The present invention relates to an assay method and compounds suitable for use in the treatment of hepatitis C virus infection.

The Hepatitis C virus (HCV) is one of the most important causes of chronic liver disease. In the United States it accounts for about 15 percent of acute viral hepatitis, 60 to 70 percent of chronic hepatitis and up to 50 percent of cirrhosis, end-stage liver disease and liver cancer. Hepatitis C is the most common blood-borne infections in the United States, infecting more than 1.8 percent of the population and causing an estimated 8,000 to 10,000 deaths annually (National Institute of Health Consensus Development Conference Statement: Management of Hepatitis C: June 10-12 (2002); http://www.consensus.nih.gov).

At least 75 percent of patients with acute Hepatitis C ultimately develop chronic infection and most of these patients have accompanying chronic liver disease. Chronic hepatitis C varies greatly in its course and outcome. At one end of the spectrum are patients who, apart from their viral load, have no signs or symptoms of liver disease and completely normal levels of serum liver enzymes. Liver biopsy usually shows some degree of damage, but the degree of injury is usually mild and the overall prognosis may be good. At the other end of the spectrum are patients with moderate to severe symptoms, high HCV RNA in serum and marked elevation of serum liver enzymes. These patients ultimately develop cirrhosis and end-stage liver disease. In the middle of the spectrum are many patients who have few or no symptoms, mild to moderate elevations in liver enzymes and an uncertain prognosis. Researchers estimate that at least 20 percent of patients with chronic hepatitis C develop cirrhosis, a process that can take 10 to 20 years. After 20 to 40 years, a smaller percentage of patients with chronic disease will develop liver cancer.

HCV is a small (50 to 60 nm in diameter), enveloped, positive, single-stranded RNA virus in the Flaviviridae family. The genome is approximately 10,000 nucleotides and encodes a single polyprotein of about 3,000 amino acids. The polyprotein is processed

by host cell and viral proteases into three major structural proteins and several non¬ structural protein necessary for viral replication (Bartenschlager and Lohmann, J. Gen. Virol. (2002) 81, 1631-1648). Because the virus mutates rapidly, changes in the envelope protein are thought to contribute to evasion of the immune system.

The virus displays extensive genetic heterogeneity: there are six known genotypes and more than 50 subtypes of hepatitis C, the different genotypes having different geographic distributions. Genotypes 1a and 1b are the most common in the United States while genotypes 2 and 3 are present in only 10 to 20 percent of patients. There is little difference in the severity of disease or outcome of patients infected with different genotypes. However, this genetic diversity impacts negatively on both treatment options and effectiveness. In particular, genotype 1 , which accounts for >70 percent of all HCV infections in the US and has infected millions of people around the world via contaminated blood transfusions, is associated with the poorest response to treatment. Patients with genotypes 2 and 3 are more likely to respond to alpha interferon treatment

(Zein, CHn. Microbiol. Reviews (2000) 13, 223-235).

Alpha interferon is a host protein that is made in response to viral infections and has natural antiviral activity. Recombinant forms of alpha interferon have been produced, and several formulations are available as therapy of hepatitis C. However, these standard forms of interferon are now being replaced by pegylated interferons (peginterferons). Peginterferon is alpha interferon that has been modified chemically by the addition of a large inert molecule of polyethylene glycol. Pegylation changes the uptake, distribution and excretion of interferon, prolonging its half-life, and therefore is more effective than standard interferon in inhibiting HCV, yielding higher sustained response rates with similar side effects.

Currently the most effective therapy appears to be a 24- or 48-week course of the combination of pegylated alpha interferon and ribavirin, an oral antiviral agent that has activity against a broad range of viruses. By itself, ribavirin has little effect on HCV, but adding it to interferon or peginterferon increases the sustained response rate by two- to three-fold. The optimal duration of treatment depends on viral genotype. Patients with

genotypes 2 and 3 have a high rate of response to combination treatment (70 to 80 percent). In contrast, patients with genotype 1 have a lower rate of response to combination therapy (40 to 45 percent).

Alpha interferon has multiple neuropsychiatry effects and strict abstinence from alcohol is also recommended during therapy with interferon. Prolonged therapy can cause marked irritability, anxiety, personality changes, depression and even suicide or acute psychosis.

Alpha interferon therapy can induce auto-antibodies and a 6- to 12-month course triggers an autoimmune condition in about 2 percent of patients, particularly if they have an underlying susceptibility to autoimmunity. Exacerbation of a known autoimmune disease (such as rheumatoid arthritis or psoriasis) occurs commonly during interferon therapy. Alpha interferon also has bone marrow suppressive effects.

Ribavirin causes red cell haemolysis to a variable degree in almost all patients. Therefore, patients with a pre-existing haemolysis or anaemia should not receive ribavirin. Similarly, patients who have significant coronary or cerebral vascular disease should not receive ribavirin, as the anaemia caused by treatment can trigger significant ischaemia. Fatal myocardial infarctions and strokes have been reported during combination therapy with alpha interferon and ribavirin. Ribavirin is excreted largely by the kidneys and patients with renal disease can develop haemolysis that is severe and even life-threatening. Ribavirin also causes birth defects in animal studies, while alpha interferon has direct antigrowth and antiproliferative effects.

Few options exist for patients who either do not respond to therapy or who respond and later relapse. Patients who relapse after a course of interferon monotherapy may respond to a course of combination therapy, particularly if they became and remained HCV RNA negative during the period of monotherapy. Another approach is the use of long-term or continual interferon, which is feasible only if the interferon is well tolerated and has a clear-cut effect on serum aminotransferases and liver histology, despite lack of clearance of HCV RNA. Therefore, new medications and approaches to treatment

are needed. Indeed, the success of HIV entry inhibitors provides a compelling case for HCV entry as a source of novel therapeutic targets.

Efforts towards the development of new therapeutic approaches against HCV infection have been hampered by the lack of an efficient, high-throughput culture system. While

HCV replicons, such as those described in EP 1043399, provide a useful alternative for the study of processes involved in HCV genome replication, they do not involve processes linked to HCV maturation or entry.

Cell-based models have been reported by others in scientific publications. However, those that appear to be physiologically relevant require production and purification of viral pseudoparticles, a technology requiring specialist skills. The models are also not easily amendable to high throughput screening. In particular, Hsu et al (PNAS (2003) 100(12), 7271-7276) describe a cell based system using pseudoparticles (also known as pseudotype virus) where the E1E2 nucleotide sequences are inserted into an envelope-defective HIV-1 proviral genome expressing a luciferase reporter gene. Huh cells were infected with pseudotype virus and the effect of antibodies was investigated.

Other cell-based models are "vaccinia" based fusion assays in which the envelope protein of the virion of interest is only transiently expressed in the virion host cell, an example of which is described by Takikawa et al (J. Virology (2000) 74(11 ), 5066-5074). While such vaccinia based cell fusion assays have been useful, they suffer from certain disadvantages. In particular, they are unsuitable for adaptation to high throughput screening.

Successful high throughput screens preferably require stable cell lines. This is in contrast to the vaccinia- or transient transfection-based methods of the prior art in which the generation of a high quantity of virus or plasmids is not compatible with a safe and robust high throughput assay. A limitation with a transfection-based method is the difficulty in obtaining highly reproducible transfections. The use of a stable cell line method, as opposed to a vaccinia-based method, allows the use of a reporter system such as the Tat and HIV LTR reporter system. In a stable cell line, Tat may be

expressed in one cell and, on fusion with a second cell, the Tat protein is able to reach the nucleus of the second cell where it will function by enabling phosphorylation of the carboxy terminal domain of RNA-pol II. A vaccinia-based method is not suitable for use with such a reporter system since the reporter system will be expressed in the cytoplasm. The reporter protein that drives expression from the reporter system will be in a different cellular compartment and so a signal will never be produced.

One of the major hurdles when designing a cell-based assay system for HCV is obtaining cell surface expression of E1 E2 since the glycoproteins are believed to be retained mainly in the endoplasmic reticulum. However, low level cell surface expression of E1 E2 has been reported to be possible, but only in conditions where the E1 and E2 are over-expressed in the cytoplasm (Voisset and Dubuisson, Biology of the Cell (2004) 96 _j , 413-420). Such expression is believed to be due to leakage from the endoplasmic reticulum. However, Deleersnyder et al (J. Virol. (1997), 71(1), 697-704) have shown that when E1 and E2 are over expressed, the glycoproteins have a high tendency of misfolding and, therefore, to be non functional.

