GB2364244A - Method of testing for agents for hepatitis c - Google Patents

Abstract

Use of a GBV-B infected common marmoset (Callifrise jacchus), or hepatocyte thereof, in a method for identifying an agent for use in the therapeutic or prophylactic treatment of hepatitis C. Agents which demonstrate anti-GBV-B activity are useful in the treatment of hepatitis C.

Description

2364244 TEST METHOD

Field of the Invention

5 The present invention relates to an animal model for identifying agents for the therapeutic and/or prophylactic treatment of hepatitis C. The invention also relates to cells and methods for testing and screening such agents.

Background of the Invention

10 It is estimated that nearly 200 million people worldwide may be infected with hepatitis C virus (HCV) with at least 85% of infected individuals expected to develop chronic hepatitis. It is estimated that a third of these patients will develop liver cirrhosis over 20-30 years with complications of end-stage liver disease, such as decompensated cirrhosis, portal hypertension and hepatocellular carcinoma.

15 Chronic HCV infection is therefore currently the major cause of orthotopic liver transplant. A vaccine to prevent HCV infection has not yet been developed. Current therapy for HCV disease involves treatment with interferon-a, alone or in combination with the nucleoside analogue ribavirin. However, in addition to some very unpleasant side effects, the treatment is only effective long term in a 20 small fraction of patients. Because of the magnitude of this disease and the lack of adequate clinical treatments, there is an urgent need to develop novel therapeutic approaches.

HCV is a positive strand RNA virus and has been classified as a genus of the Flaviviridae family. Laboratory studies with HCV are limited to molecular 25 analysis since attempts to culture the virus in vitro have so far been unrewarding.

Furthermore, apart from the chimpanzee, HCV does not infect or replicate in any other animal species. The lack of a tissue culture system and animal model has severely hampered the development of new therapies for this disease.

GB agent hepatitis was first described by Deinhardt et al (1967), J. Exp Med 30 125: 673-687, who inoculated tamarins (Saguinus species) with a serum from a surgeon affected by acute hepatitis. Senial passage of sera from these animals resulted in hepatitis. Further studies have led to the conclusion that GB agent type

2 B (GBV-B) can only infect tamarins of the Saguinus species (Parks et al (1969), J. Int. Dis. 120: 548-559).

Several groups have successfully passaged GBV-B in wild taniarin species, which are not widely used in laboratory experiments, but not in macaques nor in 5 the common marmoset (Callithrixjacchus).

The molecular characterisation of GBV-B has revealed the virus to be another member of the Flaviviridae family and to be closely related to HCV. There is a high degree of homology between GBV-B and HCV replicative processes both structurally and biochemically and the similarities in disease 10 pathogenesis indicate that GBV-B may prove a useful surrogate virus for HCV.

Summary of the Invention

The invention is based on the novel finding that GBV-B infects and replicates in the common marmoset (Callithrixjacchus). This finding was unexpected and 15 entirely contrary to the expectations of the scientists based on the teaching in the prior art that GBV-B can only infect tamarin marmosets of the Saguinus species. Common marmosets are easier to handle and manipulate than tamarin marmosets and also have the advantage of breeding more easily. A GBV-B infected mari-noset is now provided which is a screening target for the identification and development of

20 novel pharmaceutical agents. These agents may be used in the therapeutic and/or prophylactic treatment of hepatitis C.

Accordingly, the present invention provides:

a method for identifying an agent for use in the therapeutic treatment of hepatitis C, said method comprising:

25 (i) administering a test agent to a GBV-B infected marmoset of the Callithrixjacchus strain; and (ii) determining whether the test agent reduces or abolishes GBV-13 infection in said marmoset; wherein step (ii) comprises testing a sample taken from said marmoset.

- a method for identifying an agent for use in the prophylactic treatment of hepatitis C, said method comprising:

(ia) administering a test agent to a marmoset; 3 (ib) administering GBV-B to said. marmoset; and (ii) determining whether the test agent reduces or prevents GBV-B infection in said marmoset; wherein step (ii) comprises testing a sample taken from said marmoset.

