CA2131028A1 - Hepatitis c virus peptides - Google Patents

Abstract

This invention relates to a novel peptide, having the sequence as shown in SEQ ID NO's: 1 and 2, capable of binding to antibody specific for parenterally transmitted non-A non-B hepatitis (PT-NANBH), in particular (HCV) and the use of such a peptide in an immunoassay for the detection of HCV or a vaccine for its prevention.

Description

W093~17111 ~ 5 3 ~)28 Pcr/GBg3/004l0 HEPAT I T I S C Y I RUS PEPT I DES
~.
This invention relates to a novel peptide capable of ~inding to antibody specific for parenterally tran~mitted non A non B hepatitis (PT-NANBH) in particular (HCV) and the use of such a peptide in an immunoassay for the detection of HCV or a v~ccine for its prevention.

Non A non B hepatitis (NANBH) is by definition a diagnosis of exclusion and has generally been employed to describe cases of viral hepatitis infection in human bein~s that are not due to hepatitis A or B viruses. The PT-NANBH
virus-has also been referred to as Hep~titis C Virus (HCV). In ~~ the majority of such cases, the cause of the infection has not }5 been identified althoughl on clinical and epidemiological :~
grounds, a number of agents have been thought to be responsible as reviewed in Shih et al (Pro~ Liver Dis.~ 1986, 8, 433-452~.
In the USA alone, up to 10% of blood transfusions can result in NANBH which makes it a significant problem. :~
GB-A-2 239 245 discloses r.ucleotide and polypeptide sequenc s of a viral agent responsible for Post Transfusional NANBH (PT NANBH~, the core region of this viral agent responsible for PT-NANBH is disclosed within the nucleotide and peptide sequences. The core region of the disclosed viral agent responsible for PT-NANBH was not analysed in GB-A-2 239 245. Substantial portions of the core region were used in recombinant polypeptides namely BHC-ll as disclosed in GB-A-~~39 245.
-Substantial regions of the core region of the viral agent responsible for PT-NANBH disclosed in GB-A-2 239 245, were also used to detect HCV infection in British Application No.
9203803.3 filed in the name of Wellcom Foundation Limited on February 21, 1992.

EP-~-0 445 423 discloses an assay for detecting the ,t ~i3~ PCT/GB9~/00410 presence of an antibody to an HCv antigen by contacting the sample with a polypeptide containing a~ least one Ppitope of an HCV antigen. A polypPptide from the putative core region of HCV is used. Specific epitopic regions of the polypeptides are not identified.

It has surprisingly been found by ~he present inventors that several domains within the N-terminal region of ~he HCV
core protein are epitopic. It has also surprisingly been found that of the sera reactive with N-terminal region of the core protein the majority react with a single domain exclusively or i~ combination wi~h any of the other epitopic domains. This singl`e domain h s been termed the immunodominant epitope. Thls ~~ immunodominant epitope has been isolated by thP present inventors.

Accordingly, the presPnt invention provides a HCV viral -:
peptide having the foll~wing sequence (SEQ ID NO'is: 1 and 23:
Tyr-Leu-Leu-Pro-Arg-Arg (YLLPRR~
This sequence may be found at residues 35-40 of ~he core protein. The present invention also provides a DNA sequence encoding the peptide as defined above. The DNA sequence may be synthetic or cloned. Preferably the DNA sequence is as set forth in SEQ ID NO:1.

A novel synthetic peptide has also been synthesised which pep~ide has improved assay sensitivity and specificity, when compared to the natural peptide as discIosed above, in assays for HCV.

Accordingly, the present invention also provides a pepti~e of the general formula I
X1-X2~X3~x~~xs-x6 (SEQ ID NO: 3) 35 wherein; ~:
X1 represents Tyrosine, Aspartic Acid, Gluta~ic Acid, Leucine or Isoleucine;
.

W093J17111 ~ ~ 1 31 ~ 2 ~ PCT/GB93/00410 X2 represents Leucine, Cysteine, Aspartic Acid or ~lutamic ACid s X3 represents Leucine, Valine;
X4 represents Proline or Methionine;
5 X5 represents Arginine, Aspartic Acid, Glutamic Acid, Proline, Cysteine, Phenylalanine, Isoleucine, Asparagine, Glutamine or Methionine; and X6 represents Arginine, Aspartic Acid, Glutamic Acid, Serine or Cysteine and wherein the peptide is not of the formula Tyr-Leu-~eu-Pro-Arg-Arg (SEQ ID ~0: l and 2).

The present invention also provides a DNA sequence ~ ~ encoding the peptide of general formula I, wbich se~uence can be readily ascertained, bearing in mind the degeneracy of the genetic code and codon usage.

The peptides of the present invention may be used individually, as part of a larger polypeptide andfor as a mixture of peptides with other peptides from the HCV genome, a heterologous genome or topograp~ically related peptides.

The present învention also provides peptides which comprise the peptides as defined above, namely, SEQ ID NQ's: 4 and 5 and SEQ ID NO's: 6 and 7.

The present invention also provides DNA sequences encoding the peptide as set forth above. Preferably the DNA
sequences are as set forth in SEQ ID NO's: 4 and 6.
The present invention also provides expres~ion vectors - containing the DNA sequences as herein defined, which vectors being cap~ble, in an appropriate host, of expressing the DNA
' s~uence to produce the peptides as defined herein.
The expression vector normally contains control elements of DNA that effect exprescion of the DNA sequence in an WO93t17111 PCT/GB93/00410 i~3~9 appropriate host. These elements may vary according to the host but usually include a promoter, ribosome binding site, translational start and stop sites, and a transcriptional termination site. Examples of such vectors include plasmids and viruses. Expression vectors of the present invention Pncsmpass both extrachromosomal vectors and vectors that are integrated into the host cell~s chromosome. For use in E.coli, the expression vector may contain the DNA sequence of the present invention optionally as a fusion linked to either the 5'- or 3'-~nd of the DNA sequence encoding, for example, ~-galactosidase or to the 3'-end of the DNA sequence encoding, for example, the ~rp E gene. For use in the insect baculovirus (AcNPV) system, ~he DNA sequence is optionally fused to the polyhedrin coding sequence.
The present invention also provides a host c211 transformed with expression vectors as herein defined.

