WO1987005930A1 - Cellules tissulaires immortalisees a specificite virale - Google Patents

Cellules tissulaires immortalisees a specificite virale Download PDF

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WO1987005930A1
WO1987005930A1 PCT/US1987/000711 US8700711W WO8705930A1 WO 1987005930 A1 WO1987005930 A1 WO 1987005930A1 US 8700711 W US8700711 W US 8700711W WO 8705930 A1 WO8705930 A1 WO 8705930A1
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human
cells
cell
virus
infected
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Steven K. H. Foung
Linda B. Rabin
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Genelabs Inc
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Genelabs Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • 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
    • 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
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to immortalized human or non-human primate cells which are infected or infectable by tissue-specific viruses, tc methods for preparing the cells, and to uses of the cells in identifying virus-specific antigens and anti-antigen antibodies.
  • NANB hepatitis nonAnonB
  • An etiological NANB virus or viruses have been directly demonstrated by infecting chimpanzees (Bradley, Alter, Hoofnagle). but so far chimpanzees remain the only experimental animal system for studying the virus. The difficulty and expense in obtaining adequate amounts of virus-infected material have seriously limited research on this virus.
  • hepatitis B virus which can be grown in chimpanzees and marmoset monkeys, but not in inexpensive lab animals or in cell culture.
  • existing cell culture systems can be used to propagate some viruses, this approach has not been successful for many viruses which are specific for certain cell types, particularly if long propagation times are involved.
  • the reason for this limitation is that many normal human cells, such as hepatocytes, which are the specific targets of a virus of interest, such as NANB virus, cannot be -propagated in culture, and can be maintained in culture for periods of usually only 1-2 "weeks.
  • tissue-specific human cells which could be grown stably in culture would provide a convenient, and in some cases, unique host for many human infectious viruses. This is true particularly for viruses which require tissue-specific surface antigens for cell infection, and which also have relatively long incubation times.
  • the ability to propagate certain normal human cell types in culture would also provide a tool for identifying viral agents which are suspected as causative agents of diseases of that cell type.
  • diseases of the ⁇ central nervous system such as Alzheimer's disease and multiple sclerosis, are suspected of having a viral origin, and the ability to produce stable cultures of central nervous cells from
  • Stable, cultured human or non-human primate cells derived from a selected normal cell type would also be useful in studying metabolism and drug effects in specific cell types.
  • Metabolic activity in many tissue specific cell types including cells derived from liver, pancreas, pituitary, and brain tissue in short-term culture has been studied. These studies have been limited heretofore by the amount of work required to isolate cells for culture, and most studies have been confined to non-human cell cultures, for reasons of cell availability. Additionally, the length of the studies is necessarily limited to the one to several week period of cell viability. Such limitations would not apply to stable, tissue-specific cell types in culture. Further, the ability to infect the cells with tissue-specific viruses would open up new avenues for studying drug effects on infectious viruses.
  • tissue-specific cells particularly human and non-human primate cells
  • Efforts to extend the culture period of cultured hepatocytes such as by coculturing the liver epithelial cells, have produced only limited increase in culture times (Guguen-Guillouzzo) .
  • Some malignant cells associated with specific human tissues such as hepatoma cells derived from malignant liver tissue, are able to grow stably in culture.
  • tissue-specific viruses are of limited use as a host system for tissue-specific viruses, both because the cells are often deficient in specific cell receptors needed for viral infectivity, and because viral genomes are often associated with malignancies (Knowles), and therefore the identity of the infecting virus would be difficult to confirm.
  • Attempts to produce stable, tissue-specific cells in culture by fusing the cells with related myeloma cells have also been attempted. This approach is analogous to the one used for immortalizing antibody-secreting human lymphocytes, by fusion with myeloma cells under defined selection conditions_•
  • Another object of the invention is to provide methods of using virus infected immortalized cells to identify and obtain virus-specific cell-surface antigens, monoclonal antibodies specific against such antigens, viral genomic material and/or viral antigens useful in producing anti-virus vaccines.
  • Still another ob ect of the invention is to provide virus-specific cell surface antigens which are produced with viral infection of human cells, and diagnostic methods which use the antigens to detect the presence of virus-specific antibodies 'in the serum of a human infected with the viral agent.
  • the invention includes immortalized non-lymphocytic, non-malignant human or non-human primate cells derived from a selected, non-lymphocytic tissue. The cells are formed by fusion of a mouse/human hybridoma with a human or non-human primate cell derived from the selected non-lymphocytic tissue.
  • the tissue-specific cell is one which is infectable or infected with a selected viral agent in vivo, and may include, for example, hepatocytes, central nervous system cells, and synovial cells.
  • the fusion partner is produced by fusing mouse myeloma cells and human B-lymphocytes, and selecting fusion products which show stable human chromosome retention, as evidenced by continued HLA surface antigen production in culture. The fusion partner is fused with the tissue-selected cell under conditions which allow growth of successful trioma fusion products only.
  • the fusion partner is formed by fusing mouse myeloma cells and human B-lymphocytes, selecting fusion products which show immunoglobulin secretion and HLA surface antigen production in culture, and treating the selected fusion products with a mutagen.
  • the mutagenized fusion products are selected for those which retain the ability to produce HLA surface antigen, show no immunoglobulin secretion, and are unable to survive in a growth medium which allows growth of a successful product formed by fusing the fusion partner with such a human cell.
  • An exemplary fusion partner ⁇ has the characteristics of ATCC number HB 8464.
  • the immortalized cell line when infected with the selected virus, can be used to identify virus-specific cell-surface antigens, as a source of such antigens, for identifying virus-specific antibodies, and/or for identifying viral genomic material which can be used as a probe for detecting the viral agent, or for producing virus-antigen peptides for vaccine purposes.
  • triomas refers to a cell line which contains genetic components originating in three originally separate cell lineages. As used in the context of this application, these triomas are stable, immortalized cells which result from the fusion of a murine myeloma/human hybridoma with a non-lymphocytic tissue cell from a human or non-human primate source.
