EP1513930A1

EP1513930A1 - Method for culturing hvc virus in vitro

Info

Publication number: EP1513930A1
Application number: EP03760045A
Authority: EP
Inventors: Patrice Andre; Florence Komurian-Pradel; Vincent Lotteau; Glaucia Paranhos-Baccala
Original assignee: Biomerieux SA; Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Biomerieux SA; Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 2002-06-18
Filing date: 2003-06-16
Publication date: 2005-03-16
Also published as: FR2840921B1; AU2003260593A1; JP2006500918A; WO2003106665A1; AU2003260593A8; CN100500836C; US20050221464A1; FR2840921A1; CN1662646A

Abstract

The invention concerns a method for HVC virus culture in vitro comprising steps which consist in: contacting particles containing hepatitis C virus RNA with cells capable of synthesizing and secreting lipoproteins, in a suitable culture environment promoting synthesis and secretion of lipoproteins, and in collecting the virus thus obtained. The invention has diagnostic and therapeutic applications.

Description

Method for in vitro culture of the HCV virus

The present invention relates to a new method of in vitro culture of the hepatitis C virus.

Hepatitis C is the main cause of transfusion-acquired hepatitis. Hepatitis C can also be transmitted by other percutaneous routes, for example by injecting drugs intravenously. The risk of contamination of health professionals is also not negligible.

Hepatitis C is distinguished from other forms of liver disease associated with viruses, such as hepatitis A, B or D. Hepatitis C virus (HCV) infections are often chronic, resulting in diseases of the liver. liver, such as hepatitis, cirrhosis and carcinoma in a large number of cases.

Although the risk of virus transmission by transfusion has decreased due to the selection of blood donors, the frequency of hepatitis C remains high. Currently, approximately 170 million people worldwide are chronically infected with HCV. The high-risk populations are mainly found in transfusion recipients and intravenous drug users, but there are asymptomatic blood donors who do not belong to these high-risk groups and in whom circulating anti-HCV antibodies have been found. For the latter, the route of infection has not yet been identified. HCV was the first hepatotropic virus isolated using molecular biology techniques. The viral genome sequences were cloned without the viral particles being viewed.

Currently, the only natural viral particles characterized in the blood of infected patients are non-enveloped capsids, although it is known that other forms of particles containing viral RNA exist.

For convenience, the term "virus" in the present application any particle containing RNA of the HCV virus. The two terms will also be used interchangeably.

HCV is a positive single-stranded RNA virus, approximately 9.5 kb, which replicates with a copy of complementary RNA and the translation product of which is a single polyprotein of approximately 3,000 amino acids. The 5 'end of the HCV genome corresponds to an untranslated region adjacent to the genes which code for structural proteins, the core protein of the nucleocapsid and the two envelope glycoproteins, E1 and E2. The 5 'untranslated region and the core gene are relatively well conserved in the various genotypes, but the E2 envelope proteins are encoded by a hypervariable region different from one isolate to another isolate. The 3 'end of the HCV genome contains genes that code for non-structural proteins (NS) and for a well-conserved 3' non-coding region.

Because of its genomic organization and its supposed mode of replication, HCV has been classified into a new genus in the family of Flaviviridae, hepaci viruses.

The term HCV virus refers to any viral species, including strains pathogenic to humans, attenuated strains and defective strains derived from said strains. Indeed, it is known that RNA viruses have a high rate of spontaneous mutations. There may therefore be multiple strains which may be more or less virulent. It is within the ability of those skilled in the art to identify such strains, for example by homology of nucleic and / or peptide sequences with respect to a reference strain and / or by identifying a strain or an isolate with respect to to morphological and / or immunological criteria.

Many techniques have been developed to diagnose an HCV infection. For example, diagnostic immunoassays have been performed to detect antibodies to HCV proteins in patient sera. Synthesis of cDNA by reverse transcription of viral RNA and amplification by PCR. have also been used to detect the HCV genome, as an indirect measure of a potentially infectious virus in the sera of chronically infected humans or those of experimentally infected chimpanzees. Furthermore, based on the cloning of genes, hybridization techniques with a DNA probe have also been developed.

However, it is recognized that existing diagnostic techniques lack sensitivity and / or specificity and / or suffer from implementation difficulties. For example, with the hybridization method of probes it is impossible to distinguish between a virus with low infectious power and a virus with infectious power Student. It is therefore necessary, but difficult to implement, to inoculate the virus to be tested with a chimpanzee and to test the resulting infection on the animal.

