CA2079677A1 - Purified hcv and hcv proteins and peptides - Google Patents

Abstract

Intact hepatitis C virus (HCV) particles, purified HCV proteins, and glycopeptide antigens specific to HCV particles are disclosed. The virus particles, in inactivated or attenuated form, are useful in a vaccine. The purified proteins, and glycopeptide antigens are useful in a diagnostic system, for detection of human HCV
antisera, and in vaccine compositions. Also disclosed are antibodies specific against the glycopeptide antigens.

Description

~ WO91/1~74 PCT/US91/0~298 2 ~

PURIFIED HCV AND HCV PROTEINS AND PEPTIDES

; 1. Field of the Invention The present invention relates to purified hepatitis c virus (HCV), mature virus proteins and glycopeptide anti-gens isolated from the virus, and vaccine and diagnostic compositions which utilize the particles, proteins and an-tigens.
: 2. References Burk, K.H., et al., Proc. Natl. Acad. Sci. U.S.A.
81:3195-3199 (1984)).
Chomozynski et al, Anal.Biochem. 162:159 (1987).
Eichberg, J. Med. Primatol. 14:165-168 (1985).
Innis, M.A., et al, eds., PCR Protocols: A Guide to Methods and Applications, Academic Press (l990).
Enat, et al., Proc. Natl. Aca. Sci. USA 81:1411 (1984)-Feinstone et al., Infect. Immun. 41:816-821 (1983).
Harlow, E., et al., Antibodies: A Laboratory Manual (1988).
He el al., J. Infect Dis. 156:626-640 (1987).
Jacob et al., Hepatolo~y 10:921-927 (1989).
Jacob, et al., J Infect Dis, 161:1121 (1990).
Lanford, R.E., et al., In Vitro Cell. Dev. Biol.
25:174-182 (19~9) WO9l/lSS74 PCT/US91/022~

2~7~7~

Jat, et al., Mol. Cell Biol. 6:1204-1217 (1986)).
Lanford et al., Virolo~y 97:295-306 (1979).
Lanford et al., In Vitro Cell Dev. Bio., 25:174-182 (1989).
Maniatis, T., et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1982).
Maslamsky, C.J. et al., In Vitro Models for Cancer Research, Vol. II: Carcinomas of the Liver and Pancreas, M.M. Weber and L.I. Sekely (eds.), CRC Press: Bo~a Raton, 10 Fla., pp.43-60 (1985)).
Michalopoulos ,G. et al., Exptl. Cell. Res. 94:70 ` (1975) Murphy, in Virology, B.N. Fields e al., Eds., Raven Press, pp.17-18 (1985).
' 15 Portnoy, et al., J. Lab. Clin. Med., 89: 560-563, : 1977), Salas-Prato, in Growth of Cells in Hormonally Defined Media, (Book A, G.H. Sato, et al., Eds)., Cold Spring Harbor Laboratory, pp. 615-624 (1982).
Sambrook, et al. eds. Molecular Cloning. A Laboratory Manual, Vols. 1, 2, and 3, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989);
Schlesinger, S., et al, eds. in The Togaviridae and Flaviviridae, Plenum Press, New York (1986).
and Sell, M.A., et al., In Vitro_Cel. Dev. Biol.
; 21:216-220 (1985).
* Valenza, F.P., et al, J ~ed Primatol, 11:342 (1982).
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3. Backaround of the ~nvention Non-A, Non-B hepatitis has long been recognized as a virus-induced disease, distinct from other forms of viral-associated liver diseases, including hepatitis A virus , .

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~ WO91/155~4 PCT/US91/02298 2~7~7 (HAV) and B virus (HBV), and hepatitis-induced by cytomega-lovirus (CMV) or Epstein-Barr virus (EBV). NANBH virus is implicated in greater than 90% of all post-transfusion hepatitis cases, and is responsible for the induction of 5 chronic hepatitis in 40-50% of infected individuals. There is growing evidence for at least two distinct types of NANB~ viruses. One is an enterically transmitted virus, and may be seen in epidemic form where sanitation condi-tions are poor. The other is parenterally transmitted 10 NANBH virus, now commonly referred to as hepatitis C virus (HCV), which is a ma~or source of hepatitis in transfused blood.
The limited availability of an animal model (~CV
infects only chimpanzees and humans) and the absence of an 15 in vitro tissue culture model suitable for growing HCV has limited the isolation and characterization of mature HCV
virus. Such particles would be useful for production of inactivated or attenuated HCV for vaccine and diagnostic purposes, and for isolation and identi~ication of intact, 20 mature HCV proteins, and glycosylated HCV antigens.

4. Summarv of the Invention It is therefore a general object of the invention to provide isolated, intact HCV virus particles, and protein 25 and glycopeptide antigens obtainable therefrom.
The invention includes, in one aspect, purified HCV
virus particles characterized by: (a) a single-stranded RNA
genome; (b)- a flavivirus type s~ructure having virus particle sizes between about 30-60 nm, enveloped capsid 30 structures, external stalks 2-5 nm in length and width, and ` an icosahedron symmetry, and (c) immunospecific reaction with HCV-infected individuals. In one embodiment, the RNA

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2~79~77 genome of the particles contains a region with the sequence shown in Figure 4.
The particles are useful, in inactivated form, in a vaccine composition, or for generating monoclonal antibod-S ies specific against HCV virus antigens, and in particular,against glycopeptide antigens.
The protein components of the virus particles, when fractionated and purified, yield mature HCV pro*eins, such as purified, mature C, el (gp35), e2 (gp70), and the HCV
lO counterparts to NS2, NS3, NS4, or NS5, which may be sub-stantially free of HCV genomic ~NA, and non-viral serum and cell proteins normally associated with HCV virus infection in humans.
A purified protein or protein mixture is useful in 15 a vaccine composition, for generating monoclonal antibodies specific against HCV viral proteins, and in a diagnostic system for detection of human anti-HCV anti-sera.
The virus particles or proteins isolated therefrom also provide a source of ~CV-specific glycopeptide anti-20 gens, i.e., glycopeptide regions of a glycosylated HCVproteins, such as gp35 and gp70. The antigens are useful in a diagnostic system for detecting human sera from HCV-infected individuals.
In another aspect, the invention includes an antibody 25 specific against the HCV glycopeptide antigen. The anti-body is useful as a diagnostic reagent, for detecting the pxesence of ~CV antigens in HCV-infected human sera.
'/These and other objects and features of the invention will become more fully apparent when the following detailed `30 description of the invention is read in conjunction with the accompanying dxawings.

