CA2222456A1 - Vaccines against hepatitis c - Google Patents

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

W O97/01640 PCT~EP96/027~.
VACCINES A.raAINST HEPATITIS C

The present invention relates to novel vaccine fo m~ .tions, to methods of theirproduction and to their use in medicine.
3 De-O-acylated monophosphoryl lipid A is known from GB2 220 211 (Ribi).
~hPmi~lly it is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is m~nllf~ctured by Ribi Irnmunochem Montana A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in TntP.rn,.tion~Tl Patent Application No. 92/116556.
QS21 is a Hplc purified non toxic fraction of a saponin from the bark of the South ~mçric~n 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 ~ntigenic protein of hepatitis C virus has been ~lP$i,,n~t~Pd the core protein and is descAbed by, ~or exarnple, Delisse et al., J. Hepatology, 1991 ;13 (Suppl. 4): S20-S23 (for genotype lb). Particular envelope proteins of hepatitis C virus have been ~lecign,.tPr7 El and E2 ~nd are descAbed by, for example, Grakoui et al., 1993, J. Virology 67, 1385-1395;
Spacte et al.,1992, Virology 188, 819-830; l~.tsumi~ et al., J. Virology 66, 1425-1431, and Kohara et al., 1992, J. Gen. Virol; 73, 2313-2318. A majority of the HCV
genotypes identifiPd to date are described by Okamoto Hiroaki and Mishiro Shunji, tntervirology, 1994, 37: 68 et seq.
The present invention provides a vaccine composition com~ ..cing QS21, 3 25 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 immllnogenic 30 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 ~iministration to a human. Such other derivatives can be prepared by the addition, deletion, 35 substitution, or rearrangement of amino acids or by chemi~l mo-lifi~tions thereof.
Tmmunogenic fr~gm~ntc of the protein, which may be useful in the preparation of subunit vaccines, may be prepared by expression of the WO 97/01640 CA 022224~6 1997 -12 -19 PCT/~ 2764 a~plo~liate gene fr~ment~ or by peptide synthesis, for e7C~mrl~ 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 cont~ining additional sequences which can carry one 5 or more epitopes for other immunogens. Alternatively, the immunogenic derivative of the invention can be fused to a carlier polypeptide or to another carrier which has immunostimulating properties, as in the case of an adjuvant, or which otherwise enh~ncçs the immlln~ response to the protein or derivative thereof, or which is useful in expressing, purifying or formulating 10 the proteinorderivativethereof.
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, (198~) 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 thereafter, optionally, preparing a derivative thereof.
A DNA molecule comprising such coding sequence can be 20 syntheci7Pd by standard DNA synthesis techniques, such as by enzymatic ligation as described by D.M. Roberts et al in Biochemistry 1985, 24, ~090-5098, by chPmic~l synthesis, by in vitro enzymatic polymPn7~qtinn, or by a combination of these techniques.
Enzymatic polymerisation of DNA may be carried out in vitro using 25 a DNA polymerase such as DNA polymerase I (Klenow fragment) in an aL)piopliate buffer cont~ining 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 fr~ment~ may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate 30 buffer, such as O.O5M Tris (pH 7.4), O.OlM MgCl2, O.OlM dithiothreitol, lmM spermidine, lmM ATP and O.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 fr~ome~tc may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using 35 solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fr~gmentc - A Laboratory Manual' (ed. H.G. Gassen and WO 97/01640 PCT11i;~96~0t764 1~. Lang), Verlag Chemie, Weinheim (1982),or in other sci-~.ntifir;
publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, ~md R.C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D. l!~ttr~.ucci and M.H Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D. ~ttencci and M.H. C~aruthers, Journal of the American Chemical Society, 1981, 103, 3185;
S.P. Adams et al., Journal of the American Chemical Society,1983, 105, ~561;
N.D. Sinha, J. Biemat, J. McMannus, and H. Koester, Nucleic Acids Research, lg84, 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, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or deletion mutagenesis such as ri~s~ri~ed by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984, 12, 22~225.
Recombinant techniques are described in Maniatis ~. ~1., Molecular C loning - A Laboratory Manual; C~old Spring Harbor, 1982-1989.
In particular, a protein or immunogenic derivative for use in the 20 present invention can be prepared using the following steps:
i) ~,epa ing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer compri~ing a nucleotide sequence that encodes said protein or an immllnogenic derivative thereof;
i) transforming a host cell with said vector;

iii) cultllring said transformed host cell under conditions permitting ssion 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 35 an app,opriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kin"~m~n and A.J. ~ing.~m~n;