5 - a method for identifying an agent for use in the therapeutic treatment of hepatitis C, said method comprising:

(i) contacting a GBV-B infected hepatocyte from a marmoset of the Calfithr&jacchus strain with a test agent in culture; and (ii) determining whether the test agent reduces or abolishes GBV-B 10 infection in said hepatocyte; an agent identified by a method according to the invention; an agent identified by a method according to the invention for use in a method of treatment of the human or animal body by therapy; use of an agent identified by a method according to the invention in the 15 manufacture of a medicament for the treatment of hepatitis C; - a pharmaceutical composition in dosage form comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an agent identified by a method according to the invention; - a marmoset of the Callithrixjacchus strain infected with GBV-B; 20 - a GBV infected hepatocyte from a marmoset of the Callithrixjacchus strain; - use of a common mannoset infected with GBV-B or a common marmoset hepatocyte infected with GBV-B in a method for identifying an agent for the treatment of hepatitis C.

Brief Description of the Drawings

Figure I shows the titre of GBV-B in marmoset (Callithrixjacchus) sera following infection with GBV-B infectious sera.

Figure 2 shows the biochemical analysis of the liver enzyme GLDH in sera from two marmosets (Callithrixjacchus) infected with GBV-B.

Figure 3 shows the titre of GBV-B in supernatants from marmoset (Callithrix jacchus) hepatocyte cell culture following infection with GBV-B infectious sera.

Figure 4 shows the titre of GBV-B in cell pellets from marmoset (Callithrix jacchus) hepatocyte cell culture following infection with GBV-B infectious sera.

Figure 5 shows the titre of GBV-B in marmoset (Callithrixjacchus) cell culture following infection with GBV-B infectious supernatant derived from marmoset hepatocytes infected in vitro.

Figure 6 shows the titre of GBV-B in supernatants from marmoset (Callithrix jacchus) cell culture following treatment with an anti-viral agent.

Figure 7 shows the titre of GBV-B in cell pellets from marmoset (Callithrix jacchus) cell culture following treatment with an anti-viral agent.

Detailed Description of the Invention

20 Throughout the present specification and the accompanying claims the words if comprise" and "Include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

25 The present invention relates to the use of a GBV-B infected common marmoset and hepatocytes thereof in methods of identifying products for the therapeutic treatment of hepatitis C. The present invention also relates to the use of a common marmoset in methods of identifying products for the prophylactic treatment of hepatitis C wherein GBV-B is administered to the marmoset.

30 A marmoset provided by the invention is preferably of the Callithrax species and is more preferably Callithraxjacchus. A m,armoset of the invention is preferably bred in captivity. More preferably a marmoset of the invention is laboratory bred.

Typically, a marmoset of the invention will be disease free prior to administration of GBV-B. In particular, a marmoset of the invention is free of parasites especially parasites that affect the liver.

A marmoset may be infected with a GBV-B virus by exposure to a viral 5 sample by any suitable method. A typical viral sample may comprise infectious sera prepared from another infected animal, an infectious tissue culture preparation such as infected cells or supernatant from an in vitro culture or an infectious RNA preparation. Typically, a GBV-B sample is administered directly to the marmoset. A marmoset may be infected via the intravenous or intrahepatic route. Preferably, a marmoset is infected with GBV-B intravenously. GBV-13 is typically administered in a single dose. GBV-B may be administered at any appropriate dosage. A typical dose may be O.lml of GBV-B infectious sera, preferably 0.2 to 0.5ml, more preferably 0.25ml.

The invention also provides a tissue culture system for determining whether 15 an agent has anti-GBV-B properties. A tissue culture system provided by the invention may comprise hepatocytes derived from a marmoset. Typically the hepatocytes will be cultured as intact tissue, for example the hepatocytes may be present in a slice of liver in culture. Alternatively, the hepatocytes may be cultured as a primary culture. The hepatocytes may be passaged to form a cell line.

20 The cultured hepatocytes may be derived from a GBV-B infected marmoset,

i.e. they may be infected in vivo. Alternatively, the cultured hepatocytes may be infected in vitro. Examples of each are seen in Examples 2a) and 2b) below.

Hepatocytes may be infected in vitro by any suitable method. Techniques well known in the art such as electroporation, microinjection, lipofection or calcium 25 phosphate transfection may be used to introduce viral RNA into cultured hepatocytes. Another method for infecting cultured hepatocytes with GBV-B involves simply exposing cells to the GBV-B by adding GBV-B to the culture medium. Preferably, a virus sample is added to cultured hepatocytes by incubating hepatocytes with the viral sample for from 2 to 8 hours, preferably for 2 to 4 hours, 30 after which time the inoculum is removed and replaced with culture medium. The cells may then be cultured for up to 4 weeks, preferably for 2 weeks.