Based on similarities with the fusion proteins of the flaviviruses and the members of the Togaviridae family, HCV has been proposed to contain a class Il fusion protein (E2). Enveloped viruses penetrate the host cells by a process of fusion between the viral and cell membranes that is catalyzed by a fusogenic activity harboured by viral surface glycoproteins (E1 and E2 for HCV). For the class Il fusion viruses, including HCV, activation of the fusion proteins occurs in an acid pH-dependent manner, via acidified endosomal vesicles into which the virions are routed following receptor binding. Moreover, using HCV pseudo-particles (HCVpp), it has been demonstrated that the fusion-activation of HCV E1 E2 glycoproteins is pH-dependent and that cell entry of HCVpp occurs by endocytosis; i.e. the fusion of HCV virus membrane with the cell membrane occurs inside the cell where an acidification steps occurs before the fusion between the viral and the cellular membrane can occur. In contrast, HIV fusion occurs at the cell surface of infected cells and does not need acidification for infectivity.

Because endocytosis is believed to be feasible for small entities such as viruses and not for large element such as cells, a cell-cell fusion assay has not been deemed possible

using HCV envelope proteins because the envelope proteins are believed to require internalisation before fusion events can occur.

High level cell surface expression of E1 E2 is possible, typically by modifying E1 E2 in such a way that the protein is directed to the cell surface. To date, only two possibilities have been described:

Chimeric E1 E2: Takikawa et al (ibid.) designed a chimeric E1 E2 in which the native transmembrane domains of E1 E2 were replaced with those of Vesicular Stomatitis Virus G protein. Thus, the Takikawa assay uses HCV envelope protein that is not in its natural conformation.

Mutant E1 E2: mutations in the transmembrane domain of E1 and E2 have been shown to allow cell-surface expression of the glycoproteins (Cocquerel et al, J. Virol. (2000) 74(8), 3623-33). However, most mutants do not retain the ability for E1 E2 to form functional proteins when expressed at the cell surface.

It is against this background that the present invention has been conceived. In its broadest sense, the invention encompasses a cell-based assay for HCV-induced membrane fusion. In particular, the present invention relates to an assay method for determining whether an agent is capable of modulating HCV infection, the method comprising (a) contacting the agent with (i) a first cell line expressing E1 and E2 proteins on the cell surface, and (ii) a permissive second cell line, and (b) determining whether the agent inhibits fusion between the first and second cell lines, wherein the E1 and E2 proteins are full length.

For the avoidance of doubt, the term "HCV" as used herein, refers to Hepatitis C virus.

"HCV" is a generic term that embraces individual species and/or genotypes of HCV. "HCV" should thus be understood to include all HCV genome types, particularly 1b and/or 1a as appropriate, unless the context and/or specific statements indicate otherwise. An example of a suitable HCV is a 1 b subtype whose sequence may be found under the accession number AJ238799 (EMBL).

The native envelope proteins E1 and E2 of the present assay are native forms rather than chimeric constructs such as those incorporating the VSV-G transmembrane domain. The native forms are full-length sequences from approximately amino acids 192 to 383 and 384 to 748 respectively within the HCV polyprotein (for example from 192 ± 2 to 383 ± 3 for E1 and from 384 ± 2 to 748 ± 7 for E2). Expression of high-level full-length native E1 and E2 proteins at the surface of the cells unexpectedly leads to cell-cell fusion that occurs at the cell surface, rather than in the low-pH intracellular components. It is to be understood that expression of the E1 and E2 proteins includes the glycosylated forms of the E1 and E2 proteins.

In a preferred embodiment, the E1 and E2 sequences may also include some of the HCV core protein (protein C), for example the last 60 amino acids of the core protein. An example of a suitable nucleotide and corresponding amino acid sequence is given below:

Nucleotide sequence (SEQ ID NO:1): gatctcatggggtacattccgctcgtcggcgcccccctagggggcgctgccagggccctg gcgcatggcgtccgggttctggag gacggcgtgaactatgcaacagggaatctgcccggttgctccttttctatcttccttttg gctttgctgtcctgtttgaccatcccagctt ccgcttatgaagtgcgcaacgtatccggagtgtaccatgtcacgaacgactgctccaacg caagcattgtgtatgaggcagcgga catgatcatgcatacccccgggtgcgtgccctgcgttcgggagaacaactcctcccgctg ctgggtagcgctcactcccacgctc gcggccaggaacgctagcgtccccactacgacgatacgacgccatgtcgatttgctcgtt ggggcggctgctctctgctccgctat gtacgtgggagatctctgcggatctgttttcctcgtcgcccagctgttcaccttctcgcc tcgccggcacgagacagtacaggactg caattgctcaatatatcccggccacgtgacaggtcaccgtatggcttgggatatgatgat gaactggtcacctacagcagccctagt ggtatcgcagttactccggatcccacaagctgtcgtggatatggtggcgggggcccattg gggagtcctagcgggccttgcctac tattccatggtggggaactgggctaaggttctgattgtgatgctactctttgccggcgtt gacgggggaacctatgtgacagggggg acgatggccaaaaacaccctcgggattacgtccctcttttcacccgggtcatcccagaaa atccagcttgtaaacaccaacggcag ctggcacatcaacaggactgccctgaactgcaatgactccctcaacactgggttccttgc tgcgctgttctacgtgcacaagttcaa ctcatctggatgcccagagcgcatggccagctgcagccccatcgacgcgttcgctcaggg gtgggggcccatcacttacaatga gtcacacagctcggaccagaggccttattgttggcactacgcaccccggccgtgcggtat cgtacccgcggcgcaggtgtgtggt ccagtgtactgcttcaccccaagccctgtcgtggtggggacgaccgaccggttcggcgtc cctacgtacagttggggggagaat gagacggacgtgctgcttcttaacaacacgcggccgccgcaaggcaactggtttggctgt acatggatgaatagcactgggttca ccaagacgtgcgggggccccccgtgtaacatcggggggatcggcaataaaaccttgacct gccccacggactgcttccggaag

caccccgaggccacttacaccaagtgtggttcggggccttggttgacacccagatgcttg gtccactacccatacaggctttggca ctacccctgcactgtcaactttaccatcttcaaggttaggatgtacgtggggggagtgga gcacaggctcgaagccgcatgcaatt ggactcgaggagagcgttgtaacctggaggacagggacagatcagagcttagcccgctgc tgctgtctacaacggagtggcag gtattgccctgttccttcaccaccctaccggctctgtccactggtttgatccatctccat cagaacgtcgtggacgtacaatacctgta cggtatagggtcggcggttgtctcctttgcaatcaaatgggagtatgtcctgttgctctt ccttcttctggcggacgcgcgcgtctgtg cctgcttgtggatgatgctgctgatagctcaagctgaggcc

Amino acid sequence (SEQ ID N0:2):

DLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTI PASAYEVRNVSGVYHVTNDCSNASΓVYEAADMIMHTPGCVPCVRENNSSRCWVA

LTPTLAARNASVPTTTIRRHVDLLVGAAALCSAMYVGDLCGSVFLV AQLFTFSPR RHETVQDCNCSIYPGHVTGHRMAWDMMMNWSPTAALVVSQLLRIPQAWDMV

AGAHWGVLAGLA YYSMVGNW AKVLΓVMLLFAGVDGGTYVTGGTMAKNTLGIT SLFSPGSSQKIQLVNTNGSWFFLNRTALNCNDSLNTGFLAALFYVHKFNSSGCPERM ASCSPIDAFAQGWGPITYNESHSSDQRPYCWHYAPRPCGRVPAAQVCGPVYCFTPS

PVVVGTTDRFGVPTYSWGENETD VLLLNNTRPPQGNWFGCTWMNSTGFTKTCG GPPCNIGGIGNKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPYRLWHYPC TVNFΓIFKVRMYVGGVEHRI^AACNWTRGERCNLEDRDRSELSPLLLSTTEWQVL PCSFTTLPALSTGLIHLHQNWDVQYLYGIGSAVVSFAIKWEYVLLLFLLLADARV CACLWMMLLIAQAEA

A permissive cell line is one that permits or is susceptible to HCV entry and/or infection. Examples of such cell lines include Huh7, HepG2-CD81 and MOLT-4, and preferably is a hepatocyte cell line such as Huh7 and its derivatives. The first cell line may be selected from HEK293T, HeLa or any other cell line that will allow high expression of

E1 E2 on the cell surface and that will not form syncytae with permissive cells, and is preferably a HEK293T cell line.

In the assay of the present invention, the inhibition of fusion is understood to be an indication that an agent is capable of modulating HCV infection.

