WO2009030872A1 - Materials and methods for the treatment of hepatitis c - Google Patents

Materials and methods for the treatment of hepatitis c Download PDF

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Publication number
WO2009030872A1
WO2009030872A1 PCT/GB2008/000702 GB2008000702W WO2009030872A1 WO 2009030872 A1 WO2009030872 A1 WO 2009030872A1 GB 2008000702 W GB2008000702 W GB 2008000702W WO 2009030872 A1 WO2009030872 A1 WO 2009030872A1
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Prior art keywords
peptide
seq
amino acid
antibody
amino acids
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PCT/GB2008/000702
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German (de)
French (fr)
Inventor
Mats Axel Atterdag Persson
Daniel Gunnar Johansson
Jonathan Kelvin Ball
Jean Dubuisson
Cecile Voisset
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Mats Axel Atterdag Persson
Daniel Gunnar Johansson
Jonathan Kelvin Ball
Jean Dubuisson
Cecile Voisset
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Publication of WO2009030872A1 publication Critical patent/WO2009030872A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the peptide may comprise, or consist of, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
  • X 2 may be selected from the group of amino acids consisting of A, G, E, and S. Preferably X 2 is A.
  • the peptide may comprise an amino acid sequence that has at least 50, 60, 70, 80, 90, 95, 96, 97, or 98 per cent sequence identity with amino acids 488-542 of SEQ ID NO: 2.
  • nucleic acid which nucleic acid comprises a nucleotide sequence that encodes a peptide of the invention.
  • kit for immunising an individual against hepatitis C virus which kit comprises:
  • a peptide of the invention for use in diagnosing hepatitis C virus in an individual.
  • a peptide of the invention for use in measuring an antibody response to a hepatitis C virus vaccine in an individual .
  • kits for determining whether a sample contains an antibody to hepatitis C virus comprising: (i) a container comprising a peptide of the invention; and optionally (ii) instructions for incubating the peptide with a sample and observing antibody bound to the peptide.
  • kits of the invention may also include means for obtaining a blood sample or a serum sample from an individual, e.g. a syringe and hypodermic needle, and/or one or more additional reagents to observe binding of an antibody to the peptide, e.g. to perform an ELISA assay.
  • the kits of the invention may also include- one or more of: a solid support for the peptide of the invention, a washing reagent, and/or a signal generating reagent, e.g. a reagent that binds to antibodies and allows a detectable signal to be generated.
  • the signal generating reagent may catalyse a reaction that results in a substrate undergoing a colour change.
  • an antibody from the sample binds, e.g. specifically binds, to the peptide is indicative that the individual has, e.g. is infected with, hepatitis C virus.
  • a method of measuring an antibody response to a hepatitis C virus vaccine in an individual comprising the steps of:
  • the binding of an antibody from the sample to the peptide is preferably via specific binding, e.g. the antibody has specific affinity for the peptide.
  • the antibody from the sample preferably does not bind to the peptide via nonspecific binding.
  • the peptide preferably binds to an antibody from the sample via specific binding, e.g. the component has specific affinity for the peptide.
  • the sample may, for example, be any solution that is suspected of containing hepatitis C antibodies.
  • the step of determining, whether a component of the sample binds to the peptide may be carried out by using an ELISA assay.
  • FIG 5 shows neutralization of HCVpp bearing E1E2 from different genotypes by human mAbs A8, 1:7 and Ll.
  • HCVpp bearing E1E2 of genotypes 1-6 (depicted as in Figure 9) were incubated with 15ug/ml human mAbs A8, 1:7 and Ll for 2 hours at 37 0 C before 2 hrs contact with target cells. The amount of infected particles was measured after two days as Luciferase- activity. Results are given as percentages of neutralization relative to infection in the absence of antibody for each genotype (mean ⁇ SD of three independent experiments) .
  • Figure 18a shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 3 isolate UKN3A1.28. This isolate is referred to herein as gt3:l. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon .
  • Figure 18b shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 18a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734984 (GI : 58198336) .
  • Figure 26a shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 5 isolate UKN5.15.7. This isolate is referred to herein as gt5:2. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon .
  • Figure 26b shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 26a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number EF427672 (E427672.1 GI : 148645560) .
  • Figure 27 shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 5 isolate UKN5.15.7. This isolate is referred to herein as gt5:2. Isolation of this sequence is described in Lavillette
  • Z 2 may be selected from the group of amino acids consisting of R, V, K, E, A, and P. Preferably, Z 2 is R.
  • Z 3 may be selected from the group of amino acids consisting of
  • Z 6 may be selected from the group of amino acids consisting of:
  • V and F Preferably Z 10 is V.
  • Z 12 may be selected from the group of amino acids consisting of: V, I, L, and M. Preferably, Z 12 is V.
  • Z 13 may be selected from the group of amino acids consisting of: N, K, and E. Preferably Z 13 is N.
  • the E2 polypeptide may be a polypeptide having an amino acid sequence selected from the group consisting of: amino acid position 386 to position 729 of SEQ ID NO: 2, amino acid position 386 to position 729 of SEQ ID NO: 4, amino acid position 217 to position 560 of SEQ ID NO: 10, amino acid position 217 to position 560 of SEQ ID NO: 12, amino acid position 217 to position 560 of SEQ ID NO: 14, amino acid position 386 to position 733 of SEQ ID NO: 16, amino acid position 217 to position 564 of SEQ ID NO: 18, amino acid position 217 to position 564 of SEQ ID NO: 20, amino acid position 217 to position 566 of SEQ ID NO: 22, amino acid position 217 to position 566 of SEQ ID NO: 24, amino acid position 217 to position 560 of SEQ ID NO: 26, amino acid position 217 to position 561 of SEQ ID NO: 28, and amino acid position 217 to position 565 of SEQ ID NO: 36,
  • the peptides of the invention may be antigens for an antibody to E2.
  • the antibody may be the antibody clone 1:7 or A8, as described in the Examples below.
  • the antibody may be an antibody that comprises the VH chain and/or the VL chain of antibody clone 1:7 or A8.
  • the antibody may comprise the VH chain CDRl, CDR2 and CDR3 and/or the VL chain CDRl, CDR2, and CDR3 region of antibody clone 1:7 or A8.
  • the VH and VL domains of 1:7 are shown in SEQ ID NO: 37 and SEQ ID NO: 40, respectively.
  • the respective CDR regions are underlined.
  • the antibody may bind the peptides of the invention with a Kd value of less than 100OnM, 90OnM, 80OnM, 70OnM, 60OnM, 50OnM, 40OnM, 30OnM, 20OnM, 19OnM, 18OnM, 17OnM, 16OnM, 15OnM, 14OnM, 13OnM, 12OnM, HOnM, 10OnM, 9OnM, 8OnM, 7OnM, 6OnM, 5OnM, 45nM, 4OnM, 35nM, 3OnM, 25nM, 2OnM, 15nM, 1OnM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or InM.
  • an antibody elicited by a peptide of the invention may be capable of neutralizing a hepatitis C virus pseudoparticle (HCVpp) , e.g. the antibody may be capable of inhibiting the ability of the HCVpp to infect a cell.
  • HCVpp may bear an E2 and/or an E1/E2 complex from a specific HCV genotype. Production of HCVpp is described in Op De Beeck et al. (41), which is incorporated herein by reference.
  • the ability of the antibody to neutralize HCVpp may be measured by incubating the HCVpp with different concentrations of the antibody and then adding the particles to Huh-7 cells. The level of HCV infection in the cells is then measured, e.g. by luciferase assay or by western blot with anti-E2 mAB.
  • the antibody is capable of neutralizing a HCVcc with an IC 50 of less than 2000ng/ml, 1500ng/ml, 1000ng/ml,
  • the antibody is capable of neutralizing at least two HCVcc particles, wherein each particle bears an E2 and/or El/E2-complex derived from a different HCV genotype.
  • the antibody may be capable of neutralizing at least 3, 4, 5, 6, 7, or 8 HCVs, wherein each HCVcc particle bears an E2 and/or El/E2-complex derived from a different HCV genotype.
  • the HCV genotype may be selected from the six major HCV genotypes, for example from the group consisting of: Ia, Ib, 2a, 2b, 3a, 4, 5, 6, 7, or 8.
  • the nucleic acids of the invention are preferably "isolated nucleic acids".
  • An “isolated nucleic acid” is a nucleic acid that has undergone some degree of isolation, e.g. by purification.
  • the term “isolated nucleic acid” may refer to a composition in which the nucleic acid of interest is substantially free from other nucleic acids and/or other solutes.
  • an “isolated nucleic acid” is a nucleic acid that makes up at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a composition by weight, preferably disregarding any solvent.
  • Nucleic acids of the invention may be DNA or RNA. Nucleic acids of the invention also include the nucleic acids that have a complementary sequence to the nucleic acids that are described above as nucleic acids of the invention. Nucleic acids of the invention may also comprise a regulatory sequence, e.g. a promoter, that is operably linked to the nucleotide sequence that encodes the polypeptide of the invention.
  • a regulatory sequence e.g. a promoter
  • operably linked may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide (coding) sequence under the influence or control of the regulatory sequence.
  • a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide (coding) sequence which forms part or all of the selected nucleotide sequence.
  • the resulting transcript may then be translated into a desired protein or polypeptide.
  • the vectors of the invention may include one or more elements that facilitate expression of nucleotide sequence in a host cell.
  • the vector may include an element that allows the vector to replicate in a host cell.
  • the vector may include an element that allows selection of host cells that contain the vector, e.g. a marker gene.
  • antibody encompasses monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab') 2 fragments, F(ab) molecules, Fv fragments, single domain antibodies, chimeric antibodies and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule .
  • monoclonal antibody refers to an antibody composition having a homogeneous antibody population. The term is not limited by the manner in which it is made. The term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab') 2 fragments, Fv fragments, and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule.
  • the treatment may also involve treatment of an individual undergoing, or planning to undergo, liver transplantation, for example to reduce the risk of hepatitis C infecting the transplanted liver.
  • the individual to be treated may be an animal, for example a mammal.
  • the individual is a human.
  • Vaccines of the invention may be used with a current "standard therapy" for HCV, e.g. in combination with PEGylated interferon alpha and Ribavirin.
  • the preparation of vaccine compositions containing one or more peptides as the active ingredient is generally known to those of skill in the art.
  • such vaccines are prepared as injectables (e.g., either as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquids prior to injection) .
  • the compositions will generally also include one or more pharmaceutically acceptable excipients or vehicles such as water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present.
  • the vaccine compositions may be emulsified or the active ingredient may be encapsulated in liposomes.
  • Vaccines of the invention may also include one or more adjuvants, e.g. aluminium phosphate or aluminium hydroxide (alumn) .
  • adjuvants include those as referenced in Vogel FR, Powell MF, Alving CR, which is incorporated herein by reference.
  • ⁇ -interferon ( ⁇ lFN) therapy which generally entails administration of 3 million units of ⁇ -IFN three times a week subcutaneously (Causse et al. (1991) Gastroenterology 101:497-502, Davis et al . (1989) N Engl J Med 321:1501-1506, Marcellin et al . (1991) Hepatology 13:393-397), interferon ⁇ ( ⁇ -IFN) therapy (Omata et al. (1991) Lancet 338:914-915), ribivirin therapy (Di Bisceglie et al .
  • ⁇ -interferon ( ⁇ lFN) therapy which generally entails administration of 3 million units of ⁇ -IFN three times a week subcutaneously (Causse et al. (1991) Gastroenterology 101:497-502, Davis et al . (1989) N Engl J Med 321:1501-1506, Marcellin et al .
  • the peptides of the invention may can also be used in conjunction with known anti-hepatitis C virus combination therapies, e.g. a combination of PEGylated interferon alpha and Ribavirin.
  • known anti-hepatitis treatments include for example, the combination of ⁇ -IFN and ursodiol (Bottelli et al. (1993) (Abstr.) Gastroenterology 104:879, O'Brien et al. (1993) (Abstr.) Gastroenterology 104:966) and the combination of .beta. -IFN and ribivirin (Kakumu et al. (1993) Gastroenterology 105:507-512) .
  • the peptides of the invention may also be used in combination with an antibody to hepatitis C virus, e.g. an antibody having the VL amino acid sequence of SEQ ID NO: 37, 38, or 39 or an antibody having the VH sequence of SEQ ID NO: 40, 41, or 42.
  • an antibody to hepatitis C virus e.g. an antibody having the VL amino acid sequence of SEQ ID NO: 37, 38, or 39 or an antibody having the VH sequence of SEQ ID NO: 40, 41, or 42.
  • anti-HCV therapeutics with which the peptides of the invention may be used include ribavirin, amantadine (Symmetrel) , viramidine, protease inhibitors, such as VX950 and BILN2061, and polymerase inhibitors, such as NIM283,
  • Medicaments and pharmaceutical compositions may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, oral and nasal.
  • the medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body.
  • the peptides of the invention may be administered parenterally, e.g., by injection (either subcutaneously or intramuscularly) .
  • Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the peptides of the invention are administered to the subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be prophylactically and/or therapeutically effective.
  • the amount of the composition to be delivered depends on the subject to be treated, the capacity of the subject's immune system to mount its own immune-responses, and the degree of protection desired. The exact amount necessary will vary depending on the age and general condition of the individual to be treated, the severity of the condition being treated and the particular peptide selected and its mode of administration, among other factors. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual .
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated and, e.g. on the peptide or peptide combinations used.
  • Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual individual, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
  • the peptides of the invention can be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals needed to maintain or reinforce the action of the compositions.
  • the dosage regimen will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgement of the reasonably skilled practitioner.
  • a sample e.g. a sample to be analysed for the presence of antibodies to hepatitis C virus, may comprise or may be derived from: a quantity of blood; a quantity of serum derived from an individual's blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a quantity of pancreatic juice; a tissue sample or biopsy; or cells isolated from said individual.
  • a sample may be taken, or may have been taken, from any tissue or bodily fluid.
  • the sample may be a sample that has previously been removed from a human or animal body.
  • the sample may be an isolated sample, e.g. a sample that is provided in vitro. A sample that is provided as an isolated sample will already have been removed from an individual.
  • a method of the invention may not comprise a step of taking a sample from an individual.
  • the sample is taken from a bodily fluid, more preferably one that circulates through the body. Accordingly, the sample may be a blood sample or lymph sample.
  • the sample is a blood sample or blood-derived sample.
  • a blood-derived sample is a blood sample that has undergone treatment, e.g. to remove one or more blood components .
  • the blood-derived sample may be a selected fraction of a patient's blood, e.g. a selected cell- containing fraction or a plasma or serum fraction.
  • a selected serum fraction may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells.
  • Antigens bind to antibodies by presenting a particular 3- dimensional shape that is recognised by the antibody.
  • a molecule that presents a similar shape compared to the peptides of the invention may also bind to antibodies to hepatitis C virus, and may also generate an antibody response.
  • Such a molecule need not be a peptide, rather it may be any molecule that adopts the shape recognised by the antibody.
  • a mimetic may be a molecule that positions glycines, a tryptophan and an aspartic acid molecules at spatial positions that are equivalent to the spatial positions of amino acids G523, W529, G530 and D535, in the E2 polypeptide (amino acids 386-729 of SEQ ID NO: 2) .