  • the murine myeloma/human hybridoma (the "immortalizing hybridoma” or "fusion partner") is an immortal cell line which results from the fusion of a murine myeloma or other murine tumor cell with human ly phoid cells derived from a normal (preferably non-immunized) subject.
  • an immortalizing hybridoma which provides improved chromosomal stability, has human characteristics, and which does not secrete immunoglobulin is obtained.
  • hybridoma refers to a hybridoma which is capable of continuous reproduction and, therefore, is immortal but lacks the capacity to secrete immunoglobulin.
  • a hybridoma "having human characteristics" refers to a hybridoma which retains detectable human-derived chromosomes, such as those producing human HLA antigen which will be expressed on the cell surface.
  • tissue-specific cells refer to non-lymphocytic human or non-human primate cells derived from a selected tissue or organ source, such as liver, the central nervous system or synovial fluid, and which are infected or infectable with a tissue-specific virus in vivo.
  • Cell line refers to various embodiments including but not limited to individual cells, harvested cells, and cultures containing cells so long as these are derived from cells of the cell line referred to.
  • derived is meant progeny or issue. It is further known in the art that spontaneous or induced changes can take place in karyotype during storage or transfer.
  • cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • the Immortalizing Fusion Partner Cells The cells which make up the immortalizing hybridoma are murine myeloma cells and human lymphoid B cells.
  • Murine myeloma cell lines are commonly available and may be obtained through the American Type Culture Collection (ATCC), located at the National Institutes of Health (NIH) in Bethesda, Maryland.
  • Human lymphoid B cells are isolated from the blood of normal individuals using conventional techniques. Such procedures include density gradient purification and separation of B cells from T cells using standard sheep erythrocyte resetting.
  • the tissue-specific cells which are to be immortalized by fusion with the mouse/human fusion partner are isolated cells derived from a selected, non-lymphocytic tissue from a human or non-human primate.
  • the latter source includes primates, such as chimpanzees, whose phylogenetic similarity to humans (Clark) allows for successful fusion with the mouse/human fusion partner.
  • the ability of the mouse/human fusion partner to fuse with and immortalize chimpanzee cells forms the basis of an immortalized chimpanzee cell line described in a co-owned patent application for "Non-Human Primate Monoclonal Antibodies and Methods". Serial No. 767,213. filed May 1, 1985.
  • the cells are isolated from a tissue or organ which is infectable or infected with a selected virus of interest.
  • the isolated cells must also be capable of limited viability in culture, without appreciable loss of the desired tissue-specific property, for a short period until the cells have been successfully immortalized by fusion with the immortalizing partner.
  • tissue cells for culture A variety of methods for preparing tissue cells for culture have been developed, and these methods are generally suitable for isolating tissue-specific cells for fusion with an immortalizing mouse/human hybridoma according to the present invention.
  • the following cell-preparation methods for specific cell types are illustrative of general techniques which are applicable.
  • Several methods ⁇ for producing isolated hepatocytes for cell culture have been reported (Krack, Guguen-Guillouzzo) . In general, these methods involve perfusing liver tissue with a solution of collagenase, mincing the enzyme-digested tissue to release free cells, and filtering the isolated cells through a cloth mesh.
  • the isolated tissue such as fetal abortus liver tissue
  • a ground glass homogenizer having a single cell clearance
  • the . homogenized cells washed several times in a suitable culture medium, as described in Example II.
  • Similar ° methods have been used to produce short-term cell- cultures of pancreatic islet cells (Bone), anterior pituitary cells (Liang, Loughlin), and primary pituitary cells (Ben-Jonathon) .
  • CNS cell types including spinal cord neurons (Kato). neurons from human cerebral cortex (Louis), and human glial cells (Ponten) .
  • immortalized nerve cells are likely to 0 be used primarily in studying neurological disorders which are suspected of having viral origin. Therefore, the nervous-sytem cells which are isolated for immortalization, according to the method of the invention, will typically be derived from human or 5 non-human primate subjects which have been positively diagnosed as having the disease under study.
  • Fusions to form the murine-human non-secreting hybridomas and the immortalized tissue cell lines of the invention are performed by a modification of the method of Kohler and Milstein . Briefly, a mouse myeloma and human lymphocyte (to make the immortalizing hybridoma) or the hybridoma and the isolated tissue-specific cell (to make the immortalized tissue cell line) are combined in the presence of a fusogen such as polyethylene glycol under suitable conditions, e.g., 40%-50% polyethylene glycol (1000 to 4000 molecular weight) at between room temperature and 40°C. preferably about 37°C. Fusion requires about 5-10 minutes, and the cells are then centrifuged and screened. Alternatively, fusion products, such as the fusion of the immortalizing fusion partner and the human non-lymphocyte tissue cell, can be produced successfully by electrofusion, using known procedures.
  • a fusogen such as polyethylene glycol under suitable conditions, e.g., 40%-50% polyethylene glycol (1000 to 4000 molecular weight
  • screening for hybridized products is made by culturing cells centrifuged from the fusion medium in growth medium which is selective for the desired hybrids.
  • non-immortalized cells cannot survive repeated transfers on any medium, and hence will not survive repeated culturing of the centrifuged cells.
  • Commonly used lines of immortalized murine myeloma cells are incapable of growth on certain selective media which have been chosen to deprive them of their ability to synthesize DNA.
  • Two very commonly used media of this description are "hypoxanthine-aminopterin-thymidine" or "HAT" medium and azaserine-hypoxanthine medium or "AH" medium.
  • Both of these selection media take advantage of the capacity of normal cells to utilize a "salvage" pathway for DNA synthesis under circumstances where the de novo process is inhibited.
  • Aminopterin inhibits both de novo purine and pyrimidine nucleotide synthesis in normal cells and both thymidine and hypoxanthine are required for the salvage pathway.
  • Azaserine inhibits only purine synthesis, so only hypoxanthine is required for the salvage pathway.
  • the salvage process which requires hypoxanthine phosphoribosyl transferase (HPRT) is generally inoperable in the commonly used murine myeloma cells (although they retain the de novo pathway) .