Examples of host cells of use with the present invention include prokaryotic and eukaryotic cells, such as bacterial, yeast, mammalian and insect cells. Particular examples of such cells are E.coli, S.cerevisiae, P.pastoris, Chinese hamster ovary and mouse cells, and S~odoptera fruqi~erda and Trico~lusia ni. The choice of host cell may depend on a number of factors but, if post-translational modification of th~ HCV
viral peptide is important, then an eukaryotic host would be preferred.

The present invention also provides a process for preparing a peptides as defined herein which comprises isolating the DNA sequence, as herein defined, from the HCV
genome, or synthesising ~NA sequence encoding the peptides as defined herein, or generating a DNA seque~ce encoding the peptide of Formu~a I, inserting the D~A sequence into an expression vector such that it is capable, in an appropriate host, of being expressed, transforming an host cells with the expression vector, culturing the transformed host cells, and ~t3 ~ J

isolating the peptide.

The DNA sequence encoding the peptide may be synthesised using standard procedures (Gait, Oliqonucleotide Synthesis: A
Practical Approach, 1984, Oxford~ IRL Press).

The desired DNA sequence obtained as described above may be inserted into an expression vector using known and standard techniques. The exp~ession vector is normally cut using restriction enzymes and the DNA sequence inserted using blunt-end or staggered-end ligation. The cut is usually made at a restriction site in a convenient position in the expression vector such tha~, once inserted, the DNA sequences are under '~ the control of the functional elements of DN~ that effect its expression.

TransC~rmation of an host cell may be carried out using ~candard techniques. Some phenotypic marker is usually employed to distinguish between the transformants ~hat have successfully taken up the expression vect~r and those that have not. Culturing of the transformed host cell and isolation of the peptide as required may also be carried out using standard techniques.

The peptides of the present invention may be prepared by synthetic methods or by recombinant DNA technology. The peptides are preferably synthesized using automatic synthesizers.

Antibody specific to a peptide of the present invention can be raised using the peptide~ The antibody may be polyclonal or mon~clonal. The antibody may be used in quality control testing of batches of the peptides; purification of a peptide o~ viral lysate; epitope mapping; when labelled, as a conjugate in a competitive type assay, for antibody detection;
and in antigen detection assays.

WO 93/1~111 PCI/GB93/00410 ~3~9 l 6 Polyclonal antibody against a peptide of the present invention may be obtained by injecting a peptide, optionally coupled to a carrier to promote an immune response, into a mammalian host, such as a mousej rat, sheep or rabbit, and recovering the antibody thus produced. The peptide is generally administered in the form of an injectable formulation in which the peptide is admixed with a physiologically acceptable diluent. Adjuvants, such as Freund's complete adjuvant (FCA) or Freund's incomplete ad~uvant (FIA), may be included in the formulation. The formulation is normally injected into the host over a suitable period of time, plasma samples being taken at appropriate intervals for assay for anti-HCV viral antibody~
When an appropriate level of activity is obtained, the host is bled. Antibody is then extracted and purified from the blood plasma using standard procedures, for example, by protein A or ion-exchange chromatography. - ;
-Monoclonal antibody against ~ peptide of the presentinvention may be obtained by fusing cells of an immortalising cell line with cells which produce antibody against the viral or topographically related peptide, and culturing the fused immortalised cell line. Typically, a non-human mammalian host, such as a mouse or rat, is inocu~ated with the peptide. After sufficient time has elapsed for the host to mount an antibody - 25 response, antibody producing cells, such as the sp~enocytes, are removed. Cells of an immortalising cell line, such as a mouse or rat myeloma cell line, are fused with the antibody producing cells and the resulting fusions screened to identify a cell line, such as a hybridoma, that secretes the desired monsclonal antibody. The fused cell line may be cultured and the monoclonal antibody purified from the culture media in a similar manner to the purification of polyclonal antibody.

~ Diagnostic assays based upon the present invention may be used ~o determine the presence or absence of HCV infection.
They may also be used to monitor treatment of such infection, for example in interferon therapy.

WO93/17111 PCT~GB93/0~10 ~ 13 ~

In an assay for the diagnosis of viral infection, there are basically three distinct approaches that can be adopted involving the detection of viral nucleic acid, viral antigen or viral antibody. Viral nucleic acid is generally regarded as the best indicator of the presence of the virus itself and would identify materials likely to be infectious~ However, the detection of nucleic acid is not usually as straightforward as the detection of antigens or antibodies since the level of target can be ~ery low. Viral antigen is used as a marker for the presence of virus and as an indicator of infectivlty.
Depending upon the virus~ the amount of antigen present in a sample can be very low and dif~icult to detect. Antibody detection is relatively straight~orward because, in effect, the '~ host immune system is amplifying the response to an infection by producing large amounts of circulating antibody. The nature of the antibody response can often be clinically useful, for example IgM rather than IgG class antibodies are indicative of a recent infection, or the response to a particular viral antigen may be associated with clearance of the ~irus. Thus : 20 the exact approach adopted for the diagnosis of a viral infection depends upon the particulAr circumstances and the information sought. In the case of HCV, a diagnostic assay may embody any one of these three approaches.