Characteristics typical of hepatitis may be monitored to determine whether an 6 agent exhibits anti-viral activity. Characteristics of GBV-B infection may be monitored virologically and/or biochemically. A marmoset infected with GBV-B will typically demonstrate one, two, three or more of the following characteristics: a rise, which may only be transient, in the level of one or more liver enzymes in the 5 serum; an increase in viral load; production of specific antibodies to GBV-B virus; histological changes in the liver. For example a GBV-B infected marmoset will typically show one or more of the following: a rise in serum alanine transaminase level of at least 2 to 3 fold; a rise in GLDH level in the serum of at least 2 to 5 fold; at least a 3 log rise in GBV-B RNA; elevation of GBV-B levels to 10' to 10' gl/ml; 10 maintenance of elevated GBV-B levels for 50 days or longer; periportal inflammation in the liver comprising mainly lyrnphocytic infiltrate with some mononuclear cells (neutrophils and eosinophils); an increase in fibrous tissue (collagen desposition) around portal regions of the liver; evidence of abnon-nal liver histology at from 32 to 100 days post infection.

15 A method for determining whether an agent is suitable for use in the treatment of hepatitis C consists essentially of administering a test agent to a marmoset infected with GBV-B, or a marmoset hepatocyte infected with GBV- B; and monitoring GBV infection in said marmoset or said hepatocyte. An agent suitable for use in the therapeutic treatment of hepatitis C will reduce or abolish 20 GBV-B infection in said marmoset.

A method for determining whether an agent is suitable for use in the preventative treatment of hepatitis C, consists essentially of administering a test agent to a marmoset, or a marmoset hepatocyte; administering GBV-B to said marmoset, or said hepatocyte; and monitoring the effect of GBV-B administration in 25 said marmoset or said hepatocyte. An agent suitable for use in the propylactic treatment of hepatitis C will prevent, or limit the effects of GBV-B -infection. Typically there is a delay of from I minute to 3 months, for example 5 minutes, 30 minutes, I hour, 4 hours, 12 hours, I day, 3 days, I week, 2 weeks or I month between administration of the test agent and administration of GBV-B to the 30 marmoset.

Typically agents with anti-GBV-B activity will exhibit one or more of the following properties: inhibition of viral enzymes, inhibition of viral replication, 7 inhibition of fibrosis generation and/or stimulation of the immune response to clear the viral infection. GBV-B infection causes liver damage which is reflected in the increase in production of a number of liver enzymes and/or abnormal liver histology. An agent with anti-GBV-B activity will prevent, either completely or partially, or 5 reverse the GBV-B associated rises in serum levels of liver enzymes and/or GBV-B induced changes in liver histology.

GBV-B infection in marmosets may be monitored by any suitable means. Preferably, GBV-B infection is monitored by monitoring viral protein levels and/or liver enzyme levels in the sera and/or by measuring viral load and /or by monitoring 10 liver histology. More preferably GBV-B infection is monitored by analysing for the presence of GBV-B RNA and/or analysing for biochemical evidence of GBV-B infection, for example, by measuring GLDH liver enzyme levels.

The effect of a test agent on GBV-B infection is determined by testing a sample taken from a marmoset to which the test agent has been administered. Blood 15 samples and/or liver biopsies may be taken from marmosets to enable such measurements to be made. A typical blood sample is from 0.5ml to 5ml, preferably from lml to 3ml and most preferably lml. A typical liver biopsy is a needle biopsy. Preferably, a needle biopsy removes a piece of liver of from 1 to 2 millimeters in diameter by from 0.5 to 2 cm, preferably 1 cm, in length. Such samples and biopsies 20 may be taken at any suitable interval, for example at daily, weekly, fortnightly, monthly or bi-monthly intervals. Preferably samples are taken at weekly intervals. Preferably initial samples are taken at 15 minutes and I day after administration of GBV-B. Liver samples may be obtained by killing marmosets after exposure to GBV-B and/or a test agent and removing the liver post-mortem. Different 25 marmosets may be culled at different times after administrating GBV-B and/or a test agent, for example at one week, two weeks or one month post- administration.

Assays for determining whether an agent has anti-GBV-B activity may also be carried out in vitro using cultured marmoset hepatocytes (see e.g. Example 2b). AntFGBV-B activity in cultured hepatocytes may be monitored by any suitable 30 means. Typically, expression of one or more viral protein and/or viral RNA may be measured. Alternatively, the level of one or more enzymes produced by the cultured cells can be measured. Any histological changes in cultured hepatocytes may also be 8 observed.