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, e.g. binding to antibodies to hepatitis C virus, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the supernatant was precleared by the addition of 15ul Protein-G- Dynabeads 100.4 (Invitrogen) .
  • 15ul Protein-G- Dynabeads 100.4 (Invitrogen) was added to the precleared supernatant and incubated with rotation at 4°C over night.
  • 15ul Protein-G-Dynabeads 100.4 (Invitrogen) was added and incubated for 4 hours at 4 0 C. The beads were washed 3 times in RIPA-buffer and once in PBS.
  • Bound antibody was detected with anti-human immunoglobulin G antibody conjugated to alkaline phosphatase (Sigma) and pNPP substrate (Sigma) . Absorbance values were determined at 405nm. Kd values were inferred from antibody binding curves by non-linear regression using Prism 4 software (GraphPad Inc. ) .

Abstract

The disclosure relates to materials and methods for the treatment of hepatitis C. In particular, peptides are disclosed based on amino acid sequences within the E2 polypeptide of the hepatitis C virus, and to vaccine compositions comprising these peptides. Additionally, the disclosure relates to methods of treating or preventing hepatitis C infection by administration of a vaccine composition comprising the peptides.

Description

Materials and Methods for the Treatment of Hepatitis C
Field of the Invention The present invention relates to materials and methods for the treatment of hepatitis C virus, and in particular to peptides for use in vaccines against hepatitis C.
Background to the Invention Hepatitis C virus (HCV) is a member of hepacivirus genus within the Flaviviridae family (1) . It infects the human liver, and is estimated to be carried by 3 % (170 million) of the human population as a chronic infection, which if untreated often results in serious liver disease including cirrhosis and hepatocellular cancer (HCC) (2) . HCV remains a global health problem.
Virus genome replication is error prone and this, together with high virus turnover, generates extensive genetic variability. Within an infected individual HCV exists as a swarm of genetically related but distinct viruses called a quasispecies (3) . HCV can be grouped into 6 major genotypes that differ by up to 30% at the nucleotide level (4-6) . Among the most variable parts of the virus are the two envelope proteins, El and E2 (7) .
Twenty to twenty-five percent of newly infected individuals will spontaneously resolve the infection, whilst the remainder will develop a chronic infection (2) . This intriguing capacity of some individuals to eliminate the infection has prompted large efforts to study virus-host interactions, notably the native and adaptive immune responses to the virus. The interferon response and the'T-cell response have shown to be important for recovery, while NK cell response and humoral immunity have in most cases not been associated with clearance. However, antibodies to the envelope protein E2 have been shown to ameliorate the disease in chimpanzees, to correlate with protection by vaccination in the same animal species, and to reduce the rate of re-infection of the graft after liver transplantation in man (10-12,45) . Moreover, advances over the last years have provided new tools to study the virus specific antibody response, in particular antibodies that block infection. The new methods include generation of infectious retroviral pseudoparticles, bearing native HCV envelope glycoproteins on their surface (HCVpp) , and more recently, cloned HCV genomic RNA (strain JFH-I) that after transfection into appropriate cells generates infectious HCV particles (HCVcc) (13-17) .
The recent isolation of functional El and E2 genes representative of all the major genotypes of HCV has enabled assessment of the neutralizing breadth and potency of sera and monoclonal antibodies (18) . Whilst cell culture infectious virus currently represents only a limited number of HCV genotypes, this system is useful to determine the neutralizing potency of antibodies against native particles. These systems have been used to determine the neutralizing capacity and cross reactivity profile of a small number of murine monoclonal antibodies (19) . The methods are also providing important insights into the natural antibody response to HCV, such as the existence of in vitro neutralizing antibodies in humans, as well as the possible existence of virus-induced mechanisms that suppress the neutralizing antibody response in the initial, critical phase of the infection (20, 21) .
Despite these advances in the study of HCV, treatment of HCV still suffers from the drawback that up to 50 per cent of individuals treated fail to clear the virus (8), and the available treatment (a combination of PEGylated interferon- alpha and the antiviral drug Ribavirin) is not suited to all individuals. Notably, despite substantial efforts no effective vaccine has yet been developed for human use (9) .
Summary of the Invention We have previously isolated mAbs to the E2 envelope glycoprotein as a means to dissect the immune response to HCV in humans (22) . The antibodies were derived from an individual infected with HCV of genotype 2b, and isolated by their capacity to bind to E2 of genotype Ia. By their very nature, they may therefore react with divergent genotype proteins.
Indeed, we demonstrated that they bind to genotypes Ia and Ib and that they block the binding of E2 of these genotypes to CD81, a putative cell receptor utilized for virus entry (22,23) .
Recently, methodological advances have allowed investigations of neutralizing antibodies against hepatitis C virus (HCV) in vitro .
We have investigated three human monoclonal antibodies (mAbs) , previously isolated from an individual infected with HCV of genotype 2b, that are known to cross react in binding assay to the envelope E2 protein of genotypes Ia and Ib. We have surprisingly found that two of these antibodies have a neutralizing activity with a breadth not previously observed. Indeed, mAbs 1:7 and A8 recognized E2 from all the six major genotypes, and they neutralized retroviral pseudoparticles (HCVpp) carrying genetically equally diverse HCV envelope glycoproteins. Importantly, these antibodies were also able to neutralize the cell culture infectious HCV clone JFH-I m vitro, with IC50 values of 60 ng/ml and 560 ng/ml, respectively. The conformational epitopes of these two, broadly reactive antibodies were overlapping yet distinct, and involved amino acid residues in the 523-535 region of E2, known to be important for the E2-CD81 interaction. In particular, we have found that four amino acids in the E2 polypeptide that are conserved across all genotypes, G523, W529, G530 and D535, are important for E2 binding to mAbs 1:7 and A8. In addition, mutation of either of W529 and D535 to alanine completely abolished E2 binding to mAbs 1:7 and A8.
These results demonstrate that that broadly neutralizing human anti-HCV antibodies can be elicited, and that the region of amino acids 523-535 of the HCV envelope glycoprotein E2 carries neutralizing epitopes conserved across all genotypes. The surprising discovery of these conserved and broadly neutralizing epitopes provides a much needed new route for the development of vaccines against HCV.
Thus, the invention broadly relates to the provision of peptide fragments of an E2 polypeptide, and vaccines comprising these peptides for immunising against HCV.
In a first aspect of the invention, there is provided a peptide comprising the amino acid sequence WX1X2X3X4X5D (SEQ ID NO: 43), wherein each of X1, X2, X3, X4, and X5 is an amino acid, and wherein the peptide comprises less than 40 amino acids. The peptide may comprise, or consist of, less than 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 amino acids. The peptide may comprise, or consist of, more than 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, or 7 amino acids. The peptide may comprise, or consist of, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, or 7 amino acids.
Preferably, the peptide comprises the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D (SEQ ID NO: 44), wherein each of X6, X7, X8, X9, and X10 is an amino acid. Preferably, the peptide comprises the amino acid sequence GX10X9TYX6WGX2X3X4X5D (SEQ ID NO: 45), more preferably GX10PTYX6WGX2X3X4X5D (SEQ ID NO: 46) . In a further aspect of the invention, there is provided a peptide comprising, or consisting of, the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D (SEQ ID NO: 44), wherein each of X2, X3, X4, X5, X6, X7, X8, X9, and X10 is an amino acid, and wherein the peptide comprises less than 250 amino acids. The peptide may comprise, or consist of, less than 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20, 15, or 14 amino acids. The peptide may comprise, or consist of, more than 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20, 15, 14, or 13 amino acids. Preferably the peptide has no more than 150 amino acids, more preferably no more than 100 amino acids.
Preferably, the peptide comprises the sequence GX10X9TYX6WGX2X3X4X5D (SEQ ID NO: 45), more preferably GX10PTYX6WX1X2X3X4X5D (SEQ ID NO: 46) .
In any peptide of the invention:
X1 may be G.
X2 may be selected from the group of amino acids consisting of A, G, E, and S. Preferably X2 is A.
X3 may be selected from the group of amino acids consisting of
N and S. Preferably X3 is N.
X4 may be selected from the group of amino acids consisting of
D, V, and E. Preferably X4 is D. X5 may be selected from the group of amino acids consisting of
T and R. Preferably X5 is T.
X6 may be selected from the group of amino acids consisting of
S, T, D, and N. Preferably X6 is S.
X7 may be Y. X8 may be T.
X9 may be P.
X10 may be selected from the group of amino acids consisting of: A, V, E, L, Y, and N. Preferably X10 is A.
Preferably, the peptides of the invention comprise a sequence selected from the group consisting of: GEPTYDWGGSVRD, GAPTYTWGENETD, GVPTYSWGENETD, GVPTYTWGENETD, GVPTYSWGENETD, GLPTYSWGENETD, GVPTYSWGENETD, GVPTYTWGANETD, GAPTYNWGENETD, GVPTYTWGDNESD, GVPTYNWGENETD, GYPTYNWGENDTD, GYPTYNWGSNETD, GNPTYTWGENETD, and GNPTYTWGENETD (SEQ ID NO'S: 47-61) . Preferably, the peptide comprises the sequence GAPTYSWGANDTD (SEQ ID NO: 62) .
In a further aspect of the invention, there is provided a peptide, which peptide comprises, or consists of, a portion of an E2 polypeptide, which portion corresponds to amino acids
523-535 of the amino acid sequence of SEQ ID NO: 2, and which peptide comprises less than 250 amino acids of the E2 polypeptide. The peptide may comprise less than 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or 10 amino acids of the E2 polypeptide. In addition, the peptide may comprise less than 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or 10 amino acids .
The E2 polypeptide may be any E2 polypeptide from any strain of HCV.
Peptides of the invention may comprise additional N-terminal amino acids and/or C-terminal amino acids, as described in more detail below.
The peptide may comprise an amino acid sequence that has at least 50, 60, 70, 80, 90, 95, 96, 97, or 98 per cent sequence identity with amino acids 488-542 of SEQ ID NO: 2.
Preferably, the peptides of the invention are antigens for an antibody to E2, e.g. the peptides comprise an epitope of an antibody to E2. In particular, the peptides of the invention may bind to an antibody to E2. The antibody to E2 is preferably an antibody that inhibits the binding of E2 to CD81. Preferably, an antibody to E2 specifically binds to and/or targets the peptides of the invention, e.g. the peptide is recognised by the E2 binding domain of the antibody.
Preferably, the peptides of the invention are capable of eliciting, e.g. provoking and/or stimulating, an antibody response in an animal, e.g. when a peptide of the invention is administered to the animal. Preferably the antibody response is against hepatitis C virus. Preferably, the peptides of the invention are capable of eliciting an antibody response in an animal, wherein an antibody elicited by a peptide of the invention is an antibody to E2 and/or an E1/E2 complex. Preferably the antibody is an antibody to E2 and/or an E1/E2 complex from more than one hepatitis C virus genotype.
In a further aspect of the invention, there is provided an isolated nucleic acid, which nucleic acid comprises a nucleotide sequence that encodes a peptide of the invention.
Preferably, the nucleic acid comprises a nucleotide sequence that encodes a portion of an E2 polypeptide, which portion corresponds to amino acids 523-535 of the amino acid sequence of SEQ ID NO: 2. For example, the nucleotide sequence may encode an amino acid sequence selected from the group consisting of: GEPTYDWGGSVRD, GAPTYTWGENETD, GVPTYSWGENETD, GVPTYTWGENETD, GVPTYSWGENETD, GLPTYSWGENETD, GVPTYSWGENETD, GVPTYTWGANETD, GAPTYNWGENETD, GVPTYTWGDNESD, GVPTYNWGENETD, GYPTYNWGENDTD, GYPTYNWGSNETD, GNPTYTWGENETD, and GNPTYTWGENETD (SEQ ID NO'S: 47-61) . Preferably, the nucleotide sequence encodes the amino acid sequence GAPTYSWGANDTD (SEQ ID NO: 62) .
In a further aspect of the invention, there is provided a vector comprising a nucleic acid of the invention. In a further aspect of the invention, there is a provided a host cell comprising a nucleic acid and/or a vector of the invention. In a further aspect of the invention, there is provided a method of producing a peptide of the invention, which method comprises the steps of:
(a) providing a host cell comprising a nucleotide sequence that encodes a peptide of the invention, and
(b) causing the host cell to express the peptide encoded by the nucleotide sequence.
The method may also comprise step (c) isolating the peptide and/or may also comprise, prior to step (a) , introducing the nucleotide sequence that encodes the peptide of the invention into the host cell, e.g. on a vector. The step of providing the host cell may also include culturing the host cell. Methods of culturing host cells to express peptides and methods of purifying polypeptides are well known in the art. The host cell may express the peptide under the control of a constitutive promoter or inducible promoter.
In a further aspect of the invention, there is provided a peptide of the invention for use in a therapeutic method. In a further aspect, there is provided use of a peptide of the invention in the manufacture of a medicament for use in a therapeutic method.
The therapeutic method may be the immunisation of an individual, e.g. a patient, against hepatitis C virus. The method may be prophylactic, e.g. protection of an individual against hepatitis C virus infection and/or prevention of hepatitis C virus infection in a individual. The method may be curative, e.g. treatment of an individual infected with hepatitis C virus.
In particular, the therapeutic method may involve vaccinating an individual against hepatitis C virus, e.g. it may involve eliciting an immune response in the individual against hepatitis C virus. An immune response may be elicited against hepatitis C by causing the individual to produce antibodies that target hepatitis C virus, particularly the E2 of the hepatitis C virus . Preferably, the antibodies produced inhibit the infectivity of the virus, e.g. they neutralize the virus.
In a further aspect of the invention, there is provided a vaccine, e.g. a vaccine composition, comprising a peptide, e.g. a polypeptide, of the invention. The vaccine composition is preferably for immunising an individual against hepatitis C virus .
In a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide of the invention and a pharmaceutically acceptable excipient, diluent and/or carrier. The pharmaceutical composition is preferably for use in a therapeutic method as described above.
In a further aspect of the invention, there is provided a method of immunising an individual against hepatitis C virus, which method comprises administering a peptide of the invention to the individual. The method of immunising may also comprise measuring the antibody response to the peptide and/or determining whether any antibodies in an antibody response to the peptide are capable of neutralising a hepatitis C virus.
In a further aspect of the invention, there is provided a kit for immunising an individual against hepatitis C virus, which kit comprises:
(i) a container comprising a composition, e.g. a pharmaceutical composition, which composition comprises a peptide of the invention; and optionally
(ii) instructions for administering the composition to the individual.
The kit may also include means for measuring an antibody response to a hepatitis C virus vaccine in an individual. For example, the kit may comprise a container comprising the peptide of the invention and optionally instructions for using the peptide to measure the amount of hepatitis C virus antibodies in a sample taken from the individual. The kit may also include one or more additional reagents for observing an antibody bound to the peptide, e.g. to observe binding via an ELISA assay.