In a preferred embodiment, the E1 and E2 proteins are stably expressed on the cell surface. To achieve this, the first cell line comprises a coding sequence for the proteins that is stably incorporated into the cell's genome such that the proteins are expressed within the cell and directed to the cell surface. Stable expression ensures long-term expression of the proteins and is in contrast to the vaccinia based systems of the prior art. In an alternative embodiment, the envelope proteins are transiently expressed on the cell surface.

Cell-cell fusion may be measured and quantified by any suitable system. For example, since E1 and E2 are capable of supporting syncytium formation, inhibition of fusion may be determined by assaying for syncytium formation, for example using microscopy, such as low power light or phase contrast microscopy, or fluorescence activated cell analysers or sorters (FACS).

A preferred method for determining inhibition of fusion is a reporter system, in particular one in which the first cell line has one component of a stably-expressed, two- component, signal-producing system and the second cell line has the second component. The system provides a detectable signal upon fusion between the first and second cells.

Preferably the system includes a transactivating protein, such as the HIV Tat protein or any homologous protein, and a responsive expression construct, such as a Tat responsive expression construct, that encodes for a reporter. In a preferred embodiment, the Tat protein is HIV-1 Tat and the responsive expression construct is HIV-1 LTR.

The detectable signal may be by way of fluorescent proteins or a reporter enzyme such as beta-lactamase, galactosidase or luciferase. In a preferred embodiment, the detectable signal is the reporter enzyme, luciferase. It is desirable that the detectable signal or reporter enzyme is proportional to the amount of fusion that occurs between the two cell lines. By this it is meant that, for example, a smaller amount signal detected is proportional to the amount of antiviral activity of the agent being tested, such that the

amount of signal has an inverse correlation with the amount of antiviral activity of the agent.

In one configuration, the first cell line stably expresses Tat and the second cell line expresses luciferase driven by an HIVLTR promoter. In an alternative configuration, the first cell line stably expresses luciferase driven by an HIV LTR promoter and the second cell line expresses Tat.

An alternative method for determining whether the agent inhibits fusion may be by monitoring, measuring or detecting the mixing of cell contents or cell membranes. Such methods may involve the mixing of dyes loaded into one or both cell lines of the assay

(or into the cell membranes), the dyes being visually detectable or detectable by fluorescence (including detection of Fluorescent Resonance Energy Transfer or FRET, or detection by a decrease in fluorescence by quenching). Detection may also be achieved by expression of beta-lactamase enzyme in the first cell line while the second cell line is loaded with a dye such as CCF-4 that, on cleavage by beta-lactamase, yields a detectable fluorescent signal. Alternatively, the first cell line may be loaded with dye and the beta-lactamase expressed in the second cell line. Alternative configurations suitable for use in the assay of the present invention will be apparent to a person skilled in the art.

The assay of the present invention has been developed to identify one or more agents capable of modulating HCV infection. Agents may be any chemical entity, including small molecules such as synthetic or natural compounds, polymeric molecules such as oligonucleotides, peptides, polypeptide or protein molecule, macromolecules such as one or more antibodies and fragments thereof.

Such agents may be screened using the assay of the present invention to determine whether they are capable of modulating HCV infection. Modulation may be to enhance, accelerate, increase, stimulate or otherwise promote fusion, or the effect may be to retard, prevent, restrict, reduce or otherwise inhibit fusion. An agent may affect the rate of fusion, or may affect its equilibrium, or may affect the final ration of fused to unfused

cells, or may affect any combination of such factors. Preferred agents according to the invention have the effect of inhibiting and/or reducing fusion, whether by prevention, restriction, down-regulation or any other mechanism.

The Applicant has identified, and from another aspect the present invention relates to, an isolated peptide which is a subsequence of HCV E1 or E2, or a fragment, variant or homologue of such a subsequence, and which inhibits fusion in the assay of the present invention.

In one embodiment, the isolated peptide comprises a motif corresponding to the E1 fusion peptide region, the fusion peptide region which has the amino acid sequence CSAMYVGDLC (SEQ ID NO:6). Advantageously, the motif is flanked by a sequence or sequences corresponding to the sequence or sequences flanking the native E1 fusion peptide region.

A particular motif corresponds to the fusion peptide region if the motif is functionally equivalent to that region such that the motif acts as a mimic of the fusion peptide region in the natural fusion mechanism of HCV infection. Therefore, a motif that is identical to the full-length region, or to a truncated fusion peptide region, or to a variant or homologue of the full length of truncated region is encompassed, provided it has that functionality.

In a preferred embodiment, the isolated peptide contains a sequence that is at least 70% identical to all or part of amino acids 81 to 91 of the E1 amino acid sequence. Preferably the isolated peptide has 20 to 30 amino acids. An example of a suitable peptide is one having the sequence LVGAAALCSAMYVGDLCGSVFLVAQ (SEQ ID NO:3).

In another embodiment, the isolated peptide comprises a motif corresponding to the E2 hydrophobic heptad repeat region, the heptad repeat region having the sequence

LPCSFTTLPALSTGLIHLHQNIVDV (SEQ ID NO:7). Advantageously, the motif is

flanked by a sequence or sequences corresponding to the sequence or sequences flanking the native hydrophobic heptad repeat region.

A particular motif corresponds to the hydrophobic heptad repeat region if the motif is functionally equivalent to that region such that the motif acts as a mimic of the hydrophobic heptad region in the natural fusion mechanism of HCV infection. Therefore, a motif that is identical to the full length region, or to a truncated fusion peptide region, or to a variant or homologue of the full length of truncated region is encompassed, provided it has that functionality.

In a preferred embodiment, the isolated peptide contains a sequence that is at least 70% identical to all or part of amino acids 292 to 315 of the E2 amino acid sequence. Preferably the isolated peptide has 20 to 30 amino acids. Examples of suitable peptides are peptides having the sequence: QVLPCSFTTLPALSTGLIHLHQNVV (SEQ ID NO:4) or

HLHQNVVDVQYLYGIGSAVVSFAIK (SEQ ID NO: 5)

From another aspect, the present invention encompasses an isolated peptide having an amino acid sequence selected from the group comprising: LVGAAALCSAMYVGDLCGSVFLVAQ (SEQ ID NO:3);

QVLPCSFTTLPALSTGLIHLHQNVV (SEQ ID NO:4) or

HLHQNVVDVQYLYGIGSAVVSFAIK (SEQ ID NO: 5) and variants, fragments and homologues thereof.

The terms "fragment", "variant" and "homologue" include any substitution, variation, modification, replacement, deletion or addition of one or more amino acid (for example, up to 30%, up to 20%, up to 10%, up to 5% or up to 2 or 3% of the amino acids in the parent sequence) from or to the sequence, providing the resultant peptide has activity in the assay of the invention. In particular, a homologue has close identity with respect to the parent sequence structure and/or function. With respect to sequence homology, there may be at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%

homology to the sequences shown in SEQ ID NOS: 3 to 5. Most preferably there may be at least 98% homology to the sequences shown in SEQ ID NOS: 3 to 5.

"Identity" refers to sequence identity between two peptides or between two nucleic acid molecules. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acids at positions shared by these sequences. Since two polynucleotides may each (1) comprise a sequence (i.e. a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", refers to a conceptual segment of at least twenty contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least twenty contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of twenty percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1972), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Solftware Package Release 7.0, Genetics Computer Group, 575, Science Dr. Madison, W1), or by inspection. The best alignment (i.e., resulting in the highest percentage of identity over the comparison window) generated by the various methods is selected. The term

"sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.

Typically, for the variant, homologue or fragment of the present invention, the types of amino acid substitutions that may be made may be of a conserved or non-conserved nature and should maintain the hydrophobicity/hydrophilicity of the amino acid sequence. Conserved amino acid substitutions consist of replacing one or more amino acids with amino acids of similar charge, size and/or hydrophobicity characteristics, such as a glutamic acid (E) to aspartic acid (D) amino acid substitution. Non-conserved substitutions consist of replacing one or more amino acids possessing dissimilar charge, size and/or hydrophobicity characteristics, such as a glutamic acid (E) to valine (V) substitution. Amino acid substitutions may be made, for example from 1 , 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains its antiviral activity in accordance with the present invention. Amino acid substitutions may include the use of non-naturally occurring analogues.

Amino acid insertions may consist of single amino acid residues or stretches of residues. The insertions may be made at the carboxy or amino terminal end of the peptides, as well as at a position internal to the peptide. Such insertions will generally range from 2 to 10 amino acids in length. One or more such insertions may be introduced into the peptide sequences of the present invention provided such insertions result in peptides that still exhibit antiviral activity.