-~ WO91/15~74 PCT/US91/02298 2~7~77 Brief Descri~tion of the Drawings Figures lA and lB are electron photomicrographs of HCV
particles at two different magnifications, where the bars in the figures indicate 50 nm;
Figures 2A-2E are electron photomicrographs of HCV
particles, where Figures 2A-2D (bar = 23 nm) show typical variation in size and shape, with a core-like structure being evident in Figure 2C, and Figure 2E (bar = 17 nm) is taken at higher magnification and shows external surface 10 stalks on the virus particle;
Figures 3A-3C are electron photomicrographs of HCV
particles, showing bar-like structures within the virus ^ core (3A and 3C) and prominent envelope structures with external stalk projections (3B);
15Figure 4 gives the sequence of a cDNA region of an HCV
strain isolated from HCV-infected liver, also showing nucleotide divergence with earlier published HCV sequences, designated JT and PT;
Figure 5 shows electrophoretic patterns of PCR pro-- 20 ducts of RNA from various immortalized CU chimpanzee hepa-tocyte cell lînes derived from HCV-infected chimpanzee ~ hepatocytes (lanes 1-8), and from chimpanzee liver RNA
:~ during the acute phase of HCV infection (lane 9); and Figure 6 shows electrophoretic patterns of PCR pro-25 ducts of RNA from various HCV-infected CHMP cells ~lanes 1-12 and 14), from the inoculum used to infect the cells (lane 13), from chimpanzee liver RNA during the acute phase of HCV infection (lanes 15 and 18), and from an HCV cloned fragment.

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WO91/15574 PCTtUS91/022~ ;
2~7~77 Detailed Description_of the Invention I. HCV Virus Particles This section describes cell culture sources of HCV

5 particles having the characteristics:
(a) a single-strand RNA genome;
tb) a flavivirus type structure having virus particle sizes between about 30-60 nm, enveloped capsid structures, - external stalks 2-5 D in length and width, and an icosa-10 hedron symmetry, and (c) immunospecific reaction with HCV-infected individ-uals.

A. Cell culture sources.
One cell-culture source of HCV virus particles is a cultured primary hepatocytes derived from the liver of an HCV-infected chimpanzee or human, and cultured under conditions which maintain the differentiated state of the infected cells for 3-4 weeks. Methods for preparing 20 primary primate hepatocytes for culture, and culture medium conditions effective to preserve liver-specific functions r for extended periods in culture have been described by the inventors (Lanford, 1989) Details of the primary cell culture methods are given 25 in Example 1. Briefly, liver tissue obtained from an HCV-infected chimpanzee or human is perfused and hepatocytes are dislodged by treatment with collagenase. The cells are washed several times, then plated on culture plates at a density of about 5 x 105 to 5 x lo6 cells per 60 mm plate.
30 The hepatocytes are maintained in serum-free medium (SFM) which has been specifically designed to allow the cells to grow in culture in a highly differentiated state, as ., ' : -~ .
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~ WO91/15574 PCT/US91/02298 2~7~77 evidenced by the continued production and secretion in culture of liver-specific proteins.
one preferred SFM is composed of Williams' medium E
(WME) supplemented with lO mM HEPES, pH 7.4, 50 ug gentami-5 cin, and the following supplements: EGF (epidermal growthfactor), insulin, glucagon, BSA (bovine serum albumin), soybean lipids, linoleic acid, hydrocortisone, selenium, cholera toxin, LGF (liver growth factor, a glysyl-histidyl-lysine tripeptide~, ECGS (endothelial cell growth supple-lO ment), transferrin, ethanolamine, prolactin, somatotropin,and TRF (thyrotropin-releasing factor), in the proportions given in Example l. The sources of these materials are given elsewhere (Lanford). The cells are maintained in the SFM under standard cell culture conditions. The medium 15 is changed, e.g., 24 hours after isolation and every 48 hours thereafter, during the culture period. Under these conditions, the cells undergo 2-4 rounds of replication in the first several days of culture, e.g., within 7-lO days, and thereafter continue to function as liver-specific cells 20 in culture, but without appreciable signs of cell replica-tion, for 3-4 weeks total culture period. Thereafter, the virus-infected cells gradually lose hepatocyte differentia-tion, as evidenced by a decline in the production of liver-specific proteins.
The differentiation of the primary hepatocytes in : culture can be assessed by following changes in the pro-duction and secretion of liver-specific proteins. In one approach described in Example l, proteins from the culture - medium are fractionated by sodium dodecyl sulfate-poly-30 acrylamide gel electrophoresis ~SDS-PAGE~, and the frac-tionated proteins are detected by immunoblotting, using antibodies directed against the proteins of interest. In . . ', - - . . . . .
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WO91/l5~74 PCT/US91/022~

2~79~77 a second approach, also described in Example 1, radio-labeled cell culture proteins are immunoprecipitated with immobilized, protein-specific antibodiçs, and the precipi-tated antibodies are then fractionated by SDS-PAGE. In 5 both cases, analysis of the gel patterns showed that the hepatocytes produced a number of liver~specific proteins in approximate relative proportions to that found in plasma for up to 3-4 weeks, then showed a gradual decline in the amount of protein produced. The decline in liver-specific lO protein production, such as apolipoprotein production, paralleled a degeneration in the`hepatocyte cultures.
HCV particles were obtained from the HCV-infected hepatocytes in culture for up to three-seven weeks after the infected cells were placed in culture (Table 1).
Another source of HCV particles, in accordance with the invention, is chimpanzee or human primary hepatocytes which are infected in vit~o with ~CV inoculum. A method of obtaining and culturing hepatocytes from uninfected chimpanzees is given in Example 5, and generally follows 20 the culture method used to form stable, differentiated primary hepatocytes derived from HCV-infected liver cells.
The cells are infected with a pooled inoculum of plasma samples from several chimpanzees with known acute HCV
infection, as described in Example 5.
Table 1 below compares viral counts obtained from cultured hapatocytes which were derived either from HCV-infected chimpanzees tPTTx196, PTTx174, PTTx268, and : PTTx198) or from non-infected chimpanzees, whose cultured hepatocytes were infected in vitro (PTTx266 and PTTx344), 30 where culture samples were taken at the culture times indicated. The results show the ability to replicate, isolate and purify the HCV virus in hepatocytes derived ,, ~ , : . : .
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Table l ANIMAL ¦ DAY OF CULTURE
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ANIMALDAY OF CULTURE PARTICLES Xl06/ml 1 .. .. .
A third source of HCV particles, also in accordance 5 with the invention, is immortalized chimpanzee or human hepatocytes which are infected in vitro with HCV after -immortalization. Immortalization is achieved by introduc-ing an oncogene into stable, non-infected or HCV-infected primate hepatocytes in culture, as detailed in the compan-10 ion patent application for "An Immortalized Hepatocyte Cell Line", and illustrated in Example 6. Briefly, hepatocytes obtained from non-infected or H~V-infected chimpanzees (or humans) are cultured, as above, under conditions which allow expression a liver-specific proteins for extended 15 culture periods. During the first 2-4 rounds of replica-tion of the cultured cells, the culture is exposed to a - ~ . . .... ... .

