovered in the pellet. The pellet was washed twice with 10 mM phosphate pH 6.8, 2M NaCl and 4M urea; three times with 10mM phosphate pH 6.8, 0.15M NaCl and centrifuged at 17,000g for 25 minutes after each washing step. These steps were introduced in order to decrease the endotoxin content of the purified product.

Step 3: The washed pellets were resuspended in SDS-PAGE reducing sample buffer, boiled for 5 minutes, centrifuged again at 27,000g for 25 minutes and then applied on a 12% polycrylamide gel for separation the remaining proteins (Prep Cell, Biorad equipment).

Step 4: The protein was electroextracted from the gels in 25mM Tris pH8, 200mM glycine, 0.1% SDS; precipitated by 10% TCA at 0o and finally resuspended in 10mM phosphate pH6.8, 150mM NaCl and 50mM sarcosil.

The purified antigen appears as a pair, in the range 27-30 kD, both bands are recognized by an anti-NS 1 monoclonal antibody as well as by specific human monoclonal antibodies anti-nucleus and rabbit polyclonal. 1. 3 Auxiliary NSI core protein The two auxiliary 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 500nm. SB62: 5% squalene, 5% tocopherol, 2.0% tween 80; the particle size was 180nm 1 (a) Preparation of SB62 emulsion (2-fold concentrate) Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in PBS. To provide 100 ml of concentrated emulsion twice, 5 g of DL alpha tocopherol and 5 ml of squalene were stirred by whirlpool to mix thoroughly. 90 ml of PBS / Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and finally microfluidized using a M1 10S microfluidics machine. The resulting oil drops have a size of approximately 180 nm. 1 (b) Preparation of emulsion SB26 This emulsion was prepared in an analogous manner using 0.4% tween 80. 1 (c) Other emulsions as shown in the Table were made in an analogous manner. 1 (d) Preparation of the fusion protein formulation (QS21 / 3D MPL / oil in water) to the emulsion of 1 a) or b) c) an equal volume of fusion protein concentrated twice (either 20 μg or 100 μg) ) was added and mixed. This was combined with 50 μg / ml of 3D-MPL and 20 μg / ml of QS21 to give the final formulation. The buffer was adjusted according to the salt and pH content.

Example 2 2.1 Preparation of a recombinant E1 E2 oligomeric protein The oligomeric forms of the E1-E2 HCV envelope proteins can be prepared from mammalian cells infected with recombinant vaccinia virus by expressing HCV envelope sequences as a polyprotein. Ciphering sequences for a polyprotein that cover amino acids 167-1006 of the HCV genome of type 1a (H) can be inserted into vaccinia virus vectors using methods known in the art and the resulting plasmid used to prepare the vaccinia recombinant virus that will lead to the expression of the polyprotein in infected cells. The expressed polyprotrein is processed and retained intracellularly. The oligomeric form E1 -E2 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 Oligomeric E1 E2 formulations are prepared analogously to the formulations of Example 1.

Example 3 The formulations containing both the fusion protein of example 1 and the oligomer E 1 E2 of Example 2 are prepared analogously to the formulations of Example 1, each formulation containing between 50 and 100 μg of each protein.