It is therefore of paramount importance, from a public health point of view, to be able to develop specific, sensitive and practical methods for identifying and screening HCV carriers. One of the solutions could be to realize an efficient in vitro culture system of HCV which would make it possible to obtain a propagation of the virus, in particular to study its mechanisms of replication, to test neutralizing antibodies or antivirals, as well as to develop biological materials, diagnostic tests and vaccine preparations. Indeed, although the complete HCV sequence has been available since 1989 (Q. L Choo et al., Science 244, 359 (1989)), the understanding of the life cycle and mode of replication of HCV has been hampered by lack an appropriate in vitro culture system. Ito et al. (J. Gen. Virol. 77: 1043-1054 (1996)) have confirmed the maintenance of the replication of HCV in primary cultures of human hepatocytes, obtained from patients carrying HCV and for whom the disease was established. chronically, and suggested a passage of infection, but problems relating to the spread of the virus remain (impossibility of long-term culture) and the developed system is limited by the need for human liver supply and the heaviness of the technical. Furthermore, to date, there is no general consensus on the tropism of HCV and all cellular receptors for the virus have not yet been identified.

In the plasma of HCV infected patients particles containing viral RNA very heterogeneous in density are found. This heterogeneity in density of the particles containing viral RNA is attributed to their association in variable proportion with lipoproteins (Thomsen et al., 1993, Med. Microbiol. Immunol. 182: 639). In the description of the present patent application, the inventors have named these hybrid particles LVPs (lipo-viro-particles). The distribution of each of these forms along a density gradient varies from patient to patient. Existing analyzes of low density particles show densities covering those of LDLs (Low Density Lipoproteins) and VLDLs (Very Low Density Lipoproteins). To date, the nature of LVPs containing viral RNA is not precisely known. Patent application WO01 / 09289 describes a method of in vitro culture of viruses, such as HCV, from at least a fraction of LVPs obtained from serum or plasma from an infected patient, using cells which have an endocytosis pathway relayed by at least one lipoprotein receptor and modulated by an activating agent. This process is especially concerned with promoting the entry of fractions into cultured cells. However, replication of the virus remains limited and must therefore be optimized.

Patent application WO02 / 10353 describes complexes consisting of LVPs associated with human immunoglobulins, of density less than or equal to 1.063 g / ml, and containing mainly the RNA of the HCV virus, contrary to known data (Hijikata et al., 1993, J. Viral., 1953-1958). Indeed, Hijikata et al. showed that a high infectivity in the chimpanzee was found in the presence of particles of density less than 1.06 g / ml so that there could not be human immunoglobulins, very dense, in this type of particles low density. These complexes constitute a particular form of the HCV virus which is very infectious.

This patent application also describes a method of in vitro culture of the HCV virus from the LVP / immunoglobulin complexes, using cells comprising on their surface at least one type of receptor for the Fc fragment of immunoglobulins or one type of receptor having the capacity to bind immunoglobulins. Here again, this process promotes the entry of the complexes into the culture cells but it does not allow an abundant multiplication of the virus.

The present inventors have now found a new method for in vitro culture of the HCV virus which makes it possible to solve the above drawback, namely that it uses cells capable of both bringing in the particles containing the HCV RNA and improve ensure replication and production.

Indeed, the present inventors have surprisingly demonstrated that the HCV RNA particles called LVP:

- are globally spherical unit particles; they are therefore not agglomerates of virions linked to normal lipoproteins, as had been suggested in the prior art, - contain apolipoproteins B and E and triglycerides: they therefore have a lipoprotein structure, and in particular of the VLDL or chylomicron type, and

- contain the capsid and the RNA of the virus: they therefore differ from normal lipoproteins found in humans and constitute an unconventional form of the virus.

Since LVPs are hybrid particles, i.e. lipoproteins containing viral components, replication can be unexpectedly improved by using cells capable of synthesizing lipoproteins.

Thus, the subject of the present invention is a method of in vitro culture of the HCV virus comprising the steps consisting in bringing into contact particles containing the hepatitis C virus RNA with cells having the capacity to synthesize and secrete lipoproteins. , in an appropriate culture medium promoting the synthesis and secretion of lipoproteins, and in harvesting the virus thus obtained.