The peptides of the invention may also be used to detect antibodies, e.g. immunoglobulin molecules, to hepatitis C virus. Peptides that detect antibodies to hepatitis C virus will have utility in analytical applications and diagnostic applications, e.g. to diagnose whether an individual is infected with hepatitis C virus. They may also have utility in observing, e.g. measuring, an immune response to a hepatitis C virus vaccine. This may be carried out, for example, by taking a sample from an individual and detecting binding of a peptide of the invention with an antibody to hepatitis C virus in the sample. Binding of peptides to antibodies may be detected, for example, using ELISA or by using peptides that comprise a detectable marker, e.g. green fluorescent protein (GFP) . Other ways of detecting binding to an antibody will be apparent to a person skilled in the art.
In a further aspect of the invention, there is provided a peptide of the invention for use in diagnosing hepatitis C virus in an individual. In a further aspect of the invention, there is provided a peptide of the invention for use in measuring an antibody response to a hepatitis C virus vaccine in an individual .
In a further aspect of the invention there is provided use of a peptide of the invention in the manufacture of a medicament for diagnosing hepatitis C virus in an individual. In a further aspect of the invention there is provided use of a peptide of the invention in the manufacture of a medicament for measuring an antibody response to a hepatitis C virus vaccine in an individual .
In a further aspect of the invention there is provided a kit for diagnosing hepatitis C virus in an individual, wherein the kit comprises:
(i) a container comprising a peptide of the invention; and optionally
(ii) instructions for incubating the peptide with a sample taken from the individual and observing antibody bound to the peptide.
In a further aspect of the invention, there is provided a kit for measuring an antibody response to a hepatitis C virus vaccine in an individual, wherein the kit comprises:
(i) a container comprising a peptide of the invention; and optionally
(ii) instructions for incubating the peptide with a sample taken from the individual and measuring the amount of antibody bound to the peptide.
The kit for measuring an antibody response in an individual may alternatively be a kit for observing and/or determining an antibody response to a hepatitis C virus vaccine in an individual. An antibody response may be measured by measuring the amount of antibodies bound to the peptide, e.g. when the peptide is bound to a solid support. The amount of antibodies bound to the peptide may be quantitated using an ELISA assay. The individual may be been vaccinated using a peptide of the invention.
In a further aspect of the invention, there is provided a kit for determining whether a sample contains an antibody to hepatitis C virus, wherein the kit comprises: (i) a container comprising a peptide of the invention; and optionally (ii) instructions for incubating the peptide with a sample and observing antibody bound to the peptide.
The kit for determining the presence of a hepatitis C virus antibody in a sample may alternatively be a kit for measuring and/or observing the presence of a hepatitis C virus antibody in a sample. The sample may, for example, be any solution that is suspected of containing hepatitis C antibodies.
Measuring, observing and/or determining the presence of a hepatitis C virus antibody in a sample may be carried out by incubating a sample with the peptide and then observing whether the peptide has bound, e.g. specifically bound, to any antibody in the sample.
The kits of the invention may also include means for obtaining a blood sample or a serum sample from an individual, e.g. a syringe and hypodermic needle, and/or one or more additional reagents to observe binding of an antibody to the peptide, e.g. to perform an ELISA assay. For example, the kits of the invention may also include- one or more of: a solid support for the peptide of the invention, a washing reagent, and/or a signal generating reagent, e.g. a reagent that binds to antibodies and allows a detectable signal to be generated. For example, the signal generating reagent may catalyse a reaction that results in a substrate undergoing a colour change.
The kits of the invention may also include means for assessing, e.g. observing and/or determining, whether an antibody that binds to the peptide is an antibody that neutralises hepatitis C virus. For example, kits of the invention may include means for generating infectious hepatitis C virus, e.g. means for generating hepatitis C virus particles (HCVcc) or pseudoparticles (HCVpp) . The kits may include nucleic acid encoding HCVcc and/or HCVpp. Methods of using HCVcc and HCVpp to determine whether an antibody neutralises hepatitis C virus are described below.
In a further aspect of the invention there is provided a method of diagnosing hepatitis C virus in an individual, comprising the steps of:
(i) incubating in vitro a peptide of the invention with a sample taken from an individual; and
(ii) observing whether an antibody from the sample binds to the peptide.
An observation that an antibody from the sample binds, e.g. specifically binds, to the peptide is indicative that the individual has, e.g. is infected with, hepatitis C virus.
In a further aspect of the invention, there is provided a method of measuring an antibody response to a hepatitis C virus vaccine in an individual, comprising the steps of:
(i) incubating in vitro a peptide of the invention with a sample taken from an individual, which individual has been administered with a vaccine against hepatitis C virus; and
(ii) measuring the amount of antibody from the sample that binds to the peptide.
The amount of antibody that binds to the peptide may be measured using an ELISA assay, for example. The amount of antibody that binds to the peptide may be compared with a reference amount of antibody to assess, for example, whether the individual is producing an amount of antibodies sufficient to protect against hepatitis C virus infection.
The method of measuring an antibody response may also comprise determining whether an antibody that binds the peptide neutralises hepatitis C virus. This may be carried out by determining whether the antibody is capable of neutralising an HCVpp and/or an HCVpp. Neutralisation of HCVpp and HCVcc is described in more detail below.
The binding of an antibody from the sample to the peptide is preferably via specific binding, e.g. the antibody has specific affinity for the peptide. The antibody from the sample preferably does not bind to the peptide via nonspecific binding.
The sample may be a body fluid, e.g. a blood sample or a blood-derived sample. A blood-derived sample is a blood sample that has undergone treatment, e.g. to remove one or more blood components. The blood-derived sample may be plasma or serum. Preferably the sample is serum. The method of diagnosing hepatitis C and/or the method of measuring an antibody response to a hepatitis C virus vaccine may or may not also include the step of obtaining a sample of body fluid from the individual .
The step of observing and/or measuring whether an antibody from the sample binds to the peptide may be carried out using an ELISA assay. For example, the peptide may be bound to a solid support. The sample may be incubated with the peptide bound ,to the solid support and following incubation the support may be washed and subsequently incubated with a signal generating molecule. The signal generating molecule may bind to a component of the sample that binds to the peptide.
In a further aspect of the invention, there is provided a method of determining whether a sample contains a hepatitis C antibody, comprising the steps of:
(i) incubating in vitro a peptide of the invention with a sample; and
(ii) observing whether an antibody from the sample binds to the peptide. Observing that an antibody from the sample binds to the peptide is indicative that the sample contains antibodies to hepatitis C virus. The peptide preferably binds to an antibody from the sample via specific binding, e.g. the component has specific affinity for the peptide. The sample may, for example, be any solution that is suspected of containing hepatitis C antibodies. The step of determining, whether a component of the sample binds to the peptide may be carried out by using an ELISA assay.
In a further aspect of the invention, there is provided a vaccine composition comprising:
(a) a peptide comprising the sequence GX10X9X8X7X6WGX2X3X4X5D, which peptide comprises less than 40, 30, 25, 20 or 15 amino acids; and
(b) a pharmaceutically acceptable excipient, diluent and/or carrier; wherein the vaccine composition is for use in immunising an individual against hepatitis C virus.
Preferably, the vaccine composition comprises a peptide, which peptide comprises the sequence GAPTYSWGANDTD.
Brief Description of the Figures Embodiments and experiments illustrating the principles of the invention is discussed with reference to the accompanying figures in which:
Figure 1 Figure 1 shows binding curves for the three antibody clones.
The respective antibodies were incubated in microtitre wells where E1E2 of isolate H77 (gtla) had been captured on GNA.
Detection of bound antibody was performed with enzyme conjugated anti-human antiserum. Figure 2
Figure 2 shows immunofluorescence of ElE2-expressing cells stained by human mAbs A8 (upper panel), 1:7 (middle), or Ll (lower part) . Huh-7 cells expressing E1E2 of genotypes 1-6 (see Figure 9) were incubated with human MAbs. Bound antibody was detected using a FITC labeled anti-human antisera.
Figure 3
Figure 3 shows immunoprecipitations of E1E2 of all genotypes using antibodies 1:7 and A8. Immunoprecipitations of cell lysates from Huh-7 cells expressing E1E2 of genotypes 1-6 (Figure 9) were analyzed by western blot and detected with a mixture of anti-HCV-E2 antibodies. Still, this antibody mix did not equally well detect all isolates, especially gt4 detection was weaker, as shown by a direct western blot on lysates from E1E2 expressing cells (right panel) . "cl3" is a human anti-HIV-1 gpl20 antibody used as negative control. HC indicate the heavy chain of the mAb used for IP.
Figure 4
Figure 4 shows binding of human mAbs to E1E2 of different genotypes. Antibodies 1:7, A8, and Ll were incubated with saturating amounts of GNA-captured E1E2 proteins representative of all genotypes of HCV (see Figure 9) . Bound antibody was detected with alkaline phosphataseconjugated anti-human IgG antiserum.
Figure 5
Figure 5 shows neutralization of HCVpp bearing E1E2 from different genotypes by human mAbs A8, 1:7 and Ll. HCVpp bearing E1E2 of genotypes 1-6 (depicted as in Figure 9) were incubated with 15ug/ml human mAbs A8, 1:7 and Ll for 2 hours at 370C before 2 hrs contact with target cells. The amount of infected particles was measured after two days as Luciferase- activity. Results are given as percentages of neutralization relative to infection in the absence of antibody for each genotype (mean±SD of three independent experiments) .
Figure 6 Figure 6 shows neutralization of HCVcc by human monoclonal antibodies A8, 1:7 and Ll. Neutralization assays were performed by pre-incubating HCVcc (clone JFH-I) with increasing amounts of human mAbs A8, 1:7 and Ll for 2 hours at 370C before contact with target cells for two hours. At 2 days post-infection, the level of E2 in the supernatant was assessed by western blotting with mAb 3/11. The results presented are representative of three independent experiments.
Figure 7 Figure 7 shows mapping of mAb 1:7 and A8 epitopes by E2 alanine-substitution scanning. Conserved amino acids suggested to be part of the CD81 binding domain on E2, were assessed for their role in interaction with the two mAbs. Binding intensity to a panel of GNA-bound E1E2 proteins containing single alanine-substitutions was measured. Binding is expressed as a percentage relative to H77c wild-type. Residues G523, W529, G530 and D535 are important for recognition by both mAbs.
Figure 8 Figure 8 shows conserved nature of contact residues for 1:7 and A8 epitopes across functional E2 genes. Residues G523, W529, G530, D535 were observed to be important for mAb binding in the alanine-substitution epitope mapping. In agreement with the broad reactivity of these mAbs, all identified contact residues are completely conserved across functional clones representative of the six HCV genotypes (highlighted with boxes) . The sequences shown in Figure 8 are SEQ ID NO. S: 63- 79. Figure 9
Figure 9 lists the HCV isolates mentioned herein, together with the corresponding full isolate name and accession numbers, if any.
Figure 10
Figure 10a shows a nucleic acid sequence (SEQ ID NO: 1), which is a complete cds of the HCV strain H77 pCV-H77C polyprotein gene. Figure 10b shows the amino acid sequence encoded by the nucleic acid shown in Figure 10a (SEQ ID NO: 2) . This HCV isolate is referred to herein as gtla:l. The sequence is obtainable from the Genbank database under accession number AF011751.1 (GI:2327070) .
Figure 11
Figure 11a shows a nucleic acid sequence (SEQ ID NO: 3) , which is a complete cds of the HCV strain H77 pCV-Hll polyprotein gene. Figure lib shows the amino acid sequence encoded by the nucleic acid shown in Figure 11a (SEQ ID NO: 4) . The sequence is obtainable from the Genbank database under accession number AF011752.1 (GI : 2327072) .
Figure 12
Figure 12a (SEQ ID NO: 9) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype Ib isolate UKNB5.23. This isolate is referred to herein as gtlb:l. Isolation of this sequence is described in Lavillette et al. (18) and the sequence includes an engineered start codon and stop codon. Figure 12b (SEQ ID NO: 10) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 12a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77, i.e. SEQ ID NO: 2) . The sequence is obtainable from the Genbank database under accession number AY734976.1 (GI:58198320) . Figure 13
Figure 13a (SEQ ID NO: 11) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype Ib isolate UKN1B12.16. This isolate is referred to herein as gtlb:2. Isolation of this sequence is described in Lavillette et al. (18) and the sequence includes an engineered start codon and stop codon . Figure 13b (SEQ ID NO: 12) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 13a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734974.1 (GI : 58198316) .
Figure 14
Figure 14a (SEQ ID NO: 13) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 2a isolate UKN2A1.2. This isolate is referred to herein as gt2a:l. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon. Figure 14b (SEQ ID NO: 14) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 14a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734977.1 (GI : 58198322) .
Figure 15
Figure 15a (SEQ ID NO: 15) shows a nucleic acid sequence, which is the complete genome (genomic RNA) of the HCV isolate JFH-I. Figure 15b (SEQ ID NO: 16) shows the amino acid sequence encoded by the nucleic acid shown in Figure 15a. This HCV isolate is a HCV genotype 2a isolate and is referred to herein as gt2a:2. The sequence is obtainable from the Genbank database under accession number AB047639.1 (GI : 13122261) .
Figure 16 Figure 16a (SEQ ID NO: 17) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 2b isolate UKN2B1.1. This isolate is referred to herein as gt2b:l. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon . Figure 16b (SEQ ID NO: 18) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 16a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734982.1 (GI : 58198332) .
Figure 17
Figure 17a (SEQ ID NO: 19) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 2b isolate UKN2B2.8. This isolate is referred to herein as gt2b:2. Isolation of this sequence is described in Lavillette et al. (18) and the sequence includes an engineered start codon and stop codon. Figure 17b (SEQ ID NO: 20) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 17a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734983.1 (GI : 58198334) .
Figure 18
Figure 18a (SEQ ID NO: 21) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 3 isolate UKN3A1.28. This isolate is referred to herein as gt3:l. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon . Figure 18b (SEQ ID NO: 22) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 18a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734984 (GI : 58198336) .
Figure 19
Figure 19a (SEQ ID NO: 23) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 3 isolate UKN3A13.6. This isolate is referred to herein as gt3:2. Isolation of this sequence is described in Lavillette et al. (18) and the sequence includes an engineered start codon and stop codon. Figure 19b (SEQ ID NO: 24) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 19a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY894683.1 (GI : 58220839) .
Figure 20 Figure 20a (SEQ ID NO: 25) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 4 isolate UKN4.11.1. This isolate is referred to herein as gt4:l. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon. Figure 20b (SEQ ID NO: 26) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 20a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734986.1 (GI : 58198340) . Figure 21
Figure 21a (SEQ ID NO: 27) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 4 isolate UKN4.21.16. This isolate is referred to herein as gt4:2. Isolation of this sequence is described in Lavillette et al. (18) and the sequence includes an engineered start codon and stop codon . Figure 21b (SEQ ID NO: 28) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 21a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY734987.2 (GI : 109259767) .
Figure 22
Figure 22a (SEQ ID NO: 29) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 5 isolate UKN5.14.4. This isolate is referred to herein as gt5:l. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon. Figure 22b (SEQ ID NO: 30) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 22a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY785283.1 (GI : 58220847)
Figure 23
Figure 23a (SEQ ID NO: 35) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 6 isolate UKN6.5.340. This isolate is referred to herein as gt6:2. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon. Figure 23b (SEQ ID NO: 36) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 23a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number AY736194.1 (GI : 58220845) .