  • the selection procedures employed in producing the immortalizing hybricToma are aimed at selecting cells which show stable human characteristics, non-secretion of immunoglobulin. and sensitivity to a defined selection medium which can be used for selecting successful immortalized tissue cell hybrids. Briefly, the cells centrifuged from the fusion mixture of mouse myeloma and human lymphoid cells are diluted and plated in microtiter plates. Screening is done using AH or HAT medium growth, with selection of successful colonies being made on the basis of assay procedures related to stability and human character. From among the many colonies assayed, several are chosen which continue to produce immunoglobulin in the supernatant fluid for a suitable period of time, preferably in excess of six months (one criterion for stability).
  • the culture period of two-three months required to obtain a sufficient number of cells for mutagenesis may also serve as a period for gauging stability of immunoglobulin secretion.
  • the continued production of such immunoglobulin indicates that the characteristics conferred by the human lymphocyte partner have not been lost (lymphocytes which were unfused will, of course, not survive). Retention of human characteristics is assessed by assaying the cell surfaces for the presence of HLA antigen.
  • the selected colonies continue to exhibit HLA antigen expression at their cell surfaces, and in fact, continued production of immunoglobulin and HLA surface antigen appear to be linked, as the two traits are invariably found in the same cells.
  • the selected cells are then treated with a mutagen, such as 6-thioguanine, to destroy their ability to secrete immunoglobulin and confer HAT or AH sensitivity. This will make possible later fusion to give an immortalized tissue-specific cell, and subsequent use of the fusion product for protein secretion, without a contribution of immunoglobulin secretion from the fusion partner.
  • the mutagenized cells are also selected for retention of HLA antigen expression on the cell surface.
  • Example I below describes the preparation of an immortalizing hybridoma which is generally useful in producing primate triomas according to the invention.
  • the cell line, designated SBC-H20 has the selected characteristics noted in the example. The cell line was deposited with the ATCC on or about 13 December 1983 and given the designation ATCC HB 8464.
  • tissue cells -from above are fused under conditions like those outlined in Section IIC.
  • the tissue cells are mixed with the immortalizing hybridoma cells at a ratio of between about 1:1 to 1:5, and preferably about 1:3.
  • cell mixtures containing greater numbers of the tissue cells, such as in a ratio of 1:1 and 5:1 are feasible.
  • the tissue cells are usually more difficult to obtain, it is generally preferred to use an excess of the immortalizing cells in the cell fusion mixture.
  • the cell mixture is freed of serum by washing, and resuspended in polyethylene glycol to promote cell fusion.
  • the cells are washed, resuspended in culture medium, and plated on microtiter wells. Since the unfused tissue-specific cell is unable to grow in culture, the selection medium can simply be the HAT or AH medium which will discriminate against unfused immortalizing hybridoma cells. The resulting fusion products are thus easily ⁇ selected on the basis of the presence of colonies of growing cells, as seen by light microscopy.
  • the selection medium can simply be the HAT or AH medium which will discriminate against unfused immortalizing hybridoma cells. The resulting fusion products are thus easily ⁇ selected on the basis of the presence of colonies of growing cells, as seen by light microscopy.
  • hybrid cells In addition to the selection of hybrid cells based on their ability to grow in HAT or AH medium, it is generally advantageous, prior to viral infectivity studies, to confirm that the hybrid cells contain selected characteristics of the tissue cell type which has been fused.
  • one convenient confirmatory assay involves testing the hybrid cell for the'ability to secrete one or more selected tissue-specific secretory proteins.
  • the assay can be carried out by means of a solid phase reporter-linked immunoassay. In the usual solid-phase assay method, a solid surface coated with antibodies specific against the selected protein is reacted with the cell culture supernatant, binding supernatant proteins immunospecifically to the support surface.
  • HSA human serum albumin
  • the antibody can be produced by conventional serum antibody techniques for raising antibodies in rabbits or goats. These procedures generally require a source of purified or nearly purified protein for animal inoculation. Procedures for purifying many human secretory proteins have been reported, and are documented below. Alternatively, conventional monoclonal antibody techniques may be used for generating antibodies against selected human proteins.
  • the support After reacting the cell medium with the support-bound antibodies, to bind antibody-specfic secretory protein, the support is washed to remove non-specifically bound protein and then reacted with a soluble, reporter-labeled antibody which is also'" specific against the secretory protein of interest.
  • the reporter label on the soluble antibody is a fluorophore, chromophore, enzyme, or radioisotope label.
  • Techniques for labeling antibodies with reporter molecules are well known, and typically involve the use of a bifunctional coupling reagent, such as a di-N-hydroxysuccinimide, or a suitable carboxyl or amine activating agent, such as a soluble carbodiimide, to couple one or more reporter molecules to the antibody.
  • the labeled antibody may be specific against the same or a different antigenic determinant as the support-bound antibody, but in any case, must be able to bind to the selected protein, with such bound immunospecifically to the first antibody carried -on the solid support.
  • the presence of the selected protein in the cell medium is confirmed by the presence of label on the washed support.
  • Example III below illustrates an enzyme-linked immunoassay (ELISA) for detecting human serum albumin (HSA), complement C (C ). and human fibronectin (HFN) secreted by immortalized liver.
  • HSA human serum albumin
  • C complement C
  • HFN human fibronectin
  • cell type which is immortalized does not have secretory functions
  • other cell-specific characteristics such as the presence of surface antigens or cell morphological characteristics can be used to confirm that the hybrid cell line is composed of the cell line of interest.
  • Cell surface antigens can be readily assayed, according to procedures which will be detailed below, by providing an antibody which is specific against a selected tissue-type antigen, labeling the antibody with a suitable reporter, reacting the labeled antibody with the cells, and detecting the presence of reporter on the cells, after washing.
  • the cells can be subcloned to ensure monoclonality. Typically, subcloning is done by a limiting dilution technique in which parent hybrid cells are diluted to produce no more than about 1 cell per well, and the cells replated in multi-well microtiter plates. Clonal populations which show the desired tissue-specific property are then isolated.