By particles containing the HCV RNA is meant viral particles, such as in particular in the form of an LVP / immunoglobulin complex or present in the serum or the plasma of patients detected positive for HCV and containing such particles, and any inoculum containing viral RNA, regardless of the mode of preparation of viral RNA in said inoculum.

The LVP / immunoglobulin complexes and their method of purification from a plasma or serum sample from a patient infected with HCV, have already been described in patent application WO 02/10353.

However, this document neither describes nor suggests the specific nature of these lipoprotein-type LVPs, namely that they have triglycerides, of papolipoprotein B and possibly of apolipoproteins E. Apolipoprotein B is a human protein associated with the membrane of the endoplasmic reticulum and which initiates the assembly of VLDLs, the introduction of triglyceride in the presence of the microsomal triglyceride transfer protein, as well as the assembly of VLDL in the lumen of the endoplasmic reticulum. This apolipoprotein is delivered in two forms, apo B 100 and apo B 48, and is synthesized in the liver and the intestine. Apolipoprotein E can be synthesized by numerous cells and in particular by hepatocytes. Furthermore, it can be acquired by VLDLs in the blood stream.

The cells used in the method of the invention are all cells having the capacity to synthesize and secrete lipoproteins.

These cells can be either primary cells or cell lines.

The term cell line refers to established lines, immortalized spontaneously or by manipulation. In practice, to carry out a viral culture of interest, it is necessary to have permissive cells easily maintained in culture. The cell line is therefore preferably an established cell line or which results from immortalization by different methods. This can be done (i) by establishing a stable, established, continuous line, by co-cultivation of permissive cells with permissive cells of the same nature with tumors, capable of multiplying indefinitely and ensuring propagation of the virus within the culture, the viral inoculation taking place within the culture, (ii) using primary cells infected with the virus which are then co-cultured with permissive tumor cells which ensure the propagation of the virus to the within the culture of the cell line thus established (iii) by viral infection of a cell line, for example an immortalized B cell line, for example with the Epstein-Barr virus or (iv) by modification of the telomerase activity.

The expression “cells having the capacity to synthesize and secrete lipoproteins used in the process of the invention” is understood to mean cells having spontaneously this capacity and cells having acquired this capacity after induction in a culture medium promoting the differentiation or redifferentiation of cells or after transfection of genes for the synthesis of apolipoprotein apo B and of the microsomal protein for the transfer of triglycerides MTP.

Examples of cells having the ability to synthesize and secrete lipoproteins suitable for the purposes of the invention include enterocyte-type intestinal epithelium cells, brain cells and liver cells. According to one embodiment, the cells used are cells which have acquired the capacity to synthesize and secrete lipoproteins after induction in a medium promoting the differentiation or redifferentiation of cells.

Indeed, some cells, such as liver cells, have lost their ability to produce lipoproteins or other cells, such as the cells of the intestinal epithelium, have an ability that can be improved. To overcome this drawback, it is possible to induce a differentiation or redifferentiation of these cells by contacting with an appropriate medium favoring this differentiation or redifferentiation.

According to a particular embodiment, the cells suitable for the purposes of the invention are the cells of the intestinal epithelium, and in particular the cells

Caco-2 (Van Greevenbroeck, M.M.J., et al., 2000, Atherosclerosis, 25-31).

To promote their ability to synthesize and secrete lipoproteins, the cells of the intestinal epithelium, such as Caco-2, can be previously cultured for 3 weeks with DMEM medium supplemented with 10% fetal calf serum, in boxes covered with collagen or under semi-permeable membranes.

Examples of liver cells include hepatocytes (Moshage, H., et al., 1992, Journal of Hepatology, 15, 404-413) and hepatocarcinomas such as Hep G2 cells (Gherardi, E ., et al., 1992, Journal of Cell Science, 103, 531-539). According to another particular embodiment, the cells used in the method of the invention are hepatocarcinoma cells.

However, in this case, their capacity to synthesize and secrete lipoproteins can be favored by bringing these cells into contact with a modified DMEM medium, namely containing at least one agent inducing lipoprotein synthesis. Preferably, these cells are Hep G2 cells.