Figure 24
Figure 24 shows the amino acid sequences of the variable light chains of the antibody clones 1:7 (SEQ ID NO: 37), A8 (SEQ ID NO: 38) and Ll (SEQ ID NO: 39) . The CDR regions as suggested by Kabat et al . (44) are underlined. These sequences are disclosed in Allander et al . (22), US2002/0046445 Al and WO97/40176.
Figure 25
Figure 25 shows the amino acid sequences of the variable heavy chains of the antibody clones 1:7 (SEQ ID NO: 40), A8 (SEQ ID NO: 41), and Ll (SEQ ID NO: 42) . The CDR regions as suggested by Kabat et al . (44) are underlined. These sequences are disclosed in Allander et al . (22), US2002/0046445 Al and WO97/40176.
Figure 26 Figure 26a (SEQ ID NO: 31) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 5 isolate UKN5.15.7. This isolate is referred to herein as gt5:2. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon . Figure 26b (SEQ ID NO: 32) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 26a. This amino acid sequence includes an engineered N-terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number EF427672 (E427672.1 GI : 148645560) . Figure 27
Figure 27a (SEQ ID NO: 33) shows part of the nucleic acid sequence encoding the HCV polyprotein from the HCV genotype 6 isolate UKN6.5.8. This isolate is referred to herein as gt6:l. Isolation of this sequence is described in Lavillette et al . (18) and the sequence includes an engineered start codon and stop codon. Figure 27b (SEQ ID NO: 34) shows the amino acid sequence encoded by the nucleic acid sequence shown in Figure 27a. This amino acid sequence includes an engineered N- terminal methionine and represents amino acids 170 to 746 of the HCV polyprotein (referenced to the polyprotein of strain H77) . The sequence is obtainable from the Genbank database under accession number EF427671 (E427671.1 GI : 148645541) .
Detailed Description of the Invention
Peptides A peptide of the invention may comprise, or consist of, the amino acid sequence WX1X2X3X4X5D (SEQ ID NO: 43) , preferably GX10X9X8X7X6WGX2X3X4X5D (SEQ ID NO:44) , wherein each of X1, X2, X3, X4, X5, X6, X7, X8, X9 and X10 are as defined above.
A peptide of the invention may comprise, or consist of, an amino acid sequence selected from the group consisting of : GAX9TYX6WGX2X3X4X5D, GATYX6WGX2X3X4X5D, GAX9TYSWGX2X3X4X5D, GAX9TYX6WGAX3X4X5D, GAX9TYX6WGX2NX4X5D, GAX9TYX6WGX2X3DX5D, GAX9TYX6WGX2X3X4TD, GAPTYSWGX2X3X4X5D, GAPTYX6WGAX3X4X5D, GAPTYX6WGX2NX4X5D, GAPTYX6WGX2X3DX5D, GAPTYX6WGX2X3X4TD,
GAPTYSWGAX3X4X5D, GAPTYSWGX2NX4X5D, GAPTYSWGX2X3DX5D, GAPTYSWGX2X3X4TD, GAPTYSWGANX4X5D, GAPTYSWGAX3DX5D, GAPTYSWGAX3X4TD, GAPTYSWGANDX5D, GAPTYSWGANX4TD, GAPTYSWGANDTD, GX10PTYX6WGX2X3X4X5D, GX10PTYSWGX2X3X4X5D, GX10PTYX6WGAX3X4X5D, GX10PTYX6WGX2NX4X5D, GX10PTYX6WGX2X3DX5D, GX10PTYX6WGX2X3X4TD,
GX10PTYSWGAX3X4X5D, GX10PTYSWGX2NX4X5D, GX10PTYSWGX2X3DX5D, GX10PTYSWGX2X3X4TD, GX10PTYSWGANX4X5D, GX10PTYSWGAX3DX5D, GX10PTYSWGAX3X4TD, GX10PTYSWGANDX5D, GX10PTYSWGANX4TD, GX10PTYSWGANDTD, GX10X9TYSWGX2X3X4X5D, GX10X9TYSWGAX3X4X5D, GX10X9TYSWGX2NX4X5D, GX10X9TYSWGX2X3DX5D, GX10X9TYSWGX2X3X4TD, GX10X9TYSWGANX4X5D, GX10X9TYSWGAX3DX5D, GX10X9TYSWGX2X3X4TD,
GX10X9TYSWGANDX5D, GX10X9TYSWGANX4TD, GX10X9TYSWGANDTD, GX10X9TYX6WGAX3X4X5D, GX10X9TYX6WGANX4X5D, GX10X9TYX6WGAX3DX5D, GX10X9TYX6WGAX3X4TD, GX10X9TYX6WGANDX5D, GX10X9TYX6WGANX4TD, GX10X9TYX6WGANDTD, GX10X9TYX6WGX2NX4X5D, GX10X9TYX6WGX2NDX5D, GX10X9TYX6WGX2NX4TD, GX10X9TYX6WGX2NDTD, GX10X9TYX6WGX2X3DX5D,
GX10X9TYX6WGX2X3DTD, and GX10X9TYX6WGX2X3X4TD (SEQ ID NO:S 80-143) ; wherein each of X2, X3, X4, X5, X6, X9, and X10 is any amino acid, or is an amino acid selected from the respective group of amino acids defined above.
The peptide may comprise the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D, in which X2, X3, X4, X5, X6, X7, X8, X9, and X10 are as defined above, with an additional amino acid sequence adjacent to the N-terminus of the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D.
An additional amino acid sequence adjacent to the N-terminus of an amino acid sequence of interest refers, for example, to one or more additional amino acids that are bound to the amino-terminal end of the amino acid sequence of interest.
Likewise, an additional amino acid sequence adjacent to the C- terminus of an amino acid sequence of interest refers, for example, to one or more additional amino acids that are bound to carboxy-terminal end of the amino acid sequence.
For example, the additional amino acid sequence adjacent to the N-terminal of GX10X9X8X7X6WGX2X3X4X5D may be selected from the group consisting of: Z1, Z2Z1, Z3Z2Z1, TZ3Z2Z1, TTZ3Z2Z1, GTTZ3Z2Z1, VGTTZ3Z2Z1, VVGTTZ3Z2Z1, VVVGTTZ3Z2Z1, PVVVGTTZ3Z2Z1, SPVVVGTTZ3Z2Z1,
PSPVVVGTTZ3Z2Z1, TPSPVVVGTTZ3Z2Z1, FTPSPVVVGTTZ3Z2Z1, CFTPS PVVVGTTZ3Z2Z1 , YCFTPSPVVVGTTZ3Z2Z1 , VYCFTPSPWVGTTZ3Z2Z1 , PVYCFTPSPVVVGTTZ3Z2Z1 , GPVYCFTPSPVWGTTZ3Z2Z1 , CGPVYCFTPSPWVGTTZ3Z2Z1 , VCGPVYCFTPSPVVVGTTZ3Z2Z1 , Z4VCGPVYCFTPSPVVVGTTZ3Z2Z1 , Z5Z4VCGPVYCFTPSPWVGTTZ3Z2Z1 , AZ5Z4VCGPVYCFTPSPVVVGTTZ3Z2Z1,
Z6AZ5Z4VCGPVYCFTPSPVVVGTTZ3Z2Z1 , Z7Z6AZ5Z4VCGPVYCFTPSPVVVGTTZ3Z2Z1 , Z8Z7Z6AZ5Z4VCGPVYCFTPSPVWGTTZ3Z2Z1 , and z9z8z7z6AZ5z4VCGpVγcFτpSpVVVGTTZ3z2zl (gEQ jp NQ ; S 144 -181 ) , wherein each of Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, and Z9 is an amino acid.
Z1 may be selected from the group consisting of S, L, F, Q, K, and R. Preferably, Z1 is S.
Z2 may be selected from the group of amino acids consisting of R, V, K, E, A, and P. Preferably, Z2 is R.
Z3 may be selected from the group of amino acids consisting of
D and N. Preferably, Z3 is D.
Z4 may be selected from the group of amino acids consisting of:
S, N, T, Q, K and E. Preferably Z4 is S. Z5 may be selected from the group of amino acids consisting of:
K, R, A, S, and Q. Preferably Z5 is K.
Z6 may be selected from the group of amino acids consisting of:
P, S, or K. Preferably Z6 is P.
Z7 may be selected from the group of amino acids consisting of: V and I. Preferably Z7 is V.
Z8 may be selected from the group of amino acids consisting of:
I, S, T, and V. Preferably Z8 is I.
Z9 may be selected from the group of amino acids consisting of:
G, E, and D. Preferably Z9 is G.
The peptide may comprise the amino acid sequence
GX10X9X8X7X6WGX2X3X4X5D, as defined above, with an additional amino acid sequence adjacent to the C-terminus of the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D. For example, the peptide may comprise, or consist of, the amino acid sequence: Z10, Z10Z11, Z10Z11Z12, Z10Z11Z12L, Z10Z11Z12LZ13, Z10Z11Z12LZ13Z14, and Z10Z11Z12LZ13Z14Z15 (SEQ ID NO . S : 182-188) wherein Z10, Z11, Z12, Z13, Z14, Z15 is an amino acid.
Z10 may be selected from the group of amino acids consisting of
V and F. Preferably Z10 is V.
Z11 may be selected from the group of amino acids consisting of: F, L, and I. Preferably Z11 is F.
Z12 may be selected from the group of amino acids consisting of: V, I, L, and M. Preferably, Z12 is V.
Z13 may be selected from the group of amino acids consisting of: N, K, and E. Preferably Z13 is N.
Z14 may be selected from the group of amino acids consisting of
N, S, and A. Preferably, Z14 is N. Z15 may be selected from the group of amino acids consisting of
T and L. Preferably Z15 is T.
The peptide may comprise the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D, as defined above, with an additional amino acid sequence adjacent to the N-terminus, e.g. one of the sequences listed above, and an additional amino acid adjacent to the C-terminus, e.g. one of the sequences listed above. For example, a peptide of the invention may comprise the amino acid sequence Z9Z8Z7Z6AZ5Z4VCGPVYCFTPSPVVVGTTZ3Z2Z1GX10X9X8X7X6WGX2X3X4X5DZ10Z11Z12LZ1
3Z14Z15 (SEQ ID NO: 189) . A peptide of the invention may comprise the amino acid sequence
Z9Z8Z7Z6AZ5Z4VCGPVYCFTPSPVVVGTTZ3Z2Z1GX1X2TYX3WGX4X5X6X7DZ10Z11 (SEQ ID NO: 190) .
The peptide may be a cyclic peptide, e.g. at least a part or the whole of the peptide may be cyclised, e.g. the peptide may include a ring, e.g. a loop. The peptide may include functional groups, e.g. two functional groups, that may bind with each other to allow at least a part of the peptide to cyclise, e.g. to form a ring. For example, the peptide may include additional amino acids that provide functional groups to allow the peptide to cyclise, e.g. cysteine amino acids. The functional groups allowing cyclisation may be located in the peptide such that one functional group is located on the N-terminal side of the WX1X2X3X4X5D or GX10X9X8X7X6WGX2X3X4X5D motif and a second functional group is located on the C-terminal side of the motif. Preferably, functional groups allowing cyclisation are located at the N-terminus and at the C- terminus of the peptide. The functional groups may, for example, be the thiol groups of cysteine amino acids. Cysteine amino acids may be located in the peptide such that the thiol groups of the cysteine amino acids are capable of forming a disulfide bridge, thereby cyclising at least a part or the whole of the peptide. Other functional groups that may be incorporated into the peptide and that may bind with each other or with other amino acids in the peptide to cause at least part of the peptide to cyclise will be apparent to the person skilled in the art.
A peptide of the invention may comprise, or consist of, an amino acid sequence that has at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100 per cent identity to the portion of amino acids from SEQ ID NO: 2: 522-535, 521-535, 520-535, 519- 535, 518-535, 517-535, 516-535, 515-535, 514-535, 513-535, 512-535, 511-535, 510-535, 509-535, 508-535, 507-535, 506-535, 505-535, 504-535, 503-535, 502-535, 501-535, 500-535, 499-535, 498-535, 497-535, 496-535, 495-535, 494-535, 493-535, 492-535, 491-535, 490-535, 489-535, 488-535, 523-536, 522-536, 521-536, 520-536, 519-536, 518-536, 517-536, 516-536, 515-536, 514-536, 513-536, 512-536, 511-536, 510-536, 509-536, 508-536, 507-536, 506-536, 505-536, 504-536, 503-536, 502-536, 501-536, 500-536, 499-536, 498-536, 497-536, 496-536, 495-536, 494-536, 493-536, 492-536, 491-536, 490-536, 489-536, 488-536, 523-537, 522-537, 521-537, 520-537, 519-537, 518-537, 517-537, 516-537, 515-537, 514-537, 513-537, 512-537, 511-537, 510-537, 509-537, 508-537, 507-537, 506-537, 505-537, 504-537, 503-537, 502-537, 501-537, 500-537, 499-537, 498-537, 497-537, 496-537, 495-537, 494-537,
493-537, 492-537, 491-537, 490-537, 489-537, 488-537, 523-538,
522-538, 521-538, 520-538, 519-538, 518-538, 517-538, 516-538,
515-538, 514-538, 513-538, 512-538, 511-538, 510-538, 509-538, 508-538, 507-538, 506-538, 505-538, 504-538, 503-538, 502-538,
501-538, 500-538, 499-538, 498-538, 497-538, 496-538, 495-538,
494-538, 493-538, 492-538, 491-538, 490-538, 489-538, 488-538,
523-539, 522-539, 521-539, 520-539, 519-539, 518-539, 517-539,
516-539, 515-539, 514-539, 513-539, 512-539, 511-539, 510-539, 509-539, 508-539, 507-539, 506-539, 505-539, 504-539, 503-539,
502-539, 501-539, 500-539, 499-539, 498-539, 497-539, 496-539,
495-539, 494-539, 493-539, 492-539, 491-539, 490-539, 489-539,
488-539, 523-540, 522-540, 521-540, 520-540, 519-540, 518-540,
517-540, 516-540, 515-540, 514-540, 513-540, 512-540, 511-540, 510-540, 509-540, 508-540, 507-540, 506-540, 505-540, 504-540,
503-540, 502-540, 501-540, 500-540, 499-540, 498-540, 497-540,
496-540, 495-540, 494-540, 493-540, 492-540, 491-540, 490-540,
489-540, 488-540, 523-541, 522-541, 521-541, 520-541, 519-541,
518-541, 517-541, 516-541, 515-541, 514-541, 513-541, 512-541, 511-541, 510-541, 509-541, 508-541, 507-541, 506-541, 505-541,
504-541, 503-541, 502-541, 501-541, 500-541, 499-541, 498-541,
497-541, 496-541, 495-541, 494-541, 493-541, 492-541, 491-541,
490-541, 489-541, 488-541, 523-542, 522-542, 521-542, 520-542,
519-542, 518-542, 517-542, 516-542, 515-542, 514-542, 513-542, 512-542, 511-542, 510-542, 509-542, 508-542, 507-542, 506-542,
505-542, 504-542, 503-542, 502-542, 501-542, 500-542, 499-542,
498-542, 497-542, 496-542, 495-542, 494-542, 493-542, 492-542,
491-542, 490-542, 489-542, or 488-542. Preferably, the amino acid sequence has the same number of amino acids as the portion of amino acids from SEQ ID NO: 2 over which sequence identity is measured.