  • human non-lymphocytic tissue cells such as human liver cells
  • the mouse/human fusion partner described herein is capable of immortalizing human and non-human primate lymphocytes, to produce cells which are stable in culture for periods of up to several years, with little diminishment in secretory activity.
  • the fusion process is characterized by a high efficiency of successful hybrids, and of these, a high percentage of hybrid cells which are active and stable in secreting tissue-specific proteins.
  • the high efficiency of cell fusion for both lymphocytic and non-lymphocytic cells, appears to be related to the initial selection, in forming the fusion partner, of mouse/human hybrids which are stable for human chromosome retention, as evidenced by continued immunoglobulin secretion and HLA production. That is, the fusion partner is preselected for a stable arrangement of human and mouse chromosomes, which apparently favors chromosome stability in the trioma fusion product.
  • the ability of the fusion partner to form stable fusion products with such diverse cells as human lymphocytes, chimpanzee lymphocytes, and human liver cells indicates the general applicability of the method to human cell immortalization.
  • the immortalized cells are used as an infectable host for certain human or non-human primate viruses, and particularly those which can not otherwise be grown in culture.
  • parent hybrid tissue cells such as fused liver cells
  • the parent cells are then assayed for infectability by the virus.
  • virus infection is detected by the steps- of (a) infecting the cells with a known source of the active virus and (b) assaying the cells during a one to several week incubation period, for the presence of viral infection.
  • the trays containing the fused cell colonies are duplicated, so that a stock of uninfected cells will be available after the virus-infected cell colonies are identified.
  • the virus-induced cell change is preferably one which is readily and unambiguously detected in the culture of immortalized tissue cells.
  • the viral infection may be characterized by cytopathic effects (CPE), such as cell lysis, multinucleation or cell clumping.
  • CPE cytopathic effects
  • Other types of virus-induced cell responses included metabolic changes, such as a pH shift or " release of one or more cellular enzymes. Both of these types of metabolic changes can be detected by the use of suitable indicator dyes or substrates.
  • viral infection can be determined by the presence of virus-specific surface or intracellular or extracellular antigens which are expressed as a result of viral infection.
  • cell surface antigens can be detected readily by conventional labeled antibody techniques.
  • the cultured cells are first reacted with the serum from a human or non-human primate infected with the virus. After washing the cells to remove non-specifically bound material, the cells are incubated with a reporter-labeled anti-human antibody, preferably a fluorescent-labeled antibody. Infected cells are then easily detected by the presence of reporter on the cell surface. This method is illustrated in Examples V and VI for detecting immortalized cells infected with NANB hepatitis virus.
  • a specific liver hybrid cell which has been infected with NANB virus from an infected chimpanzee, and which shows virus-specific cell surface antigens has been deposited with the American Type Culture Collection, and is identified by ATCC number HB 9027.
  • the hybrid liver cell which is infectable with NANB from an infected chimpanzee is also infectable with NANB from human plasma, and with hepatitis B virus from an infected human, also as described in Example V.
  • hybrid cells formed in accordance with the invention can be infected with, and used to propagate tissue-specific viruses which are otherwise not capable of being propagated in culture.
  • the general methods for infecting and culturing the hybrid cells with a selected human-infectious virus generally follow the procedures outlined above and described in Example V. Briefly, plasma from a human or other infected source, such as chimpanzee, is used to infect the cells, and viral infection is followed by monitoring a virus-related cell change over time in culture. Where virus infection is characterized by the appearance of virus-specific antigens, as in the case of the two hepatitis viruses propagated as in Example V, the viral infection is preferably followed by immunological methods for detecting antigens.
  • the virus can be harvested, if desired, by conventional methods for releasing and purifying viral particles from cells.
  • the infectability of the cells, and the ability of serially passage a viral agent from infected to non-infected cell line is demonstrated in Example VI.
  • each of two immortalized liver cell lines infected with NANB viral agent were cultured under normal growth conditions, then lysed by one of a variety of means to release a supernatant fraction.
  • the supernatant fractions or itomycin-treated cells
  • the infected cells showed immunofluorescent reactivity (surface antigen appearance) within ten weeks of infection.
  • the supernatant from a second generation infected cell line was similarly passaged to uninfected cells in culture, yielding infected third generation cells.
  • antigens are specific to the viral infection.
  • the antigens may be intracellular, bound to the surfaces of the infected cells, and/or expressed in extracellular form.
  • One general method for determining the presence of virus-specific antigens which is outlined above and illustrated in Example V, involves immunospecific binding between the antigen and anti-antigen antibody from the serum of a human or experimental animal which has the infection. This method of course depends on the presence of such antibodies in the infected subject.
  • the assay typically - involves reacting the serum with the cells, washing the cells to remove unbound -serum components, then reacting the cells with a labeled anti-immunoglobulin antibody which can bind specifically with the anti-antigen antibody.
  • a labeled anti-immunoglobulin antibody which can bind specifically with the anti-antigen antibody.
  • Intracellular and extracellular antigens can be assayed by conventional immunoassays which may involve immunoprecipitation or Ouchterlony immunodiffusion techniques, using the above serum and a cell-free antigen preparation.
  • Example VII The detection and partial isolation of a virus-specific surface antigen, in response to infection with NANB viral agent, is illustrated in Example VII.
  • Plasma membrane fractions of the three cell lines were prepared by the methods described below, and the three membrane fractions were dot blotted onto filters by conventional methods. The dot blots were reacted with IgG antibody fractions obtained from either normal or NANB-infected individuals, then examined for antibody binding by addition of a fluorescent-labeled anti-human IgG antibody. The results are given in Table 4 of Example VII.
  • both of the infected cell lines gave greater immunoreactivity with NANB(+) IgG fraction than with the normal fraction, demonstrating that the antigen fraction can be used to assay for NANB infection, based on the presence of NANB-specific antibodies which are present in the infected individual and reactive with the fraction.