As an agent inducing the synthesis of lipoproteins, mention may be made of different lipids such as fatty acids, preferably C ₁₈ or C ₂₀ , for example oleate (Luchoomun, J., et al., 1999, The Journal of Biological Chemistry, Vol

274 (28), 19565-19572), and phospholipids such as lysophosphatidylcholine (Zhou, Z., 1998, Biochimica and Biophysica Acta, 1391, 13-24). The use of oleate in the medium for redifferentiation of hepatocarcinomas constitutes a particular embodiment of the invention.

According to one embodiment of the invention, the modified DMEM medium consists, in addition to DMEM (Gibco BRL) and an agent inducing lipoprotein synthesis, 1% HEPES (Gibco), 1% glutamine (Gibco ), gentamycin

0.25 mg / ml (Gibco), Ultroser-G 1.5% (Gibco), Forskolin 5.10 ^'6 M (ICN),

PMA 1.6.10 ^"7 M (4-α-phorboll2-myristatel3-acetate) (Sigma), retinol acetate

5.6 IU / ml (Sigma), 0.5.10 M sodium butyrate (Sigma), 10 ^" M niacidamide

(ICN), 2.10 ^"6 g / ml polybrene (Sigma), 2.9.10 ^{" 8} M sodium selenite (ICN) and 1.10 ^"9 M sodium triiodo-L-thyronine (ICN).

Another type of cell which can be used in the process of the invention consists of cells obtained after transfection of genes for the synthesis of apolipoproteins apo B and optionally apo E and of the microsomal protein for the transfer of triglycerides. By way of example of such cells, mention may be made of transfected insect cell lines as described by Gretch, D.G., et al. in The Journal of Biological

Biochemistry, 1998, Vol 271 (15), 8682-8691.

The particles containing the HCV RNA, when they are brought into contact with cells having the capacity to synthesize and secrete lipoproteins, enter these cells either via the lipoprotein receptors of said cells, of the LDL receptor type, as in the case of LVP for example, or by internalization by transfection according to techniques known to those skilled in the art, as in the case of preparations of viral RNA for example.

The culture medium used in the process of the invention is such that it promotes the synthesis and the secretion of lipoproteins, preferably of VLDL or chylomicron type. A suitable medium retained is a modified DMEM medium supplemented with agents promoting the metabolism of the cell in culture, as well as with at least one agent inducing the synthesis of lipoproteins.

As agents promoting the metabolism of the cell, mention may be made of dexamethasone and insulin. A preferred modified DMEM medium is as defined above and supplemented with dexamethasone and insulin. As agents inducing the synthesis of lipoproteins, mention may be made of different lipids such as fatty acids, preferably C ₁₈ or C ₀ , for example Poleate, phospholipids such as lysophosphatidylcholine, as well as 22 or 25 OH -cholesterol. The use of the oleate as an agent inducing the synthesis of lipoproteins, optionally in combination with 22 or 25 OH-cholesterol, constitutes another particular embodiment of the invention.

The virus thus cultivated by the method of the invention can be harvested by various methods such as centrifugation, for example on a density gradient, and immunoprecipitation using anti-apo B and / or anti-apo E antibodies.

The invention also relates to a diagnostic composition comprising at least the viral particles obtained according to the method of the invention or one of its components as a source of antigen.

Those skilled in the art will easily determine the amount of viral particles to be used depending on the diagnostic technique used.

The invention also relates to a method for screening and / or selecting at least one antiviral molecule, comprising the step of bringing said antiviral molecule into contact in the culture medium during the culture method of the invention.

The selection of the antiviral molecules is carried out by techniques well known to those skilled in the art such as the reduction in the number of intracellular viral RNA and / or of infectious viral particles secreted in the supernatant in the presence of variable concentrations of the various inhibitors.

These inhibitors can be nucleotide or nucleoside analogs, inhibitors of viral proteases or other molecules interfering with the functions of other viral proteins.

However, the invention also opens up other therapeutic perspectives in that it makes it possible to develop a therapeutic composition capable of influencing in a qualitative and / or quantitative manner the propagation and the in vivo replication of HCV, which is characterized in that it comprises inter alia an agent capable of modulating, repressing or inhibiting the synthesis of lipoproteins, such as for example an inhibitor of the microsomal protein for the transfer of triglycerides. The term agent capable of modulating, repressing or inhibiting the synthesis of lipoproteins means any molecule making it possible, respectively, to control, reduce or suppress the propagation and replication in vivo of HCV.

These agents can be selected by screening from a pool of molecules having recognized pharmacological activity on lipid metabolisms.