In an assay for the diagnosis of HCV in~ol~ing detection of viral nucleic acid, the method may comprise hy~ridising viral RNA present in a test sa~ple, or cDNA synthesised from such viral RNA, with a DNA sequence corresponding to the nucleotide sequence of SEQ ID NO: l, 4 or 6 or encoding the peptide of formula I and screening the resulting nucleic acid hybrids to identify any HCV viral nucleic acid. The application of this method is usually restricted to a test sample of an appropriate tissue, such as a liver biopsy, in which the viral RNA is likely to be present at a high level.
The DNA sequence corresponding to the nucleot}de sequence of SEQ ID NO: l, 4 or 6 or encoding the peptide of Formula I may take the form of an oligonucleotide or a cDNA sequence ~-~ 8 PCTIGB93/~lU

sequence optionally contained within a plasmid. Screening of the nucleic acid hybrids is preferably carried out by using a labelled DNA sequence. Preferably the peptide of the present invention is part of an oligonucleotide wherein the label is situated at a sufficient distance from the peptide so that binding of the peptide to the viral nucleic acid is not interfered with by virtue of the label being too close to the binding site. One or more additional rounds of screening of , one kind or another may be carried out to charac~Prise further the hy~rids and thus identify any HCV viral nucleic acid. The steps of hybridisation and scre~ning are carried out in accordance with procedures known in the art~ -:

~ The present invention also provides a test kit for the -detection of HC~ viral nucleic acid, which comprises iJ a labelled oligonucleotide comprising a DNA sequence corresponding to the nucleotide sequence of SEQ ID NO:
1, 4 or 6 or encoding the peptid~ of formu~a I; and ~
optionally; ~;

ii) washing solutions, reaction buffers and a substrate, if ~-~
the label is an enzyme.
~ ,;, Advantageously, the test kit also contains a positive control sample to facilitate in the identification of viral nucleic acid. -In an assay for the diagnosis of HCV involving detection of viral antigen or antibody, the method may eomprise contacting a test sample with a peptide of the present invention or a polyclonal or monoclonal antibody against the peptide and determining whether there is any antigen-antibody binding contained within the test sample. For this purpose, a test kit may be provided comprising a peptide, as defined herein, ~r a polyclonal or monoclonal anti~ody thereto and means for determining whether there is any binding with ~ WO93~17111 PCT/GB93~00410 ~13 ~02~

antibody vr antigen respectively contained in the test sample.
The test sample may be taken from any of the appropriate tissues and physiological fluids mentioned above for the de~ection of viral nucleic acid. If a physiological fluid is obtained, it may optionally be concentrated for any viral antigen or antibody present.

A variety of assay formats may be emp~oyed. The peptide can be used to capture selectively antibody against HC~ from solution, to label selectively the antibody already captured, or both to capture and label the antibody. In addition, the peptide may be used in a variety of homogeneous assay formats in which the antibody reactive with the peptide i5 detected in solution with no separation of phases.
The types of assay in which the peptide is used to cap~ure antibody from solution involve immobilization of the peptide on to a solid surface. This surface should be capable of being w~shed in some way. Examples of suitable surfaces include polymers of various types (moulded into microtitre wells; beads; dipsticks of various types; aspiration tips;
electrodes; and optical devices), particles (for example latex;
stabilized red blood cells; bacterial or fungal cells; spores;
gold or other metallic or metal-containing sols; and proteinaceous colloids) with the usual size of the particle being from 0.02 to 5 microns, membranes (for example of nitrocellulose; paper; cellulose acetate; and high porosity/high surface area membranes of an organic or inorganic material).
The attachment of the peptide to the surface can be by passive adsorption from a solution of optimum composition which may include surfactants, solvents, salts and/or chaotropes; or by active chemical bonding. Active bonding may be through a ~ariety of reactive or activatible functional groups which m~y he exposed on the surface (for example condensîng agents;
active acid esters, halides and anhydrides; amino, hydroxyl, or WO~3/171~1 PCT/~93/00410 .,~3~ o carboxyl groups; sulphydryl ~roups; carbonyl groups; diazo groups; or unsaturated groups). Optionally, the active bonding may be through a protein (itself attached to the surface passively ol through active bonding), such as albumin or casein, to which the viral peptide may be chemically bonded by any of a variety of methods. The use of a protein in this way may confer advantages because of isoelectric point, charge, hydrophilicity or other physico-chemical property. The viral peptide may also be attached to the surface (usually but not necessarily a membrane) following electrophoretic separation of a reaction mixture, such as immuno precipitation.
:~
- After contacting (reacting) the surface bearing the ~ peptide with a test sample, allowing time for reaction, and, where necessary, removing the excess of the sample by any of a variety of means, (such as washing, centrifugation, ~iltration, magnetism or apilliary action) the captured antibody is detected by any means which will give a detectable signal. For example, this may be achievPd by use of a labelled molecule or particle as described above which will react with the captured antibody (for example protein A or protein G and the li~e;
anti-species or anti-immunoglobulin-sub-type; rheumatoid factor; ~r antibody to the peptide, used in a competitive or blocking fashion), or any molecule containing an epitope contained in the peptide.

The detectable signal may be optical or radioactive or physico-chemical and may be provided directly by labelling the molecule or particle with, for example, a dye, radiolabel, electroactive speci~s, magnetically resonant species or fluorophore, or indirectly by labelling the molecule or particle with an enzyme itself capable of giving rise to a measurable change of any sort. Alternatively the detectable signal may be obtained using, for example, agglu~ination, or through a diffraction or birefringent effect if the surface is in the form of particles.

~0 93/17tll ~ 3 1 3 ~ 3 P~/GB93/00410 Assays in which a peptide itself is used to label an already captured antibody require some form c~f labelling of the peptide which will allow it to be detected. The labelling may be direci by chemically or passively attaching for example a 5 radio label, magnetic resonant species, particle or enzyme label to the peptide; or indirect by attaching any form of label to a m~lecule which will itself react with the peptide.
The chemistry of bondin~ a label to the peptide can be directly through a moiety already present in the peptide, such as an amino group, or through an intermediate moiety, such as a maleimide group~ Cap~ure of the antibody may be on any of the surfaces already me~tioned by any reagent including passive or activated adsorption which will result in specific antibody or ~ immune complexes being bound. In particular, capture of ~he antibody could be by anti-species or anti-immunoglobu~in-sub-type, by rheumatoid factor, proteins A, G and the like, or by any molecule containing an epitope contained in the peptide.