  • the method allows for detection of infection in humans of a viral agent which provokes a virus-specific antibody response on infection.
  • the method involves isolating from immortalized tissue cells which are infected with the viral agent, a fraction containing a cell surface antigen which shows greater immunological reactivity with antibodies obtained from an infected individual.
  • the fraction is reacted with a blood sample, e.g., a serum sample or IgG fraction, from the test individual, and the binding of antibody to the antigen is detected.
  • a typical assay procedure follows the method in Example VII, where analyte human serum is reacted with the cells, and the cells are exposed, after washing, to a labeled anti-human immunoglobulin antibody.
  • This general approach may also be suitable for detecting the presence of viral infection of certain immortalized cell types, such as central nervous system cells, where viral etiology is suspected.
  • the immortalized cells are exposed to serum derived from the cell donor or another individual having the disease, with the presence of cell antigens being determined by a second labeled antibody, as above.
  • the specificity of the antigen must be confirmed by suitable controls involving an immortalized control (non-infected cells).
  • the virus-specific antigen from above is also useful for generating virus-specific murine monoclonal antibodies (Mabs).
  • the method follows standard Mab procedures, in which a mouse is inoculated with cell or cell-free material containing the antigen, and B-lymphocytes obtained from the inoculated animal are fused with mouse myeloma cells, according to fusion procedures like those detailed above. Successful fusion products can be determined by assaying microtiter wells for the presence of the desired Mab, using assay techniques similar to those for detecting serum antibodies which are specific against the antigen.
  • the Mabs are useful in diagnosing virus infection, where infection is evidenced by the presence of the virus-related antigen in the serum of the infected individual.
  • This assay can be performed, for example, as a solid-phase sandwich-type assay, in which the antigen is first reacted with the Mab attached to a solid support, followed by attachment of a soluble labeled anti-antigen Mab to the support through the antigen.
  • Another application of the Mabs is for purifying the virus-specific antigen, using conventional affinity chromatography techniques.
  • the purified antigen would be valuable, for example, in designing an anti-virus vaccine, based on the known amino-acid sequence of the purified antigen.
  • An important advantage of the present invention is the ability to propagate relatively large amounts of viral genomic material in the hybrid cell cultures.
  • the genomic material may be either in the form of self-replicating viruses, or, if the viral genome is integrated into the cell genome, as part of the replicating cell DNA. In either case, the presence and identity of active viral- genes can be determined by DNA subtraction enrichment methods which have been reported (Kunkel). or by antigen expression in a suitable expression system.
  • the latter method is performed, for example, by preparing a cDNA library of polyA RNA isolated from the virus-infected cells, cloning the library fragments in a suitable cloning vector, transforming host cells, typically a bacterial host, with the vector, and using the anti-antigen antibody described above to detect the expression of the antigen by the host.
  • the identified viral genomic material may be used as a probe for identifying further the presence and or localization of viral genome in the infected cell, as a diagnostic tool for detecting the presence of virus in a blood or tissue sample from an individual, or in • molecular cloning procedures, for producing antigen or antigen fragment peptides which are useful for vaccine purposes.
  • Example I Preparation of Immortalizing Hybridoma SBC-H20
  • Mouse myeloma cell line SP20/08A2 was obtained for use as the immortalizing partner from Frank Fitch, University of Chicago. This cell line is freely available and can be used without restriction. Other mouse myeloma lines are also readily available.
  • Human peripheral B lymphocytes were isolated from the heparinized blood of a normal human donor by Ficoll-Hypaque gradient as described in reference 11. The peripheral B lymphocytes and myeloma cells were mixed at a 1:1 ratio, washed once in RPMI 1640 medium (Gibco), and pelleted at 250 x g for 10 min.
  • the pellet was gently resuspended in 1 ml of RPMI with 40-45% (V/V) polyethylene glycol solution. MW 1430-1570 (BDH Chemicals, Poole, England) which was pre-warmed to 37°C. After two min at room temperature, the cell suspension was diluted to 6 ml with RPMI. centrifuged at 500 x g for 3 min, and. beginning 8 min from the onset of fusion, the cell pellet was washed with RPMI containing 10% fetal calf serum (FCS). The pelleted cells were plated in multi-well trays using suitable dilutions to obtain individual clones.
  • FCS fetal calf serum
  • the colonies were grown on AH selection medium containing 2 ⁇ g/ml azaserine and 100 ⁇ M hypoxanthine, and successful clones were assayed for immunoglobulin production and fo -HLA surface proteins using the assay methods - described in reference 6.
  • This murine-human hybridoma includes: sensitivity to HAT and AH media, resistance to ouabain (Sigma) to a concentration of 10 ⁇ M, non-secretion of immunoglobulins, human chromosomal stability over time, and production of HLA surface protein.
  • IMDM Iscove's Medium
  • FCS 20% fetal calf serum
  • Isolated hepatocytes from Example II were suspended to a final cell concentration of about 3 x 7 ⁇ o cells/ml in IMDM.
  • the isolated cells were mixed with the hybridoma cell line SBC-H20 (Example I), at a cell ratio of about 3:1.
  • the cells were washed three times in IMDM without serum, with pelleting at 200 x g for 10 minutes, and resuspended gently in 1 ml of 55% IMDM:45% polyethylene glycol (v/v) MW 1430-1570 (BDH
  • the fused cells were resuspended in IMDM containing 10% FCS and 100 ⁇ M hypoxanthine, 19 ⁇ M thymidine (HT medium) and plated in microtiter wells at
  • HAT medium HT medium with 800 nm aminopterin (HAT medium).
  • HAT selection medium was used for 14 days prior to switching to HT medium, at which point unfused SBC-H20 cells and unfused hepatocytes were 100% nonviable, as evidenced by trypsin blue inclusion.
  • Hybrid cells formed by fusion of the SBC-H20 fusion partner with the hepatocytes were observed in. 9 of the 120 microtiter wells (Group A) and 45 of the 48 wells in the 24-well trays (Group B), as evidenced by the presence of colonies which were visible by light microscopy. Some of the wells or trays contained two or more distinct colonies.