A peptide of the invention may comprise, or consist of, a portion of an E2 polypeptide, which portion is, and/or corresponds to, the portion of amino acids: 522-535, 521-535,
520-535, 519-535, 518-535, 517-535, 516-535, 515-535, 514-535, 513-535, 512-535, 511-535, 510-535, 509-535, 508-535, 507-535,
506-535, 505-535, 504-535, 503-535, 502-535, 501-535, 500-535,
499-535, 498-535, 497-535, 496-535, 495-535, 494-535, 493-535,
492-535, 491-535, 490-535, 489-535, 488-535, 523-536, 522-536,
521-536, 520-536, 519-536, 518-536, 517-536, 516-536, 515-536,
514-536, 513-536, 512-536, 511-536, 510-536, 509-536, 508-536,
507-536, 506-536, 505-536, 504-536, 503-536, 502-536, 501-536,
500-536, 499-536, 498-536, 497-536, 496-536, 495-536, 494-536,
493-536, 492-536, 491-536, 490-536, 489-536, 488-536, 523-537,
522-537, 521-537, 520-537, 519-537, 518-537, 517-537, 516-537,
515-537, 514-537, 513-537, 512-537, 511-537, 510-537, 509-537,
508-537, 507-537, 506-537, 505-537, 504-537, 503-537, 502-537,
501-537, 500-537, 499-537, 498-537, 497-537, 496-537, 495-537,
494-537, 493-537, 492-537, 491-537, 490-537, 489-537, 488-537,
523-538, 522-538, 521-538, 520-538, 519-538, 518-538, 517-538,
516-538, 515-538, 514-538, 513-538, 512-538, 511-538, 510-538,
509-538, 508-538, 507-538, 506-538, 505-538, 504-538, 503-538,
502-538, 501-538, 500-538, 499-538, 498-538, 497-538, 496-538,
495-538, 494-538, 493-538, 492-538, 491-538, 490-538, 489-538,
488-538, 523-539, 522-539, 521-539, 520-539, 519-539, 518-539,
517-539, 516-539, 515-539, 514-539, 513-539, 512-539, 511-539,
510-539, 509-539, 508-539, 507-539, 506-539, 505-539, 504-539,
503-539, 502-539, 501-539, 500-539, 499-539, 498-539, 497-539,
496-539, 495-539, 494-539, 493-539, 492-539, 491-539, 490-539,
489-539, 488-539, 523-540, 522-540, 521-540, 520-540, 519-540,
518-540, 517-540, 516-540, 515-540, 514-540, 513-540, 512-540,
511-540, 510-540, 509-540, 508-540, 507-540, 506-540, 505-540,
504-540, 503-540, 502-540, 501-540, 500-540, 499-540, 498-540,
497-540, 496-540, 495-540, 494-540, 493-540, 492-540, 491-540,
490-540, 489-540, 488-540, 523-541, 522-541, 521-541, 520-541,
519-541, 518-541, 517-541, 516-541, 515-541, 514-541, 513-541,
512-541, 511-541, 510-541, 509-541, 508-541, 507-541, 506-541,
505-541, 504-541, 503-541, 502-541, 501-541, 500-541, 499-541,
498-541, 497-541, 496-541, 495-541, 494-541, 493-541, 492-541,
491-541, 490-541, 489-541, 488-541, 523-542, 522-542, 521-542,
520-542, 519-542, 518-542, 517-542, 516-542, 515-542, 514-542, 513-542, 512-542, 511-542, 510-542, 509-542, 508-542, 507-542, 506-542, 505-542, 504-542, 503-542, 502-542, 501-542, 500-542, 499-542, 498-542, 497-542, 496-542, 495-542, 494-542, 493-542, 492-542, 491-542, 490-542, 489-542, or 488-542 of the amino acid sequence of SEQ ID NO: 2. Preferably, the portion of the E2 polypeptide shares at least 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100 per cent sequence identity with the portion of SEQ ID NO: 2.
A peptide of the invention may comprise, or consist of, a portion of an E2 polypeptide as indicated above, and which peptide comprises, or consists of, less than 250 amino acids of the E2 polypeptide. In particular, the polypeptide may comprise, or consist of, or have more than or less than, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 amino acids of an E2 polypeptide.
In all aspects of the invention, a peptide of the invention may comprise, or consist of, or have less than or more than 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,
271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,
331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,
391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,
451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 amino acids.
The peptides of the invention are preferably "isolated peptides". An "isolated peptide" is a peptide that has undergone some degree of isolation, e.g. by purification. For example, the term "isolated peptide" may refer to a composition in which the peptide of interest is substantially free from other peptides and/or other solutes. Preferably, an "isolated peptide" is a peptide that makes up at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a composition by weight, preferably disregarding any solvent.
The terms "peptide" and "polypeptide" are used herein interchangeably.
Peptides of the present invention may be linked, e.g. joined and/or bound, to molecules that have a particular function, e.g. molecules for adjuvant or other immunogenic purposes, or for detection purposes. The linkage may be via a covalent bond or a non covalent bond. For example, the molecule may be covalently linked to the peptide using synthetic chemistry techniques, by specific non-covalent binding e.g. using an antibody binding domain, or as a fusion protein, e.g. the molecule linked to the peptide may be a peptide or polypeptide .
A molecule to which the peptide is linked may be an adjuvant molecule, e.g. aluminium phosphate or aluminium hydroxide. The molecule may allow detection of the peptide, e.g. it may be a signalling molecule, e.g. GFP. The molecule may facilitate purification of the peptide, e.g. it may be a 6-histidine tag. The molecule to which the peptide is linked may be a polypeptide . E2 polypeptides
Peptides of the invention may comprise, or consist, of a portion of an E2 polypeptide.
SEQ ID NO: 2 shows the polyprotein amino acid sequence of hepatitis C virus strain H77. A host signal peptidase mediates cleavage of the polyprotein into various components, including E2. The E2 polypeptide of hepatitis C strain H77 is located at amino acids 386-729 of SEQ ID NO: 2.
An E2 polypeptide may be any E2 polypeptide from any strain of HCV. The skilled person is able to identify the E2 polypeptide and nucleic acid encoding the E2 polypeptide from a hepatitis C virus strain, e.g. by polymerase chain reaction (PCR) amplification. This is the methodology used in Lavillette et al . (18) to identify E2 amino acid and nucleic acid sequences from a panel of hepatitis C virus strains. In particular, the E2 polypeptide may be an E2 polypeptide selected from one from one of the six major HCV genotypes, for example, any one of genotypes 1 to 6, for example any one of genotypes Ia, Ib, 2a, 2b, 3, 4, 5 and 6.
An E2 polypeptide may be a polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequence from amino acid position 386 to position 729 of SEQ ID NO: 2, and the amino acid sequences corresponding to amino acid position 386 to amino acid position 729 of SEQ ID NO: 2 in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 36.
For example, the E2 polypeptide may be a polypeptide having an amino acid sequence selected from the group consisting of: amino acid position 386 to position 729 of SEQ ID NO: 2, amino acid position 386 to position 729 of SEQ ID NO: 4, amino acid position 217 to position 560 of SEQ ID NO: 10, amino acid position 217 to position 560 of SEQ ID NO: 12, amino acid position 217 to position 560 of SEQ ID NO: 14, amino acid position 386 to position 733 of SEQ ID NO: 16, amino acid position 217 to position 564 of SEQ ID NO: 18, amino acid position 217 to position 564 of SEQ ID NO: 20, amino acid position 217 to position 566 of SEQ ID NO: 22, amino acid position 217 to position 566 of SEQ ID NO: 24, amino acid position 217 to position 560 of SEQ ID NO: 26, amino acid position 217 to position 561 of SEQ ID NO: 28, and amino acid position 217 to position 565 of SEQ ID NO: 36,
Reference to "E2" preferably refers to an E2 molecule, e.g. an E2 glycoprotein. Reference to an "E2 polypeptide" preferably refers to the amino acid sequence of E2.
E1/E2 complexes
"E1/E2 complex" or "E1E2" refers to a complex of El and E2. When co-expressed and secreted, El and E2 can form a complex spontaneously in the media. Formation of an "E1/E2 complex" is readily determined using standard protein detection techniques such as polyacrylamide gel electrophoresis and immunological techniques such as immunoprecipitation.
The El polypeptide is also a component of the hepatitis C virus polyprotein that is cleaved by host signal peptidase. For example, the hepatitis C virus strain 77 El polypeptide is from amino acid position 193 to amino acid position 382 of SEQ ID NO: 2.
Amino acids and amino acid sequences
The term "amino acid" includes all natural and non-natural amino acids, i.e. amino acids that are encoded by the genetic code and amino acids that are not encoded by the genetic code, respectively. However, where an "X" and/or a "Z" is specified as an amino acid, preferably it is a natural amino acid. A "natural amino acid" is an amino acid encoded by the genetic code, e.g. the amino acid is selected from the group consisting of: Alanine, Argenine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and Valine.
The "N-terminus" amino acid of an amino acid sequence is the amino acid at the amino-terminal end of the amino acid sequence. Likewise, the "C-terminus" amino acid is the amino acid at the carboxyl-terminal end of the amino acid sequence.
Amino acid sequences are numbered such that the N-terminal amino acid is at the first position. For example, an amino acid at position 523 refers to the 523rd amino acid in the E2 polypeptide counting from the N-terminal amino acid.
Where a peptide is specified as comprising, or consisting of, particular amino acids in an amino acid sequence, e.g. amino acids 523-535 of SEQ ID NO: 2 (or amino acid position 523 to amino acid position 535 of SEQ ID NO: 2), this refers to a peptide that comprises, or consists of, an amino acid sequence that starts at the earlier position, e.g. position 523, and ends at the later position, e.g. 535. The amino acid sequence includes the earlier and later positions, e.g. the amino acid sequence includes the amino acids at positions 523 and 535.
Where a peptide is specified as comprising a portion of an E2 polypeptide that corresponds to a portion of amino acids in SEQ ID NO: 2, the portion of the E2 polypeptide is determined as follows: the E2 amino acid sequence is pairwise aligned with SEQ ID NO: 2, for example using the ClustalW 1.82 software, and using the default parameters. The ClustalW software may be accessed from the European Bioinformatics institute at http://www.ebi.ac.uk/clustalw/. The default parameters of ClustalW are: Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein/DNA ENDGAP = -1, Protein/DNA GAPDIST = 4.
The pairwise alignment will result in amino acids in the E2 polypeptide being paired with the amino acids of interest in SEQ ID NO: 2. Thus, "a portion of an E2 polypeptide that corresponds to a portion of amino acids in SEQ ID NO: 2" refers to the amino acids in the E2 polypeptide that are paired with the amino acids in the portion of interest in SEQ ID NO: 2.
Preferably, the E2 polypeptide has a sequence identity of at least 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent compared with SEQ ID NO: 2.
Sequence Identity
In certain aspects the invention concerns compounds which are isolated peptides/polypeptides comprising an amino acid sequence having a sequence identity of at least 70% with a given sequence. Alternatively, this identity may be any of 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity.
Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequence (referred to by the SEQ ID No.) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences. Where the aligned sequences are of different length, sequence identity of the shorter comparison sequence may be determined over the entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined over the entire length of the shorter given sequence.
For example, where a given sequence comprises 100 amino acids and the candidate sequence comprises 10 amino acids, the candidate sequence can only have a maximum identity of 10% to the entire length of the given sequence. This is further illustrated in the following example:
(A) Given seq: XXXXXXXXXXXXXXX (15 amino acids) Comparison seq: XXXXXYYYYYYY (12 amino acids)
The given sequence may, for example, be SEQ ID NO: 2
% sequence identity = the number of identically matching amino acid residues after alignment divided by the total number of amino acid residues in the longer given sequence, i.e. (5 divided by 15) x 100 = 33.3%
Where the comparison sequence is longer than the given sequence, sequence identity may be determined over the entire length of the given sequence. For example:
(B) Given seq: XXXXXXXXXX (10 amino acids)
Comparison seq: XXXXXYYYYYYZZYZZZZZZ (20 amino acids)
Again, the given sequence may, for example, SEQ ID NO: 2 % sequence identity = number of identical amino acids after alignment divided by total number of amino acid residues in the given sequence, i.e. (5 divided by 10) x 100 = 50%.
Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW, T-coffee or Megalign (DNASTAR) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. Preferably, ClustalW 1.82 is used to align the sequences using the default parameters .
Peptide function
The peptides of the invention may be antigens for an antibody to E2. The antibody may be the antibody clone 1:7 or A8, as described in the Examples below. The antibody may be an antibody that comprises the VH chain and/or the VL chain of antibody clone 1:7 or A8. The antibody may comprise the VH chain CDRl, CDR2 and CDR3 and/or the VL chain CDRl, CDR2, and CDR3 region of antibody clone 1:7 or A8. The VH and VL domains of 1:7 are shown in SEQ ID NO: 37 and SEQ ID NO: 40, respectively. The respective CDR regions are underlined. The VH and VL domains of A8 are shown in SEQ ID NO: 38 and SEQ ID NO: 41, respectively. The respective CDR regions are also underlined. These antibodies are also described in Allander et al. (22), WO97/40176 and US2002/0016445 Al, which are incorporated herein by reference.
A peptide of the invention that comprises an epitope of an antibody to E2 may adopt a particular conformation, e.g. three-dimensional structure, which is recognised by an E2 antibody. Preferably, an antibody to E2 specifically binds and/or targets the peptides of the invention, e.g. the peptide is recognised by the binding domain of the antibody. Preferably, an antibody to E2 binds the peptides of the invention with an EC50 value of less than 5μg/ml, 4μg/ml, 3μg/ml, 2μg/ml, 1.8μg/ml, 1.7μg/ml, 1.6μg/ml, 1.5μg/ml, 1.4μg/ml, 1.3μg/ml, 1.2μg/ml, l.lμg/ml, l.Oμg/ml, 0.9μg/ml,
0.8μg/ml, 0.7μg/ml, 0.6μg/ml, 0.5μg/ml, or 0.4μg/ml. The EC50 value is the concentration of antigen (peptide) at which half of the antibodies have bound the antigen.
The antibody may bind the peptides of the invention with a Kd value of less than 100OnM, 90OnM, 80OnM, 70OnM, 60OnM, 50OnM, 40OnM, 30OnM, 20OnM, 19OnM, 18OnM, 17OnM, 16OnM, 15OnM, 14OnM, 13OnM, 12OnM, HOnM, 10OnM, 9OnM, 8OnM, 7OnM, 6OnM, 5OnM, 45nM, 4OnM, 35nM, 3OnM, 25nM, 2OnM, 15nM, 1OnM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or InM.