Deletions or truncations are also contemplated within the scope of the present invention. Such deletions consist of the removal of one or more amino acids from the

^■ sequences of the present invention. Such deletions may involve a single or contiguous or greater than one discrete portion of the peptide sequences. One or more deletions may be introduced, provided the resulting peptide exhibits antiviral activity.

Derivatives of the peptides of the invention are also contemplated. Examples of such derivatives include peptides that have been modified, for example to enhance solubility

or formulation. Examples of modifications are the attachment of fusion sequences or pegylation.

The peptides of the invention may also be used as inhibitors of viral membrane fusion- associated events, in particular HCV. In other words, the peptides may also be used to inhibit or reduce the level of membrane fusion between two or more cells relative to the level of membrane fusion that occurs between the cells in the absence of the peptide.

The present invention also contemplates a pharmaceutical composition including a peptide of the invention, or variants, fragments and homologues thereof, together with one or more pharmaceutically acceptable excipients, diluents or carriers. The pharmaceutical composition may include one or more additional therapeutic agents.

Also encompassed by the invention is the use of the peptides of the invention, or a composition thereof, in the manufacture of a medicament for the treatment of HCV infection. Alternatively, the invention encompasses a method of treatment of a mammal suffering from HCV infection which comprises treating said mammal with a therapeutically effective amount of a peptide of the invention, or a pharmaceutical composition thereof.

Use of the peptides in an assay, such as a competition binding assay, to identify antiviral compounds that may be useful in the treatment of HCV infection is also contemplated.

The term "treating", as used herein means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above.

The phrases "therapeutically effective amount" and "effective amount" are intended to mean the amount of an inventive agent that, when administered to a mammal in need of

treatment, is sufficient to effect treatment for injury or disease conditions alleviated by the inhibition of HCV infection. The amount of a given HCV-inhibiting agent used in the method of the invention that will be therapeutically effective will vary depending upon factors such as the particular HCV-inhibiting agent, the disease condition and the severity thereof, the identity and characteristics of the mammal in need thereof, which amount may be routinely determined by artisans.

Administration of the peptides of the invention and pharmaceutical compositions thereof may be performed according to any of the accepted modes of administration available to those skilled in the art. Illustrative examples of suitable modes of administration include oral, nasal, parenteral, topical, transdermal, rectal, inhalation, injection and implantation.

Pharmaceutical compositions of the present invention may be administered in any suitable pharmaceutical form, including solid, semisolid, liquid, or lyopholized formulations, such as tablets, powders, capsules, suppositories, suspensions, liposomes, and aerosols.

Acceptable methods of preparing suitable pharmaceutical forms of the pharmaceutical compositions are known or may be routinely determined by those skilled in the art. For example, pharmaceutical preparations may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate, to give the desired products for oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural, and/or rectal administration.

Pharmaceutical compositions of the invention may also include suitable excipients, diluents, vehicles, and carriers, as well as other pharmaceutically active agents, depending upon the intended use. Solid or liquid pharmaceutically acceptable carriers, diluents, vehicles, or excipients may be employed in the pharmaceutical compositions. Illustrative solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba,

sucrose, talc, gelatin, pectin, acacia, magnesium stearate, and stearic acid. Illustrative liquid carriers include syrup, peanut oil, olive oil, saline solution, and water. The carrier or diluent may include a suitable prolonged-release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., solution), or a nonaqueous or aqueous liquid suspension.

In view of the biological nature of peptides, specific formulation is typically required to prevent digestion in the stomach or intestines. For example, peptides may be formulated with excipients such as cyclic lactones and cyclic ketones which enhance permeability of the skin or mucosa, while iontophoretic and piezoelectric devices may be used for transdermal delivery. Dispersal of peptides in liposomes, microvesicles, or micelles, or encapsulation in nanoparticles enables administration by a variety of routes, including transdermal, transmucosal, intranasal and intrabuccal.

Bioadhesive polymers formulated with peptides, or applied as coatings to standard encapsulation, may be used to retain a therapeutically effect dose within the mouth, throat and/or oesophagus where the peptide may be absorbed. Alternatively, starch, gel-based lozenges that dissolve in the mouth may be used. Coating of peptides with an acid-resistant vehicle, carrier or coating also facilitates oral absorption.

Lyophilisation, or spray drying, or dispersal with nano-particulate additives or GRAS (Generally Recognised As Safe) excipients, such as magnesium stearate, lecithin, hyaluronic acid, octyl phenoxypolyethoxyethanol, glycolic acid, lactic acid, citric acid, organic acids, amorphous glass-forming materials especially polyhydroxy compounds, or leucine enable intra-oral delivery via inhalation. Examples of polyhydroxy compounds are sugars, such as sucrose, trehalose and lactose, or carbohydrate polymers, such as dextran, inulin and polyhydric alcohols (e.g. mannitol). Other glass-forming materials include proteins such as albumin and hydrolysed gelatine, as well as polymers such as polyvinyl pyrrolidine) (PVP).

Encapsulation of peptides within biodegradable, non-silicone-based, hydrophilic polymer matrices, foams and microspheres, or printing onto microchip devices enables implantation, or administration by injection, and slow release over time. Such administration may be site-directed. For example, the therapeutic dose may be administered directly to the liver. Examples of suitable polymers include poly(lactic acid- co-glycolic acid), polyactide co-glycolide, poly(DL-lactide-co-glycolide, poly(DL-lactide), poly(DL-lactide-co-caprolactone), or polyvinyl pyrrolidine). An example of a suitable hydrogel is the thermo-sensitive ReGel® injectable system. Below body temperature, ReGel® is an injectable liquid while at body temperature it forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

The attachment of polyethylene glycol (PEG; pegylation) enhances the size of a peptide as well as increasing its half life within the body. An alternative to PEGylation is polysialic acid. A similar method of formulation is fusion of peptides to the Fc or CH region of human antibodies, such as IgGI and lgG4.

A dose of the pharmaceutical composition may contain at least a therapeutically effective amount of one or more peptides of the present invention and preferably is made up of one or more pharmaceutical dosage units. The selected dose may be administered to a mammal, for example, a human, in need of treatment mediated by inhibition of HCV activity, by any known or suitable method of administering the dose, including topically, for example, as an ointment, cream or transdermal patches or devices; orally; rectally, for example, as a suppository; parenterally by injection or implant; intravenously; subcutaneously; intramuscularly; or continuously by intravaginal, intranasal, intrabronchial, intraaural, or intraocular infusion.

Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be apparent, to those skilled in this art. For examples, see Remington's Pharmaceutical Sciences. Mack Publishing Company, Easter, Pa., 15 ^th

Edition (1975).

It will be appreciated that the actual dosages of the peptides used in the pharmaceutical compositions of this invention will be selected according to the properties of the particular peptide being used, the particular composition formulated, the mode of administration and the particular site, and the host and condition being treated. Optimal dosages for a given set of conditions can be ascertained by those skilled in the art using conventional dosage-determination tests. For oral administration, e.g., a dose that may be employed is from about 0.001 to about 1000 mg/kg body weight, or from about 0.1 to about 100 mg/kg body weight, or from about 1 to about 50 mg/kg body weight, or from about 0.1 to about 1 mg/kg body weight, with courses of treatment repeated at appropriate intervals. The dosage forms of the pharmaceutical formulations described herein may contain an amount of a peptide of the present invention deemed appropriate by one of ordinary skill in the art. For example, such dosage forms may contain from about 1 mg to about 1500 mg of a peptide of the present invention, or may contain from about 5 mg to about 1500 mg, or from about 5 mg to about 1250 mg, or from about 10 mg to about 1250 mg, or from about 25 mg to about 1250 mg, or from about 25 mg to about 1000 mg, or from about 50 mg to about 1000 mg, or from about 50 mg to about 750 mg, or from about 75 mg to about 750 mg, or from about 100 mg to about 750 mg, or from about 125 mg to about 750 mg, or from about 150 mg to about 750 mg, or from about 150 mg to about 500 mg of a peptide of the present invention.