Typically, observations and measurements may be made before and after administration of GBV-B and/or before and after application of the test agent to the marmoset or cultured hepatocytes. The factors indicative of viral infection may be 5 compared between control marmosets or hepatocytes which have not received the test agent and marmosets or hepatocytes which have been contacted with the test agent.

Preferably levels of one, two or more of the following liver enzymes are monitored: glutamate dehydrogenase (GLDH), alanine transaminase (ALT), gamma- 10 glutarnyltransferase (GGT), isocitrate dehydrogenase (ICD), alpha glutathione Stransferase (alpha GST) and/or sorbitol dehydrogenase (SDH).

Preferably levels of one, two or more viral proteins are monitored. For example, the presence of non-structural viral proteins such as NS2, NS3 NS4a, NS4b, NS5a and/or NS5b and/or structural viral proteins such as core protein, El 15 and/or E2 may be determined. A typical assay for monitoring viral proteins may be immunofluorescence or ELISA using specific antibodies to one or more of the proteins.

Inhibition of viral enzymes can typically be monitored directly by assaying for enzyme activity in an in vitro cell culture system. GBV-B enzymes are typically 20 involved in the processing of the viral polyprotein. Inhibition of viral enzymes may therefore be monitored by analysing the viral proteins produced in infected marmoset cells. Typically, such analysis will involve electroporesis and western blotting of proteins expressed in virally infected cells using one or more antibody specific for viral proteins. Analysis of the immunolabelled bands on the western blot will reveal 25 the extent of processing of the viral polyprotein. Comparison with the results of a parallel experiments with and without addition of the test agent will reveal whether a viral enzyme is inhibited, Processing of the viral polyprotein is essential for viral maturation. Therefore, inhibition of viral enzymes will also lead to inhibition of viral maturation which will be reflected in a decreased increase in viral load compared to 30 control marmosets to which the test agent has not been administered.

Inhibition of viral replication can be monitored by measuring the viral load. Preferably viral load is measured using quantitative polymerase chain reaction 9 (PCR). Preferably RNA is extracted from marmoset serum, liver and/or hepatocytes and the amount of GBV-B specific mRNA is measured by quantitative reverse transcriptase TaqManTm PCR.

A typical TaqMan'm PCR assay utilizes a standard solution containing a 5 known number of GBV-B RNA molecules. The GBV-B RNA is converted into cDNA using reverse transcriptase and a primer specific for GBV-13 RNA and the cDNA is amplified by PCR using GBV-B primers plus a probe oligonucleotide which is also specific for GBV-B. Degradation of the probe oligonucleotide occurs during the PCR reaction and results in the release of a fluorescent moiety such that 10 the increase in fluorescence is directly proportional to the level of GBV-13 RNA in the starting material. By including standards of known concentrations of GBV-B RNA in the assay, the absolute levels of GBV-B RNA in the starting material can be estimated.

Inhibition of fibrosis generation may typically be monitored in liver sections 15 which have been stained specifically for collagen and/or reticulin. Sirius red stain is an example of a specific stain that may be used. The change in collagen staining observed in the liver of GBV-B infected, marmosets is qualitative rather than quantitative. Preferably a scoring system is used to quantify the degree of fibrosis in the liver.

Stimulation of the immune response can typically be monitored by detecting antibodies directed against viral proteins. Antibodies specific for viral proteins may be detected using an ELISA assay.

In a typical ELISA assay GBV-B proteins are bound to A solid phase, such as a microtitre plate. The test sample, preferably marmoset sera, is added. Any anti- 25 GBV-B antibodies present in the sample bind to the GBV-B proteins. Material which has bound non-specifically is removed by washing and bound antibodies are detected by the addition of secondary antibody that specifically binds to marmoset immunoglobulin. The secondary antibody is conjugated to an enzyme which catalyses a colour change when a specific substrate is added. The colour reaction 30 (optical density) is related to the concentration of anti-GBV-B antibody and can be measured using a spectrophotometer.

Administration of an agent suitable for use in a method of therapeutic treatment of hepatitis C will typically result in,a reduction in viral load and/or a reduction.in elevated enzyme levels and/or histological improvements, typically a reduction in fibrous tissue and/or a reduction in inflammation in the liver.