Table 1 Concentrated vehicles twice SEQ ID NO 1 1 MDPNTVSSFQ VDCF HVRX RVADQE GDA PFLORLRRDQ KSURGRGST 51 GLDIETATRA GKQIVERII-K EESDEA KMT MSTNPKPQRK TKROTNRRPQ 101 DVKFPGGGQI VGGVYI-LPRR GPRLGVRATR KTSERSQPRG RRQPIPKARQ 151 PEGRA ACjPG YP P YGNEG MGWAGWLLSP KGSRPSWCPT DPRRRSRNLG 201 KVIDTLTCGF ADLMGYIPI.V GAPPGGAARA I-AHGVRVLED GVNYAT SEQ ID NO 2 1 GAATTCGTAC C ACATCTCT CACCTACCAA ACAATGCCCC CCTGCAAAAA 51 ATAAATTCAT ATAAAAAACA TACAGATAAC CAICTGCOGT GATAAATTAT 101 C CTGGCGGT GTTGACATAA ATACCACTGG CGGTGATACT GAGCACATCA 151 GCAGGACGCA CTGACCACCA TGAAGGTGAC GCTCTTA? AA ATTAAGCCCT 201 GAAGAAGGCC AGCATTCAAA GCAGAAGGCT TTGGGGTGTC TGATACß? AA 251 CGAACCATTG GCCG AAGTG CGATTCCGGA TTAGCTGCCA ATGTGCCAAT 301 CGCGGGGGG TTTCGTTCAG GACTACAACT GCCACACACC ACCAAAGCTA 351 ACTGACAGGA GAATCCAGAT GGATGCAC ?? ACACGCCGCC GCGAACCTCG 401 CGCAGAGAAA CAGGCTCAAT GGAAAGCAGC AAATCCCCTG TTGCTTGGGG 451 TAAGCGCAAA ACCACTTCCG AAAGAT? TT TTAACTATAA ACG GGATGC 501 AAGCGTTTAT GCGGAAGAGG TA? AGCCCTT CCCGAGTAAC AAAAAAACAA 551 CAGCATAAAT AACCCCGCTC TTACACATTC CAGCCCTGAA AAACGGCATC 601 AAATTAAACC ACACCTTAAG GAGGATATAA CATATGGATC CAAACACTGT 651 GTCAAGCTTT CAGGTACAT GCTTTCTTTG GCATGTCCGC AAACGAGTTG 701 CAGACCAAGA ACTAGGTGAT GCCCCATTCC TTGATCGGC TCGCCGAGAT 751 CAGAAATCCC TAAGAGGAAG GGGCAGCACT CTTGGTCTGG ACATCGAGAC 801 AGCCACACGT CCTGG AGC AGATAGTGGA GCGGAT CTG AAAGAAGAAT 851 CCGATGAGGC ACTTAAAATG AcCATGAGCA CAAATCCtAA ACCCCAAAGA 901? AA? CC? AAC GTAACACCAA CCGTCGCCCA CAGGACGTTA AGTTCCCGGG 951 CGGTGGTCAG ATCGTtGGTG GAGTTTACcT GTTGCCGCGC AGGGGCCCCA 1001 GGTTGGGTGT GCGcGCGACT AGGAAGACTT CCGAGCGGTC GCAACCTCCT 1051 GGAAGGCGAC AgCC ATCCC CAAGGCTCGC CaGCCCGAGG GCAGGgCC G 1101 GGCaCAGCCc GGGTATCCTT GGCCCCTCTA TGGCAATGAG GGCaTGGGGT 1151 GGGCAGGATG GCTCCTGTCA CCCCGCGGCT CcCGGCCTAG GGCGCCCC 1201 AcgGACCCCC GGCGTAGGTC GCCTAATt G CGTAAGGTCA TCCATACCC 1251 cACgTGCGGC TTCGCCGACC TCATGGGGTA CATTCCGCTC CTCGCCGCCC 1301 CCccAGGGGG CGCTGCCAGG CCCtTCGC? C ATsGTCTCCC CCTTCTCGAC 1351 GACGGCGTGA ACTATGCAAC ACaaTCTAGA ATCGATAAGC TTCGACCGATGAGA GCCTTCAACC CACTCAGCTC CTTCCGGTGG GCGCGGGGCA 1451 TGACTATCGT CGCCGCACTT ATGACTGTCT TCTTTATCAT GCAACTCGTA 1501 GGACAGGTGC CGGCAGCCCT CTGGGTCATT TTCGGCGAGG ACCGCTTTCG 0 1551 CTGGAGCGCG ACGATGATCG GCCTGTCGCT TGCGGTATTC GGAATCTTGC 1601 ACGCCC CGC TCAAGCCTTC G CACTGGTC CCGCCACCAA ACGTTTCGGC 1651 GAGAAGCAGG CCATTATCGC CGGCATGGCG CCCGACGCGC TGGGCTACGT 1701 CTTGCTGGCC TTCGTCCAGT AATCACCTCA GAACTCCATC TGGATTTGTT 1751 CAGAACGCTC GGTTGCCGCC GGGCGTTTTT TATTGGTGAG AATCGCAGCA 1801 ACTTGTCGCG CCAATCGAGC CATGTCGTCG TCAACGACCC CCCATTCAAG 18S1 AACAGCAAGC AGCATGAGA ACTTTGGAAT CCAGTCCCTC TTCCACCTCC 1901 TGACGACGCG