The degree of binding of an E2 antibody to the peptides of the invention may be determined by ELISA. This may involve immobilising the peptide on a surface and adding the antibody to form a detectable complex with the peptide. Such methods are well known to the person skilled in the art, and are described, for example, in Examples 9 and 11 of US2002/0016445 Al, which is incorporated herein by reference.
The peptide may also comprise additional amino acids that are not involved in forming the epitope. For example, the eptiope may exist as a domain in the peptide. Additional amino acids may be useful for isolating the peptide, e.g. a 6-histidine tag.
The peptides of the invention may bind to an antibody that inhibits the binding of E2 to CD81. We have found that the epitopes of the broadly reactive 1:7 and A8 antibodies involve amino acids in the 523-535 region of E2, which is known to be important for the E2-CD81 interaction. An antibody to E2 that inhibits the binding of E2 to CD81 may be selected using a "neutralization of binding" (NOB) assay, e.g. as described in Allander et al. (22), Rosa et al . (42) and US2002/0016445 Al, which are incorporated herein by reference. See in particular Example 12 of US2002/0016445 Al, which describes using the NOB assay to measure the ability of antibodies to block the binding of HCV E2 to target cells. Antibody selection may involve measuring the antibody's ability to inhibit the binding of E2 to a cell, e.g. MOLT-4 cells expressing CD81. MOLT-4 is a human cell line reported to allow low-level HCV replication in vitro, as described by Shimizu et al . (43) . The inhibition may be measured by incubating the cells and E2 with different concentrations of the antibody and detecting the amount of E2 bound to the cell. Preferably the concentration of antibody that inhibits 50% of E2 binding to the cell is less than 2.0μg/ml, l.Oμg/ml, 0.9μg/ml, 0.8μg/ml, 0.7μg/ml, 0.6μg/ml, 0.5μg/ml, 0.4μg/ml, or 0.35μg/ml.
Preferably, the peptides of the invention are capable of eliciting, e.g. provoking and/or stimulating, an antibody response in an animal, e.g. when a peptide of the invention is administered to the animal.
A peptide capable of eliciting an antibody response in an animal may be identified, for example, using ELISA. For example, a blood sample may be taken from an animal following administration of the peptide. ELISA may be used to determine whether any antibodies in the blood sample bind to E2 molecules or E1/E2 complexes bound to a solid support.
For example, the peptides of the invention may elicit an antibody response in an animal. Preferably, the peptides of the invention are capable of eliciting an antibody response, wherein an antibody elicited by a peptide of the invention is an antibody to E2 and/or an E1/E2 complex.
Preferably, an antibody elicited by a peptide of the invention binds, and/or is capable of binding, to at least two E2 molecules or E1/E2 complexes, wherein each E2 molecule or E1/E2 complex is derived from a different genotype of HCV. For example the antibody elicited may have E2 and/or E2/E2-complex binding activity. The antibody may be an antibody to at least 3, 4, 5, 6, 7, or 8 E2 molecules or E1/E2 complexes, wherein each E2 molecule or E1/E2 complex is derived from a different HCV genotype. The hepatitis C virus genotype may be selected from the six major HCV genotypes, for example from the group consisting of: Ia, Ib, 2a, 2b, 3a, 4, 5, or 6, . Preferably, an antibody binds at least one, preferably at least 2, 3, 4, 5, 6, 7, or 8 E2 molecules or E1/E2 complexes, with an EC50 value of less than 5μg/ml, 4μg/ml, 3μg/ml, 2μg/ml, 1.8μg/ml, 1.7μg/ml, 1.6μg/ml, 1.5μg/ml, 1.4μg/ml, 1.3μg/ml, 1.2μg/ml, l.lμg/ml, l.Oμg/ml, 0.9μg/ml, 0.8μg/ml, 0.7μg/ml, 0.6μg/ml, 0.5μg/ml, or 0.4μg/ml, wherein each E2 molecule or E1/E2 complex is derived from a different HCV genotype.
An antibody elicited by a peptide of the invention may be capable of neutralizing HCV, e.g. the antibody may have HCV neutralizing activity. An antibody that is capable of neutralizing E2 HCV is, for example, an antibody that is capable of inhibiting the ability of a hepatitis C virus to infect a cell, e.g. an Huh-7 cell. Preferably the antibody is capable of neutralizing at least two HCVs, wherein each HCV bears an E2 and/or E1/E2 complex derived from a different HCV genotype. The antibody may be capable of neutralizing at least 3, 4, 5, 6, HCVs, wherein each HCV bears an E2 and/or E1/E2 complex derived from a different HCV genotype. The HCV genotype may be selected from the six major HCV genotypes, for example from the group consisting of: Ia, Ib, 2a, 2b, 3a, 4, 5, 6, .
In particular, an antibody elicited by a peptide of the invention may be capable of neutralizing a hepatitis C virus pseudoparticle (HCVpp) , e.g. the antibody may be capable of inhibiting the ability of the HCVpp to infect a cell. The HCVpp may bear an E2 and/or an E1/E2 complex from a specific HCV genotype. Production of HCVpp is described in Op De Beeck et al. (41), which is incorporated herein by reference. The ability of the antibody to neutralize HCVpp may be measured by incubating the HCVpp with different concentrations of the antibody and then adding the particles to Huh-7 cells. The level of HCV infection in the cells is then measured, e.g. by luciferase assay or by western blot with anti-E2 mAB.
Preferably, the antibody is capable of neutralizing a HCVpp by at least 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 95%, e.g. in a neutralization assay as described in the Examples under "HCV retroviral pseudoparticle neutralization assay" . The percent neutralization is as compared to a negative control, i.e. an assay in which the antibody is not incubated with the HCVpp.
Such a neutralization assay may involve incubating HCVpp with 15μg/ml antibody for 1 hour at 37 °C, adding the HCVpp to Huh-7 cells and incubating the cells for 2 hours. The supernatant may then be removed and the cells incubated for a further 48 hours. The level of HCV infection may then be measured.
Preferably the antibody is capable of neutralizing a HCVpp with an IC50 of less than 2000ng/ml, 1500ng/ml, lOOOng/ml, 900ng/ml, 800ng/ml, 700ng/ml, 600ng/ml, 500ng/ml, 550ng/ml, 500ng/ml, 450ng/ml, 400ng/ml, 350ng/ml, 300ng/ml, 35010ng/ml, 200ng/ml, 250ng/ml, 200ng/ml, 150ng/ml, lOOng/ml, 90ng/ml, 80ng/ml, 70ng/ml, 60ng/ml, 50ng/ml, 40ng/ml, 3010ng/ml, 20ng/ml, or even 10ng/ml. The IC50 is the concentration of antibody that results in 50% neutralisation, i.e. the number of cells infected by the HCVpp is reduced by 50% compared to negative control in which antibody is not incubated with the HCVpp.
Preferably, the antibody is capable of neutralizing at least two HCVpp, wherein each HCVpp bears an E2 and/or an E1/E2 complex derived from a different HCV genotype. The antibody may be capable of neutralizing at least 3, 4, 5, 6, 7, or 8
HCVpp, wherein each HCVpp bears an E2 and/or an E1/E2 complex derived from a different HCV genotype. The HCV genotype may be selected from the six major HCV genotypes, for example from the group consisting of: Ia, Ib, 2a, 2b, 3a, 4, 5, 6, 7, or 8.
In addition, or alternatively, an antibody elicited by a peptide of the invention may be capable of neutralizing a cell culture infectious hepatitis C virus particles (HCVcc) , e.g. the antibody may be capable of inhibiting the ability of a HCVcc to infect a cell. The HCVcc may bear an E2 and/or E1/E2 complex from a specific HCV genotype. Construction of HCVcc particles are described in Wakita et al . (15) and Rouille et al. (25), which are incorporated herein by reference. For example, a full-length cDNA HCV isolate is transcribed in vitro and the transcribed RNA is delivered to Huh-7 cells, e.g. by electroporation . Viral stocks may be harvested from the cell culture supernatant, e.g. after 3-4 days post transfection. The ability of the antibody to neutralize HCVcc particles may be measured by incubating the HCVcc particles with different concentrations of the antibody and then adding the particles to Huh-7 cells. The level of HCV infection in the cells is then measured, e.g. by luciferase assay or by western blot with anti-E2 mAB.
Preferably, the antibody is capable of neutralizing a HCVcc particle by at least 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 95%, e.g. in a neutralization assay as described in the Examples under "Infectious HCV neutralization assay". The percent neutralization is as compared to a negative control, i.e. an assay in which the antibody is not incubated with the HCVcc.
A HCVcc neutralization assay may involve incubating HCVcc particles with antibody at a concentration of 15μg/ml for 1 hour at 370C, adding the HCVcc particles to Huh-7 cells and incubating the cells for 2 hours at 370C. The supernatant may then be removed and the cells incubated for a further 48 hours. The level of HCV infection may then be measured.
Preferably the antibody is capable of neutralizing a HCVcc with an IC50 of less than 2000ng/ml, 1500ng/ml, 1000ng/ml,
900ng/ml, 800ng/ml, 700ng/ml, 600ng/ml, 500ng/ml, 55010ng/ml, 500ng/ml, 450ng/ml, 400ng/ml, 350ng/ml, 300ng/ml, 350ng/ml, 200ng/ml, 250ng/ml, 200ng/ml, 150ng/ml, 100ng/ml, 90ng/ml, 80ng/ml, 70ng/ml, 60ng/ml, 50ng/ml, 40ng/ml, 30ng/ml, 20ng/ml, or even 10ng/ml.
Preferably, the antibody is capable of neutralizing at least two HCVcc particles, wherein each particle bears an E2 and/or El/E2-complex derived from a different HCV genotype. The antibody may be capable of neutralizing at least 3, 4, 5, 6, 7, or 8 HCVs, wherein each HCVcc particle bears an E2 and/or El/E2-complex derived from a different HCV genotype. The HCV genotype may be selected from the six major HCV genotypes, for example from the group consisting of: Ia, Ib, 2a, 2b, 3a, 4, 5, 6, 7, or 8.
Nucleic acids
The nucleic acids of the invention are preferably "isolated nucleic acids". An "isolated nucleic acid" is a nucleic acid that has undergone some degree of isolation, e.g. by purification. For example, the term "isolated nucleic acid" may refer to a composition in which the nucleic acid of interest is substantially free from other nucleic acids and/or other solutes. Preferably an "isolated nucleic acid" is a nucleic acid that makes up at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a composition by weight, preferably disregarding any solvent.
Nucleic acids of the invention may be DNA or RNA. Nucleic acids of the invention also include the nucleic acids that have a complementary sequence to the nucleic acids that are described above as nucleic acids of the invention. Nucleic acids of the invention may also comprise a regulatory sequence, e.g. a promoter, that is operably linked to the nucleotide sequence that encodes the polypeptide of the invention.
Where the peptide of the invention is linked to a molecule having a particular function as a fusion protein, the nucleic acid may encode both the peptide of the invention and that molecule.
The term "operably linked" may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide (coding) sequence under the influence or control of the regulatory sequence. Thus a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide (coding) sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.
Vectors The vectors of the invention may include one or more elements that facilitate expression of nucleotide sequence in a host cell. The vector may include an element that allows the vector to replicate in a host cell. The vector may include an element that allows selection of host cells that contain the vector, e.g. a marker gene. Methods of introducing nucleic acid such as vectors into cells, e.g. by transformation or transfection, are well known in the art.
Host cells
The invention relates to host cells comprising a nucleic acid of the invention. The host cell may be a eukaryotic cell, e.g. a yeast or a CHO cell, or a prokaryotic cell, e.g. E. coli. The host cell may be a cell that is suitable for culturing, i.e. it may be a cell that is not part of a living human or animal body. The nucleic acid of the invention may be present in the host cell as part of the host cell genome.
Alternatively, it may be present in the host cell as an autonomously replicating entity, e.g. a plasmid. Preferably the nucleic acid is heterologous to the host cell, i.e. the nucleic acid is not naturally present in the host cell. For example, the nucleic acid may be inserted into the genome, e.g by genetic engineering.
Antibodies
An antibody defined as being an antibody to a particular molecule or as binding to a particular molecule, e.g. E2, is an antibody that is specific, e.g. immunologically specific, for that molecule, e.g. the antibody selectively targets that molecule .
The term "antibody" encompasses monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab')2 fragments, F(ab) molecules, Fv fragments, single domain antibodies, chimeric antibodies and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule . As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited by the manner in which it is made. The term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab')2 fragments, Fv fragments, and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule.
Therapeutic methods
The peptides of the invention are useful for eliciting an antibody response in an individual against hepatitis C virus. The peptides may be used to treat an individual who is not infected with hepatitis C virus. In this case, treating the individual with the peptides of the invention will reduce the risk of the individual becoming infected with hepatitis C virus. For example, the treatment may prevent and/or alter the disease course, e.g. the severity of disease. The peptides may also be used to treat an individual who is infected with hepatitis C virus, an individual who has been diagnosed with hepatitis C virus infection, and/or treatment of an individual who is displaying symptoms and/or signs of hepatitis C virus infection .
Treatment may involve using peptides of the invention with a current "standard therapy" for HCV, e.g. in combination with PEGylated interferon alpha and Ribavirin.
The treatment may also involve treatment of an individual undergoing, or planning to undergo, liver transplantation, for example to reduce the risk of hepatitis C infecting the transplanted liver.
Methods of diagnosing Hepatitis C virus infection are known in the art, e.g. by detecting the virus in the blood of an individual and/or by detecting antibodies to hepatitis C virus in the individual.
The individual to be treated may be an animal, for example a mammal. Preferably the individual is a human.
Vaccines
Therapeutic and prophylactic vaccines, e.g. vaccine compositions, are provided herein, which generally comprise one or more peptides of the invention as described above. The prophylactic vaccines can be used to prevent hepatitis C virus infection, and the therapeutic vaccines used to treat individuals following hepatitis C virus infection. Prophylactic uses include the provision of increased antibody titer to hepatitis C virus in a vaccinated subject. In this manner, a subject, for example a subject at high risk of contracting hepatitis C virus infection (e.g., immunocompromised individuals, organ transplant individuals, individuals obtaining blood or blood product transfusions, and individuals in close personal contact with hepatitis C virus - infected individuals) can be provided with passive immunity to the hepatitis C virus agent. Other prophylactic uses for the present hepatitis C virus vaccines includes prevention of hepatitis C virus disease in an individual after exposure to the infectious agent. Therapeutic uses of the present vaccines involve both reduction and/or elimination of the infectious agent from infected individuals, as well as the reduction and/or elimination of circulating hepatitis C virus and the possible spread of the disease.
Vaccines of the invention may be used with a current "standard therapy" for HCV, e.g. in combination with PEGylated interferon alpha and Ribavirin. The preparation of vaccine compositions containing one or more peptides as the active ingredient is generally known to those of skill in the art. Typically, such vaccines are prepared as injectables (e.g., either as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquids prior to injection) . The compositions will generally also include one or more pharmaceutically acceptable excipients or vehicles such as water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. Additionally, minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. The vaccine compositions may be emulsified or the active ingredient may be encapsulated in liposomes. Vaccines of the invention may also include one or more adjuvants, e.g. aluminium phosphate or aluminium hydroxide (alumn) . Other suitable adjuvants include those as referenced in Vogel FR, Powell MF, Alving CR, which is incorporated herein by reference. A Compendium of Vaccine Adjuvants and Excipients, 2nd ed. as well as others as are known by those having ordinary skill in the art. Amounts and concentration of adjuvant are readily determined by those having ordinary skill in the art.