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virus or plasmid vector containing a suitable oncogene, such as the SV40 large T antigen oncogene, and immortalized cells are selected on the basis of continued growth beyond initial 2-4 rounds of replication, when non-immortalized 5 cells are essentially non-replicative.
To select an immortalized cell line capable of supporting HCV infection and replication, several immortal-ized cell lines from above are infected with an HCV
inoculum, and the individual cell lines are assayed for the 10 presence of HCV RNA, using PCR methods such as detailed in Example 6.
Figure S shows a gel analysis of the PCR products from HCV-infected CU~cell lines (produced by immortalization of hepatocytes from an ~CV-infected chimpanzee). Lanes 1-8 15 are CUl, CU3, CU4, CU5, CU6, CU8, CU9 and CU12, respective-ly. Lane 9 is a positive control of chimpanzee x198 liver RNA during the acute phase of HCV infection and was processed identically as the CU RNA samples. Lanes lO and 11 are the cDNA and PCR negative controls to demonstrate 20 the lack of contamination during the PCR assay. Lane 12 is lambda DNA cleaved with HindIII as size markers. Lane 5 (CU6) and 9 (PCR positive control) show a positive reac-tion. All lanes have a lower band that represents the primers used in the PCR reaction. Positive reactions were 25 obtained with CU6 cell line, the inoculum used to infect the cell lines, and each of the positive controls. The negative controlled were negative indicating that no contamination occurred during the PCR reaction.
i Figure 6 shows a gel analysis of PCR products from 30 HCV-infe~ted CHMP cell lines (produced by immort lization of hepatocytes derived from non-infected chimpanzees hepatocytes). Lanes 1-12 represent CHMP 1.21, 1.22, 1.23, ~ . . . . .

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W091~1~574 PCT/US91/02~?n~

2~7~7 }~,.24, 1.25,1.26, 1.27, 1.28, 1.29, 1.30, 1.31 and 1.32, respectively. Lane 13 is the PCR analysis of the inoculum used to infect both the CU and CHMP cell lines. Lane 14 is CHMP 2.02. Lane 15, 18 and 19 are PCR positive controls.
5 Lane 15 and 18 are PTTx198 liver RNA as described for Figure 6. Lanes 16 and I7 are cDNA and PCR negative controls, respectively. Lane 19 is a P~R positive control consisting of a gel purified band from a cloned fragment of HCV homologous to the PCR primers used in this assay. Lane 10 20 is HindIII digested lambda DNA as size markers.
Positive reactions were obtained with CHMP 1.27 and CHMP 2.02 cell lines, the inoculum used to infect the cell - lines, and each of the positive controls. The negative - controls wexe negative indicating that no ontamination 15 occurred during the PCR reaction.
Thus, of the 20 CU and CHMP cell lines tested, three were permissive for infection with and replication of HCV.
The cell lines are CHMP 1.27, CHMP 2.02 and CU6. These results demonstrate that immortalized chimpanzee hepato-20 cytes, whether derived from non-infected or HCV-infected animals, are infectable with HCV, and support replication - of HCV, for use in the production of HCV.
. . .
B. - Isolation of Virus Particles Virus particles can be isolated from HCV infected chimpanzee or human hepatocytes in culture by gradient centrifugation methods, as described in Example 2. In one preferred method, culture ~edium is clarified by low-speed centrifugation, then separated from soluble culture-medium ' 30 components by centrifugation through a 20% sucrose layer by high-speed centrifugation. The material is further .i :.

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_WOgl/tS574 PCT/US91/02298 ~7~7 purified by centrifugation onto a 68% sucrose cushion at high speed.
Other methods for separating vixus particles from soluble culture-medium components may be used. For 5 example, clarified culture medium can be passed through a size-exclusion matrix, to sepa~ate soluble components by size exclusion.
Alternatively, the clarified culture medium can be passed through an ultrafiltration membrane having a 10-20 10 nm pore size capable of retaining virus particles, but passing solute (non-particulate) culture medium components.

C. Virus Particle Characteristics Purified HCV virus particles from above were examined 15 for morphological features, as detailed in Example 2.
Figures lA and lB are electron photomicrographs of HCV
particles from cells derived from PTTx174 chimpanzee, at two different magnifications, where the bars in the figures indicate 50 nm. Figures 2A-2E are electron photomicro-20 graphs showing further structural features and variationsin HCV particles (composite of HCV's from liver derived from different HCV-infected chimpanzees), where the bars in ; Figures 2A-2D represent 23 nm. A core-like structure is evident in Figure 2C. Figure 2E (bar = 17 nm) is taken at 25 higher magnification and shows external surface stalks on the virus particle;
The electron micrographs of HCV particles (from hepatocytes derived from the liver of PTTx266 chimpanzee) in Figures 3A-3C show bar-like structures within the virus 30 core (3A and 3C) and prominent envelope structures with extern~l stslk projections (3~

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Summarizing the structural features, the HCV particles have:
(a) an approximate average outside diamPter of 39-46 nm, but with a wide range in particle size (30-60nm);
(b) an internal core structure approximately 35-4Onm in diameter;
(c) a dense intra-core bar-like structure within some of the particles;
(d) external "stalks" and "knobs" protruding from the envelope, which measure approximately 2 to 5 nm in length, and 2 to 5 nm in width, respectively;
(e) particle envelopes; and (f) icosahedron symmetry.
The virus particles isolated and visualized electron 15 microscopically from the tissue cultured hepatocyte medium displayed a morphology similar to the genus flaviviruses of . the Togaviridae family. Togavirus virions consist of a lipid-containing envelope with surface projections sur-rounding a spherical nucleocapsid with proven or presumed 20 icosahedral symmetry. Virions are 40 to 70 nm in diameter.
The genome consists of one molecule of positive-sense infectious ssRNA of MW 4x106. The viruses exhibit pH-dependent hemagglutinating activity. Replication takes place in the cytoplasm, and assembly involves proven or :25 presumed budding through host cell membranes. [See, for example, Murphy or Schlesinger).
Similarly, the chloroform sensitive nature of the HCV
virus, indicative of a lipid-containing envelope (Fein-stone), as well as the apparent size distribution of the 30 HCV agent (30-60nm) determined by selective filtration techniques (He), are features compatible with our ultra--W091~15~74 PCT/US91tO2298 2~7~$7~

structural observations of an enveloped virus, whose size range is observed to be 39-60nm.

D. Infective HCV Particles The HCV particles isolated as disclosed above are also infectious. This is evidenced by the ability of cell culture medium from the HCV-infected primary or immortal-ized hepatocytes to produce HCV infection in chimpanzees.
Details of one study in which culture medium from HCV-lO infected primary hepatocytes is used to infect chimpanzees are given in Example 4. Weekly blood samples and periodic liver biopsies showed active hepatitis infection at 16-20 weeks after initial infection.
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15 D. Virus Particle Genome The virus particles described in Example 2 were isolated from primary hepatocytes derived from the liver of a chimpanzee infected with the Hutchinson strain of HCV.
In order to detect and sequence the RNA genome of the 20 infective HCV, total RNA isolated from the biopsied liver sample was amplified by polymerase chain reaction (PCR) methods, using HCV-specific primers, and the amplified fragment was cloned and sequenced, according to methods detailed in the Example 4.
~ 25 The amplified, cloned HCV sequence (termed BTR 623) r,' includes 623 nucleotides of HCV specific sequence. This ; isolate was compared to sequences of previously published JI and PT HCV clones (Kubo), as shown in Figure 4. The !, sequences given in this figure indicate that the HCV strain ~; 30 used in these studies has significant seguence divergence with the published isolates. The greatest level of divergence was seen with the Jl sequence. BTR 623 had ' .
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WO9l/15~4 PCT/US91/02~
2~79~7 96.2~ nucleotide and 97.4% amino acid homology with PT, and 79.8~ nucleotide and 93.8% amino acid homology with Jl.