AGGCTGCATC CCCTTCCCCA TTATGATTCT TCTCGCTTCC 1951 GGCGGCATCG GGATGCCCGC GTTGCAGGCC ATGCTGTCCA GGCAGGTAGA 2001 TGACGACCAT CAOGGACAGC TTCAACGATC GCTCGCGGCT CTTACCAGCC 2051 TAACTTCGAT CAC GGACCG CTGATCGTCA CGGCGATTTA TGCCGCCTCG 2101 GCGAGC? CAT GGAACGGCTT GGCATGGATT GTAGGCGCCG CCCTATACC 2151 TGTCTCCCTC CCMOGTTGC GTCGcccTGC ATCGAGCCGG CCCACCTCGA 2201 CCTGAATGGA AGCCGGCGGC ACCTCGCTAA CGGATTCACC ACTCCAAGAA 22S1 TTGGAGCCAA TCAATTCTTG CGGACAACTC TGAATCCGCA AACCAACCC 2301 TGGCAGAACA TATCCATCGC GTCCGCCATC TCCAGCAGCC GCACGCCSGCG 2351 CATCTCGGGC AGCGTrsGsT CCTCCCCACG GGTGCCCAG ATCG GCTCC 2401 TGTCGTTGAG GACCCGGCTA GGC GGCGGG GTTGCCTTAC GGTTAGCAC 2451 AATGAATCAC CGATACGCGA GCGAACGTGA AGCGACTGCT GCTGCAAAAC 2501 GTCTGCGACC TGAGCAACAA CATGAATGGT CTTCGGTTTC CG GTTTCC 2551 AAAGTCTGGA AACGCGGAAG TCAGCGCCCT GCACCATTAT GTTCCGGATC 2601 TGCATCGCAG GATGCTGCTG GCTACCCTGT GGAACACC TO CATCTGTATT 2651 AACGAAGCGC TGGCATTGAC CCTGAGTGAT TTTTCTCTGG TCCCGCCGCA 2701 TCCATACCGC CACTTCTTTA CCCTCACAAC GTTCCAGTAA CCGGCCATGT 27S1 TCATCATCAG TAACCCGTAT CGTGAGCATC CTCTCTCGTT TCATCGGTAT 2801 CATTACCCCC ATGAACAGAA ATTCCCCCT ACACGGAGGC ATCAAGTCAC 2851 CAAACAGGAA AAAACCGCCC TTAACATGGC CCGCTTTATC AGAAGCCAGA 2901 CATTAACGCT TCTGGAGAAA CTCAACGAGC TCGACGCGGA TGAACAGGCA 2951 GACATCTGTG AATCGCTTCA CGACCACGCT GATGAGCTTT ACCGCAGCTG 3001 CCTCGCGCGT TTCGGTGATG ACGCTGAAAA CCTCTGACAC ATGCAGCTCC 3051 CGGAGACGGT CACAGCTTGT CTGTA? GCGG ATGCCGGGAG CAGACAAGCC 3101 CGTCAGGGCG CGTCAGCGGC TGTTGGCGGG TGTCGssGCG CAGCCATGAC 3151 CCAGTCACGT AGCGATAGOG GAG GTATAC GGCTTAACT ATGCGGCATC 3201 AGAGCAGATT GTACTGAGAG TGCACCATAT ATGCGGTGTG AAATACCGCA 3251 CAGATGCGTA AGGAGAAAAT ACCGCATCAG GCGCTCTTCC GCTTCCTCGC 3301 TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT 3351 CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG 3401 AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG 3451 CCGCGTTGCT GGCGTTTTTC CAT? OGCTCC GCCCCCCTGA CGAGCATCAC 3501 AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG 3551 ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA 3601 CCCTGCCGCT TACCGGATAC CTGTCCOCCT TTCTCCCTTC sGGAAGCCTG 3651 GCGCTTTCTC AATGCTCACC CTGT? OCTAT C CAGTTCGG TGTAGGTCGT 3701 TCGCTCCAAG CTGGGCTGTG TGC? CGAACC CCCCGTTCAG CCCGACCGCT 3 51 GCGCCT ATC CGGTAACTAT CCTCTTGAGT CCAACCCGGT AAGACACGAC 3801 TTATCGCCAC TGGCAGCAGC CACTGGTJ? C AGGATTAGCA GAGCGAGGTA 3851 TGTAGGCGGT GCTACAGAGT TCTTGAAGTG CTGGCCTAAC TACGGCTACA 3901 CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC 3 51 GGAAAAAGAG TTGGTAGCTC TTGATCCCGC AAACAAACCA CCGCTGGTAG_4001_CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT 4051 CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTCACGC TCAGTGGAAC 4101 GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT 4151 CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA 4201 TATATGAGTA AACTTGGTCT GACAGTTACC 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 CTSC? ACTTT 4451 ATCCGCCTCC ATCCAGTCTA tTAATTGTTG CCGGGAAGCT AGAGTAAGTA 4501 GTTCGCCAGT TAATAGTTTC CGCAACGTTG TTGCCATTGC TGCAGGTCGA 4551 CGGATCAGCC TCGAGGTGAG GTCTGCCTCG TGAAGAAGGT CTTGCTGACT 4601 CATACCAGGC CTGAATCGCC CCATCATCCA GCCAGAAAGT GAGGGAGCCA 4651 CGGTTGATGA GAGCTTTGTT GTAssTGGAC CAGTTGGTCA TTTTGAACTT 4701 TTCCTTTGCC ACGGAACGGT CTGCCTTGTC GGGAAGATGC GTGATCTGAT 4751 CCTTCAACTC AGCAAAAGTT CGATTTATTC AACAAACCCA CGTTCTCTCT 4801 CAAAATCTCT GATGTTACAT TGCACAAGAT AAAAATATAT CATCATGAAC 4851 AATAAAACTG TCTGCTTACA TAAACAGTAA TACAAGGGGT GTTATGAGCC 4901 ATATTCAACG GGAAACGTCT TGCTCGAGGC CGCGATTAAA TTCCAACATG 4951 GATGCTGATT TATATCGGTA TAAATGGGCT CGCGATAATG TCGGGCAATC 5001 AGGTGCGACA ATCTATCGAT TGTATGGGAA GCCCGATGCG CCAGAGTTGT 5051 TTCTGAAACA TGGCAAAGCT AGCGTTGCCA ATGATGTTAC AGATGAGATG 5101 GTCAGACTAA ACTGGCTGAC GGAATTTATG CCTCTTCCGA CCATCAAGCA 5151 TTTTATCCGT ACTCCTGATG ATGCATGGTT ACTCACCACT GCGATCCCCG 5201 GGAAAACAGC ATTCCAGGTA TTACAAGAAT ATCCTGATTC AGGTGAAAAT 5251 ATTGTTGATG CGCTGGCAGT GTTCCTGCGC CGGTTGCATT CGATTCCTGT 5301 TTGTAATTGT CCTTTTTAACA GCGATCGCGT ATTTCGTCTC GCTCAGGCGC 5351 AATCACGAAT GAATAACGGT TTGGTTGATG CGAGTGATTT TGATGACGAG 5401 CGTAATGGCT GGCCTGTTGA ACAAGTCTGG AAAGAAATGC ATAAGCTTTT 5451 GCCATTCTCA CCGGATTCAG TCGTCACTCA TGGTGATTTC TCACTTGATA 5501 ACCTTATTTT TGACGAGGGs AAATTAATAG GTTGTATTGA TsTTGGACCA 5551 GTCGGAATCG CAGACCGATA CCAGGATCTT GCCATCCTAT GGAACTGCCT 5601 CCGTGACTTT TCTCCTTCAT TACAGAAACC GCTTTTTCAA AAATATGGTA 5651 TTGATAATCC TGATATGAAT AAATTGCAGT TTCATTTGAT GCTCGATGAG 5701 TTTTTCTCAT CAGAATTGCT TAATTGGTTG TAACACTGGC AGAGCATTAC 5751 GCTGACTTGA CGGGACGGCG GCTTTGTTGA ATAAATCGAA CTTTTGCTGA 5801 GTTGAAGGAT CAGATCACGC ATCTTCCCGA CAACGCAGAC CGTTCCGTGG 5851 CAAAGCAAAA GTTCAAAATC ACCAACTGGT CCACCTACAA CAAAGCTCTC 5901 ATCAACCGTG GCTCCCTCAC TTTCTGGCTG GATGATGGGG CGATTCAGGC 5951 CTGGTATGAs TCAGCAACAC CTTCTTCACG AGGCAGACCT CACCTCGAGG 6001 CTGATCCCCG SEO IV NO 3 1 MSTNPKPQRK TKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR GPRLGVRATR 51 KTSERSOPRG RRQPIPKARR PEGRT AQPG YPWPLYGNEG CGWAGWI-I 101 RGSRPS GPT DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GAPLGGAARA 151 LAHGVRVLED GVNYATGNLP GCSFSIF -A SCLTVPAS AYQVRNSSCL 201 YHVTNDCPNS SIVYEAADAI LHTPGCVPCV REGNASRC V AVTPTVATRD 251 GKLPTTQ RR HIDI-VGSAT LCS? LYVGDL COSVTLVGOL FTFSPRRHWT 301 TQDCNCSIYP GHITGHRMA CMHHN SPT? A WAQ LRI PQAIMDHIAG 351 AHWGVI? CIA YFSMVGNH? K V WL LPAG VDAETHVTGG NAGRTTAGLV 401 GLLTPCAKQN IQLDÍTNGS HINSTA NCN Efi NTGWLAG LFYQHKFNSS 451 GCPERI-ASCR R TDFAQGWG PISYANGSGH DERPYCWHYP P PCGIVPAK 501 SVCGPVYCFT PSPVWCTTD RSGAPTYSWG ANDTDVFV N NTRPPLGNWF 551 GCTWMNSTGF TKVCGAPPCV IQGVGN T L CPTDCFRKHP EATYSROGSG 601 P GGPRCMVD YPYRLWHYPC T8YTIFKVR HYVGGVEHR EAAOWTRGE 651 RCDLEDRDRS ELSPLL- STT QWQVLPCSFT TLPA STGLI HLBQNIVDVQ. 