Administration of an agent suitable for use in the prophylactic treatment of 5 hepatitis C will typically prevent an increase in viral load following exposure to GBV-B or will reduce the increase in viral load compared to a marmoset to which the test agent has not been administered. Preferably, administration of an agent suitable for use in preventing hepatitis C will prevent a rise in the serum level of one or more liver enzyme and/or will prevent or limit any histological changes normally io associated with GBV-B infection.

A marmoset of the invention may also be used to assess the potential side effects of an agent for the treatment of hepatitis C. For example, the metabolism and toxicity of an agent demonstrating anti-viral activity may be assessed using a marmoset of the invention. This has the advantage of enabling efficacy, toxicity, 15 pharmacokinetic properties and/or potential therapeutic effects of metabolites of the test agent to be assessed using the same animal model.

Suitable test agents which can be tested in the above assays include combinatorial libraries, defined chemical entities, peptide and peptide mimetics, oligonu'cleotides and natural product libraries, such as display (e.g. phage display 20 libraries) and antibody products. Preferred test agents include interferon and other cytokine mimetics or agonists. Preferred test agents for determining suitability for use as a vaccine in the prophylactic treatment of hepatitis C include GBV- B subunit proteins, HCV subunit proteins and DNA molecules encoding such subunit proteins.

Such subunit proteins include -core, El, E2, NS2, NS3, NS4a, NS5a and NS5b.

25 The terin 'agent' is intended to include a single substance and a combination of two, three or more substances. For example, the term agent may refer to a single peptide, a mixture of two or more peptides or a mixture of a peptide and a defined chemical entity. The term 'agent' also refers to a composition comprising an immunogenic substance and one or more suitable adjuvants.

30 Marmosets may be dosed with the test agent prophylactically or therapeutically on one or more occasions. Typically test agents may be administered bi-weekly, weekly, twice weekly, daily or two, three or more times a day, for example, at hourly or at two, three or four hourly intervals.

The test agents may be formulated with standard carriers and/or excipients as is routine in the pharmaceutical art, and as fally described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania 17"' Ed.

1985.

A test agent may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, nasal, vaginal, intravenous, intraarterial, intrahepatic, intramuscular, intraperitoneal, subcutaneous or other appropriate administration routes. A test agent may be present in the food or drinking water or may be administered using an osmotic minipump.

Test agents may be administered at any appropriate dosage. A typical dose may be from 0. 1 to 50mg: per kg of body weight, for example from 0.5 to 30mg per kg of body weight, I to 20mg per kg of body weight or I to I Omg per kg of body weight.

15 Cultured hepatocytes may be contacted with a test agent by any suitable method. The cultured hepatocytes may be perfused with the test agent or the agent may be added to the culture medium bathing the cells. The test agent may be introduced into the cells directly. Any suitable technique known in the art may be used to introduce the test agent into the cultured cells. Such well-known techniques 20 include microinjection, electroporation and methods involving the use of transfection agents such as lipofectants, DEAE-dextran and calcium phosphate.

Test substances may be used at a concentration of from I nM to 1 000N, preferably from I N to 1 00tM, more preferably from 1 tM to I OgM.

An agent identified by a method of the invention may be used in a method of 25 therapeutic or prophylactic treatment of the human or animal body by therapy. The invention provides a method for treating a patient infected with hepatitis C virus (HCV), the method comprising administering to the said patient a therapeutically effective amount of an agent identified by a method using a GBV-B infected marmoset or a GBV-B infected marmoset hepatocyte to determine anti-viral activity 30 of the agent. Preferably the patient infected with HCV is suffering from hepatitis C.

The invention provides a method for treating a subject at risk of contracting 12 hepatitis C, the method comprising administering to the said subject a prophylactically effective amount of an agent identified by a method wherein GBV-B is administered to a common marmoset or marmoset hepatocyte in culture to determine whether the agent has anti-GBV-B activity. A subject at risk of 5 contracting hepatitis C is typically at risk of HCV infection.

It is preferred that therapeutic treatment is administered in the early stages of hepatitis C. Cirrhosis, portal hypertension, hepatocellular carcinoma and other complications of hepatitis C may also be treated using an agent identified by a method of the invention.

10 An agent for use in a method of treatment of hepatitis C by therapy will typically improve the condition of a patient suffering from hepatitis C and/or ameliorate the symptoms of hepatitis C.