As an example of an agent capable of inhibiting the synthesis of lipoproteins, it is possible to cite the inhibitor of the microsomal protein for the transfer of triglycerides.

The invention will be better understood with the aid of the following examples given by way of non-limiting illustration, as well as with the aid of the appended figures 1 to 3, in which: - Figure 1 represents a graph showing the influence of culture conditions (modified DMEM medium plus oleate compared to the standard medium) of Hep G2 cells on the secretion of the HCV viral particles, secretion evaluated by the quantification of the viral RNA in the culture supernatant as a function of time,

FIG. 2 represents a graph showing the influence of the addition of oleate in the modified DMEM culture medium on the secretion of the viral particles evaluated by the quantification of HCV RNA in the culture supernatant as a function of time, and

- Figure 3 shows a graph showing the production of the HCV virus by Caco-2 cells, after differentiation for 3 weeks of culture, said production being demonstrated by the number of viral RNA in the culture supernatant as a function of time .

Example 1: Characterization of LVP

1.1 Demonstration of the globally spherical shape of the LVPs The LVPs were purified from sera from patients detected positive for the hepatitis C virus, as described in patent application WO 02/10353.

The low density fraction (LDL) freed of the purified LVPs was diluted, as well as the purified LVPs in PBS and were visualized using an electron microscope (JEOL device, Laennec Joint Imaging Center, Lyon, France) after floating drops of the sample on 200 mesh copper grids coated with a Formvar support film (Electron Microscopy Science, PA) for 3 min at room temperature, colored for 3 min by flotation on a medium of photungstic acid at 4% (mass / vol), buffered to pH 7.2 with NaOH, then dried.

The LDL fraction consisted of particles of homogeneous spherical structure, with an average diameter of 25 nm, in agreement with normal LDL. In contrast, the purified LVPs were unusually large spherical structures, with an average diameter of 100 nm. 1.2 Determination of the constituents of the LVPs a) Determination of the lipid concentration of the LVPs

The total cholesterol, phospholipid and triglyceride concentrations were determined using the Cholesterol RTU, Phospholipids Enzymatic PAP 150 and Triglyceride Enzymatic PAP 150 kits (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's recommendations, and in establishing standard curves. b) Determination of the apo B concentration of the LVPs

The apolipoprotein apoB concentration in the purified LVPs was determined using an ELISA assay. To do this, 96-well flat bottom ELISA plates (Maxisorb; Nunc) were coated with 100 μl of human anti-apo B monoclonal antibody.

(5 μg / ml; clone 1609; Biodesign, Saco, Maine) in PBS (phosphate buffered saline). It was left to stand overnight at 4 ° C, then the reaction was blocked with 2% BSA (bovine serum albumin). The samples were first incubated for 30 min at room temperature in a PBS-0.2% BSA mixture supplemented with 10 μg of human IgG / ml, then they were distributed on the plates at a rate of 100 .mu.l / well.

After 2 h of incubation at 37 ° C and washing with PBS-0.05% Tween medium

20, human goat anti-apo B antibodies conjugated with peroxidase (1.6 μg / ml; Biodesign) in a PBS-0.2% BSA mixture were added at a rate of 100 μl / well. left incubated for 90 min at 37 ° C.

The plates were washed and the o-phenylenediamine substrate (Sigma) was added at the rate of 150 μl / well. The development of the reaction was left for 10 min and the plates were read at 490 nm. c) Results The results, in terms of triglyceride / cholesterol (TG / Chol) and triglyceride / apo B (TG / apo B) ratio are shown in the table below. For comparison, this table also contains these same reports for the patient's normal lipoproteins, that is to say the lipoproteins obtained after extraction of the LVPs.

Board

: p <0.04: p <0.01

The LVPs, which are found in the two fractions of different density, namely low and very low density, contain more triglyceride per apo B molecule than normal lipoproteins of the same fractions. These data confirm that:

- the LVPs contain many lipids, - the lipid concentration of the LVP is different from that of normal lipoproteins; this therefore excludes any contamination, and

- LVP contains apo B.