II. HCV Antiaens 5 A. HCV proteins Heretofore, HCV virus proteins have been obtained only in recombinant form, using expression vectors with known HCV coding sequences to express HCV proteins or peptides in a suitable expression system. Such recombinant proteins 10 are likely to differ from mature, intact virus proteins in glycosylation, acetylation, and phosphorylation modifica-tions, as well as terminal residue modifications or cleavages. These modifications, particularly glycosylation features, are likely to be important in virus interactions 15 with host cells (Schlesinger), and in the host's immune response to the virus.
The present invention allows glycosylation and other post-translation modifications in intact HCV virus proteins to be identified and isolated. The glycosylation sites can 20 be identified by standard Western blotting procedures (Harlow), in which isolated HCV virus is fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the virus protein bands are probed with HCV
antisera, to identify im~unoreactive viral proteins.
25 Similarly, the viral proteins can be cleaved by selected proteases, prior to Western blotting, to identify immunore-active peptide fragments. The immunoreactive proteins or protein fragments can be identified by amino acid sequenc-ing of the Western blot bands, either directly, or after 30 additional band purification, if necessary.
~ rom the identification of the proteins, and from known consensus sequence~ for glycosylation sites, e.g., .. . . , . . . . . : . . ~ ................ - , .

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- WO91/15574 PCTtUS91/0229X
2 ~ 7 ~ ~ 7 rl Asp-X-Ser/Thr, proteins or fragments thereof which contain glycosylated residues can be identified. Alternatively, such proteins or fragments can be identified by comparing 2-dimensional gel electrophoresis patterns of virus 5 proteins or peptide fragments before and after treatment with selected glycosida6es. Two RCV proteins, gp35, and gp70, have been identified by others from coupled in vitro translation end studies, as containing glycosylation sites based on their sensitivity to endoHglycosidase.
After identifying glycosylated proteins or peptides of interest, the isolated viral particles are used as a source of the selected glycoprotein or peptide. Protein or pep-tide isolation from the viral particles can be carried out by standard methods, such as ion exchange and size-exclu-15 sion chromatography, and HPLC purification. T h e present invention contemplates in particular, mature gp35 (El) and gp70 (E2) HCV proteins with native glycosylation, and mature glycosylated peptides from these two proteins.
The proteins and peptides are useful in a diagnostic system 20 and in a vaccine composition, as described in Section IV
below.

III. Anti-HCV Antibodies i In another aspect, the invention includes polyclonal 25 or monoclonal antibodies specific against mature HCV
particles and protein components thereo~. The antibodies are defined by specific immunoreactivity with features of HCV particles, or proteins or peptide fragments thereof, due to normal post-translational modifica~ion. That is, 30 the antibodies are immunoreactive only with recombinant RCV
proteins which contain normal virus post-translational modifications.

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WO9l/15574 PCT/US91/02~A8 2~7~77 Polyclonal antibodies can be prepared, in accordance with one embodiment, by affinity chromotography, using the glycopeptide antigens identified from above immobilized on a solid support, for extracting immunoreactive antibodies 5 in naturally-infected human or chimpanzee HCV anti-sera or antisera generated specifically against the glycopeptide antigen.
Alternatively, the glycosylated proteins or peptides from above can be used to produce monoclonal antibodies, 10 employing standard methods (Harlow~. Briefly, the protein or peptide antigen is used to elicit an immune response in an animal, such as a mouse or rabbit, B lymphocytes from the spleen of the immunized animal are immortalized with a suitable hybridoma partner, and selection of desired 15 hybridomas is made on the basis of immunoreactivity with ~ the glyc~protein or peptide of interest. The antibodies - made by the selected hybridoma are useful in a diagnostic method, for screening human sera for HCV infection, and in a vaccine composition, for producing active immunity, as 20 discussed in Section 4.
. ~
IV. Utilitv A. Detection of HCV Antisera The virus particles, and proteins and glycosylated 25 peptides derived therefrom are useful as diagnostic reagents for detecting anti-HCV antibodies present in HCV-infected sera. As noted above, the mature particles, proteins and glycosylated peptides o~fer the advantage over recombinantly prepared HCV peptides and proteins in that in 30 addition to peptide antigens, the agents provide potential-ly unique antigenic sites associated with mature viral proteins, such as glycosylated peptides.

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WOgl/15574 PCT/US9t/02298 2~7~'77 In one preferred diagnostic configuration, test serum is reacted with a solid phase reagent having surface-bound viral proteins or peptides. After binding anti-HCV
antibody to the reagent and removing unbound serum compo-5 nents by washing, the reagent i5 reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-HCV antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter 10 associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate.
The solid surface reagent in the above assay prepared 15 by known technigues for attaching protein material to solid ` support material, such as polymeric beads, dip sticks, or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically 20 through a free amine group, to a chemically reactive qroup on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
In a second diagnostic configuration, known as a hompgeneous assay, antibody binding to a solid support 25 produces some change in the reaction medium which can be ` directly detected in the medium. Known general types of homogeneous assays proposed heretofore include (a) spin-; labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of 30 the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects .

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WO91/15~74 PCT/US91/022~
207~7~

enzyme/substrate interactions, and ~d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter. The adaptation of these methods to the protein antigen of the present invention 5 follows conventional methods for preparing homogeneous assay reagents.
In each of the assays described above, the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the lo presence of bound antibody. The examining may involve attaching a labeled anti-human antibody to the antibody being examined, either IgM (acute phase) or IgG (convales-cent or chronic phase), and measuring the amount of reporter bound to the solid support, as in the first 15 method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
Also forming part of the invention is an assay system or kit for carrying out the assay method just described.
20 The kit generally includes a support with surface-bound mature virus particle, protein or peptide, and a reporter-labeled anti-human antibody for detecting surface-bound - anti-409-1-1 antibody.

25 B. HCV Vaccine The virus particles, or mature processed proteins or antigenic peptides therefrom can be formulated for use in a HCV vaccine. The vaccine can be formulated by standard ~ethods, for example, in a suitable diluent such as water, 30 saline, buffered salines, complete or incomplete adjuvants, and the like. The immunogen is administered using standard t-chniqu-s for antibody induction, suoh as by subcutaneous , . , . . - . . . ~

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_~WO91/15574 PCT/US91/02298 2$7~77 administration of physiologically compatible, sterile ; solutions containing inactivated or attenuated virus particles or antigens. An immune response producing amount of virus particles is typically administered per vaccini-5 zing injection, typically in a volume o* one milliliter or less.
A specific example of a vaccine composition includes, - in a pharmacologically acceptable adjuvant, intact virus particles. This vaccine contains a combination of core and 10 envelope antigens. Another specific example includes, in - a pharmaceutically acceptable adjuvant, a purified mature virus protein, such as the gp35 or gp70 protein, or a combination of C ~core) protein with envelope protein, such as the gp35 or gp70 proteins.
Although the invention has been described with respect to particular methods, cell line, HCV strains, and applica-tions, it will be apparent that various changes and modification can be made without departing from the invention.
Example 1 ; Primary HCV-Infected Chimpanzee HeDatocytes A. Liver Sa~ples A parenteral ~CV virus infection was induced in 25 chi~panzee PTTx7, a 14-year old female, by inoculation with 5 ml of a 20-fold concentrate of acutiei phase plasma of unknown titer derived from a second chimpanzee passage of the Hutchinson strain of HCV (obtained from Dr. K Burk, Biotech Resources, Inc., San Antonio, TX~) was monitored by 30 ALT/AST (alanine aminotransferase/aspartate aminotransfer-ase) enzyme fluctuations from weekly ~lood samples and by histopathologic examination of periodic liver needle punch ,., - . . : ,: . .. . , . . - . : ,: . . : , - :, , :, .. . :. . . . . . . .. . ,... . , .: .. ... :. : . . - . .. ,. , :