The labelled peptide may be used in a competi~ive binding fash~pn in which its binding tv any specific m~lecule on any of the surfaces exemplified above is blocked by antigen in the sample. Alternati~ely, it may be used in a non-competiti~e fashion in which antigen in the sample is bound specifically or non-specifically to any of the surfaces above and is also bound ~5 to a specific bi- or poly-valent molecule (e~g~ an antibody) with the remaining valencies being used to capture the labelled peptide.

Often in homogeneous assays the peptide and an antibody are separately labelled so that, when the antibody reacts with the recombinant peptide in free solution, the two labels interact to allow, for example, non-radiative transfer o~
energy captured by one label to the other label with appropriate de~ection of the excited second label or quenched first label (e~g. by fluorimetry, magnetic resonance or enzyme measurement). Addition of either viral peptide or antibody in a sample results in restriction of the interaction of the W~93/17~ 3~ 12 PcT/G893/oo4lo labelled pair and thus in a different level of signal in the detector.

A suitable assay format for detecting HCV antibody is the direct sandwich enzyme immunoassay (EIA) format. A peptide is coated onto microtitre wells. A test sample and a peptide to which an enzyme is coupled are added simultaneously. Any HCV
antibody present in the test sample binds both to the peptide coating the well and to the enzyme-coupled peptide. Typically, the same peptide are used on both sides of the sandwich. After washing, bound enzyme is detecLed using a specific substrate involving a colour change. A test kit for use in such an EIA
comp~ises:

~l) a peptide-, as herein defined labelled with an enzyme;

(2) a substrate for the enzyme;

(3) means providing a surface on which a pep~ide is ~0 immobilised; and (4) optionally, washing solutions and/or buffers.

It is also possible to use IgG/IgM antibody capture ELISA
wherein an antihuman antibody is coated onto microlitre wells, a test sample is added to the well. Any IgG or IgM antibody present in the test sampl~ will then bind to the anti-human antibody. A peptide of the present invention, which has bsen labelled, is added to the well and the peptide will bind to any IgG or IgM antibody which has resulted due to infection by HCV.
The IgG or IgM antib~dy can be visualizPd by virtue of the label on the peptide.

It can thus be seen that the peptides of the present invention may be used for the detection of HCV infection in many formats, namely as free peptides, in assays including classic ELISA, competition ELISA, membrane bound EIA and WO93/17111 ~ :t 3 ~ . PCT/GB93/00410 immunoprecipitation. Peptide conju~ates may be used in amplified assays and IgG/IgM antibody capture ELISA.
~.
The peptide of the present invention may be incorporated --~
into a vaccine formulation for inducing immunity to HCV in man.
For this purpose the peptide may be presented in association with a pharmaceutically acceptable carrier. ~;

For use in a vaccine formulation, the peptide may optionally be presented as part of an hepatitis B core fusion particle, as described in Clarke et al ~Nature, 1987, 330, 381-384), or a polylysine based polymer, as described in Tam (PNAS, }988,- 85, 5409-5413)~ Alternatively, the peptide may bf optionally be attached to a particulate structure, such as liposomes or ISCOMS.

Pharmaceutically acceptable carriers include liquid media suitable for use ~s vehicles to introduce the peptide into a pa~ient. An example of such liquid media is saline so1ution.
The peptide may ~e dissolved or suspended as a solid in the carrier.

The vaccine formulation may also contain an adjuvant for stimulating the immune response and thereby enhancing the effect of the vaccine. Examples of adjuvants include aluminium hydroxide and aluminium phosphate.

The vaccine formulation may contain a final concentration of peptide in the rang from 0.0l to 5 mg/ml, preferably from 0.03 to 2 mg/ml. The ~accine formulation may be incorporated into a sterile container, which is then sealed and stored at a low temperature, for example 4~C, or may be freeze-dried.

In order to induce immunity in man to HCV, one or more doses of the vaccine formulation may be administered. Each dose may be 0.l to 2 ml, preferably 0.2 to l ml. A method for inducing immunity to HCV in man, comprises the administration WO93J171t1 ~ PCT/GB93/00410 ~ l4 of an effective amount of a vaccine formu~ation, as hereinbefore defined.

Tne present invention also provides the use of a peptide as herein defined in the preparation of a va~cine for use in the induction of immunity to HCV in man.

Vaccines of the present invention may be administered by any convenient method for the administration of vaccines including oral and parenteral (e.g. intravenous, subcutaneous or intramus~ular~ iniection. The treatmen~ may consist of a sin~le dose of vaccine or a plurality of doses over a period of time.-, -- ~

In the sequence listing, there are listed SEQ ID NO: l to 29 to which reference is made in the description and claims.

The embodiments of the invention will now be illustrated.

Exam~le l:_ Identification of E~ito~ic domain Qf the HCV CoreProtein The N-terminal 97 amino acid residues of HCV Core protein SEQ ID NO: 8 was mapped with PEPSCAN analysis. This process involved the serological analysis of sequentially overlapping peptides which spanned the region of interest, to identify epitopic domains. Ninety overlapping octameric peptides were synthesized on polypropylene pin supports according to the manufacturer's instructions (Cambridge Research Biochemicals], such that each peptide overlapped with the preceding sequence by the seven N-terminal residues, and with the subsequent peptide by the seven C-terminal residues. Serum or IgG

purified from the serum of individuals confirmed to be seropositive for HCV were used to screen the peptides.

3~

~131~23 :
Example_2: Screeninq of the Peptides .. - ,:
The assa~ procedure followed was performed basically as described in the manufacturers instruction manual. The time and temperature of the test antibody incubation differed from that prescribed, thereby decreasing the time taken f or each assay.