CA 022224~6 l997-l2-l9 Blackwell ~çit-ntific PUblic~tionc: Oxford, Fn~l~nf~, 1988. The term 'transformed' or 'transformant' will hereafter apply to the reslllting host cellco~t~ining and expressing the foreign gene of interest.
The replicable expression vector may be prepared by cleaving a 5 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
molecul~s 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 ~letçrrnined 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 ç~ 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 desçribe~l in, for example, ~ni~tic et ~ cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL
Press Ltd, 1985.
The choice of transforrning conditions is ~l~tçrmin~d by the host cell.
Thus, a b~rtPri~l host such as E. cQli may be treated with a solution of CaC12 (Cohen ~ al. Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnC12, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol.
M~mm~ n cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells.
Culturing the transformed host cell under conditions pçrmitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis ç~ 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. ~ it may be W O 97/01640 PCT~EP96/02764 lysed physically, chPmi~lly or enzym~ti~lly and the protein product isolated from the resulting lysate. Where the host cell is m~mm~ n, the product may generally be isolated from the nutrient medium or from ceLL free extracts.
Convention~l protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
7 PrRferably, 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 cont~inin~ foreign epitopes. The polypeptide is preferably an influenza protein, such as the NS 1 protein, or an immunogenic denvative thereof. DNA coding for such a novel compound, vectors cont~ining said DNA, host cells transformed with said vectors, and their use in producing said novel compound, forrn still further aspects of the invention claimed.
The vaccines of the present invention are preferential stirnulators of IgG2a production and THl cell response. This is advantageous, because of the known impli~atic)r~ of THl response in cell mediated response. Indeed in mice in-luction of ][gG2a is correlated with such an immllnP response.
The vaccines of the invention enh~nce induction of cytolytic T lyrnphocyte responses. Induction of CTL is easily seen when the target antigen is synthPci~ed intr~çllnl lrly, ie during infection by the virus, because peptides genPr~te~l by proteolytic breakdown of the antigen can enter the appropriate proceccin~ pathway, ]Rading to presentation in association with class I molecules on the cell membrane.
]However, in genera]L, pre-formed soluble antigen does not reach this procescing and presentation pathway, and does not elicit class I restricted CTL. Therefore conventional non-living vaccinPs, while eliciting antibody and T helper responses, do not generally induce CTL me~i~tPd immunity. The combination of the two adjuvantsQS21 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 iimmllnP responses.
In certain systems, the combination of 3D-MPL and QS21 together with an oil ", iin water emu]Lsion have been able to synergistically enh~nce interferon ~ production.
Additiona]Lly the oi]L in water emulsion may contain span 85 and/or lecithin. A
35 Ipreferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in TntPrn~tion~l patent application published under No. 92116556 - SmithKlLine Beecham Biologica]Ls s.a.

CA 022224~6 1997-12-19 W O97/01640 PCT~EP96tO2764 Tlle oil in water emulsion may be utilised on its own or with other adjuvants or immunQ-stim~ ntc 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 subst~nti~lly 1: 1. The preferred range for optimal synergy is 2.5: 1 to 1: 1 3D MPL: QS21. Typically for human ~lmini ~tration QS21 and 3D MPL will be present in a vaccine in the range 1 llg - 100 ~g, preferably 10 ~g - 50 ~Lg 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 emlllcion 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. Fn~ar s~ tion within liposomes is clesçrihed, for ex~mrlP, 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 ~ignifi~nt adverse side effects in typical vaccinees. Generally, it is expected that each dose will compri.ce 1 - 1000 ~Lg of protein, preferably 2-100 ~Lg. An optimal amount for a particular vaccine can beascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunisation ade~uately 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 tre~tment comprising atiminictering an effective amount of a vaccine of the present invention to a patient.
The following examples illustrate the invention.

W O 97/lD1640 PCT~EP96/02764 lF.Y~-nrl~ .l ]L.1 Construction and expression of a recomhin~nt HCV core fusion protein Plasmid pMG81 a derivative of pMG27 (Gross et al 1985, Mol.Cell. Biol. ~: 1015) in which: (i) the 81 firstcodons of the NSl coding region from influen7~ strain A/PR18/34 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 recict~nce gene has been replaced by the kanamycin re~i~t~nce gene from transposon lrn902, 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 TCM128-2.
l:~e nucleotides sequences corresponding to amino acids 2-166 of the core protein ~were amplified from TCM128-2. During the polymerase chain reaction, NcoI and XbaI res~iction 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 pRlT 14129.
pRlT 14129 contains the coding sequence for the fusion protein NSl (flu)-core(HCV) and expresses the polypeptide described in SEQ ID NO. 1. The coding sequence forthe fusion protein NS 1 (flu)-core(HCV) is cont~inPd in SEQ ID NO 2. SEQ ID NO 3shows 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) cont~ining the thermosensitive repressor of the ApL promoter.
I'he recombin~nt bacteria were grown in a 20 Litters fermentor under fed-batch condi~ions at 30~. The expression of the NSl-Core protein was ind~lced by raising the temperature to 38-42~C. The cells were then harvested and mech~nic~lly disrupted.