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biopsies, according to published methods (Valenza). The "PTT" animal designations used herein identify individual chimpanzees housed at the Southwest Foundation for 3iomedi-cal Research, San Antonio, TX.
All biopsies were processed identically using conven-tional techniques. Immediately after harvesting, the liver biopsies were fixed for 1-3 hours in neutral buffered 3.7%
formalin, processed manually according to standard proce-dures, embedded in paraffin, sectioned at 4 microns and 10 stained with hematoxylin and eosin. All sections were examined histologically by the same board certified veterinary pathologist.
Since the onset of clinical hepatitis was significant-ly delayed, a second inoculation of 1.5 ml (1025 CID50) of the original HCV virus Hutchinson inoculum was administered at week 10 to assure infection. The appearance of elevated ALT on week 12 indicated that the second inoculum either potentiated the primary infection or was not required. The ALT profile of the animal exhibited 20 a rise above normal values from 12-19 weeks post inocula-tion, and a second ALT elevation occurred on week 39.
A liver punch biopsy taken after ALT elevations (week 19) revealed an increased number of lymphocytes in portal areas and in the parenchyma of the liver. Associated with 25 the parenchymal lesions were necrotic hepatocytes. The hepatocytes around central vein areas were often lightly stained and granular with minimal swelling of the cyto-plas~. All these changes described indicated minimal, lymphocytic, multifocal, viral hepatitis.
Liver wedge surgery was performed on week 14 at the onset of definitive ALT elevation. Ketamine hydrochloride was used as the immobilizing and preanesthetic agent.

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~WO91/15574 PCT/US91/02298 2~7~77 Surgery was performed under general anesthesia with non-hepatotoxic sodium pentobarbital. A liver wedge of approximately 10 g was perfused using a modification of established protocols (Maslansky). Microscopic examina-5 tion of liver tissue taken at this time revealed occasional collections of lymphocytes and macrophages in hepatic triads and in focal parenchymal areas. There were no other changes indicating a significant inflammatory response.
Although minimal inflammation was present, this finding 10 could be representative of normal liver tissue.
A two-step perfusion procedure was employed with all solutions maintained at 37C throughout the perfusion procedure. The initial perfusion lasted 10 minutes using l liter of Ca++, Mg++ -free Hanks Balanced salt solution 15 supplemented with lO mM HEPES (pH 7.4), 0.5 mM EDTA, and lO0 ~g/ml gentamicin sulfate. The next perfusion was for r 20 minutes at approximately 60 ml/min. of Williams Medium ` E (WME) supplemented with lO mM HEPES (pH 7.4), 100 ~g/ml gentamicin sulfate, and 200 units/ml collagenase Type I
; 20 (300 units/mg, Sigma, St. Louis. M0).
The liver capsule from above was removed with fine forceps and hepatocytes were dislodged by gentle agitation - in 100 ml of the above collagenase solution. The hepato-cyte suspension was filtered through several layers of 25 gauze pads into an equal volume of cold Williams Medium E
(WME) containing 5% fetal bovine serum (FBS), 10 mM HEPES
(pH 7.4), and 100 ~g/ml gentamicin sulfate. Hepatocytes - were sedimented at 50 x g for 5 minutes and cell pellets were resuspended in WME 5% FBS. Sedimentation was repeated 30 twice, pellets were resuspende~ in lO ~l WM~ 5~ FBS, and viability and cell density were determined by trypan blue exclusion.

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WO9l/15574 PCT/US91/0~8 2979~7 B. Cell Culture Conditions PRIMARIA plates ~Falcon, Becton-Dickinson, Lincoln Park, NJ) were coated with rat tail collagen (Michalopou-los) for 6 minutes at room temperature, the excess collagen 5 was removed, and plates were dried overnight under U.V.
light. Viable cells were plated at a density of 3-4 x 106 cells/60mm dish. Cell attachment occurred during a 3-hour incubation at 37C, 10~ CO2 in WME, 5% FBS, at which time cell monolayers were gently washed one time with WME and 10 re-fed with the serum-free medium formulation described below. The medium was changed 24 hours after isolation and at 48 hour intervals thereafter.
The cultured hepatocytes displayed a typical hepato-cyte morphology as observed by phase-contrast microscopy on 15 day 5 of culture. This morphology was maintained until days 21-28 when the cultures exhibited a degenerative process.
In this and the other examples below, the serum-free media (SFM) formulation utilized a basal medium supplement-20 ed with 10 mM HEPES, pH i.4, 2.75 mg/ml NaHC03, and 50 ~g/mlgentamicin, together with the supplements as listed below.
In the described media of Table 2, Williams Medium E (WME) served as a basal medium. Although WME is presently preferred as the basal medium of the serum-free medium 25 other commercial media formulations can be expected to give satisfactory results. For instance, a mixture of Dulbec-co's modified Eagle's medium and Ham's Fl2 medium (Salas- -; Prato) or RP~I 1640 (Gibco) (Enat, Sell should give satisfactory results when supplemented with the supplements 30 llsted in Table 2.

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Table 2 . SupplementMedium Concentration EGF lO0 ng/ml 5 Insulin 10 ~g/ml ~lucagon 4 ~g/ml BSA 0.5 mg/ml Linoleic Acid5 ~g/ml Hydrocortisone10~ M
10 Selenium lO~ M
: Cholera Toxin2 ng/ml LGF 20 ng/ml Transferrin5 ~g/ml : Ethanolamine10~ M
: 15 ProlactinlO0 ng/ml Somatotropin1 ~g/ml TRF lO~ M

' To prepare the media, the supplements were added in : 20 the following quantities in Table 2 to 500 ml of WME in a ; sterile plastic bottle:

5 ml 50 mg/ml BSA (bovine serum albumin), 500 ~g/ml Linoleic Acid 25 0.5 ml 5 mg/ml Insulin 0.5 ml 5 mg/ml Insulin, 5 mg/ml Transferrin, and 5 ~g/ml : .
Selenium (ITS) 50 ~l 102 N Hydrocortisone 5 ~l 200 ~g/ml Cholera toxin -, 30 0.5 ml 100 ~g/ml EGF (epidermal growth factor) : 50 ~l 10-2 ~ Ethanolamine 0.5 ml 1 mg/ml Somatotropin `

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2~7~77 50 ~l l mg/ml Prolactin 0.5 ml 10'3 M Thyrotropin Releasing Hormone 50 ~l 200 ~g/ml LGF (liver growth factor, i.e., glycyl-histidyl-lysine) 5 l ml 2.0 mg/ml Glucagon WNE was purchased with L-glutamine and without NaHC03 from Hazelton Research Products, Inc. (Denver, Pennsylvania). Supplements, including growth factors and hormones were obtained from Sigma (St. Louis, M0) or 10 Collaborative Research (Bedford, MA).