Sera: A total of 16 sera were assayed by PEPSCAN. These consisted of 12 sera from paid U.S. Donors which were identified as being positive for antibodies against HCV in routine screening at the site of donation. The remaining four sera were from pa~ients diagnosed .as having non-A non-B
~' hepatitis, and which were subsequently shown ~o contain antibodies to HCV. Sera were screened either as the crude serum fraction, or.as a caprylic acid preparation. Caprylic acid purification of IgG from the sera was performed as described by Stienbuch and Audran (1969) Arch. Biochem.
Biophys. 134, 279-284.
Assay: The assay format involves sequentially incubating the various pins in microwell plates containing blocking, test antibody, conjugate, and substrate solutions. The immune complex builds up on the pin with the enzyme labelled conjugate being immobilized on the bound anti-peptide antibody from the test serum. When the pin is then immersed in substrate, the enzyme brings about the colour chang~ whir-h is used to quantify ~he amount of anti-peptide antibody bound.

Blocking: Pins are immersed in 200 ~1 of Supercocktail (1% Ovalbumin, 1% bovine serum albumin in 0.15M phosphate buffered saline pH 7.2 + 0.1~ Tw~en 20) and left to stand at `~
room temperature for 1 hour.

Test antibody: The test antibody is diluted between 1/200 (IgG) and 1/1000 (serum) in supercocktail. The pins are then stood in this solution ~175~1fwell) at 373C for 1 hour.

W~93/171-11 3~ ^PCT/~Bg3/00410 After this incubation the pins are washed four times in 0.15M
phosphate buffered saline pH 7.2 + 0.05~ Tween 20, each at room temperature for lO minutes.
: ::
Conjugate: Rabbit anti-human IgG coupled to horseradish peroxidase is diluted l/lOOO in supercorktail. The peptide bearing pins are then stood in this solution at roo~
temperature for l hour. Once again the pins are then washed four times as above.
Substrate: The substrate solution is made up fresh for each assay. To lOOml of O.lM phosphate, 0.08M citrate buf~er pH 4.0 is dissolved 50mg of 2,2' AZINO-bis~3 ethylbenzthiaæo-~ linsulphonic acid (ABTS) and 0.3~l~ml 30% hydrogen peroxide is added. The pins are immersed in the wells containing substrate solution (150~1/well) and incubated a~ room temperature in the dark. Once sufficient colour has developed the reaction is stopped by removing the pins. The ptates are then read i~nediately in a spectrophotometric plate reader at a wavel ngth of 405~m. The absorbance values are then plotted against pin number. The epitopic domains are then defined as the continuous sequence of amino acids common to all positive pins in the domain i.e.

25 Positive Pin l Gly-Val-Tyr-Leu-Leu-Pro-Arg-Arg (SEQ ID
( G V Y L L P ~ R~ NO: 9) Positive Pin 2 Val-Tyr-Leu-Leu-Pro-Arg-Arg-Gly (SEQ ID
( V Y ~ L P R R ~) NO: lO) Positive Pin 3 Tyr-Leu-Leu-Pro-Arg-Arg-Gly-Pro (SEQ ID
( Y L L P R R G P) NO~

In this example the epitope has the sequence Tyr-Leu-Leu-Pro-Arg-Arg (YLLPRR) (SEQ ID NO's: 1 and 23.

Cleaning: After use the pins are cleaned by ultrasonication in disruptiQn buffer (1% sodium dodecyl sulphat~, 0.1% 2-mercaptoethanol in O.lM sodium dihydrogen phosphate) heated to W0~3M7111 ~J l 3 ~ ~ ~ ~ PCT/GB93/0~10 60~C. Pins are sonicated for 30 minutes and then washed twice in distilled water at 60C and once in boiling methanol. Pins can then be stored at rQom temperature sealed in ba~s containing silica gel, or can be re-used immediately for the next assay.

Several domains within the N-tenminal region of HCV Core protQin were thus identified as being epi-'opic. One of these regions, the immunodominant epitope, was detected to a greater or lesser extent by all of the sera tested. The immunodominant epitope is situated fro~ ~esidues 35-40 ~f the Core protein having the amino acid sequence Tyr-Leu-Leu-Pro-Arg-Arg (YLLPRR) SEQ ID NO's l and 2. Sample assay resul~s.are shown in Table ~-~ 1 .
lS
Table l: .

Absorbance Units for different ~era Peptide SEOI D 65221 65241652?6 65234 L
NO

GGGQI~GG 12 137 121 127 134 121 GGQIVGGV - 13 323 122 ll9 179 ll9 GQIVGGVY 14 216 112. 128 l65 138 IVGGVYLL 16 268 l26 183 l86 158 GGVYLLPR 18 132 311 246 344 l9l GVYLLPRR l9 613 511 350 229 3?8 ~YLLPRRG 20 769 641 440 280 306 YLLPRRGP 21 332 36? 24~ 177 242 LLP~RGPR 22 142 129 98 lOl 133 PRRGPRLG 24 85 73 l29 109 llO
~RGP~LGV 25 145 85 150 14~ l23 GPRLGVRA 27 lll 60 146 75 lOl P~LGVRAT 28 165 79 155 88 ll5 RLGVRATR 29 81 l80 177 lOO 124 , 3~
~ 18 The amino acid residues are denoted by the single letter code (Eur. J. Biochem. 138, 9-37. 1984). The underlined characters represent the immunodominant regionr Example 3: _Prepar tion of novel_mimic of oriqinal E~itoPe The Replacement Net ~ethod was used to sequentially replace each of the constituent amino acid residues in the epitope with each of the other genetically encoded amino acids.