  • HSA human serum albumin
  • C complement C
  • HFN fibronectin
  • the enzyme-linked iramunoassay used to detect the presence of the proteins is described in Example IV below.
  • Table I The results obtained for the 45 fused cell colonies in Group A, and the 49 fused cells in Group B are shown in Table I. As seen, several of the fused cell lines were active in secretion of one or more of the liver proteins, and 5 colonies in Group A and 8 colonies in Group B secreted all three proteins.
  • hybridomas were cloned by limiting dilution to ensure monoclonalit .
  • Multiple monoclonal cell lines were isolated which continued to secrete HSA, complement C , and HFN.
  • Example IV Identification of Liver Cell Products Selected hybridomas from the Group A and Group B colonies in Example III were subcloned by limiting dilution to insure monoclonality, and reassayed for parenchymal secretory proteins.
  • Goat anti-human antibodies specific against human albumin, complement C , and fibronectin were obtained from Cappell Labs (Malvern, PA) .
  • Microtiter trays were coated with one of the three different anti-sera overnight with 50 nl/well of a 1:1000 dilution (1 ⁇ g/ml)at 4 C in a moist chamber. The following day, 150 nl of a 0.2 % gelatin solution were added to each well for one hour at room temperature, to saturate non-specific binding sites in the wells. The trays were then washed four times with cold PBS /0.05% Tween-20.
  • Analyte and control samples (50 ⁇ l) were added to the trays and incubated for 1 hour at room temperature.
  • the analyte samples were undiluted medium obtained from the growing monoclonal cultures.
  • the control samples contained human plasma diluted 1:20,000 (complement control), purified human fibronectin at 5 ⁇ g/ml. and human serum albumin, at 25 ⁇ g/ml. After incubation, the trays were washed three times with the above Tween solution.
  • Peroxidase-conjugated goat antibodies against human C , albumin, or fibronectin were purchased from Cappell Labs.
  • the peroxidase-conjugat_ed antibody 50 ⁇ l
  • the peroxidase-conjugat_ed antibody 50 ⁇ l
  • substrate mixture was added and the peroxidase enzyme reactions were allowed to develop for about thirty minutes at room temperature before arresting the reaction by the addition of 100 ⁇ l of 10% SDS.
  • the substrate mixture was prepared by mixing 7.2 ml of a 0.1M citrate/HCL buffer. pH 4.2, 4.8 ml of 2.5 mM ABTS, and 80 ⁇ l of a 1 M H O solution. The mixture was prepared about 30 minutes prior to addition to the washed trays. The assay mixture/SDS solutions were read 415 or 405 nm. As seen from the results in Table II, each of the parent hybrids B3, D4, and D8. (all HFN secretors) yielded multiple subclones which were also competent in HFN secretion only. Similarly parent hybrid cell line D4, which was active in producing all three liver proteins, yielded multiple subclones, each of which was competent in secreting all three proteins assayed.
  • Hybrid liver cells prepared as in Example III were subcloned by limiting dilution and a subclonal colony which secreted HFN was isolated.
  • the hybrid cells were plated at 1 x 10 cells/well in a 24-well tray and overlaid with 100 ⁇ l of plasma from one of the following three sources: (a) chimpanzee plasma known to contain NANB viral agent(s) by passage into a -second chimpanzee, (b) human plasma from an individual with acute post-transfusion NANB hepatitis, and (c) chimpanzee plasma known to contain hepatitis B virus.
  • Goat anti-human IgG and IgM conjugated with flu ⁇ rescein isothiocyanate was obtained from a commercial source (Zymed Labs) and diluted with PBS to a final concentration of about 1 ⁇ g antibody/ml. Either anti-IgM or anti-IgG FITC conjugated antibody (70 ⁇ l) was added to the washed cells, and the slides were incubated at room temperature for 30 minutes. After washing with PBS and distilled water as above, the slides were mounted with one drop of 50% glycerol in PBS and observed under a fluorescence microscope. The cells were scored for weak (+), intermediate (++), and strong (+++) fluorescence.
  • the first indications of immunofluorescence occurred at about 4-6 weeks after initial cell infection with all three virus sources.
  • the results shown in Table 3 were obtained after 6 weeks infection with chimpanzee plasma known to contain NANB agent(s). As seen in the righthand columns in Table 4, specific immunofluorescence was observed only with sera from NANB-infected animals, and not with other known sera.
  • the results indicate that (a) the liver hybrid cells are infectable by NANB virus, (b) the infected hybrid cells are expressing a virus-specific surface antigen which is recognized by NANB serum antibody from chimps with known NANB infection, and (c) an incubation period of between about 4-6 weeks is required for surface antigen expression.
  • the results shown in Table III were obtained with anti-IgG antibody. No immunofluorescence was observed with the FITC conjugated anti-IgM antibody, as would be expected if the chimp anti-NANB antibodies are IgG type antibodies.
  • the human plasma from an individual with acute post-transfusion NANB gave a similar result.
  • infected liver hybrids showed specific immunofluorescence with a serum from a NANB-infected chimpanzee, but not with control chimpanzee serum.
  • the NANB-infected hybrid cells were also examined for the presence of NANB virus capable of further cell infection.
  • Infected hybrid cells from above, after 12 weeks incubation, were pelleted and . washed three times with PBS. The cell's were resuspended in PBS to about 5 x 10 cells/ml and sonicated to clarity.
  • the supernatants (0.5 ml/well) were then inoculated on uninfected hybrids and cultured in the manner described above for cell infection by chimp plasma. After about 4-6 weeks in continuous culture, specific immunofluorescence was observed with chimp NANB serum, but not with serum from uninfected chimps.
  • Hybrid liver cells infected with plasma from a human with hepatitis B infection showed specific immunofluorescent staining with a goat antiserura to hepatitis B surface antigen six weeks after the hybrid cell culture was infected. Furthermore, the hybrid liver cells secreted hepatitis antigens that can be detected with a commercially available kit that detects hepatitis B surface antigens. This firmly established the ability of the hybrid cells to propagate hepatitis B virus and to secrete hepatitis B specific antigens.