C. Secretory Protein Production The synthesis and secretion of albumin, apolipoprotein A-I and apolipoprotein E were monitored by immunoblotting 15 of sequential aliquots of tissue culture medium, according to standard methods (Haslow). Briefly, proteins were separated by sodium dodecyl sulfate-polyacrylamide qel electrophoresis (SDS-PAGE), and were electrophoretically transferred to Nylon-X nitrocellulose filters (Fisher) at 20 lO0 mA current for 16 hours at 4C. Unoccupied binding sites were blocked in 10% nonfat dry milk in phosphate ;~ buffered saline (PBS) for 2 hours at 37 C in PBS-milk-Tween ( PBS containing 5% nonfat dry milk, 0.3% Tween-20), using r~ primary antibodies directed against each of the specific 25 proteins. Membranes were washed three times with PBS-Tween and incubated l hour at 37C in PBS-milk-Tween with tl~I]-protein A (8.5 ~Ci/~g, New En~land Nuclear, Boston, MA).
` Membranes were washed three times with PBS-Tween and air dried. Immunoblots were autoradiographed at -85 C on XAR-5 30 film (Kodak, Rochester, N.Y.) with intensifying screens.
The levels of apolipoproteins A-I and E increased in the cultures up to day 13, remained constant from day 13-28, and declined from day 28-45. Albumin detected by this .
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W091/15~74 PCT/US91/02298 2~7~7 immunoblot procedure remained at constant levels throughout the culture period. Although albumin is a marker for differentiated hepatocytes, it is not as stringent of a marker for the differentiated state as is lipoprotein 5 synthesis.
- The decline in lipoprotein synthesis after 28 days in culture paralleled a degeneration in the hepatocyte cultures. The degeneration of primary hepatocytes after 3-4 weeks of culture was evid~nt in cultures derived form two lO different HCV-infected chimpanzees. Normal hepatocyte cultures generally survive more than lOO days in the serum free media. It thus appears that the degenerative process of HCV-infected primary hepatocytes may be due to viral induced cytopathic effect.
To further characterize the differentiated state of the hepatocytes in vitro, the de novo synthesis of liver specific plasma proteins was analyzed. On day 17, cultures were labeled for 24 hours with [35S] methionine (>800 ~Ci/m~ol, ICN) for 24 hours. Medium was filtered and mixed 20 with l/lO volume of lOx CHAPS extraction buffer [final concentration 1.0% CHAPS (CalBiochem), 0.25mM phenylmethyl ~ulfonyl fluoride, lOmM EDTA, 0.05 M Tris (pH 8.0), O.l M
NaCl, lOO ~M leupeptin] and incubated for l hour at 4 C
with agitation. Commercially obtained antibodies (CalBio-25 chem, San Diego, CA and Boehringer Mannheim, Indianapolis, ~ IN) directed against human plasma proteins (20~1) were bound i to protein A-agarose beads (50 ~ epligen) for l hour in CHAPS extraction buffer on ice. The beads were washed two times with detergent wash buffer [CHAPS extraction buffer ` 30 plus 1% deoxycholic acid and 0.1% SDS~ and were incubated with the labeled medium overnight at 4C with agitation.

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WO9l/lSS74 PCT~US9l/02~
2~7~77 The beads were pelleted and washed three times with detergent wash buffer~
Bound proteins from the culture medium were eluted with 50 ~l electrophoresis sample buffer containing 2% SDS
5 and 2% 2-mercaptoethanol, heated at 100C ~or lO min. and analyzed by SDS-PAGE. Gels were processed for fluorography with Autofluor (National Diagnostics, Somerville, NJ), dried, and autoradiographed at -85 C on XAR-5 film.
Analysis of the gel patterns indicates that the amount 10 of plasma proteins synthesized in vitro reflects the concentrations found in plasma. The intensities of the polypeptide bands in descending order were albumin, o-l antitrypsin, plasminogen, fibrinogen, transferrin, apo A-I
`~ and E, beta-2 macro~lobulin, pre-albumin, apo A-II and A-15 III, complement components C3, C4 and C5, C-reactive protein, and apo C-2 and C-3. All markers examined were detected with the exception of o-fetoprotein, which is a marker for poorly differentiated hepatocytes.
:: -Exam~e 2 HCV_Particles A. Virus Particle Isolation Infected primary hepatocyte cells from above were grown on coverslips and analyzed at various times during 25 the culture period for the presence of a novel HCV virus-associated antigen that can be detected by immunocytochemi-cal staining (Burk). Typical cytoplasmic staining was observed in all samples examined, with a tendency for the percentage of cells expressing this marker to increase with 30 time in culture. ~owever, the number of cells with definitive staining never increased above 10%.
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A sample of culture medium was clarified by centrifu-gation at 12,000 x g, 30 min. at 4C. The clarified medium (23 ml) was layered over a discontinuous sucrose gradient formed of 5 mls of 68~ sucrose in phosphate buffered saline ; 10 (PBS), and 10 ml of 20% sucrose. The layered material was centrifuged at 27,000 rpm (131,000 x g) in a Beckman SW28 rotor for 3.8 hours. A 2 ml sample at the 68%/20% sucrose interface was drawn off and diluted to a final sucrose concentration of 20% with PBS. Several gradients (4-6) 15 were prepared from the media collected from each time point.
The diluted material (12.5 ml) was layered over 1 ml of the 68% sucrose solution and centrifuged at 30,000 rpm .(154,000 x g) for 16 hours in a Beckman SW41 rotor. The ;20 interface material (at the top of the 68% sucrose layer) was collected by bottom puncture, collecting 1-1.5 ml of material. The isolated material, containing purified HCV
virus, was frozen at -85C.
' 25 ~. Morphology `-Purified HCV virus specimens from above were examined by a modification of the pseudoreplication technique (Portnoy). Briefly, 10 ~l of virus-containing fluid was pipetted onto agar disks (2% in 0.15~ NaCl, 0.01~ merthio-30 late). The agar disk surfaces were covered with a Parlo-dion film, 0.75% in amyl acetate (Mallinckrodt, Paris, KY).
The fil~ containing the HCV viral agent was floated onto a , .