Administration The peptides of the invention can be administered in conjunction with one or more additional therapeutic agents e.g. ancillary immunoregulatory agents, such as cytokines, lymphokines, and chemokines, including but not limited to IL- 2, modified IL-2 (cysl25 : serl25) , GM-CSF, IL-12, γ-interferon, IP-10, MlPlβ and RANTES. When the peptides of the invention, are used as therapeutic agents, e.g. as therapeutic vaccines, they can be administered in conjunction with known anti- hepatitis C virus therapeutics, such as α-interferon (αlFN) therapy which generally entails administration of 3 million units of α-IFN three times a week subcutaneously (Causse et al. (1991) Gastroenterology 101:497-502, Davis et al . (1989) N Engl J Med 321:1501-1506, Marcellin et al . (1991) Hepatology 13:393-397), interferon β (β-IFN) therapy (Omata et al. (1991) Lancet 338:914-915), ribivirin therapy (Di Bisceglie et al . (1992) Hepatology 16:649-654, Reichard et al . (1991) Lancet 337:1058-1061) and antisense therapy (Wakita et al . (1994) J Biol Chem 269:14205-14210) .
The peptides of the invention may can also be used in conjunction with known anti-hepatitis C virus combination therapies, e.g. a combination of PEGylated interferon alpha and Ribavirin. Other known anti-hepatitis treatments include for example, the combination of α-IFN and ursodiol (Bottelli et al. (1993) (Abstr.) Gastroenterology 104:879, O'Brien et al. (1993) (Abstr.) Gastroenterology 104:966) and the combination of .beta. -IFN and ribivirin (Kakumu et al. (1993) Gastroenterology 105:507-512) .
The peptides of the invention may also be used in combination with an antibody to hepatitis C virus, e.g. an antibody having the VL amino acid sequence of SEQ ID NO: 37, 38, or 39 or an antibody having the VH sequence of SEQ ID NO: 40, 41, or 42.
Other anti-HCV therapeutics with which the peptides of the invention may be used include ribavirin, amantadine (Symmetrel) , viramidine, protease inhibitors, such as VX950 and BILN2061, and polymerase inhibitors, such as NIM283,
Albuferon, Zadaxin, and DAPY.
In a further aspect of the invention, there is provided a peptide of the invention and a second pharmaceutical for use in a therapeutic method.
In a further aspect of the invention, there are provided products containing a peptide of the invention and a second pharmaceutical as a combined preparation for simultaneous, separate or sequential use in a therapeutic method. Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body.
The peptides of the invention, e.g. formulated as vaccines, may be administered parenterally, e.g., by injection (either subcutaneously or intramuscularly) . Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
The peptides of the invention, e.g. as formulated as vaccine compositions, are administered to the subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be prophylactically and/or therapeutically effective. The amount of the composition to be delivered depends on the subject to be treated, the capacity of the subject's immune system to mount its own immune-responses, and the degree of protection desired. The exact amount necessary will vary depending on the age and general condition of the individual to be treated, the severity of the condition being treated and the particular peptide selected and its mode of administration, among other factors. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual . The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated and, e.g. on the peptide or peptide combinations used. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual individual, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
In addition, the peptides of the invention, e.g. formulated as vaccine compositions, can be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals needed to maintain or reinforce the action of the compositions. Thus, the dosage regimen will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgement of the reasonably skilled practitioner.
Samples
A sample, e.g. a sample to be analysed for the presence of antibodies to hepatitis C virus, may comprise or may be derived from: a quantity of blood; a quantity of serum derived from an individual's blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a quantity of pancreatic juice; a tissue sample or biopsy; or cells isolated from said individual. A sample may be taken, or may have been taken, from any tissue or bodily fluid. The sample may be a sample that has previously been removed from a human or animal body. The sample may be an isolated sample, e.g. a sample that is provided in vitro. A sample that is provided as an isolated sample will already have been removed from an individual. A method of the invention may not comprise a step of taking a sample from an individual.
In preferred arrangements the sample is taken from a bodily fluid, more preferably one that circulates through the body. Accordingly, the sample may be a blood sample or lymph sample.
In a particularly preferred arrangement the sample is a blood sample or blood-derived sample. A blood-derived sample is a blood sample that has undergone treatment, e.g. to remove one or more blood components . The blood-derived sample may be a selected fraction of a patient's blood, e.g. a selected cell- containing fraction or a plasma or serum fraction. A selected serum fraction may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells.
Alternatively the sample may comprise or may be derived from a tissue sample, biopsy or isolated cells from said individual.
Mimetics
Antigens bind to antibodies by presenting a particular 3- dimensional shape that is recognised by the antibody. Thus, a molecule that presents a similar shape compared to the peptides of the invention may also bind to antibodies to hepatitis C virus, and may also generate an antibody response. Such a molecule need not be a peptide, rather it may be any molecule that adopts the shape recognised by the antibody. For example, a mimetic may be a molecule that positions glycines, a tryptophan and an aspartic acid molecules at spatial positions that are equivalent to the spatial positions of amino acids G523, W529, G530 and D535, in the E2 polypeptide (amino acids 386-729 of SEQ ID NO: 2) .
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals. There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore". In this case, we have shown that amino acids G523, W529, G530 and D535, and in particular W529 and D535, in the E2 polypeptide affect binding of E2 to broadly neutralising hepatitis C antibodies. The spatial position of W529 and D535, or G523, W529, G530 and D535, in the E2 polypeptide may be taken as the pharmacophore.
Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, e.g. binding to antibodies to hepatitis C virus, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
The invention includes the combination of the aspects and preferred features described in the summary of the invention and in the detailed description except where such a combination is clearly impermissible or expressly avoided.
Any heading used herein is for organizational purposes only and is not to be construed as limiting the subject matter described.
Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Examples
Abbreviations: BSA, bovine serum albumin; GNA, Galanthus nivalis lectin; HCV, hepatits C virus; HCVpp, HCV pseudo-particles; HCVcc, cloned HCV from cell culture; HIV, human immunodeficiency virus; gt, genotype; mabs, monoclonal antibodies; PBS, phosphate buffered saline; WB, western blot. Results
Determination of EC50 for binding E1E2 of genotype Ia (isolate H77c)
The three mAbs investigated, clones 1:7, A8 and Ll, were selected for the present study because earlier results indicated that they were binding to distinct epitopes and blocked the binding of soluble E2 to CD81 expressing cells (NOB assay) . The MAbs were expressed as full length human IgGl from the vector pMThlgGl in stably transfected Drosophila S2 cells (24) . Approximately 10-15ug mAb/ml media could be purified 10 days post induction.
EC50 values for each mAb were determined for the reference H77c (gtla:l, Figure 9) protein in GNA capture ELISA (Fig. 1) . mAb A8 showed slightly lower EC50 than mAb 1:7 (0.44ug/ml vs. 0.67ug/ml), while mAb Ll had considerably lower affinity to isolate H77 (EC50 2.31ug/ml) . These equate to Kd values of approximately 1.5 x 10-8 M (Ll), 2.7 x 10-9 M (A8), and 4.1 x 10-9 M (1:7) .
mAbs 1:7 and A8 display a broad reactivity The reactivity of mAbs 1:7, A8, and Ll against different genotypes was first determined by immunofluorescence. HCV envelope glycoproteins from different genotypes were expressed in Huh-7 cells, and the permeabilized cells incubated with these mAbs. As previously observed, HCV-infected cells stained with mAbs 1:7, A8, and Ll displayed a pattern of specific fluorescence in a network of cytoplasmic membranes and the nuclear envelope, which likely correspond to endoplasmic reticulum membranes (Fig. 2) (25) . Clone Ll stained cells expressing E1E2 of genotypes Ib, 2a, 2b, 5, and 6 (Fig. 2) , but not genotypes Ia, 3, and 4 (isolates gtla:l, gt3:l, and gt4:l (Figure 9)) (data not shown) . Interestingly, both mAb 1:7 and A8 immunostained cells expressing E2 of all the tested isolates, indicating a broad reactivity for these mAbs (Fig. 2) . The broad reactivity of mAbs 1:7 and A8 was confirmed by immunoprecipitation . Indeed, mAbs 1:7 and A8 immunoprecipitated E2 of all isolates tested (Fig. 3) . However, genotypes 3 and 4 gave significantly weaker signals in the final western blot than the other genotypes. This is probably due to differences in affinity of the antibodies used to develop the blotted membrane for the different E2 genotypes, as shown by a direct western blot on lysates from E1E2 expressing cells (Fig. 3, right panel) .
mAb binding to GNA captured HCV envelope glycoprotein from all genotypes
To further characterize the capacity of our mAbs to recognize different isolates, the relative binding of mAb 1:7, A8 and Ll to different genotypes was tested in an ELISA with E1E2 representing different genotypes (Fig. 4) (18, 26) . mAb 1:7 and A8 showed binding to all genotypes while mAb Ll had a more limited binding range. Interestingly, mAb Ll did not bind gtla:l (H77c) , but did bind gtla:3 (UKN1A20.8) emphasizing the diversity within genotypes. In a similar fashion, mAb Ll bound to gt2a:l (UKN2A1.2), but did not bind gt2a:2 (JHF-I) .
mAb 1:7 and A8 are broadly neutralizing antibodies
To establish additional characteristics about the human mAbs, we analyzed their potential neutralizing activity. We first tested the ability of our antibodies to neutralize HCVpp bearing the HCV El and E2 envelope glycoproteins of HCV genotypes Ia, Ib, 2a, 2b, 3a, 4, 5 and 6 (13,18) . Interestingly, both 1:7 and A8 neutralized HCVpp bearing El and E2 from all genotypes tested (Fig. 5) . However, some ■ variations were observed and a lower neutralizing activity was observed for mAb A8 against the Ib isolates tested (gtlb:l and gtlb:2) . In contrast to mAbs 1:7 and A8, mAb Ll had a more varied behavior (Fig. 5) . Indeed, this antibody showed a neutralizing activity against gtlb:l, gtlb:2, gt2a:2 (JFH-I), gt3:l, gt4:2, gt5:l and gt6:2 isolates, but no neutralizing activity was detected against gtla:4, gt2b:l and gt4 : 1 isolates. It is worth noting that differences in the neutralizing activity of mAb Ll could be observed between gt4 : 1 and gt4:2, which belong to the same genotype, again indicating different reactivities within the same genotype.
We also assessed the neutralizing capability of our mAbs using the recently described JFH-I HCVcc system (gt2a) (15) . Both mAbs 1:7 and A8 neutralized HCVcc JFH-I in a dose-dependent manner (Fig. 6) . This was shown by analyzing the level of E2 in the lysate of infected cells 2 days post infection. The approximate IC50 values were estimated by performing density measurements of E2 after western blotting. This indicated approximate IC50 values of 560 ng/ml and 60 ng/ml for clones A8 and 1:7, respectively (Fig. 6) . Surprisingly, Ll had no neutralizing activity in this HCVcc system, whereas it neutralized HCVpp containing the envelope glycoproteins of the JFH-I isolate. Since these particles differ in their assembly process, it is possible that the epitope recognized by Ll is accessible at the surface of HCVpp but not on HCVcc. Differences between HCVpp and HCVcc have indeed already been reported for HCV entry (27) . Altogether, our results indicate that mAbs 1:7 and A8 are broadly potent neutralizing antibodies .
Epitope Mapping of mAb 1:7 and A8
We have earlier observed that mAb 1:7 and A8 bind to conformational epitopes on E2 and block the interaction with CD81 (22) . Therefore, to map their epitope (s) we utilized a panel of full length E1E2 mutants of the gtla H77c isolate, containing single-point alanine substitutions of conserved residues within regions of E2 shown to be involved in CD81 binding (28) . Reactivity of the antibodies to the E2 mutants was measured to GNA captured E1E2 in an ELISA. Both mAbs showed a similar pattern with residues critical for binding being located within region 523-535 (annotated according to the amino acid positions occurring in the H77 sequence) . Substitutions G523A, W529A, G530A and D535A all distinctly abolished binding of these two mAbs to E2 (Fig. 7) . Interestingly, three of these residues are also critical for CD81 binding and are conserved over a wide range of isolates (Fig. 8) (28) .
Discussion
In the present report, we have assessed the breadth of the anti-viral response in vitro of three human mAbs derived from an infected individual (22) . Importantly, two of these antibodies bound to E1E2 proteins and neutralized pseudoparticles bearing the HCV envelope glycoproteins of all the six major genotypes. The third clone, representing a dominant population in the initial screen for these antibodies, reacted to some but not all isolates/proteins of genotypes Ia, Ib, 2b, 4 and 5. The two broadly neutralizing antibodies were titred for neutralization of the cloned JFH isolate (gt2a) , and their epitopes at least partially mapped using a panel of single point mutated E2 proteins based on the H77 isolate (gtla) .
In the binding studies, we observed a slightly higher affinity for clones 1:7 and A8 for binding to E1E2 of the H77 isolate (gtla) than the previous assessment using E2 of the HCV- 1 strain (also gtla) (22) . Although we cannot exclude that this is related to isolate-specific differences, our antibodies likely recognize E2 with a higher affinity in our GNA captured E1E2 complexes, which are likely to have a more native conformation than the E2 alone. Indeed, it has been shown that the binding of CD81 is better to E1E2 than to E2 alone (29), and there are reports of anti-E2 antibodies (e.g. clone CBH2) that only recognize their epitope when E2 is co-expressed with El (30, 31) . Accordingly, we regard the present data as more accurate . A panel of pseudoparticles bearing the HCV envelope glycoproteins of different genotypes allowed us to determine the neutralization profile in vitro for the three mAbs . The results agreed well with the biochemical binding data as well as the cell staining patterns for cells transiently expressing E1E2 constructs. The pseudoparticle neutralization data provided the antiviral genotype specificity, and the sensitivity was determined by using the HCVcc system. Here, the titres observed in the neutralization of the cloned JFH-I virus showed that clone 1:7 was about 10 times more active than clone A8, despite that they had similar binding affinities to H77 proteins.
In a final set of experiments, we wanted to map the binding site for the three antibodies. From earlier experiments, we knew that they bound to conformational epitopes (22) . Therefore epitope mapping using overlapping peptides was unlikely to provide conclusive results. Instead, we assessed antibody binding to a panel of full length E1E2 molecules derived from the H77 isolate carrying single point alanine mutations in regions of E2 implicated CD81 binding (28) . Given that one antibody (Ll) was not broadly reactive and did not react well with the H77c wild-type sequence, we did not attempt to map the epitope of this antibody. However, the broadly neutralizing clones 1:7 and A8 were mapped. Within the constraints of the amino acids investigated, they appeared to share the same epitope, with E2 residues 523, 526, 527, 529, 530 and 535 being particularly important for the antibody-ElE2 interaction. However, as the two clones react somewhat differently with different individual isolates (Figs. 2 and 5) , it is likely that other residues outside the conserved amino acids play a role in recognition by these antibodies. It is possible that these two antibodies have slightly different epitopes and/or different structural features affecting binding. The region critical for binding of clones 1:7 and A8 to H77 has been shown to be equally critical for the E1E2 - CD81 interaction, supporting our earlier results (22, 28) . The residues now found to be involved are conserved in all genotypes (Fig 8) . However, antibodies binding to this region have been shown to be affected by the presence of HDL (32-34) . In preliminary tests, the neutralization of pseudo particles by clones 1:7 and A8 was about 10-20% reduced in presence of serum (data not shown) . Additional work has to be performed to elucidate the extent of this reduction, if any. Still, we note that mAb 1:7 demonstrated a high titre compared to other neutralizing antibodies found in the literature.