1.2 Purification of the NSl-Core fusion protein I'he antigen was purified in a denatured form by preparative electrophoreses:
., W 097/01640 PCT~EP96/02764 Step 1: Bacterial cells were broken (Rannie-2 x 14,500 pi) in a 20 mM phosph~tP
buffer pH7 COIlt~inin,, protease inhibitors (lmM pefabloc, 0.5mg/leupeptin, 0.1%aprotinin).

Ste~ 2: Lysate was centrifuged for 25 minutPs, at 17,000g. At this stage ~e 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 minutPs 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 sa-m--ple buffer, boiled for 5 minutP~s, centrifuged again at 27,000g for 25 minutP,s and then applied on a 12%
polyacrylamide gel for separation of the rem~ining 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.
1'he purified antigen appears as a doublet, in the 27-30 kD range, both bands are recognised by an anti-NS 1 monoclonal antibody as well a,s by anti-core specific hurnan monoclonal and rabbit polyclonal antibodies.

1.3 Adjuvantation of the NSI-Core Protein 1'he two adjuvant formulations were made each compricing the following oil in water emulsion component.

SB26: 5% squalene 5% tocopherol 0.4% tween 80; the particle size was 500 nm sizeSB62: 5% Squalene 5% tocopherol 2.0% tween 80; the particle size was 180 nm l(a) Preparation of emulsion SB62 ~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 ~ml of squalene are vortexed to mix thoroughly. 90ml of PBSlTween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe WO 97/01640 PCTlEP96102764 alnd finally microflni~Ti~d by using an M110S microfl-litli~s m~-~hin~o. The resllltin~ oil clroplets have a size of approxim~t~ly 180 nm.

1 (b) Preparation of emulsion SB26 ~i~is emulsion was prepared in an analogous manner utili.~ing 0.4% tween 80.

1 (c) Other emulsions as depicted in the Table were made in an analogous manner.
l (d) Preparation of fusion protein/QS21/3D MPL/ oil in water formIll~tion To the emulsion of 1 a) or b) or c) an equal volume of twice concentrated fusionprotein(ei~ler 20,ug or 100~1g) was added and mixed. This was combined with S0~g/ml of 3D-MPL and 20,ug/ml of QS21 to give the final fcrm~ tion. Buffer was set according to salt content and pH.

F.Y~mpIe 2' 2.~1 Preparation of a recomhin~nt ElE2 oligomeric ~l~tei,, Oligomeric forms of El-E2 HCV envelope proteins can be prepared form m~mm~ n 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 pl~mi~l used to prepared vaccinia recombinal1t virus that will lead to expression of the polyprotein in infected cells. The expressed poly~ te-,l is processed and retained intracellularly. El-E2 oligomeric form can be purified from cell extracts in which the ElIE2 protein complex has been solubilized using specific detergent (Ralston et al, 1993, J. Virology 67:6753) ~0 (Dubuisson et al 1994, J. Virology 68:6147).

2.2 Preparation of vaccine formulations e E~ormnl~tions of oligomeric ElE2 are prepared analagously to the formulations of 35 FY~mple 1.

WO 97/01640 CA o 2 2 2 2 4 5 6 19 9 7 - 1 2 - 19 PCT/E~96/02764 F.Y~mpl~ 3 Formnl~tions cont~ining both the fusion protein of Example 1 and the ElE2 oligomer 5 of Exarnple 2 are prepared analagously to the formlll~tions of F.Y~mple 1, each formulation cont~ining between 50 and lOO~g of each protein.

WO 97101640 PCT/13:P96/02764 Table 1 Vehicles two fold concentrated ~-Fmll1ci~Dc SB Tocophe~ol % Squalene ~oTween 80 Yo Span 85 9~ Lecithin % SIZe 26 5 5 0.4 0 0500 nm 800 nm 10-0%
26.1 5 5 0.4 0 0.1500 nm 63 5 5 0.6 0 0 SOOnm 64 5 5 0.8 0 0500 nm 61 5 5 1 0 0250-300 nm 62 5 5 2 0 0180 nm 0.4 1 0500 nm 80-100%
800 nm 20-0%
40.1 5 5 0.4 1 0.1500 nm 1 1 0300 nm 0.4 1.5 0500 nm 66 5 5 0.4 2 0 500nm CA 022224~6 l997-l2-l9 WO 97/01640 PCT~EPg6/02764 45 7Sl CAGAAATCCC TAAGAGGAAG GGGCAGCACT CTTGGTCTGG ACATCGAGAC