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2~7~7 liquid surface in a 1~ phosphotungstie acid, pH 7.0, and retrieved by immersio~ onto copper grids t3mm). After drying, the specimens were examined by transmission electron microscopy without further treatment.
sVirus particles were observed in the samples obtained from the purified tissue culture medium. Figures 1-3 are electron micrographs of the observed material. The morphological features of the particles are described above.
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Example 3 HCV Particles from Cultured Cells are Infectious ; The production of infectious virus in the hepatocyte cultures was assayed by inoculation of a chimpanzee with 15 tissue culture medium and monitoring the animal for signs of disease. Tissue culture medium as described above was c~llected from PTTx7 hepatocyte cultures at two-day inter~als, and passed through 0.45 ~ filters and stored at -`` 100 C. A pool of media from days 3 through 31 were 20 collected (190 ml total) and concentrated by pressure dialysis under N2 gas at 4C with an exclusion membrane of 30,000 MM (YM30, Amicon, Beverly, MA)). An 8-fold concen-trate (22 ml) was stored at -100C until use. The concen-trated material (10 ml) was used to inoculate an HBV-immune 25 chimpanzee (PTTx196).
Weekly blood samples and periodic liver needle biopsies were obtained from PTTx196 for analysis. A slight increase in ALT occurred durin~ week 4 and microscopic exa~ination of a li~er needle biopsy at that time revealed 30 minimal changes similar to those observed in normal tissue, but of interest under these conditions. Liver needle biopsles t-ken during weeks 8 and 12 exhibited essentially i;

, ~WOgl/15574 PCT/US91/02298 2~7~77 normal tissue with no microscopic lesions recognized. Due to the delay in onset of clinical hepatitis, a second injection of the same inoculum (7ml) was administered at week 12. This was followed by an elevation in ALT values 5 three weeks later. Peak ALT was exhibited 16-20 weeks after the first inoculation. Histologic examination of a liver needle biopsy taken at week 14 showed early signs of hepatitis, including foci of inflammatory cells in the hepatic parenchyma, and hydropic generation of hepatocytes 10 with occasional necrotic hepatocytes. Electron microscopic examination of the biopsy revealed the presence of cyto-plasmic tubules which are typical of HCV-infected tissue.
Plasma samples taken from PTTx196 during weeks 0, 18 and 23 of this experi~ental HCY infection were analyzed for - 15 an increase in antibody titer to cytomegalovirus, Epstein-Barr virus, herpes simplex virus, HBV surface and core an-tigens (B sAG, HBcAG), and spumavirus, since these agents may cause hepatitis or could be transmitted by this methodology. No change in the antibody response to these 20 agents was detected in the plasma samples from PTTx196.
` These results demonstrate that the disease transmitted to PTTx196 was caused by an HCV agent.
~6 Example 4 Genori_~çgyence of the ~CV Particles Chimpanzee PTTx198, an 8 year old male chimpanzee, had been inoculated wit~ the Hutchinson strain of HCV virus - used in Example 2. During the acute-phase of the infec-, tion, a liver wedge was used to isolate hepatocytes, which -s 30 were cultured as HCV-infected primary hepatocytes as described in Example 1. The culture medium was used to purify virus as detailed in Example 2. HCY particles, of , WO 91/15574 P(~/US91tO22qs~
2 ~ 7 ~ 7 the type seen in Figures 1-3 and reported in Example 2, were observed.
A second portion of the biopsied liver from the HCV-infected animal was used to isolate total RNA by the S conventional guanidinium isothiocyanate extraction and ultracentrifugation through a cesium chloride gradient (Sambrook). The RNA was used for cDNA synthesis with a specific hepatitis C virus (HVC) oligodeoxyribonucleotide as a primer for reverse transcription. The primer for cDNA
10 synthesis was derived from a previously reported primer (Kubo), and has the sequence:
(5'-GGAAGCTTGACATGCATGTCATGATGTA-3') The primer includes 20 nucleotides of HCV specific sequence and 8 nucleotides at its 5' end containing a 15 HindIII restriction site for subsequent cloning purposes.
The reverse transcription was performed as described (Sambrook) in the presence of S ~g of RNA, 0.5 ug of 3' primer, 2 units of reverse transcriptase (E. Anglian Biotech, Cambridge, MA) in a 10 ~l reaction volume contain-; 20 ing 50 mM Tris-HCL pH 8.2, 6mM MgCl2, 10 mM dithiothreitol (DTT) and 500 ~M of each of the four deoxyribonucleotide triphosphates (dNTP).
After incubation ~or 40 minutes at 42C, 1 ~l of the reaction mixture was added to a PCR reaction mixture 25 provided in a commercial PCR kit ~Perkin-Elmer/Cetus), as described by the manufacturer. The above 3'-end primer and a 5'-end primer having the se~uences (5'-GGGAATTCGGCTATACCGGCGACTTCG-3') which includes 20 nucleotides of HVC sequence (Kubo) and an 30 additional 8 nucleotides at its 5' end and an EcoR1 site, were added to the reaction mixture. The PCR reaction was allowed to proceed for 30 cycles.

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, - WO9l/15574 PCT/US91/02298 2S7~77 The 623 basepair cDNA fragment amplified by PCR was visualized on an agarose gel by ethidium staining. This fragment was gel purified and amplified again by 30 cycles of PCR. The resulting DNA was gel purified, digested with 5 EcoR1 and HindIII and cloned into the EcoRI/HindIII site of plasmid pGEMX1 (Promega, Madison, WI). The nucleotide sequence was determined by dideoxy chain termination method on double stranded DNA using the SP6 and T3 promoter ;~ primers (Promega).
Example 5 HCV Particles from In Vitro Infected Primarv Cells In Example 2, HCV particles were obtained from primary hepatocytes which were infected in vivo, i.e., prior to 15 culturing as primary hepatocytes. In the present example, uninfected chimpanzee primary hepatocytes in culture were infected with HCV in culture, and the virus was allowed to replicate in the infected cells.
Liver wedge biopsies were obtained from healthy, 20 uninfected chimps identified as PTTx256, a 5 year old male chimpanzee, and PTTx344, a 1 year old female chimpanzee.
The liver biopsies were used to produce primary cultured hepatocytes, according to the methods detailed in Example 1. The cells were infected with a pool of acute phase 25 plasma from HCV-infected chimpanzees. The virus stock was a pool of acute-phase plasma from HCY-infected chimpanzees.
The stock was diluted five-fold in SF~ and added to the cultures. The cultures were incubated for 3 hr at 37C with the inoculum, and then 1.5 ml of SFM was added to the 30 cultures and the incubation was continued for 16 hr. The cultures were washed three times with WME to remove residual inoculu~ and changed to SFM.

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2 0 7 9 ~ 7 7 A sample of culture medium was taken at days 1, 2, 3, 6, 9, 12, 15, 18, and 21 for the PTTx266 animal, and at days 1, 3, 5, 7, 9, and 12 for the PTTx344 animal. Virus particles were isolated from culture medium by sucrose 5 gradient centrifugation, as detailed in Example 2. The samples were examined by electron microscopy to determine - viral counts. The results are shown in Table 1 above, - expressed as virus particles per ml of culture medium.
The table also shows virus counts observed for culture 10 medium obtained at various times after initial culturing, for cell cultures derived from liver cells of HCV-infected chimpanzees, PTTx196, PTTx174, PTTx268, and PTTx198.
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Example 6 Im~orta~ized Uninfected Hepatocytes Uninfected primary chimpanzee hepatocytes derived from PTTx266 were cultured in SFM, substantially as described in Example 1 for HCV-infected cells. The cells were immortal-ized with a retrovirus derived from the U19-5 cell line 20 which constitutively produces the U19 amphoteric retrovi-rus. The U19-5 cell line was a gift from Drs. P.S. Jat and P.A. Sharp, M.I.T. (Cambridge, MA). The retrovirus recombinant plasmid construct has been described in detail (Jat). The plasmid construct produces a large T antigen 25 protein defective for binding to the SV40 origin of replication.
- The U19-5 cell line was grown in DMEM medium with 10%
` fetal bovine serum (FBS) under standard culture conditions ; (Jat). Culture medium was collected at 24-hour intervals 30 and passed through a 0.45 ~m filter (Amicon, Beverly, CA)) ;i prior to use for infection of primary hepatocyte cultures.