1.3 Presence of apoliproteins B and E

The presence of apo B and apo E has been demonstrated by preventing the entry of LVP into PLC cells after blocking the binding sites for the apoB and apoE receptors of said cells as follows:

5 x 10 ⁵ cells of human hepatoma cell lines were grown

PLC / PFR / 5 (ATCC CRL 8024) (Alexander Cells) (Human Hepatoma) in 96-well plates (Maxisorb, Nunc?) For 24 h with DMEM culture medium (Gibco, BRL) supplemented with 10% "of fetal calf serum (Biowhittaker, Emerainville, France), 2mM HEPES (Gibco / BRL), 1% non-essential amino acids (Gibco / BRL) and 50 IU of penicillin / streptamycin (Gibco / BRL) / ml at 37 ° C.

On the one hand, the apo B receptor binding sites have been blocked with monoclonal antibodies directed against the apo B receptor binding site (4G3 and 5 ^B 11; Ottawa Heart Institute Research Corporation, Ottawa, Ontario Canada ) and on the other hand the binding sites to the apo E receptors with monoclonal antibodies 1D7 (Ottawa Heart Institute Research Corporation). The PLC cells were washed three times with PBS and incubated for 3 h with purified LVP. The cells were washed, then they were harvested in 350 μl of lysis buffer from the Rneasy kit (Qiagen) and the RNA was extracted as indicated above.

Blocking the recognition of LVP by blocking the recognition sites of apo B and E indicates that they contain apolipoproteins and highlights the lipoprotein structure of LVP.

1.4 Presence of the capsid consisting of the core protein and the RNA of the virus in the LVPs

The presence of the capsid was demonstrated by delipidating the purified LVPs as follows: the LVPs were incubated for 30 min while stirring gently, in a solution of 85%> ether-15% butanol. The presence of the capsid was visualized using an electron microscope (JEOL device, Center Commun d'Imagerie de Laennec, Lyon, France) as indicated in point 1.1 above.

The presence of the core protein of the HCV virus was confirmed by Western blot by contacting the delipidated LVPs as described above, with anti-HCV core protein monoclonal antibodies (19D9D6; Jolivet-Reynaud, CP, et al. , 1998, J. Med. Virol., 56, 300-309) and secondary antibodies labeled with 10 nm gold and visualization using the grids indicated above by immunodetection after negative staining by flotation on l uranyl acetate 3%.

Finally, the presence of the virus RNA was demonstrated by extraction of the purified and defatted LVPs using a QIAamp kit (Qiagen SA, Courtaboeuf, France). Example 2 Influence of the Culture Conditions on the Production of the HCV Virus by Hep G2 Cells

First of all, Hep G2 cells were cultured either in a medium consisting of DMEM and 10% fetal calf serum (standard medium), as in patent application WO01 / 09289, or in a modified DMEM medium, c ie containing DMEM supplemented with PHEPES 1%, glutamine 1%, gentamycin 0.25 mg / ml, PUloser-G 1.5%, Forskoline 5.10 ^"6 M, PMA 1.6.10 ^"7 M, retinol acetate 5.6 IU / ml, sodium butyrate 0.5.10 ^" M, niacidamide 10 ^" M, polybrene 2.10 ^{" 6} g / ml, sodium selenite 2.9.10 ^"8 M and triiodo-L-thyronine sodium l.lO ^{" 9} M.

Culture plates of 24 Falcon wells at 150,000 cells per well were then seeded with standard medium or modified DMEM medium and left in culture for 24 h.

The medium was aspirated and the cells were incubated in the presence of the virus, at the rate of 500,000 HCV RNA per well, for 6 h using DMEM supplemented with 0.2% BSA.

The cells were then washed with PBS and cultured either in standard medium or in modified medium, as indicated above, but also supplemented with hexamethasone and insulin and a mixture of oleate and BSA 0.15 mM oleate.

All cultures were carried out in an atmosphere at 5% CO ₂ and at 37 ° C. The supernatant was then removed from the wells (in triplicate) and the HCV RNA was quantified by RT-PCR according to the protocol described by Komurian-Pradel, F. in J. Virol. Methods, 2001, 95, 111-119. The results, copies of RNA / ml of supernatant as a function of time, are shown in FIG. 1 in which the diamonds represent the use of the standard medium and the squares the use of the medium promoting the synthesis and the secretion of lipoproteins.

This figure highlights that the replication of the virus and its secretion is improved by using cells having the capacity to synthesize and secrete lipoproteins and by putting them under favorable conditions. EXAMPLE 3 Importance of Adding Lipids to the Medium Promoting the Synthesis and Secretion of Lipoproteins

Hep G2 cells were first cultivated for 24 h in a modified medium as described in Example 2 above, and they were added to the wells of the Falcon 24 well culture plates at a rate of 150 000 cells per well.