  • Example VI Serial Passage Infection by NANB Virus
  • One measure of viral infection in cultured cells is by serial passage of the infecting agent from infected to non-infected immortalized culture cells.
  • This example describes the serial passaging of a NANB viral agent from NANB-infected liver cells, designated RO2 and RSI, to uninfected cell line GL424. All of the immortalized cell lines were prepared as in Example III.
  • the R02 cell line was produced by infection with chimpanzee plasma known to contain NANB viral agent by passage into a second chimpanzee, substantially as described in Example V.
  • the RSI cell line was produced by infection with the serum from a human who died of NANB hepatitis.
  • Example V Infection by the NANB viral agent, in serial passage, was confirmed by reacting the newly infected cells with a NANB-specific human IgG antibody, then with fluorescein-labeled goat anti-human IgG antibody, as. in Example V.
  • the antibody was derived from the serum of a human who died of NANB hepatitis, and was specifically reactive with chimp NANB infected tissue, as assayed in Example V.
  • the antibody was contained in an IgG fraction of the serum prepared' conventionally by HPLC.
  • the following four methods for releasing the viral agent from the two infected cell lines were employed: a. The cells were washed 2 times with phosphate biffered saline (PBS).
  • PBS phosphate biffered saline
  • the cell pellet was resuspended in a minimum volume of distilled water (1-2 ml), and the cells were observed microscopically for cell lysis.
  • lysis 90-100% complete, an equal volume of 2X PBS was added, and the lysis suspension was centrifuged at 1500 rpm for 5 minutes. The supernatant was added to the culture medium of uninfected GL424 cells.
  • the infected cells were treated with 100 ⁇ g/1 of mitomycin C for 24 hours. After two washings with PBS, the cells were co-cultivated with uninfected GL424 cells.
  • the infected cells were sonicated with two 15 second bursts, while cooled in an ice bath.
  • Cell lysis was confirmed microscopically.
  • the cell lysis suspension was pelleted as in (a) above and the supernatant added to the culture medium of the uninfected GL424 cells.
  • the infected cells were subjected to two freeze/thaw cycles, then pelleted and as in (a) above, before adding the supernatant to uninfected GL424 cells.
  • the exposed cells were monitored weekly by surface immunofluorescence for binding to the above NANB-specific antibody. All four methods showed transmission of the viral agent, as evidenced by immunofluorescence acitvity, within ten weeks of exposure to the supernatant fractions from each of the two infected cell types. No immunofluorescence activity was observed in control GL424 cells which were exposed to the supernatant from uninfected GL424 cells.
  • GL424 cells infected with the water-lysis were themselves subject to a water-lysis step, and the supernatant added -to- the culture medium of uninfected GL424 cells.
  • immunofluorescence was observed, evidencing passage of the viral agent from a second to a third generation.
  • Example VII NANB-Specific Cell Surface Antigen
  • the cell lines examined in this example are uninfected line GL424 and infected cell lines R02 and RSI. Both the R02 and RSI lines gave immunofluorescent activity when reacted with the IgG antibodies from NANB-infected human serum and fluorescent-labelled anti-human antibodies, whereas no immunofluorescence was seen with the non-infected GL424 line.
  • GL424, R02, and RSI cell lines from above were harvested during log phase, using approximately 1 x
  • the homogenate was centrifuged twice at 1000 x
  • Immunodot blots were run using insoluble membrane preparations from GL424, R02, and RSI at concentrations of approximately 100 ⁇ g to 0.1 ⁇ g per well. Blots were probed with normal IgG or NANB IgG diluted twofold from 1/8 to 1/512. There was nonspecific reactivity evident in all reactions of membranes with greater than 1 ⁇ g/well protein and IgG concentrations greater than 1/512 IgG. The results of the immunodot blot data are summarized in Table 3 below.
  • Results are from Immunoblot Blots probed with 1/512 NANB IgG or normal IgG.
  • R02 membrane was reactive with NANB IgG at a concentration of 1 ⁇ g/well, while control GL424 was not. A lower amount of nondifferentiated reactivity was evident in all three membrane preparations at 1 ⁇ g/well with 1/512 normal IgG.
  • the results from dot blots indicate differences in the immunoreactivity of the NANB-infected line R02 as compared to uninfected GL424. These results support the finding of antigenic differences indicated by the differential immunofluorescence reactivity of the three cell lines with NANB plasma.
  • the invention encompasses a broad range of human and non-human immortalized tissue cells which can be used in stable cell culture and which are infected or infectable with a variety of human-infectious viruses.

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Abstract

Une lignée de cellules tissulaires humaines immortalisées est formée par fusion d'une cellule tissulaire sélectionnée non lymphocytique de primate humain ou non humain avec une cellule immortalisante d'hybridome d'être humain/de souris. La cellule tissulaire ainsi que la lignée de cellules résultant de la fusion sont infectées ou peuvent être infectées par un agent viral sélectionné, ce qui permet l'identification des antigènes de surface des cellules à spécificité virale et leur utilisation comme une source servant à produire des antigènes à spécificité virale, des anticorps anti-antigéniques et des compositions de vaccins.