For Replacement Net analysis, a parent sequence is entered into the Epitope Mapping Software provided by Cambridge ~ Research 8ioche~icals, and ~ set of replacemPnt peptides i5 generated. This process was performed for the six amino acid residue immunodominant ~ore epitope of HCV. The peptides were synthesized on polypropylene pins as directed by the manufacturer (CRB). The Assay format was identical to that ::
followed for PEPSCAN above.
~he Replacement Net of the core immunodominant epitope sequence Tyr-Leu-Leu-Pro-Arg-Arg (Y~LPRR~ (SEQ ID NO: 1 AND 2) wi~h the 12 paid donor human sera as described above was considered.
:~
For each position within the epitope, the frequency of each replacement residue as an acceptable replacement for the parent s~quence is given. Replacement residues were deemed acceptable when the signal of the new peptide was 80% of the 30 parent sequence or greater. ~:

Based on these results, the substitute amino acid residues at each position, which are most li~ely potentiating substitutions, can be obtained. These are shown in Table 2 The residue in italics is the parent sequence.

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o ~ o In WO93/17111 ~J 1 3 ~ ~ 2 ,3 PCT/GBg3/~lo Example 4: Preparation of AC 22 Fully protected peptide-resin was assembled on a p~
hydroxymethylphenoxymethyl (HMP~ resin by stepwise solid pha~e synthesis (proceeding from the carboxyl terminal residue towards the amino terminal r sidue) according to the general procedure set out in Applied Biosystems User Bulletin No 33, November 1990. HMP resin was transferred into the reaction vessel of an Applied Biosystems Peptide Synthesizer Model 431A.
The carboxyl-terminal amino acid residue was coupled to the resin using the standard Applied Biosystems Fastmoc cycle protocol. Unreacted coupling sites on the resin were th~n blocked by capping with benzoic anhydride. All subsequent ~ amino acids were then added in a stepwise manner moving from the carboxyl terminus to the amino terminus using ~he Fastmoc cycle protocol. The N-~-a~ino group of all peptides was protected by the 9-fluorophenylmethoxycarbonyl (Fmoc) l~nkage.
Side chain functional groups of t~e relevant amino acids were protected by the following groups : 20 Cys-Trt t~rityl) G~y-Trt (Trityl) Tyr-tBu (Tert. Butyl~
Arg-Pmc t2,2,5,7,8-pentamethylchroman-6-sulphonyl) The fully protected peptide-resin was transferred to a pear-sh~ped flask and tre~ted with ethanedi~hiol (0.25 ml~, thio~nisole (0.5 ml), water (0.5 ml) and TF~ ~8~75 ml~. The mixture was stirred for 90 minutes at room temperature, then filtered through glass wo~l in a Pasteur pipette. The filtrate was dropped int~ ice-cold ether, contained in a Corex centrifuge tube, whereupon the peptide precipitated. The tub~
was centrifuged and the liquor aspira~ed to leave the peptide pellet in the tube. The peptide was washed with ether (usin~
a spatula to dis-agqregate the pellet and the peptide recovered by centrifugation. This was repeated a total of six times.
The peptide was ~inally dried by centrifugation in vacuo.

W093/17111 - PCT/GB93/~10 ~ 3~ 22 ExamPle 5: Vaccine Formulation ~ vaccine formulation may be prepared by conventional techniquPs using ~he follcwing constituents in the indicated amounts.

HCV viral peptide > 0.36 mg ~whether alone, as a longer :~
polypeptide or with a cocktail of other peptides) Thiomersal 0.04 - 0.2 mg Sodium Chloride c 8.5 mg -.
Water < 1 ml ~ ~ ' .

~'dl~ 1 02~

-SEQUENCE LISTI~G

(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) ~AME: The Wellcome Foundation Limited tB) STREET: Unicorn House, 160 Euston Road, (O~ ~ITY: London (E) COUNTRY: Great Britain (F) POSTAL CODE (ZIP): N~l 2BP
(ii) TITLE OF INYENTION: Novel Peptides (iii) NUMBER OF SEQUENCES: 2g ~iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk . (B) COMPUTER: IBM PC compatible 2 ~ . (C3 OPERA~ING SYSTEM: PC-~OS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Version #1.25 (EPO) ~:~
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 18 base pairs -(B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D~ TOPOLOGY: linear :
(ii) MOLECULE TYPE: ~DNA

(ix) EEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.. 18 ~.
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Tyr Leu Leu Pro Arg Arg ~:
l 5 (2) INFORMATION FOR SEQ ID NO: 2: :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) ~YPE: amino acid (D) TOPOLOGY~ ear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Tyr Leu Leu Pro Arg Arg l 5 W O ~3/17111 PCT/GB93/0041Q~

~ 24 (2~ INFORMATION EOR SEQ ID NO: 3: .. -`
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 6 amino acids ~-(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ~:

(ix) FEATURE: ~:
(A) NAME/KEY: Modified-site (B) LOCATION: one-of(l) :-(D) OTHER INFORMATION: Inote= "Xaa represents Tyr, Asp, : :
Glu, Leu or Ile" `
(ix) FEATURE: :.
~A) NAMEIKEY: Modified-site - (B) LOCATION: one-of(2) . (D) OTHER INFORMATION: fnote= "Xaa represents Leu, Cys, Asp or Glu" ::~
. .
(ix~ FEATURE: .
- (A) NAME/KEY: Modified-site (B) LOCATION: one-of(3) ~D) OTHER INFORMATION- /note= "Xaa represents Leu or Yal"
(ix) FEATURE: -.
(A~ NAME/KEY: Modified-site (B) LOCATION: one-of~4) :~
(D3 OT~ER INFORMATION: /note= "Xaa represents Pro or Met" ~-~
:
(ix) FEATURE: ~:
(A) NAME/KEY: Modified-site :~.
(B) LOCATION: one-of(5) :~
- (D) OTHER INFORMATION: /note= "Xaa represents Arg, Asp, Glu, Pro, Cys, Phe, Ile, Asn, Gln or Met" .~
(ix) FEATURE: :
(A) NAMEIKEY: Modified-site (B) LOCATION: ~ne-of~6) (D) OTHER INFORMATION: /note= "Xaa represen~s Arg, Asp, Glu, Ser or Cys"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Xaa Xaa Xaa Xaa Xaa Xaa l 5 W O 93/17111 ~ PCT/GB93/00410 3 ~ Q
~5 ~2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single - -(D) TOPOLOGY: linear -:
(ii) MOLECULE TYPE: cDNA
1~ :
(ix) FEATURE: ~ -(A) NAMEIKEY: CDS `;
(B) LOCATION: 1..51 lS ~D) OTHER INFORMATION: /note= "Nucleotides 1-3 are of ~r`
non-HCY origin" -~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: .
~^ TGC CAG ATC GTT GGT GGA GTT TAC TTG TTG CCG CGC AGG GGC CCT AGA 48 Cys Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg l 5 l0 15 Leu -~ ...
30 (2) INFORMATION FOR SEQ ID NO: 5: ~ :
(i) SEQUENCE CHARACTERISTICS:
(A) LENCTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY~ ear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Cys Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg l 5 l0 15 Leu ~.