The cells were then incubated in the presence of the virus, at a rate of 400,000 HCV RNA per well, for 6 h in DMEM medium supplemented with 0.2% BSA alone or with 0.1 μM of 25 OH cholesterol. The cells were then washed as indicated in Example 2 above and cultured in a modified medium.

An oleate-BSA mixture with 0.15 mM oleate was added 3 days after inoculation of the cells.

The supernatant was collected and the HCV RNA was quantified by RT-PCR. The results, highlighting the importance of the use of oleate, are given in FIG. 2 in which the diamonds represent the use of the modified medium alone and the squares the use of the modified medium supplemented with P oleate and 25 OH cholesterol.

EXAMPLE 4 Culture of the Virus Using Caco-2 Cells

Caco-2 cells are differentiated on semi-permeable membranes (Transwell, Costar) contained in 24-well plates, for 3 weeks in standard medium (DMEM supplemented with 10% fetal calf serum).

The cells thus prepared were incubated with the viral particles at the rate of 200,000 HCV RNA / well for 6 h.

The cells were washed and cultured in DMEM medium supplemented with 10% o fetal calf serum and 0.15 mM oleate-taurocholate.

The supernatant was removed above Pinsert and the viral RNA was quantified by RT-PCR. The results are shown in FIG. 3 which highlights the culture of the HCV virus by cells of the intestinal epithelium in a medium which promotes the synthesis and secretion of lipoproteins.

Claims

1. A method of in vitro culture of the hepatitis C virus, characterized in that it comprises the stages consisting in bringing into contact particles containing the RNA of the hepatitis C virus with cells having the capacity to synthesize and secrete lipoproteins, in an appropriate culture medium promoting the synthesis and secretion of lipoproteins, and in harvesting the virus thus obtained.

2. Method according to claim 1, characterized in that the particles containing the hepatitis C virus RNA are the lipo-viro-particles LVP.

3. Method according to one of claims 1 or 2, characterized in that the cells having the capacity to synthesize and secrete lipoproteins are chosen from cells having spontaneously this capacity and the cells having acquired this capacity after induction in a medium of culture favoring the differentiation or redifferentiation of cells or after transfection of genes for the synthesis of apolipoprotein B and of the microsomal protein for the transfer of triglycerides.

4. Method according to claim 3, characterized in that the cells used are cells having acquired the capacity to synthesize and secrete lipoproteins after induction in a medium favoring the differentiation or redifferentiation of the cells.

5. Method according to any one of claims 1 to 4, characterized in that said cells are cells of the intestinal epithelium.

6. Method according to claim 5, characterized in that said cells are Caco-2 cells.

7. Method according to one of claims 4 to 6, characterized in that the cells are previously treated for 3 weeks with a DMEM medium supplemented with 10% o of fetal calf serum.

8. Method according to any one of claims 1 to 4, characterized in that said cells are hepatocarcinoma cells.

9. Method according to claim 8, characterized in that the hepatocarcinoma cells are the Hep G2 cells.

10. Method according to one of claims 8 or 9, characterized in that said cells are previously brought into contact with a modified DMEM medium containing an agent inducing the synthesis of lipoproteins.

11. Method according to claim 10, characterized in that the agent inducing the synthesis of lipoproteins is P oleate.

12. Method according to any one of the preceding claims, characterized in that the medium promoting the synthesis and secretion of lipoproteins is a medium derived from the modified DMEM medium, supplemented with agents promoting the metabolism of the culture cell, as well as 'with at least one agent inducing lipoprotein synthesis.

13. The method of claim 12, characterized in that said medium comprises oleate as an agent inducing the synthesis of lipoproteins.

14. Method according to claim 13, characterized in that said medium also contains 22 or 25 OH-cholesterol as another agent inducing the synthesis of lipoproteins.

15. Method for screening and / or selecting at least one antiviral molecule, characterized in that it comprises the step of bringing said antiviral molecule into contact in the culture medium during the culture process according to any one of claims 1 to 14.

16. Therapeutic composition capable of qualitatively and / or quantitatively influencing the propagation and replication in vivo of HCV, characterized in that it comprises inter alia an agent capable of modulating, repressing or inhibiting the synthesis of lipoproteins .