PCT/US1987/000711 1986-04-01 1987-04-01 Cellules tissulaires immortalisees a specificite virale Ceased WO1987005930A1 (fr)

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WO1989004669A1 (fr) * 1987-11-18 1989-06-01 Chiron Corporation Diagnostic et vaccins du virus d'hepatite non-a non-b
WO1990000597A1 (fr) * 1988-07-06 1990-01-25 Genelabs Incorporated Virus et antigenes d'hepatite serique non a et n b
EP0414475A1 (fr) * 1989-08-25 1991-02-27 Chiron Corporation Procédé de culture du HCV dans des cellules eucaryotes
WO1991015574A1 (fr) * 1990-04-03 1991-10-17 Southwest Foundation For Biomedical Research Virus purifies de l'hepatite c et proteines et peptides de ces virus
EP0460056A4 (en) * 1989-02-24 1992-06-24 Southwest Foundation For Biomedical Research Non-a, non-b hepatitis hepatocyte cell culture
US5218099A (en) * 1986-04-01 1993-06-08 The United States Of America As Represented By The Department Of Health And Human Services Post-transfusion, non-A, non-B hepatitis virus polynucleotides
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
WO1994025064A1 (fr) * 1993-05-04 1994-11-10 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMANSERVIC ES Propagation de cellules virales de l'hepatite c et procedes associes
US5436318A (en) * 1990-04-06 1995-07-25 Genelabs Technologies, Inc. Hepatitis C virus epitopes
WO1996024662A1 (fr) * 1995-02-10 1996-08-15 Consiglio Nazionale Delle Ricerche Procede de propagation 'in vitro' du virus de l'hepatite c (vhc) dans les cultures de cellules animales non lymphoblastoides, et produits obtenus
US5698390A (en) * 1987-11-18 1997-12-16 Chiron Corporation Hepatitis C immunoassays
US5712088A (en) * 1987-11-18 1998-01-27 Chiron Corporation Methods for detecting Hepatitis C virus using polynucleotides specific for same
US5714596A (en) * 1987-11-18 1998-02-03 Chiron Corporation NANBV diagnostics: polynucleotides useful for screening for hepatitis C virus
US6027729A (en) * 1989-04-20 2000-02-22 Chiron Corporation NANBV Diagnostics and vaccines
US6171782B1 (en) 1987-11-18 2001-01-09 Chiron Corporation Antibody compositions to HCV and uses thereof
US6861212B1 (en) 1987-11-18 2005-03-01 Chiron Corporation NANBV diagnostics and vaccines
WO2013064833A1 (fr) * 2011-11-03 2013-05-10 Queen Mary And Westfield College University Of London Procédé de réplication du vhc in vitro
WO2023239928A1 (fr) * 2022-06-09 2023-12-14 CHO Plus Inc. Hybrides cellulaires comme cellules hôtes pour la production à haut rendement de vecteurs de thérapie génique et de vaccins viraux

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US5218099A (en) * 1986-04-01 1993-06-08 The United States Of America As Represented By The Department Of Health And Human Services Post-transfusion, non-A, non-B hepatitis virus polynucleotides
US5714596A (en) * 1987-11-18 1998-02-03 Chiron Corporation NANBV diagnostics: polynucleotides useful for screening for hepatitis C virus
US5698390A (en) * 1987-11-18 1997-12-16 Chiron Corporation Hepatitis C immunoassays
US6861212B1 (en) 1987-11-18 2005-03-01 Chiron Corporation NANBV diagnostics and vaccines
US6171782B1 (en) 1987-11-18 2001-01-09 Chiron Corporation Antibody compositions to HCV and uses thereof
US6096541A (en) * 1987-11-18 2000-08-01 Chiron Corporation Cell culture systems for HCV
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
US5712088A (en) * 1987-11-18 1998-01-27 Chiron Corporation Methods for detecting Hepatitis C virus using polynucleotides specific for same
US6074816A (en) * 1987-11-18 2000-06-13 Chiron Corporation NANBV diagnostics: polynucleotides useful for screening for hepatitis C virus
WO1989004669A1 (fr) * 1987-11-18 1989-06-01 Chiron Corporation Diagnostic et vaccins du virus d'hepatite non-a non-b
CN100397082C (zh) * 1987-11-18 2008-06-25 希龙股份有限公司 检测样品中hcv的方法
US7790366B1 (en) 1987-11-18 2010-09-07 Novartis Vaccines And Diagnostics, Inc. NANBV diagnostics and vaccines
WO1990000597A1 (fr) * 1988-07-06 1990-01-25 Genelabs Incorporated Virus et antigenes d'hepatite serique non a et n b
EP0460056A4 (en) * 1989-02-24 1992-06-24 Southwest Foundation For Biomedical Research Non-a, non-b hepatitis hepatocyte cell culture
US6027729A (en) * 1989-04-20 2000-02-22 Chiron Corporation NANBV Diagnostics and vaccines
EP0414475A1 (fr) * 1989-08-25 1991-02-27 Chiron Corporation Procédé de culture du HCV dans des cellules eucaryotes
WO1991015574A1 (fr) * 1990-04-03 1991-10-17 Southwest Foundation For Biomedical Research Virus purifies de l'hepatite c et proteines et peptides de ces virus
US5436318A (en) * 1990-04-06 1995-07-25 Genelabs Technologies, Inc. Hepatitis C virus epitopes
US5843639A (en) * 1990-04-06 1998-12-01 Genelabs Technologies, Inc. Hepatitis C virus epitopes
WO1994025064A1 (fr) * 1993-05-04 1994-11-10 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMANSERVIC ES Propagation de cellules virales de l'hepatite c et procedes associes
WO1996024662A1 (fr) * 1995-02-10 1996-08-15 Consiglio Nazionale Delle Ricerche Procede de propagation 'in vitro' du virus de l'hepatite c (vhc) dans les cultures de cellules animales non lymphoblastoides, et produits obtenus
WO2013064833A1 (fr) * 2011-11-03 2013-05-10 Queen Mary And Westfield College University Of London Procédé de réplication du vhc in vitro
WO2023239928A1 (fr) * 2022-06-09 2023-12-14 CHO Plus Inc. Hybrides cellulaires comme cellules hôtes pour la production à haut rendement de vecteurs de thérapie génique et de vaccins viraux
US12252713B2 (en) 2022-06-09 2025-03-18 CHO Plus, Inc. Cell hybrids as virus packaging cells for high efficiency production of gene therapy vectors and viral vaccines

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AU7238387A (en) 1987-10-20
AU613310B2 (en) 1991-08-01
EP0261233A1 (fr) 1988-03-30
EP0261233A4 (fr) 1988-08-29
JPS63502962A (ja) 1988-11-02

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