,9~ :

(2) INFORMATION FOR SEQ ID NO: 6:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs ~-(B) TYPE: nucleic acid . - :
(C) STRANDEDNESS: single : -~
(D) T~POLOGY: linear ~
(ii) MOLECULE TYPE: cDNA .~.
.,;,., (ix) FEATURE:
(A~ NAME/KEY: CDS
. (B) LOCATION: l.. 51 ~- :
(D) OTHER INFORMATIQN: /note= "Nucleotides 49 to 51 are of ~ -:
non-HCV origin" ~- .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ~-2~

Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu l 5 l~ 15 C~s 30 (2) INFORMATION FOR SEQ ID NO: 7: :
~i) SEQUENCE CHARACTERISTICS: ::
(A) LENGTH: 17 amino acids (B) TYPE: amino acid ~-. 35 ~D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein :::
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu l 5 lO l~ ;
Cys ~YO 93/17111 P~T/GB93/00410 (2) INFO~MATION FOR SEQ ID NO: 8: `~
~i) SEQUENCE CHARACTERISTICS- ~:
~A~ LENGTH: 97 amino acids (B~ TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ~xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 8~
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn l 5 10 l5 --.
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly ~Q, Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 ~-~
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 - 55 60 ~ ~:
-~
Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ths Trp Ala ~ln Pro Gly 65 70 75 ~ 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp Leu (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHAXACTERISTICS:
(A) LENCTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unkDown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Gly Val Tyr Leu Leu Pro Arg Arg l 5 W ~ 93/t7111 3~ PCT/GB93/00410 (2~ INFORMATION FOR SEQ ID NO: lO:
(i) SEQUENCE CHARACTERISTICS
~A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown -(ii) MOLECULE TYPE: peptide ~xi) SEQUENCE ~ESCRIPTION: SEQ ID ~0: lO: ;~
Val Tyr Leu Leu Pro Arg Arg Gly l 5 `:~
(2) INFORMATION FOR SEQ ID NO: ll~
(-i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 8 amino acids (B~ TYPE: amino acid ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO~
Tyr Leu Leu Pro Arg Arg Gly Pr~
l 5 (2) INFORMATION FOR SEQ ID NO: l~:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ~ .

(xi) SEQUENCE DESCRIPTIO~: SEQ ID NO: 12:
Gly Gly Gly Gln Ile Val Gly Gly l 5 ::

WO 93~17111 PCT~GB93/00410 3~

(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ~ :~
10' (xi~ SEQUE~CE DESCRIPTION: SEQ ID NO: 13 Gly Gly Gln Ile Val Gly Gly Val -~
l 5 (2) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS: . ~
20~, (A) LENGTH: ~ amino acids O
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptid~ -~5 ~xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 14 Gly Gln Ile Val Gly Gly Val Tyr (2) INFORMATION FQR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B~ TYPE: amino acid (D~ TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
~5 ::
Gln Ile Val Gly Gly Val Tyr Leu l 5 W O Q3/171tl PCTJ~B93J00410 ~ 30 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
~A~ LENGTH: 8 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: unknown -~
~ii) MOLECULE TYPE: peptide 10' -(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: ~
Ile Yal Gly Gly Yal Tyr Leu Leu --(2) INEORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
20~. (A) LENGTH: 8 amino aci~s tB) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLEGULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Val Gly Gly Yal Tyr Leu Leu Pro (2~ INFORMATION FOR SEQ ID NO: 18:
(i) 5EQUENCE CHARACTERISTIGS:
~A) LENGTH: 8 amino acids (B) TYPE: amino aciZ
(D) TOPOLOGY: unknown (ii~ MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME~KEY: Region (B3 LOCATION: 4.. 8 (D) OTHER INFORMATION: Inote= "Immunodominant region"

(xi3 SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Gly Gly ~al Tyr Leu Leu Pro Arg W 0 ~3/17111 ~ J 3 1 ~ ~ J PCT/GB93/OW10 (2) INFOR~ATION FOR SEQ ID NO: 19: ::
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 8 amino acids (B) TYPE: amino acid :
~D) TOPOLOGY: unknown ~-:
tii) MOLECULE TYPE: peptide 10' (ix) FEATURE:
(A) NAMEIKEY: Region (B) LOCATION: 3..B
(D) OTHER INFORMATION: /note= "Immunodominant region" ~ -(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Gly Val Tyr Leu Leu Pro Arg Arg 20~, 1 5 (~) INFORMATION EOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids (B) TYPE: amino acid (D) TOPOL05Y: unknown (ii~ MOLECULE TYPE: peptide (ix~ FEATURE:
(A) NAME/KEY: Region (B) LOCATION: 2..7 (D) OTHER INFORMATION: /note= "Immunodominant region"

.
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: :
Val Tyr Leu Leu Pro Arg Arg Gly ~:

~3~ PCT/GB~3~00410 ~2) INFORMATION EOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS: ::
(A) LENGTH: 8 amino acids :~
~B) TYPE: amino acid :~
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAMEIKEY: Region `
(B) LOCATION: 1..6 (D) OTHER INFORMATION: /note= "Immunodominant region"

~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: ~:
Tyr Leu Leu Pro Arg Arg Gly Pro 2~0~.~ 1 5 : ~`
(2) I~FORMATION FOR SEQ ID NO 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D~ TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide :

(xi) SEQ'JENCE DESCRIPTION: SEQ ID NO: 22:
Leu Leu Pro Arg Arg Gly Pro Arg :`

(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids ~::
(B~ TYPE: amino acid .~ :
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: pep~ide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
~.
Leu Pro Arg Arg Gly Pro Arg Leu 1 5 .