The subject invention also includes isotopically-labelled peptides, which are identical to those recited in the peptides of the present invention, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into peptides of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ CI, respectively. Peptides of the present invention and prodrugs thereof which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled peptides of the present invention, for example those into which radioactive isotopes such as ³ H and ¹⁴ C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³ H, and carbon-14, i.e., ¹⁴ C, isotopes are particularly preferred for their

ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ² H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances, lsotopically labelled peptides of the present invention and prodrugs thereof can generally be prepared by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

The active peptides may be applied as a sole therapy or may involve one or more other antiviral substances, for example those selected from, for example, HCV inhibitors such as interferon alphacon-1 , natural interferon, interferon beta-la, interferon omega, interferon gamma-1b, interleukin-10, BILN 2061 (serine protease), amantadine

(Symmetrel), thymozine alpha-1 , viramidine. Other antiviral substances include HIV inhibitors such as nelfinavir, delavirdine, indinavir, nevirapine, saquinavir, and tenofovir. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.

The present invention will be further described by way of example and with reference to the following figures: Figure 1 : E1 and E2 expression at the cell surface; cell surface levels of E1 and E2 were measured by FACS scan using either HCM-081-5 antibody (recognises E1) or AP33 antibody (recognises E2) on a HEK239T parental cell line that does not express E1 E2 (Figure 1A) or on a clone, HEK293TcE1 E2TAT6 known to express E1 E2 at the cell surface (Figure 1 B), under the following condition: Shaded histogram: cells were incubated with no antibody,

Mid grey line: cells incubated with a phycoerythrin (PE) labelled secondary antibody only,

Dark grey line: cells incubated with an anti-E1 antibody and a PE-labelled secondary antibody that recognise the anti-E1 antibody, Pale grey line: cells incubated with AP33 and a PE-labelled secondary antibody that recognises AP33;

Figure 2: Schematic principle of HCV cell-cell fusion assay; The HCV fusion assay is based on either the transient or stable expression of E1 E2 and Tat in HEK293T cell line (or any other cell line easy to transfect). In this assay, E1 E2 envelope proteins need to be expressed at the cell surface. The corresponding cells are then mixed together with a permissive cell line (e.g. Huh7) expressing a reporter

(luciferase in this example) under the control of HIV-1 LTR. In this system an HIV- LTR-reporter stable cell line would have been selected for a minimal expression of the reporter in absence of the transactivator Tat and for an up-regulation in the presence of Tat. If the two cell lines fuse, Tat (expressed in HEK293T cell background) can reach and transactivate HIV LTR (expressed in the Huh7 cell background), resulting in the expression of the reporter;

Figure 3: Correlation between E2 cell surface expression and fusion activity; HEK293T cells were transfected with E1 E2 (strain 1a). Thirty-six hours post- transfection, selection medium (zeocin) was added to the cells. Approximately six weeks later, clones grown from individual cells were selected and subjected to

FACS analysis for E2 expression. Twelve clones were also analysed for their respective fusion activity using the HCV cell-cell fusion assay. The plot shows a correlation between the log E2 expression and the log fusion activity for these twelve HEK293T-E1 E2 clones; Figure 4: Native E1 E2 is needed for HCV cell-cell fusion activity; HEK293T cells were transiently transfected with Tat and either the empty vector (pBud, used as a negative control), a vector expressing Vesicular Stomatitis Virus envelop protein G (pVSV-G, used as a positive control), a vector expressing mutated E1 E2 (pBudcE1 E2 (MLJTpmut), E1 E2 with two point mutations in the trans-membrane domain), a vector expressing native E1 E2 from strain 1b (pBudcE1 E2, native

E1E2) or a vector expressing chimeric E1 E2 envelop protein (pBudVSVGtmd- E1 E2, E1 E2 proteins with the authentic transmembrane domain of E1 E2 replaced with that of VSV-G protein). The resulting cells were mixed respectively with Huh7 cells expressing HIV-1 LTR-luciferase. This experiment was carried out in triplicate and the measure of the resulting luciferase expressions shown in the histogram includes the standard deviations;

Figure 5: Cell-cell fusion activity is modulated by the pH of culture medium used to cultivate the cells expressing E1E2; Four clones expressing E1E2 at the cell surface (the parental HEK293TcE1 E2c1.5 clone does not expresses Tat, while the three clones HEK293TcE1 E2TAT6, HEK293TE1 E2cE1 E2TAT10/1/14 and HEK293TcE1 E2TAT 10/2/7 stably express Tat) have been grown in two different mediums (similar medium adjusted to either pH5.5 or pH7.0) for two days. The resulting cells were mixed with Huh7-LTR-luciferase and, two days later, luciferase expression was measured. The data is plotted in a histogram showing that fusion activity is enhanced when the cells expressing E1 E2 are grown at low pH; Figure 6: screening for cells permissive for fusing with HEK293TE1 E2 cell line;

Seven different cell lines were assayed in the HCV cell-cell fusion assay. Using two different plates, either HEK293T parental cell line expressing Tat or HEK293T cells expressing E1 E2 and Tat were mixed with one of the following cell line: Huh7, HepG2, Vero, HeLa, HEK293T, CHOK1 or MOLT-4 respectively. Forty-eight hours later, the level of luciferase was measured in both plates and the ratio between the luciferase counts obtained in the first plate (containing HEK293T parental cell line) and the second plate (containing HEK293T cell expressing E1 E2) was calculated; Figure 7: Inhibition of HCV cell-cell fusion by a known Scavenger Receptor BI (SBRI) antagonist; Using two different plates, Hek293T cells expressing E1 E2 and Tat were mixed with Huh7-LTR-luciferase cells, together with increasing concentration of BLT1 (BLT1 is a known antagonist of SRBI; Nieland et al, PNAS (2002) 9S ₁ 15422-15427). Fourty-eight hours later, the level of luciferase was monitored in the first plate, while cytotoxicity was monitored in the second plate using a wst-1 kit; Figure 8: HCV cell-cell fusion is blocked by a monoclonal antibody against E2;

Using two different plates, Hek293T cell expressing E1 E2 and Tat were mixed with Huh7-LTR-luciferase cells together with two concentrations of AP33, a blocking antibody directed against E2 (Clayton et al, J. Gen. Virol. (2001) 82, 1877-1883). Forty-eight hours later, the level of luciferase was monitored in the first plate, while cytotoxicity was monitored in the second plate using a wst-1 kit;

Figure 9: Inhibition of HCV cell-cell fusion by peptide derived from E1 region; Overlapping peptides (25mers) covering the entire E1 region were tested in the

cell-cell fusion assay of the invention. Figure 9A shows a consensus sequence for E1. This consensus sequence was made using the following strains (HCV-BK, accession number M8335; H-1, accession number AB107944; H-77 accession number AF009606; HC-J2, accession number D10074; HC-J5, accession number D10075; HC-J7, accession number D10077; HC-J8, accession number D10988;

HCV 476, accession number D10688; HCV KF , accession number D10687; HCV JT , accession number D11168) and using AlignX ^® software. The sequence of the peptides used in this study and their position relative to the consensus sequence is shown. Figure 9B is a histogram representing the % inhibition each peptide achieves in the cell-cell fusion assay using two different peptide concentrations

(10μg/ml and 1mg/ml respectively); and

Figure 10: Inhibition of HCV cell-cell fusion by peptide derived from E2 region; Overlapping peptides (25mers) covering the entire E2 region were tested in the cell-cell fusion assay of the invention. Figure 10A shows a consensus sequence for E2. This consensus sequence was made using the following strains (HCV-BK, accession number M8335; H-1 , accession number AB107944; H-77 accession number AF009606; HC-J2, accession number D10074; HC-J5, accession number D10075; HC-J7, accession number D10077; HC-J8, accession number D10988; HCV 476, accession number D10688; HCV KF , accession number D10687; HCV JT , accession number D11168) and using AlignX ^® software. The sequence of the peptides used in this study and their position relative to the consensus sequence is shown. Figure 10B is a histogram representing the % inhibition each peptide achieves in the cell-cell fusion assay using two different peptide concentrations (10μg/ml and 1 mg/ml respectively). Histogram bar 30 relates to peptide number 30 which a 25mer that covers part of a hydrophobic heptad repeat region of E2 and which shows inhibitory activity in the assay of the present invention.

EXAMPLES

Example 1

One ^" of the principles of a fusion assay is the expression of viral envelope proteins on the surface of the transfected cell. In the case of the HCV envelope proteins, E1 and E2

include a carboxy-terminal signal sequence that retains the proteins in the endoplasmic reticulum. Previous attempts at developing an HCV fusion assay have deleted the signal sequence, thus enabling translocation of truncated E1 and E2 to the cell surface.

However, the present invention provides full-length E1 and E2 which have been shown to be expressed in their natural conformation on the surface of HEK293T cells.