In summary, the broad reactivity of human antibody clones 1:7 and A8 was observed both in biochemical binding assays, cell staining and neutralization of pseudoparticles and HCVcc. Their binding was dependent on residues in the a. a. 523-535 region of E2, but we note that they had slightly different profiles of reactivity to individual isolates, indicating that they have overlapping, but distinct epitopes. In this region of E2, there are several residues conserved across all genotypes. Taken together, our combined data lend support for the existence of a conserved, neutralizing epitope in the mentioned region of E2, and that antibodies to this region can be raised in the course of a natural infection. Broadly neutralizing antibodies have been suggested as important goals for prophylactic immunizations against other viruses, and anti-E2 antibodies correlated with protection in HCV vaccine experiments in chimpanzees (12, 35) . Accordingly, this region of E2 is of particular interest to explore for vaccine design. In addition, the human antibodies characterized in this report are potential candidates, single or in combinations with other human anti-HCV antibodies, for passive immunization to complement other pharmaceutical measures against HCV infection. Materials and Methods
Cell culture
Human Huh-7 hepatoma cells (36) were grown in Dulbecco' s modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 1% PEST (lOOU/ml penicillin, lOOμg/ml streptomycin) (Gibco BRL) .
Transfections and plasmids for expression of HCV E1E2 glycoproteins Huh-7 cells were transfected using Fugene 6 (Roche) according to manufacturer's recommendations. Plasmids used for expression of Hepatitis C Virus (HCV) E1E2 protein in the immunofluorescence and immunoprecipitation assays were: H77c (genotype (gt) Ia; Genbank Accession Number AF001751) , UKN1B5.23 (gtlb; AY734976) , UKN2A1.2 (gt2a; AY734977), UKN2B1.1 (gt2b; AY734982) , UKN3A1.28 (gt3; AY734984), UKN4.11.1 (gt4; AY734986) , UKN5.14.4 (gt5; AY785283) , UKN5.15.7 (gt5; EF427672) and UKN6.5.8 (gt6; EF427671) (18) . To facilitate reading, the E1E2 clones used have been renamed in the present illustrations. A list of the full name of the isolates, accession numbers, if any, and the temporary names used in our figures can be found in Figure 9.
Production of antibodies cDNA encoding the human mAbs 1:7, A8 and Ll were subcloned from the original Fabexpressing vector (22) into the vector pMThlgGl allowing expression of full length human IgGl in Drosophila S2 cells (37) as previously described (24) . In brief, stable cell lines were established using the pCoBlast vector (Invitrogen) conferring blasticidin resistance to stably transfected cells. Stable cell lines were grown in Drosophila Serum Free Media supplemented with 16.5mM L- glutamine and 25ug/ml Blasticidin (all Invitrogen) . mAb expression was induced by the addition of 50OuM CuSO4. 10 days post induction, media was harvested and immunoglobulins were purified using HiTrap Protein A column (Amersham Pharmacia) . Immunofluorescence
Huh-7 cells expressing E1E2 proteins representing a range of genotypes were immunostained using either mAb 1:7, A8, or Ll as described previously (24) . All antibodies used were diluted to lug/ml in 1% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) .
Immunoprecipitation 48h post transfection with E1E2 cDNA, 2 x 106 Huh-7 cells were washed in PBS and lyzed in 1.5ml RIPA buffer (1% Nonidet P-40, 0.5% Sodium deoxycholate, 0.1% Sodium dodecyl sulphate (all Sigma) in PBS with protease inhibitors added (30μL/mL Aprotinin, ImM Sodium orthovanadate (both Sigma) ) for 10 min at 40C. The chromosomal DNA was aggregated by agitation of the culture dish and the lysate was centrifuged for lOmin, 10,000 x g at 40C to remove cell debris. To reduce background, the supernatant was precleared by the addition of 15ul Protein-G- Dynabeads 100.4 (Invitrogen) . After lhour of rotation at 4°C, the beads were removed using a magnetic rack, lug of mAb 1:7, A8 or Clone3 (anti-HIV negative control (38) ) , was added to the precleared supernatant and incubated with rotation at 4°C over night. 15ul Protein-G-Dynabeads 100.4 (Invitrogen) was added and incubated for 4 hours at 40C. The beads were washed 3 times in RIPA-buffer and once in PBS. The beads were prepared as a normal reduced protein sample and run on a 4-12% NuPAGE gels in MES buffer (Invitrogen) followed by electroblotting onto an Immobilon-P Transfer Membrane (Millipore) according to manufacturer's recommendations. The membrane was incubated with mAbs H52 (39), ALP98 and AP33 (40) for 1 hour at room temperature, washed in PBS twice for 5 minutes, and incubated with AP-conjugated goat-anti-mouse IgG F(ab')2 antibody (#31324, PIERCE) for 1 hour at room temperature. All antibodies were diluted 1:500-1:2000 in 1% Bovine Serum Albumin (Sigma) in PBS with 0.05 % Tween 20. After washing the membrane as described above, enzyme activity was detected using BCIP and NBT (Sigma) .
HCV retroviral pseudoparticle neutralization assay Hepatitis C virus pseudoparticles (HCVpp) were produced as described previously (41) with plasmids kindly provided by B. Bartosch and F. L. Cosset (INSERM U758, Lyon, France) . Plasmids encoding HCV envelope glycoproteins of genotypes Ia plasmid (strain H), Ib (UKN1B5.23), 2b (UKN2B1.1), 3a (UKN3A1.28), 4 (UKN4.11.1), 5 (UKN5.14.4) and 6 (UKN6.5.340) were used to create HCVpp displaying E1E2 of the major genotypes (Figure 9) . These HCV envelope glycoprotein encoding plasmids have been previously described (13,18) . The genotype 2a plasmid (strain JFH-I) was kindly provided by T. Pietschamnn and R. Bartenschlager (University of Heidelberg, Germany) .
Supernatants containing the pseudotyped particles were harvested 48 h after transfection and filtered through 0.45 urn pore-sized membranes. HCVpp were incubated with 15ug mAb /ml for Ih at 37°C and added to Huh-7 cells seeded the day before in 24-well plates and incubated for 2 h at 370C. The supernatants were then removed and the cells were incubated at 37°C. At 48h post infection, Luciferase assays were performed as indicated by the manufacturer (Promega) .
Infectious HCV neutralization assay
The plasmid pJFH-1, containing the full-length cDNA of JFH-I isolate and kindly provided by T. Wakita (National Institute of Infectious Diseases, Tokyo, Japan) , was used to generate HCV particles from cell culture (HCVcc) as previously described (15, 25) . Briefly, the pJFHl plasmid was linearized and used as a template for in vitro transcription with the MEGAscript kit from Ambion. In vitro transcribed RNA was delivered to Huh-7 cells by electroporation and viral stocks were obtained by harvesting cell culture supernatants at three to four days post-transfection. HCVcc were incubated with antibodies for Ih at 370C and then added to Huh-7 cells seeded the day before in 24-well plates and incubated for 2 h at 370C. The supernatants were then removed and the cells incubated at 37°C. At 48h postinfection, HCVcc infection level was analyzed by western blot with anti-E2 mAb 3/11 (39) .
GNA capture ELISA
An ELISA to detect mAb binding to E2 was performed as previously described (26) . In brief, HEK293FT cells were transfected with plasmids encoding the glycoproteins El and E2 from all genotypes. Clarified lysates from transfected cells were captured on to GNA {Galanthus nivalis) lectin (Sigma) - coated Maxisorp enzyme immunoassay plates (NUNC) . Antibody titrations were performed on the H77c glycoproteins between concentrations of 50- 0.016μg/mL mAb 1:7, A8 or Ll. For cross genotype detection, the 50% binding concentration to H77c was used. Bound antibody was detected with anti-human immunoglobulin G antibody conjugated to alkaline phosphatase (Sigma) and pNPP substrate (Sigma) . Absorbance values were determined at 405nm. Kd values were inferred from antibody binding curves by non-linear regression using Prism 4 software (GraphPad Inc. ) .
Antibody epitope mapping by alanine-scanning Alanine substitution mutants of gtla E1E2 glycoprotein
(isolate H77c) were captured using GNA coated plates and detected with monoclonal antibodies, as described above.
Protein amounts were normalized using western blot analysis of monomeric E2 proteins as described previously (28) . Captured proteins were detected using human monoclonal antibodies at concentrations that gave 50% binding of the wild type H77c.
Mutant E2 glycoproteins are annotated according to the amino acid position in the H77c seguence. References
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38. Samuelsson A, Yari F, Hinkula J, Ersoy O, Norrby E, Persson MAA (1996) Eur J Immunol 26:3029-3034. 39. Flint M, Maidens C, Loomis-Price LD, Shotton C, Dubuisson J, Monk P, Higginbottom A, Levy S, McKeating JA (1999) J Virol 73:6235-6244.
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41. Op De Beeck A, Voisset C, Bartosch B, Ciczora Y, Cocquerel L, Keck Z, Foung S, Cosset FL, Dubuisson J (2004) J Virol 78:2994-3002.
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Sequences of Proteins of Immunological Interest, 5th edn. Bestheda MD, US Department of Health and Human Services.
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Claims

Claims :
1. A peptide comprising the amino acid sequence WX1X2X3X4X5D (SEQ ID NO: 43), wherein each of X1, X2, X3, X4, and X5 is an amino acid, and which peptide comprises less than 40 amino acids.
2. The peptide of claim 1, wherein the peptide comprises the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D (SEQ ID NO: 44), wherein each of X6, X7, X8, X9, and X10 is an amino acid.
3. A peptide comprising the amino acid sequence GX10X9X8X7X6WGX2X3X4X5D (SEQ ID NO: 44), wherein each of X6, X7, X8, X9, and X10 is an amino acid, and which peptide comprises less than 250 amino acids.
4. The peptide of claim 3, wherein the peptide comprises the sequence GX10X9TYX6WGX2X3X4X5D (SEQ ID NO: 45) .
5. The peptide of any one of the preceding claims wherein:
X1 is G;
X2 is selected from the group of amino acids consisting of A,
G, E, and S;
X3 is selected from the group of amino acids consisting of N and S;
X4 is selected from the group of amino acids consisting of D,
V, and E;
X5 is selected from the group of amino acids consisting of T and R; X6 is selected from the group of amino acids consisting of S,
T, D, and N;
X7 is Y;
X8 is T;
X9 is P; and X10 is selected from the group of amino acids consisting of A,
V, E, L, Y, and N.
6. The peptide of any one of the preceding claims, which peptide comprises a portion of an E2 polypeptide, which portion corresponds to amino acids 523-535 of the amino acid sequence of SEQ ID NO: 2, and which peptide comprises less than 250 amino acids of the E2 polypeptide.
7. The peptide of any one of the preceding claims, wherein the peptide comprises an amino acid sequence selected from the group consisting of: GAPTYSWGANDTD (SEQ ID NO: 62),
GVPTYTWGGNDTD (SEQ ID NO: 191), GEPTYDWGGSVRD (SEQ ID NO: 47), GAPTYTWGENETD (SEQ ID NO: 48), GVPTYSWGENETD (SEQ ID NO: 49), GVPTYTWGENETD (SEQ ID NO: 50), GVPTYSWGENETD (SEQ ID NO: 51), GLPTYSWGENETD (SEQ ID NO: 52), GVPTYSWGENETD (SEQ ID NO: 53), GVPTYTWGANETD (SEQ ID NO: 54), GAPTYNWGENETD (SEQ ID NO: 55), GVPTYTWGDNESD (SEQ ID NO: 56), GVPTYNWGENETD (SEQ ID NO: 57), GYPTYNWGENDTD (SEQ ID NO: 58), GYPTYNWGSNETD (SEQ ID NO: 59), GNPTYTWGENETD (SEQ ID NO: 60), and GNPTYTWGENETD (SEQ ID NO: 61) .
8. The peptide of claim 7, wherein the peptide comprises the amino acid sequence GAPTYSWGANDTD (SEQ ID NO: 62) .
9. The peptide of any one of the preceding claims, wherein the peptide comprises an amino acid sequence that has at least
70 per cent sequence identity with amino acids 488-542 of SEQ ID NO: 2.
10. The peptide of claim 9, wherein the peptide comprises amino acids 488-542 of SEQ ID NO: 2.
11. The peptide of any one of the preceding claims, wherein the peptide is an antigen for an antibody to E2.
12. The peptide of any of the preceding claims, wherein the peptide is capable of eliciting an antibody response in an animal .
13. The peptide of claim 12, wherein the peptide is capable of eliciting an antibody response in an animal against hepatitis C virus.
14. An isolated nucleic acid comprising a nucleotide sequence, which nucleotide sequence encodes a peptide as defined in any one of claims 1 to 13.
15. A peptide, as defined in any one of claims 1 to 13, for use in a therapeutic method.
16. A vaccine comprising a peptide as defined in any one of claims 1 to 13.
17. The peptide or vaccine, according to claim 15 or claim 16, for use in immunising an individual against hepatitis C virus .
18. Use of a peptide, as defined in any one of claim 1 to 13, in the manufacture of a medicament for use in immunising an individual against hepatitis C virus.
19. The peptide, vaccine, or use according to claim 17 or claim 18, wherein immunising an individual against hepatitis C virus includes administering PEGylated interferon alpha and/or Ribavirin to the individual.
20. A method of immunising an individual against hepatitis C virus, which method comprises administering a peptide, as defined in any one of claims 1 to 13, to an individual.
21. The method according to claim 20, wherein the method comprises administering PEGylated interferon alpha and/or Ribavirin to the individual .
22. A kit for immunising an individual against hepatitis C virus, which kit comprises:
(i) a container comprising a composition, which composition comprises a peptide as defined in any one of claims 1 to 13; and optionally (ii) instructions for administering the composition to the individual.
23. A kit for diagnosing hepatitis C virus in an individual, wherein the kit comprises: (i) a container comprising a peptide as defined in any one of claims 1 to 13; and optionally
(ii) instructions for incubating the peptide with a sample taken from the individual and observing antibody bound to the peptide.
24. A kit for measuring an antibody response to a hepatitis C virus vaccine in an individual, wherein the kit comprises:
(i) a container comprising a peptide as defined in any one of claims 1 to 13; and optionally (ϋ) instructions for incubating the peptide with a sample taken from the individual and measuring the amount of antibody bound to the peptide.
PCT/GB2008/000702 2007-09-07 2008-02-29 Materials and methods for the treatment of hepatitis c WO2009030872A1 (en)

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