WO 93/17111 ~ ~ PCI`/GB93~0041U

(2) INFOR~lATION FOR SEQ ID NO: 24.
~i) SEQUENCE CHARACTERISTICS.
(A~ LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: un~nown (ii) MOLECULE TYPE: pepcide (xi) SEQUENCE DESCRIPTION: SEQ ID NQ: 24:
Pro Arg Arg Gly Pro Arg Leu Gly l 5 (2~ I~FORMATION FOR. SEQ ID NO: 25:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids ~~ (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) ~OLECULE TYPE. pep~ide (xi) SEQUENCE DESGRIPTION: SEQ ID NO: 25:
Arg Arg Gly Pro Arg Leu Gly Val (2) INFORMATION FOR 5EQ ID NO: 26 (i) SEQUENCE CHARACTERISTICS: -`
~A) LENGTH: 8 amino aoids ~8) TYPE: amino arid ~:
(D3 TOPOLOGY~ unknown f (ii) MOLECULE TYPE: pep~ide (xi) SEQUE:NCE ~ESGRIPTION: SEQ ID NO: 26:
Arg Gly Pro Arg l,eu Gly Val Arg ~3~' 34 (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE C~ARACTERISTICS: ~:
(A) LENGTH: 8 amino acids :-(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ~xi) SEQUE~CE DESCRIPTION: SEQ ID NO: 27:
Gly Pro Arg Leu Gly Val Arg Ala t2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
20~. (A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: pep~ide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Pro Arg Leu Gly Val Arg Ala Thr (2) INFORMATION FOR SEQ ID NO: 29: ~ `~
(i) SEQUENCE CHARACTERISTICS~
~A) LENGTH: 8 amino acids . -~
(B) TYPE: amino acid (D) TOPQLOGY: unknown (ii) ~OLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2g:
-Arg Leu Cly Yal Arg Ala Thr Arg

Claims (15)

Claims

1. A HCV viral peptide having the sequence:
(SEQ ID NO's: 1 and 2)

2. A peptide having the sequence of the general formula I
X1-X2-X3-X4-X5-X6 (SEQ ID NO: 3) I
wherein;
X1 represents Tyrosine, Aspartic Acid, Glutamic Acid, Leucine or Isoleucine;
X2 represents Leucine, Cysteine, Aspartic Acid or Glutamic Acid;
X3 represents Leucine, Valine;
X4 represents Proline or Methionine;
X5 represents Arginine, Aspartic Acid, Glutamic Acid, Proline, Cysteine, Phe ylalanine, Isoleucine, Asparagine, Glutamine or Methionine; and X6 represents Arginine, Aspartic Acid, Glutamic Acid, Serine or Cysteine and wherein the peptide is not of the formula (SEQ ID NO: 1 or 2).

3. A viral peptide having as set forth in SEQ ID NO's: 4 and 5 or SEQ ID NO's: 6 and 7.

4. A DNA sequence encoding a peptide as claimed in claim 1, 2 or 3.

5. A DNA sequence as claimed in claim 4 as set forth in SEQ
ID NO: 1, 4 or 6.

6. An expression vector containing a DNA sequence as claimed in claim 4 or 5 being capable in an appropriate host of expressing the DNA sequence to produce a peptide.

7. A host cell transformed with an expression vector as claimed in claim 6.

8. A process for preparing a peptide as claimed in any of claims 1 to 3 which comprises isolating the DNA sequence, as set forth in SEQ ID NO: 1, 4 or 6, from the HCV genome, or synthesising a DNA sequence encoding the peptide, as set forth in SEQ ID NO: 1, 4, 6, or generating a DNA sequence encoding for the peptide of Formula I, inserting the DNA sequence into an expression vector such that it is capable, in an appropriate host, of being expressed, transforming an host cell with the expression vector, culturing the transformed host cell, and isolating the peptide.

9. A process for preparing the peptide as claimed in any of claims 1 to 3 characterized in that it is prepared synthetically.

10. A polyclonal or monoclonal antibody against a peptide as claimed in any of claims 1 to 3.

11. A method of the detection of HCV viral nucleic acid which comprises hybridizing viral RNA present in the test sample or cDNA synthesized from such RNA, with a DNA sequence corresponding to SEQ ID NO: 1, 4, 6, or encoding the peptide of formula I and screening the resulting nucleic acid hybrids to identify any HCV viral nucleic acid.

12. A method for the detection of HCV viral antibody which comprises contacting a test sample with a peptide as claimed in any of claims 1 to 3 or a polyclonal or monoclonal antibody as claimed in claim 10 and determining whether there is any antigen-antibody binding contained within the test sample.

13. A test kit for the detection of HCV viral antibody which comprises the peptide as claimed in any of claims 1 to 3 or a polyclonal or monoclonal antibody as claimed in claim 10 and means for determining whether there is any antigen-antibody binding contained in the test sample.

14. A vaccine formulation which comprises a peptide as claimed in any of claims 1 to 3 in association with a pharmaceutically acceptable carrier.

15. A method for inducing immunity in man to HCV which comprises the administration of an effective amount of a vaccine formulation according to claim 14.