Materials and methods Cell lines: HEK293T: a HEK293Tcell line was maintained in Dulbecco Modified Eagles Medium

(DMEM) and glutamax-l (Gibco 61965-026) containing 10% foetal calf serum (FCS; PAA A15-043), 1 mM sodium pyruvate (Sigma S8636) and 0.4mg/ml G418 (Gibco 10131-027). Cells were split 1 :5 - 1 :20 twice a week using Accutase (ITC, AT104) to avoid clumping of the cells. HEK293TcE1 E2TAT6: HEK293T cells were stably transfected to express HCV E1 and

E2 glycoproteins and the HIV Tat transactivator by Zeocin selection (0.2mg/ml) (selection agent for E1E2 expression; Invitrogen 45-0430) and 10μg/ml blasticidin (selection agent for Tat expression; Invitrogen 46-1120). The E1E2 domain expressed comprises the last 60 amino acids of the core protein and the complete E1 and E2 sequences. The sequence was derived from a 1 B consensus strain and the expression vector used was pBud. HEK293TcE1E2 cells were stably transfected to express the HIV Tat peptide by Blasticidin selection (10μg/ml) using the pTAT plasmid. Cells were maintained in DMEM and glutamax-l containing 10% FCS, 1mM sodium pyruvate, 0.4mg/ml G418, 0.2mg/ml zeocin and 10μg/ml blasticidin. Cells were split 1 :5 - 1 :20 twice a week using Accutase to avoid clumping of the cells.

Antibodies:

AP33 was provided by Arvind Patel and is a monoclonal antibody that specifically recognise the amino acid region 412 to 423 in E2 (Clayton et al, J. Virol. (2002) 76(15). 7672-7682).

HCM-081-5: monoclonal antibody that recognise HCV E1 (Austral biologicals, # HCM- 081-5).

Secondary antibody: R-phycoerythrine labelled goat anti mouse monoclonal antibody (Sigma, P9287).

Determination of E1 and E2 expression on the cell surface by flow cytometric fluorescence-activated cell sorting (FACS) analysis:

HEK293T and HEK293TcE1 E2 TAT6 cells were washed once with 5ml/flask PBS, then detached using trypsin and diluted to a final volume of 10ml with ice cold sterile PBS containing 0.1% BSA. The cells were dispersed by repeated vigorous pipetting using a 10ml pipette and bulb to minimise clumping of the cells. 1ml aliquots of each suspension were counted using an automated cell counter (CEDEX™) to determine cell density and viability. After centrifugation at IOOOrpm for 5min, the cells were re- suspended at 2.2 x 10 ⁷ cells/ml in ice-cold sterile PBS containing 0.1% bovine serum albumen (BSA). Aliquots of 45μl were transferred to 1.5ml eppendorf tubes and 5μl of either AP33 or HCM-081-5 or PBS were added to the tubes and mixed. The tubes were left to incubate for 1hour on ice. The cells were then spun down (1 min, 5000rpm), washed twice in PBS containing 0.1% BSA and the pellet was re-suspended in 45μl of PBS containing 0.1% BSA. Either 5μl of the secondary antibody or of PBS containing 0.1% BSA was then added to the corresponding eppendorf and left for 1 hour on ice. The cells were then spun down (1min, 5000rpm), washed twice in PBS containing 0.1% BSA and the pellet was re-suspended in 1 ml of PBS containing 2% formaldehyde. The cells were then subjected to a FACS analysis.

Results

Figure 1 shows the result of the FACS analysis of E1 and E2 expression on the parental HEK293T cells and on HEK293TcE1 E2TAT6 clone cells respectively. Looking at Figure

1 a, the parental HEK293T cells show no difference in fluorescence between the cells incubated with no antibody (shaded histogram), with the secondary antibody only (pale grey line) or with the cells incubating with the primary and the secondary antibody (dark and pale grey lines, respectively), indicating that there is no expression of either E1 or E2 on surface of these cells. In contrast, HEK293TE1 E2 cells incubated with either anti-

El antibody or anti-E2 antibody show a shift in fluorescence when compared to

incubation in the absence of any antibody or in the presence of the secondary antibody (Figure 1b).

These results show that the HEK293TcE1 E2 TAT6 clone expresses both E1 and E2 on the cell surface.

Example 2

As shown in Figure 2, the cell-cell fusion assay of the present invention requires the combination of a cell expressing viral envelope protein on its cell surfaces with a second cell expressing at least one of the human receptor that mediate viral infection. Thus the assay mimics viral infection in a non-infectious environment. In particular, recombinant

HEK293 cells expressing HCV envelope proteins E1 and E2 on their cell surface promote fusion with permissive cell lines such as the hepatocyte cell line, Huh7. A reporter assay for fusion is achieved by expressing a transactivator protein (HIV-1 Tat) in one cell line whilst the other cell line contains a reporter system (HIV-1 LTR) under the control of HIV-1 Tat.

Materials and methods

HEK293TcE1 E2 TAT6: HEK293T cells were stably transfected to express HCV E1 and E2 proteins and the HIV Tat transactivator by Zeocin selection (0.2mg/ml) as described in Example 1.

Huh7tl_TR-luc: Huh7 cells were stably transfected to contain a luciferase reporter under the control of a truncated version of the HIV LTR using G418 selection (0.8mg/ml). Cells were maintained in Modified Eagles Medium (MEM; Gibco 21090-022) containing 10% FCS, 2mM L-glutamine (Sigma G7531), 1mM sodium pyruvate, 1x Non Essential Amino

Acids (NEAA; Gibco 11140-035) and 0.8mg/ml Geneticin (G418 (selection agent for maintenance of the LTR-luciferase reporter); Gibco 10131-027). Cells were split 1 :3 - 1 :10 twice a week using Accutase Il (PAA, L11-012) to avoid clumping of the cells.

Fusion medium: MEM + 10% FCS +2mM L-glutamine + 1mM sodium pyruvate + 1x

NEAA (Huh7tLTR-luc growth medium without G418) and 1% penicillin-streptomycin (Gibco 15140-122).

HEK293TcE1 E2 TAT6 cells and Huh7tLTR-luc cells were washed once with 5ml/flask PBS, then detached using trypsin and diluted to a final volume of 10ml with fusion medium. Cells were then dispersed by repeated vigorous pipetting using a 10ml pipette and bulb to minimise clumping of the Huh7tLTR-luc cells. 1ml aliquots of each suspension were counted using an automated cell counter (CEDEX™) to determine cell density and viability.

Huh7tLTR-luc cells and HEK293TcE1 E2 TAT6 cells were mixed in fusion medium to give a suspension containing 3 x 10 ⁵ Huh7tLTR-luc and 3 x 10 ⁵ HEK293TcE1 E2 TAT6 per ml of medium, i.e. a total cell density of 6 x 10 ⁵ viable cells/ml.

Cell suspensions were added to 96-well flat-bottom tissue culture plates (Corning Costar 3585) to give 100μl/well containing 6 x 10 ⁴ total cells/well (3 x 10 ⁴ Huh7tLTR-luc + 3 x 10 ⁴ HEK293TcE1 E2 TAT6). This cell density provided a near-confluent layer of cells. The plates were then incubated for 24 to 48 hours at 37°C, 5% CO ₂ .

After incubation, the fusion medium removed was from the wells (by careful inversion of plate and draining on gauze squares) and the cells washed once with 100μl/well PBS. The PBS was drained and 25μl/well Promega passive lysis buffer (Cat # E194A) added.

The plate was incubated for ~10mins at 37 ⁰ C before being placed on a plate shaker (e.g. Heidolph Titramax 100, speed setting 4) for ~30sec to detach cell debris from the plate. 20μl aliquots were then transferred from each well to wells on a white 96-well plate (Packard picoplate-96 Cat # 6005162). 20μl passive lysis buffer was then added to empty wells for determination of "reagent background" (20μl/well lysis buffer +

100μl/well luciferase substrate). 100μl Promega luciferase substrate (Cat # E1501) was added to each well and the plate read immediately on a Victor 2™ plate reader using the manufacturer's luminescence protocol. The reagent background reading was subtracted from all other readings.

The assay was also carried out using transient transfection of HEK293T cells. The protocol was as set out above with the exception that preparation of the cells was begun

two days before they were required for experiments. In particular, on day -2, a T75 flask was inoculated with -3x10 ⁶ HEK293T cells in 15ml medium without antibiotics (no G418, zeocin or penicillin/streptomycin). On day -1 , the cells were transfected by adding 24μg plasmid EV279 (pcDNA3.1 containing the HIV tat exon 1 ; provided by Eric Verdin) DNA to 1.5ml Gibco Optimem-1 reduced serum medium (Cat # 11058-021).