851 CCGATGAGGC ACTTAAAATG AcCATGAGCA CAAATCCTAA ACCCCAAAGA

~51 CGGTGGTCAG ATCGTtGGTG GAGTTTACcT GTTGCCGCGC AGGGGCCCCA
55 1 o o 1 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
65 1251 cACgTGCGGC TTCGCCGACC TCATGGGGTA CATTCCGCTC GTCGGCGCCC
1301 CCccAGGGGG CGCTGCCAGG GCCtTGGCAC ATGGTGTCCG GGTTCTGGAG

CA 022224~6 l997-l2-l9 WO !97/lDI640 PCT/E;P9610t764 1351 GACGGCGTGA ACTATGCAAC AtaaTCTAGA ATCGATAAGC TTCGACCGAT
lgOl GCCCTTGAGA GCCTTCAACC CAGTCAGCTC CTTCCGGTGG GCGCGGGGCA

1.501 GGACAGGTGC CGGCAGCGCT CTGGGTCATT TTCGGCGAGG ACCGCTTTCG
0 1.551 CTGGAGCGCG ACGATGATCG GCCTGTCGCT TGCGGTATTC GGAATCTTGC
1.601 ACGCCCTCGC TCAAGCCTTC GTCACTGGTC CCGCCACCAA AC~l~llCGGC

lg51 GGCGGCATCG GGATGCCCGC GTTGCAGGCC ATGCTGTCCA GGCAGGTAGA

2351 CATCTCGGGC AGC~llGG~l CCTGGCCACG GGTGCGCATG ATCGTGCTCC

2501 GTCTGCGACC TGAGCAACAA CATGAATGGT CTTCGGTTTC C~~ llCGT

~5 2701 TCCATACCGC CA~ll~lTTA CCCTCACAAC GTTCCAGTAA CCGGGCATGT

6~

CA 022224~6 l997-l2-l9 W 09710~640 PCT~EP96/02764 3451 CCGCGTTGCT GGC~ll"l'll'C CATAGGCTCC GCCCCCCTGA CGAGCATCAC

4001 CG~l'G~l"l"l"l''l"l"l'~'l"l"l'GCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT

~551 CGGATCAGCC TCGAGGTGAG GTCTGCCTCG TGAAGAAGGT GTTGCTGACT

4651 CGGTTGATGA GAG~lll~ll GTAGGTGGAC CAGTTGGTGA TTTTGAACTT

CA 022224~6 l997-l2-l9 WO 971'01640 PCTIEP961Q2764 t.~ 1 4751 CCTTCAACTC AGCAAAAGTT CGATTTATTC AACAAAGCCA C~ll~l~lCT
a 801 CAAAATCTCT GATGTTACAT TGCACAAGAT AAAAATATAT CATCATGAAC
~a 851 AATAAAACTG TCTGCTTACA TAAACAGTAA TACAAGGGGT GTTATGAGCC
.~901 ATATTCAACG GGAAACGTCT TGCTCGAGGC CGCGATTAAA TTCCAACATG

5051 TTCTGAAACA TGGCA~AGGT AGCGTTGCCA ATGATGTTAC AGATGAGATG

5201 GGAA~ACAGC ATTCCAGGTA TTAGAAGAAT ATCCTGATTC AGGTGAAAAT

S401 CGTAATGGCT GGCCTGTTGA ACAAGTCTGG AAAGAAATGC ATAAGcTTTlr 30 5,451 GCCATTCTCA CCGGATTCAG TCGTCACTCA TGGTGATTTC TCACTTGATA

5601 CGGTGAGTTT TCTCCTTCAT TACAGAAACG G~lllllCAA AAATATGGTA

S:EQ ID NO 3 :151 LAHGVRVLED GVNYATGNLP GCSFSIFLLA LLSCLTVPAS AYQVRNSSGL
:201 YHVTNDCPNS SIVYEAADAI LHTPGCVPCV REGNASRCWV AVTPTVATRD

CA 022224~6 l997-l2-l9 W O 9710~640 PCT~EP96/02764 301 TQDCNCSIYP ~L~l~K~Aw DMMMNWSPTA AL W AQLLRI PQAIMDMIAG
3Sl AHWGVLAGIA YFSMVGNWAK VL W LLLFAG VDAETHVTGG NAGRTTAGLV

~01 LALPQRAYAL DTEVAASCGG VVLVGLMALT LSPYYKRYIS WCMWWLQYFL

gSl NHLTPLRDWA HNGLRDLAVA VEPW FSRME TKLITWGADT AACGDIINGL

Claims (15)

Claims

1. 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.