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WO91~15574 PCT/US91/02298 2~ 7~ ~ 77 Subconfluent cultures of primary hepatocytes (Example 1) were infected one day post-plating by the addition of 1 ml of Ul9-5 culture medium to the cells in the presence of PolybreneTM (8 ~g/ml). The plating density was such as to 5 allow the cells several rounds of cell division to occur after introduction of the oncogene. After incubation overnight, cells were washed three ti~es with WME and maintained in SFM until colony outgrowths were observed, typically about 1 month after infection.
The cells were selected for G418 (Geneticin, GIBC0, Gaithersburg, MA) resistance by addition to the culture medium of G418 (400 ~g/ml). The cells were then treated by a collagenase/dispase (Boehringer Mannheim) solution at a concentration of lO0 ~g/ml in phosphate-buffered saline 15 (PBS, pH7.2) for 10 minutes at 37C. Following dissocia-tion, a five-fold excess of 5% fetal bovine serum in Williams medium E (5~ FBS/WME) was added to the solution.
Cells were pelleted at 50 x g for six minutes, resuspended ` in a minimal volume of 5% FBS/WME and allowed to attach 20 during a 2-3 hour period at 37C under 10~ CO2. Ihe o~s were plated at a low cell density so that single colony outgrowths could be isolated and subcloned. From over 100 colonies, over 70 were picked based upon differences in morphological appearance. The cell lines are designated 25 CHNP cells, and are assigned cell line numbers, such as -` CHMP 1.21, CHMP 1.22, etc.

Example 7 HCV Infectivity_of Immortalized Hepatocytes Immortalized chimpanzee hepatocytes derived from HCV-infected primary hepatocytes were prepared substantially as described in Example 6, but using hepatocytes obtained from s ` .

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2 $ 7 ~ ~ 7 7 a liver biopsy of a chimpanzee (PTTx198) with acute-phase HCV. The cell lines are designated CU cell lines.
Several CHMF~ (Example 6) and CU cell lines were cultivated on collagen coated 25 fCm2 Primaria fla,~ks in SFM
5 under normal conditions (37C, 10% Co2 atmosphere). When the cultures reached a level of 90S confluency, they were inoculated with chimpanzee plasma known to contain HCV.
The inoculum was a pool of plasmas obtained from three ; chimpanzees (P~Tx7, PTTx268, and PTTx174) during the acute 10 phase of a HCV infection and did not contain any other infectious agent. The plasmas were diluted 5-fold in SFM
and 1 ml was added to the cultures. After incubation for ~; 3 hr at 37C, another 3 ml of SFM was added to the cultures and the incubation was continued for 16 hr. : The cultures 15 were washed three times with WME to remove the inoculum and SFM was added. The medium was changed every other day and on the 11th day after infection the cultures were harvested '' for analysis.

20 B. RNA CharactP,rization The cells were washed three times with phosphate ,i buffered saline (PBS) and the cellular RNA was extracted '~ and purified using a standard GITC extraction procedure ';' (Chomozynski). The cells were lysed with a solution ~, 25 containing 4M guanidine isothiocyanate, 0.18~ 2- mercaptoe-~,' thanol, and 0.5% sarcosyl. The cell lysate was extracted several times with acidic phenol-chloroform- isoamyl alcohol, and the RNA was precipitated wi~h isopropanol.
The purified RNA was resuspended in water and one tenth of 30 each sample was used for polymerase chain reaction (PCR) amplification to detect the HCV RNA genome. , f ' , ~
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PCR was conducted using standard methodology, as detailed above (Innis). The first step involved a cDNA
reaction in which a DNA copy of the ~CV ~NA was made using reverse transcriptase and an oligonucleotide primer 5 designated 6A that is complementary to the strain of HCV
used in our studies. The four primers used for cDNA and PCR were derived from the putative nonstructural region of HCV designated NS3 and their sequences are given below.
Primers:
10 5A 5' TCTGTGATAGACTGCAACACG 3' 6A 5' TTTGGTGATTGGGTGCGTCAG 3' 5B 5' GATGCTGTCTCCAGGACTCAA 3' 6B 5' AACAGCGCCCAGTCTGTATAGCAG 3' The sequence of these primers was derived from the 15 sequence of a cDNA clone of a strain of HCV as previously described (Jacob, 1991). A portion of the cDNA reaction mixture (1/4th) was PCR amplified for 35 cycles using the Taq polymerase and the oligonucleotide primers SA and 6A.
A portion of the first round of PCR (1/50th) was used for 20 a second round of PCR using the primers 5B and 6B.
Figure 5 shows a gel analysis of the PCR products from HCV-infected CU cell lines. Figure 6 shows a gel analysis of PCR products from HCV-infected CHMP cell lines. The results of the gel studies are described above.
Although the invention has been described with respect to particular cell lines and modes of HCV infection, it will be apparent various changes and modifications can be made without departing from the invention.

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Claims (14)

IT IS CLAIMED:

1. A purified hepatitis C (HCV) virus composition comprising virus particles characterized by:
(a) a single-strand RNA genome;
(b) a flavivirus type structure having virus particle sizes between about 30-60 nm, enveloped capside structures, external stalks 2-5 nm in length and width, and an icosa-hedron symmetry, and (c) immunospecific reaction with HCV-infected individ-uals.

2. The composition of claim 1, wherein the RNA genome contains a region with the sequence shown in Figure 4.

3. The composition of claim 1, in which the RNA
genome is inactivated.

4. The composition of claim 3, for use as a vaccine, which further includes a suitable vehicle for injecting the virus in a suspended form.

5. A protein composition containing a mature hepati-tis C virus (HCV) protein selected from the group of C, e1 (gp35), e2 (gp70), and the HCV counterparts to NS2, NS3, NS4, and NS5, substantially free of HCV genomic RNA, and non-viral serum and cell proteins normally associated with HCV virus infection in humans.

6. The composition of claim 5, wherein the selected protein is substantially purified from the other HCV
proteins in the group.

7. The composition of claim 6, for use in a system for detecting the presence of HCV-specific antibodies in human sera, wherein the protein is bound to a solid support, and the system further includes means for detect-ing the presence of human antibody bound to the support.

8. The composition of claim 7, wherein the protein is gp35 or gp70.

9. The composition of claim 5, for use as a vaccine, which further includes a suitable vehicle for injecting the protein in a suspended form.

10. The composition of claim 9, wherein the protein is gp35 or gp70.

11. A hepatitis C virus (HCV) antigen containing a glycopeptide epitope contained in hepatitis C virus gp35 or gp70 proteins derived from intact HCV particles.

12. The antigen of claim 11, for use in a system for detecting the presence of HCV-specific antibodies in human sera, wherein the antigen is bound to a solid support, and the system further includes means for detecting the presence of human antibody bound to the support.

13. An antibody immunospecific against a hepatitis C
virus (HCV) glycopeptide epitope contained in hepatitis C
virus gp35 or gp70 proteins derived from intact HCV
particles.

14. The antibody of claim 13, for use for use in detecting HCV infection in human sera.