60μl Iipo2000 reagent (lipofectamine; Invitrogen Cat # 12566-014) was added to a separate 1.5ml aliquot of Optimem-1. The tubes were gently mixed for 5 minutes before being combined and remixed. After 20 minutes at room temperature, the 3ml of DNA/lipofectamine solution was added to the flask of cells. The flask was then incubated overnight at 37°C, 5% CO2 and the cells ready for use the following day.

Results

To achieve a reproducible and robust HCV cell-cell fusion assay, the generation of a stable cell line expressing E1 E2 and Tat was preferred. Initial experiments involved transfecting E1 E2-expressing plasmid into a HEK293T cell line. Two days after transfection, 0.2mg/ml zeocin was added to the medium to select for clones expressing E1 E2. Several clones were selected as resistant to zeocin and were expanded to grow in T75 flasks. Twelve clones were further characterised in parallel in the HCV cell-cell fusion assay and for cell surface E2 expression. One day before the assay, the clones were transfected with Tat expressing plasmid. Two days after, the HEK293T E1E2 stable clones transfected with Tat were mixed with Huh7-LTR luciferase cells. One plate was subjected to luciferase analysis, while a duplicate plate was subjected to FACS analysis for E2 expression using AP33 monoclonal antibody (see Example 1 for assay description). The logged E2 expression was then fitted individually to the logged fusion activity using an ordinary least squares weighting. The correlation of the plot gave an r=0.915 demonstrating there is a good correlation of E2 (strain 1a) cell surface expression and the ability of the HEK293T cell to fuse with Huh7 cells.

In further experiments, the effect of introducing two point mutations in the transmembrane domain of E2 or by replacing the transmembrane domains of E1 E2 by that of VSV-G transmembrane domain (as described in Takikawa et al (ibid.)) was investigated.

As shown in Figure 4, mutagenesis of the key transmembrane domains in E2 prevents protein export to the cell surface and renders the envelope-expressing cells incapable of fusing with the hepatocytes. In particular, a two-point amino acid mutation (from MetLeu to ThrPro) in the C terminal transmembrane domain of E2 substantially reduced the luciferase signal. Figure 4 also shows that full length E1 and E2 amino acid sequences are required for fusion since replacement of the transmembrane domains with that of Vesicular Stomatitis Virus rendered the luciferase signal undetectable.

While optimising the assay of the present invention, it was found that HEK293TcE1 E2

TAT cells that were allowed to grow in over-confluence gave a higher signal than usual (data not show). These results were investigated further by incubating these cells in either their normal media (at ph 7.0) or at pH 5.5.

As shown in Figure 5, cells grown for 2 days at pH5.5 showed a higher fusion activity with Huh7LTR-luc compared to cells grown at pH 7.0. HCV envelope proteins are believed to belong to the class Il of fusion envelope protein which need to be internalised in an acidic environment before they can drive the fusion of the viral and the cellular membrane (Jardetzky and Lamb, Nature (2004) 427, 307-308). Cultivating the cells for 2 days at pH 5.5 is believed to mimic this acidification step needed for HCV envelope to fuse and therefore gives a higher fusion activity compared to cells grown at pH 7.0.

In conclusion, these results confirm that cell-cell fusion is mediated by the E1 and E2 glycoproteins, that the presence of both E1 and E2 is required for fusion to occur and that fusion activity is dependent on the presence of functional E2 protein. Low pH treatment also enhances E1 E2-driven fusion.

Example 3 In the literature (Seipp et al, J. Gen. Virol. (1997) 78(10), 2467-76), Huh7 cells have been shown to be permissive for HCV infection and to express the putative receptors

CD81, LDL-R and SR-BI. Although the HCV cell-cell fusion of the present invention uses Huh7 cell line, alternative cell lines have been investigated (Figure 6).

Materials and Methods Cells:

Huh7: Cells were maintained in MEM containing 10% FCS, 2mM L-glutamine, 1mM sodium pyruvate and 1x NEAA. Cells were split 1 :3 — 1 :10 twice a week using Accutase

Il to avoid clumping of the cells.

HepG2: Cells were maintained in MEM containing 10% FCS, 2mM L-glutamine, 1mM sodium pyruvate and 1x NEAA. Cells were split 1 :3 - 1 :10 twice a week using Accutase

Il to avoid clumping of the cells.

Vero: Cells were maintained in MEM containing 10% FCS, 2mM L-glutamine, 1mM sodium pyruvate and 1x NEAA. Cells were split 1 :5 - 1 :20 twice a week using Accutase

Il to avoid clumping of the cells. HeLa: Cells were maintained in DMEM and glutamax containing 10% FCS. Cells were split 1 :5- 1 :20 twice a week using Accutase Il to avoid clumping of the cells.

HEK293T: Cells were maintained in DMEM and glutamax-l containing 10% FCS, 1mM sodium pyruvate. Cells were split 1 :5 - 1 :20 twice a week using Accutase to avoid clumping of the cells. CHOK1 : Cells were maintained in DMEM and glutamax containing 10% FCS. Cells were split 1 :5 - 1 :20 twice a week using Accutase Il to avoid clumping of the cells.

MOLT-4: Cells were maintained in RPMI 1640 (Gibco 31870-025) containing 10% FCS,

2mM L-glutamine, 1 mM sodium pyruvate and 1x NEAA. Cells were split 1 :3 - 1 :10 twice a week using Accutase Il to avoid clumping of the cells. HEK293TcE1 E2 TAT6: HEK293T cells were stably transfected to express HCV E1 and

E2 proteins and the HIV Tat transactivator by Zeocin selection (0.2mg/ml) as described in Example 1.

Fusion assay: The protocol was carried out as described in example 2 for transient transfection. In particular, on day -2, Huh7, HepG2, Vero, HeLa, HEK293T, CHOK1 and MOLT-4 cells were dispensed respectively into T75 flasks with -3x10 ⁶ cells in 15ml of their respective

medium. On day -1 , the cells were transfected by adding 24μg of a plasmid expressing luciferase under the control of HI V- 1 LTR Xo 1.5ml Gibco Optimem-1 reduced serum medium (Cat # 11058-021). 60μl Iipo2000 reagent (lipofectamine; Invitrogen Cat # 12566-014) was added to a separate 1.5ml aliquot of Optimem-1. The tubes were gently mixed for 5 minutes before being combined and remixed. After 20 minutes at room temperature, the 3ml of DNA/lipofectamine solution was added to the flask of cells. The flask was then incubated overnight at 37°C, 5% CO ₂ and the cells ready for use the following day.

Each of the cell lines to be tested (i.e. Huh7, HepG2, Vera, HeLa, HEK293T, CHOK1 and MOLT-4) was mixed respectively with HEK293TcE1 E2 TAT6 cells in fusion medium to give a suspension containing 3 x 10 ⁵ of cells to be tested and 3 x 10 ⁵ HEK293TcE1 E2 TAT6 per ml of medium, i.e. a total cell density of 6 x 10 ⁵ viable cells/ml.

Cell suspensions were added to 96-well flat-bottom tissue culture plates (Corning Costar 3585) to give 100μl/well containing 6 x 10 ⁴ total cells/well (3 x 10 ⁴ cells to be tested + 3 x 10 ⁴ HEK293TcE1 E2 TAT6). This cell density provided a near-confluent layer of cells. The plates were then incubated for 24 to 48 hours at 37°C, 5% CO ₂ .

After incubation, the fusion medium removed was from the wells (by careful inversion of plate and draining on gauze squares) and the cells washed once with 100μl/well PBS. The PBS was drained and 25μl/well Promega passive lysis buffer (Cat # E194A) added. The plate was incubated for ~10mins at 37 ⁰ C before being placed on a plate shaker (e.g. Heidolph Titramax 100, speed setting 4) for ~30sec to detach cell debris from the plate. 20μl aliquots were then transferred from each well to wells on a white 96-well plate (Packard picoplate-96 Cat # 6005162). 20μl passive lysis buffer was then added to empty wells for determination of "reagent background" (20μl/well lysis buffer + 100μl/well luciferase substrate). 100μl Promega luciferase substrate (Cat # E1501) was added to each well and the plate read immediately on a Victor 2™ plate reader using the manufacturer's luminescence protocol. The reagent background reading was subtracted from all other readings.