An agent for use in a method of prophylatic treatment of hepatitis C will typically lessen the severity of one or more of the symptoms resulting from HCV 15 infection and/or may prevent the onset of one or more symptom of HCV infection.

An agent identified according to a screening method outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art, and as fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania 17" Ed.

20 1985, the disclosure of which is included herein of its entirety by way of reference.

Compositions and medicaments for use in a method of treating liver disease may be formulated in dosage form. Medicaments comprising a therapeutic agent identified by a method of the invention may be in a form suitable for administration to a patient, for example in tablet, capsule or liquid form, or may be in a concentrated 25 forrn suitable for preparation by a pharmacist.

The agents may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, nasal, vaginal, intravenous, intraartenial, intrahepatic, intramuscular, intraperitoneal, subcutaneous or other appropriate administration routes.

30 A therapeutically effective amount of an agent is administered to a patient.

An amount of an agent sufficient for preventing infection is administered to a subject at risk of hepatitis C. The dose of a therapeutic agent may be determined according 13 to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from 5 about 0. 1 to 50 mg per kg of body weight, according to the activity of the specific agent, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

An agent that is capable of preventing hepatitis C infection by stimulating the 10 immune system to produce antibodies specific for proteins of HCV is preferably administered in a single dose. One or more further doses may be required for long term protection against hepatitis infection. Further doses may be administered after a period of I to 15 years after the initial dose, for example after 1, 2, 3, 4, 5, 8, 10, 12 or 15 years. Regular doses may be administered at regular intervals after the first 15 dose, for example at 3, 5, 8, 10 or 15 yearly intervals.

An agent that is capable of preventing hepatitis C infection by stimulating the immune system of a mammal to produce antibodies specific for proteins of a HCV is preferably a nucleic acid, Nucleic acid, such asRNA or DNA, and preferably, DNA, is provided in'the form of a vector which may be expressed in the cells of the 20 mammal.

Such nucleic acids may be administered to the animal by any available technique. For example, the nucleic acid may be introduced by injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic, acid may be delivered directly across the skin using a nucleic acid delivery device such as 25 particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to the mucosal surfaces for example by intranasal, oral, intravaginal, intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.

30 Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectarn. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic, 14 acid is administered in the range of I pg to I mg, preferably to I pg to I O[tg nucleic acid for particle mediated gene delivery and 1 ORg: to I mg for other routes. The following Exwnples illustrate the invention.

Example 1: GBV-V Infection in Common Marmosets Two laboratory bred, disease-free common marmosets (380 and 384) were infected intravenously in the right femoral vein with 0.25ml each of GBV- B 5 infectious sera (stock G 17 originally isolated from an infected tamarin, Saguinus labiatus, in August 1999). Iml blood samples were taken from either the left or right femoral vein at 15 minutes, I day and then weekly thereafter. The sera were then analysed for the presence of GBV-B RNA by quantitative PCR (TaqMan") and for biochemical evidence of hepatitis by measuring GLDH liver enzyme levels.

10 The results shown in table I below indicate that both animals were PCR positive for the GBV-B genome 71 days post infection.

More detailed quantitative PCR shows that in both animals the virus load dropped 0.5 - I log immediately after infection as would be e xpected (Table I and Figure 1). However, over the next 2 weeks virus levels increased by 1.5 logs in 15 animal 380 and by 3 logs in animal 384.

The results obtained from biochemical analysis of the liver enzyme GLDH (Table 2 and Figure 2) show that the levels of GLDH in both animals rose slowly and transiently. The level of GLDH in marmoset sera is typically from 3 to 30 IU/L.

The base line pre-infection level. in marmoset 380 was 15.6 IU/L. Following 20 infection the peak GLDH level in marmoset 380 was 31.4 IU/L which was two fold higher than the baseline. The baseline pre-infection level of GLDH is marmoset 384 was 12.9 IU/L. The level of GLDH rose to four times the baseline level (53 IU/L) following GBV-B infection in marmoset 384. The observed increases in GLDH levels following administration of GBV-B infected sera is indicative of early stage hepatitis.