701 YLYGVGSSIA SWAUC EYVV L FU-LADAR VCSC WMMLL SSQAEAA EN 751 LVII-NAASI-A GTHSLVSFLV FFCFAWY KG RWVPGAVYAL YGM PU-L L 801 I? PQRAYA DTEVAASCGG WLVG MA T LSPYYKRYIS WCKWW1-QYFL 851 TRVEAQLHV VPPLNV OGR D? VI MCW HPILVFOITK U-AIFGPLW 901 ILQASU-KVP YFVRVQGU-R ICAU-RKI? G OHYVOMAIIK I-OAI-TGTYVY 951 NHLTP RDWA HNSLROL? V? VEPWFSRME TKLITWGADT AACGDII G 1001 PVSARR

Claims (1)

1. CLAIMS A vaccine composition comprising: QS21; Lipid A De-O-acylated monophosphoryl (3D-MPL); an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: a metabolizable 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 of the same. A vaccine composition according to claim 1, wherein the HCV protein or the immunogenic derivative thereof is chemically conjugated to a carrier molecule. A vaccine composition according to claim 1 or 2 wherein the immunogenic derivative is a fusion polypeptide. 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. A vaccine composition according to claim 4 wherein the influenza protein is the NS1 protein. A compound which comprises an HCV core protein, or an immunogenic derivative thereof, fused to a polypeptide containing foreign epitopes. A compound according to claim 6, wherein the polypeptide containing foreign epitopes is an influenza protein or an immunogenic derivative thereof. A compound according to claim 7, wherein the influenza protein is the NS1 protein. A method for 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 of claims 6 to The use of a composition according to any one of claims 1 to 5 or a compound according to any of claims 6 to 8 in the manufacture of a medicament for use in the prevention or treatment of HCV infection. A process for the preparation of a composition according to any of claims 1 to 5, which process comprises mixing the constituents thereof in the required proportions. A process for the preparation of a compound according to any of claims 6 to 8, which process comprises expressing DNA encoding said compound in a recombinant host cell and recovering the product. A DNA molecule encoding a compound according to any of claims 6 to 8. A recombinant vector comprising the DNA of claim 13. A host cell transformed with the recombinant vector of claim 14.