Results

In the search for a cell line that was able to provide a suitably high signal to noise ratio, when used in combination with HEK293T cells expressing E1 E2, the human hepatocellular carcinoma Huh7 cells was found to give a sufficient signal over background luciferase count for the requirements of the assay of the invention (Figure 6). This cell line has already been reported as being highly permissive for HCV pseudoparticle infection (Bartosch et al, J. Exp. Med. (2003) 197(5), 633-642). In addition and as shown in Figure 8, the present experiment confirms that the other well- characterised human hepatocellular carcinoma cell line, HepG2, is not permissive for HCV entry as it lacks CD81 receptor (Bartosch et al, J. Biol. Chem. (2003) 278(43). 41624-30). In the assay of the present invention, the human lymphoblastic leukaemia Molt-4 cell has also been shown to be permissive for HCV entry, consistent with observations made by Laggings et al. (J. Virol. (1998) 72(5), 3539-3546) but different from those made by Bartosch et al (ibid). Finally, the present experiment also shows

Vero, HeLa, Hek293T and CHOK1 cell lines to be non-permissive to HCV entry in the assay of the present invention.

Example 4 Development of the cell-cell fusion assay has enabled high-throughput screening for inhibitors of HCV virus entry. In particular, the assay has been developed in a format suitable for use with 384-well plates or more.

Materials and Methods Compounds were diluted from 4mM stock (100% DMSO) 1 :20 in PBS/0.05% Pluronic to give 200//M compound in PBS/0.05% Pluronic/5% DMSO. This stock was then diluted aseptically 1 :3 to give a 67μM stock in PBS/0.05% Pluronic with a final DMSO concentration of -1.67%. 60μM compound in PBS/0.05% Pluronic/1.67% DMSO was used to demonstrate 100% inhibition, while PBS/0.05% Pluronic/1.67% DMSO was used as control. Compound at 60//M in PBS/0.05% Pluronic/1.67% DMSO was used as a standard.

Assay plates were prepared with compound by adding 10μl/well compound to white opaque tissue culture treated plates (Greiner 781080). On receipt of cells at 2x10 ⁶ cells/ml, the same volume of 2% FCS HCV fusion media (see example 2) was added to each cell line to obtain solutions at 1x10 ⁶ cells/ml. 10//I of each cell line was added per well. Plates were lidded and incubated at 37°C and 5% CO2 in a humidified incubator for 48 hours. Bright-Glo Luciferase reagent (Promega E2620) was prepared as described by the manufacturer and, after 48 hours, 10//I was added to each well. The plates were shaken for 3 minutes before being read on a Leadseeker™ plate reader. The data were exported into Microsoft ^® Excel 97 SR-2 (k) and the %inhibition calculated and plotted against the compound concentration. These percent inhibition values were used to obtain IC50 values.

Results

In the search for the receptor(s)/co-receptor(s) involved in HCV entry, the scavenger receptor class B type I (SR-BI) has recently been shown to play an important role in this process. Indeed, antibody directed against SR-BI reduced the infectivity of HCVpp. SR- Bl is a 82 kDa glycoprotein involved in the selective uptake of cholesteryl esters uptake from high density lipoprotein and low density lipoprotein. Nieland et al (ibid.), have identified small molecule inhibitors of the selective transfer of lipids mediated by SR-BI. It was postulated that an inhibitor of SR-BI function should inhibit SR-BI entry and, on this basis, one of these compounds (BLT-1) was evaluated to determine its ability to act as an inhibitor of HCV entry using the cell-cell fusion assay of the invention. As shown in Figure 7, BLT-1 inhibited HCV cell-cell fusion in a dose-dependent manner and displayed an IC50 of 6.9μM. No significant cell toxicity was observed at the IC ₅₀ concentration.

The ability of the assay to detect inhibitors of E2 binding was confirmed using two concentrations of the E2 monoclonal antibody, AP33. As seen in Figure 8, 10μl and 25μl of AP33 inhibited fusion activity by more than 60% and 70% respectively, demonstrating that the assay is dependent on the presence of E2. The results also show that the assay of the invention is suitable for the identification of potential inhibitors of HCV entry that act by binding to E2.

Example 5

The development of the cell-cell fusion assay has enabled the screening of peptides libraries to identify domains in E1 and E2 that are essential for virus entry and potentially useful as therapeutics. In particular, overlapping (25mers) covering the E1 E2 sequence were tested in the cell-cell fusion assay of the present invention at concentrations of 10μg/ml and 100μg/ml.

Garry and Dash (Virology (2003) 307, 255-265) suggest that amino acids 272 to 281 of the full length HCV polyprotein represent a putative fusion peptide. As shown in Figure 9, use of a 25-amino acid peptide spanning this putative domain (see histogram bar number 5) saw a -45% reduction in cell-cell fusion at a concentration of 10μg/ml (4μM).

Drummer and Poumbourios (J. Biol. Chem. (2004) 279(29), 30066-30072) suggest that a hydrophobic heptad repeat region in E2 is essential for HCV pseudovirus infection. As shown in Figure 10, significant inhibition of fusion (-60%) was seen when a 25mer having a sequence identical to part of the hydrophobic heptad repeat region (see histogram bar number 30) was screened in the assay of the present invention at a concentration of 10μg/ml.

SEQUENCE LISTING FREE TEXT

SEQ ID NO:1

C terminal of protein C, E1 and E2 from Hepatitis C Virus 1 b

SEQ ID NO:2

C terminal of protein C, E1 and E2 from Hepatitis C Virus 1b

SEQ ID NO:3 Peptide derived from SEQ ID NO:2 residues 134 to 158

SEQ ID NO:4

Peptide derived from SEQ ID NO:2 residues 542 to 566

SEQ ID NO:5

Peptide derived from SEQ ID NO:2 residues 560 to 584

SEQ ID NO:6

Peptide derived from SEQ ID NO:2 residues 141 to 150

SEQ ID NO:7 Peptide derived from SEQ ID NO:2 residues 544 to 568

SEQ ID NO:8

C terminal of protein C, partial sequence of E1 protein from Hepatitis C Virus 1b

SEQ ID NO:9

Peptide derived from SEQ ID NO:8 residues 1 to 25

SEQ ID NO:10

Peptide derived from SEQ ID NO:8 residues 18 to 42

SEQ ID NO:11

Peptide derived from SEQ ID NO:8 residues 36 to 60

SEQ ID NO:12 Peptide derived from SEQ ID NO:8 residues 61 to 85

SEQ ID NO:13

Peptide derived from SEQ ID NO:8 residues 79 to 103

SEQ ID NO:14

Peptide derived from SEQ ID NO:8 residues 96 to 128

SEQ ID NO:15

Peptide derived from SEQ ID NO:8 residues 122 to 146

SEQ ID NO:16 Peptide derived from SEQ ID NO:8 residues 158 to 182

SEQ ID NO:17

Peptide derived from SEQ ID NO:8 residues 176 to 199

SEQ ID NO:18

Peptide derived from SEQ ID NO:8 residues 194 to 218

SEQ ID NO:19

C terminal of E1 protein, E2 protein from Hepatitis C Virus 1b

SEQ ID NO:20

Peptide derived from SEQ ID NO:19 residues 1 to 26

SEQ ID NO:21 Peptide derived from SEQ ID NO:19 residues 21 to 45

SEQ ID NO:22

Peptide derived from SEQ ID NO:19 residues 39 to 63

SEQ ID NO:23

Peptide derived from SEQ ID NO:19 residues 57 to 81

SEQ ID NO:24

Peptide derived from SEQ ID NO:19 residues 75 to 99

SEQ ID NO:25

Peptide derived from SEQ ID NO: 19 residues 93 to 117

SEQ ID NO:26

Peptide derived from SEQ ID NO:19 residues 111 to 135

SEQ ID NO:27

Peptide derived from SEQ ID NO:19 residues 129 to 153

SEQ ID NO:28

Peptide derived from SEQ ID NO:19 residues 147 to 171

SEQ ID NO:29

Peptide derived from SEQ ID NO:19 residues 165 to 189

SEQ ID NO:30 Peptide derived from SEQ ID NO:19 residues 183 to 207

SEQ ID NO:31

Peptide derived from SEQ ID NO:19 residues 201 to 226

SEQ ID NO:32

Peptide derived from SEQ ID NO:19 residues 220 to 244

SEQ ID NO:33

Peptide derived from SEQ ID NO:19 residues 238 to 262

SEQ ID NO:34

Peptide derived from SEQ ID NO:19 residues 256 to 280

SEQ ID NO:35 Peptide derived from SEQ ID NO:19 residues 274 to 297

SEQ ID NO:36

Peptide derived from SEQ ID NO:19 residues 291 to 315

SEQ ID NO:37

Peptide derived from SEQ ID NO:19 residues 345 to 369

SEQ ID NO:38

Peptide derived from SEQ ID NO: 19 residues 358 to 382