TABLE 1

Level of GBV-B RNA (genomes /ml) Time post infection Marmoset 380 Marmoset 384 Pre < 1 X 104 < 1 X 104 mins 1.52 x 10' 6.74 x 10' 8 days 3.5 x 10' 1.92 x 10' days 1.64 x 107 1. lox lo, 22 days 6.82 x 107 8.06 x 10' 36 days 8.32 x 107 4.9 x 10' 43 days 3.4 x 107 1.4.4 x 109 days 1.29 x 10' 3.5 x 10' 57 days 5.14 x 107 2.02 x 10' 64 days 1.38 x 10' 3.76 x 106 71 days 1.2 x 104 1.58 x 10' 78 days 4.4 x 106 5.6 x 10' days 6.65 x 106 3.83 x 106 93 days 1.31 X 106 1.9 X 106 99 days 1.54 x 106 7.94 x K NB:Ilmlt of detection of assay = 1 X 104 genorne equivalents per nil 17 TABLE2

Levels of GLDH (IU/L) Time post infection 380 384 Pre 15.6 12.9 8 days 17 22.5 days 12.4 23.8 22 days 11.7 16.3 36 days 31.4 22.1 43 days 13.6 35.2 days 15.2 53 57 days 11.8 43.5 64 days 2.3 8.9 71 days 18.4 18 78 days 16.7 13.9 days 18.7 12.6 93 days 22.4 37.5 99 days 11.3 17.3 Example 2: Marmoset Hepatocyte Cell Cultures And Their Use In Screening a) In Vitro Hepatocyte Infections.

Primary cultures of uninfected marmoset hepatocytes were infected with a GBV-B 10 infectious tamarin serum. After a lag phase of between 1-4 days, progeny virus could be detected in the cell culture supernatant and inside the infected cells. Titres of 10' genomes per ml were detected in the culture medium. UV irradiation of the inoculum prior to infection prevented viral replication. (Figs. 3 and 4) 15 Cultured supernatant from cells 6 days post infection can be used to infect further cultures. (passage experiment, Fig. 5) b) Ex Vivo Infected Hepatocyte Cultures 20 Cultures of infected hepatocytes isolated from infected marmosets were treated with a protease inhibitor active against both HCV & GBV-B protease. This treatment reduced viral titres by 1-2 logs (Figures 6 and 7). Infected cultures treated with 18 interferon-alpha, a currently approved treatment for Hepatitis c infections, show no loss of viral titre.

19

Claims (1)

1. A method for identifying an agent for use in the therapeutic treatment of hepatitis C, said method comprising:

5 (i) administering a test agent to a GBV-B infected marmoset of the Callithrixjacchus strain; and (ii) determining whether the test agent reduces or abolishes GBV-B infection in said marmoset, 2. A method for identifying an agent for use in the prophylactic 10 treatment of hepatitis C, said method comprising:

(ia) administering a test agent to a marmoset; (ib) administering GBV-B to said marmoset; and (ii) determining whether the test agent prevents or limits the effects of GBV-B infection in said marmoset.

15 3. A method according to claim I or 2 wherein step (ii) comprises monitoring the level of GBV-B RNA and/or the level of a liver enzyme in serum samples taken from said marmoset.

4. A method according to claim I or 2 wherein step (ii) comprises monitoring liver histology in liver biopsies taken from said marmoset.

20 5. A method for identifying an agent for use in the therapeutic treatment of hepatitis C, said method comprising:

G) contacting a GBV-B infected hepatocyte from a marmoset of the Callithrixjacchus strain with a test agent in culture; and (ii) determining whether the test agent reduces or abolishes GBV-B 25 infection in said hepatocyte.

6. A method according to claim 5 wherein said hepatocyte has been infected with GBV-B in vitro.

7. A method according to claim 5 or 6 wherein step (ii) comprises monitoring GBV-B RNA and/or a GBV-B protein and/or the 10 production of a liver enzyme.

8. An agent identified by a method according to any preceding claim.

9. An agent according to claim 8 for use in a method of treatment of the human or animal body by therapy.

10. Use of an agent according to claim 8 in the manufacture of a medicament for the therapeutic or prophylactic treatment of hepatitis C.

5 11. A pharmaceutical composition in dosage form comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an agent according to claim 8.

12. A marmoset of the Callithrixjacchus strain infected with GBV-B.

13. A cultured GBV-B infected hepatocyte from a marmoset of the 10 Callithrixjacchus strain.

14. A hepatocyte according to claim 13 which has been infected in vitro.

15. Use of a marmoset according to claim 12 or a hepatocyte according to claim 13 or 14 in a method for identifying an agent for the therapeutic or prophylactic treatment of hepatitis C.