# Patent

## Title
Tissue culture system for production of hepatitis C virus

## Abstract

A tissue culture system for production of infectious hepatitis C virus is described. In particular, the invention provides recombinant monocistronic and bicistronic genomic constructs for production of virus, including constructs for production of wild-type HCV type 2a strain JFH1 and constructs for production of chimeric viruses comprising HCV proteins from strain JFH1 and a second HCV isolate. Constructs of the invention also include a reporter gene to facilitate measurement of RNA replication and viral infectivity in cultures. The cell culture system may also include various factors that improve viral replication or infectivity. In addition, a neutralization assay using HCV grown in cell culture is described.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a §371 filing from PCT/US2007/018591, filed Aug. 22, 2007, and claims the benefit under 35 U.S.C. §119(e)(1) of U.S. Provisional Application No. 60/839,608, filed Aug. 22, 2006, which applications are incorporated herein by reference in their entireties and from which applications priority is claimed pursuant to the provisions of 35 U.S.C. §§119/120.


TECHNICAL FIELD
The invention relates to a tissue culture system for production of infectious hepatitis C virus.
BACKGROUND
Hepatitis C virus (HCV) was identified over a decade ago and is now known to be the leading cause of non-A and non-B viral hepatitis (Choo et al., Science (1989) 244:359-362; Armstrong et al., Hepatology (2000) 31:777). HCV infects approximately 3% of the world population, an estimated 200 million people (Cohen, J., Science (1999) 285:26). About 30,000 newly acquired HCV infections occur in the United States annually. Additionally, there is a large incidence of HCV infection in developing countries. Although the immune response is capable of clearing HCV infection, the majority of infections become chronic. Most acute infections remain asymptomatic and liver disease usually occurs only after years of chronic infection.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having about 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715).
In particular, several proteins are encoded by the HCV genome. The order and nomenclature of the cleavage products of the HCV polyprotein is as follows: NH2—C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Another protein (F) has also been identified and results from translational frame-shifting within the C gene. Branch et al., Semin. Liver Dis. (2005) 25:105-117. Initial cleavage of the polyprotein is catalyzed by host proteases which liberate three structural proteins, the N-terminal nucleocapsid protein (termed core) and two envelope glycoproteins, gpE1 (also known as E) and gpE2 (also known as E2/NS1), as well as nonstructural (NS) proteins that encode the viral enzymes and other activities. The NS regions are termed NS2, NS3, NS4 and NS5. NS2 is an integral membrane protein with proteolytic activity and, in combination with NS3, cleaves the NS2-NS3 junction. The NS3 protease, along with its NS4a cofactor, serves to process the remaining polyprotein. In these reactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b and NS5a), and an RNA-dependent RNA polymerase (NS5b). Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease.
Development of anti-viral drugs and vaccines against HCV infection has been hindered by the lack of a suitable animal model or cell culture system for HCV replication. HCV has been found to grow poorly in cell culture, and the use of subgenomic replicons, which replicate more efficiently in cultured cells, has failed to produce infectious viral particles (Bartenschlager et al. (2000) J. Gen. Virol. 81:1631-1648; Lohmann et al. (1999) Science 285:110-113; Ikeda et al. (2002) J. Virol. 76:2997-3006; Pietschmann et al. (2002) J. Virol. 76:4008-4021). Furthermore, existing replication systems have been found to be inadequate for testing some antiviral strategies. Replication systems based on self-replicating subgenomic replicons that only express nonstructural viral proteins do not emulate all steps of the HCV virus life cycle that may be targeted for antiviral intervention. Cell lines that are stably transfected with HCV precursors, but that are incapable of HCV RNA replication, cannot be used to screen for antiviral drugs that block viral RNA replication. Recently, Pietschmann et al. demonstrated that infectious virus could be produced from a bicistronic artificial HCV genomic construct in cell culture utilizing a chimeric construct comprising on one cistron, sequences from HCV type 2a strain JFH1 and sequences from a second HCV strain, and on a separate cistron, a reporter gene for monitoring transcription and infection (PNAS (2006) 103:7408-7413).
There remains a need, however, for improved tissue culture systems for production of HCV and for development of a tissue culture system that can cost-effectively and efficiently produce infectious HCV particles that can be used in testing antiviral therapeutics.
SUMMARY OF THE INVENTION
The present invention is based on the development of a reliable cell culture system for production of infectious HCV particles. In particular, the invention provides methods of growing wild-type and chimeric HCV viruses in cells that are capable of being infected with HCV, such as hepatocytes or other liver derived primary cells or tumors cell lines. The methods utilize recombinant monocistronic and bicistronic genomic constructs for production of virus, including constructs for production of the wild-type HCV type 2a strain JFH1 and constructs for production of chimeric viruses comprising NS3 to NS5b and a C-terminal portion of NS2 from strain JFH1, and core to p7 and an N-terminal portion of NS2 from a second HCV isolate (e.g., type 1a H77C, type 1b Con1, or type 3a NZ1 or 452). Constructs of the invention may also include a reporter gene to facilitate measurement of RNA replication and viral infectivity in cultures. The cell culture system may also include various factors that improve viral replication or infectivity, such as an RNA silencing suppressor (e.g., an influenza virus NS1 polypeptide) to inhibit host defense pathways or CD81 to facilitate viral entry into host cells. In addition, a neutralization assay using HCV grown in cell culture is described and neutralization titers measured by this assay have been found to correlate well with actual protection from HCV infection in vaccinated animals.
Thus, the subject invention is represented by, but not limited to, the following embodiments:
In one aspect, the invention includes a recombinant HCV bicistronic genomic construct comprising a first cistron comprising a coding sequence for an N-terminal fragment of the JFH1 core protein comprising at least the first 12 residues and up to the first 18 residues of the JFH1 core protein, including any number of residues in between, such as residues 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, or 1-18 of the JFH1 core protein, wherein the coding sequence for the N-terminal fragment of the JFH1 core protein is linked to and translated in frame with a reporter gene to generate a single polypeptide fusion product. The reporter gene is followed by an internal ribosomal entry site (IRES) for translation of a second cistron. The second cistron comprises a coding sequence for an HCV polyprotein comprising open reading frames for core, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, and NS5b regions, wherein the C-terminal portion of NS2, and the NS3, NS4a, NS4b, NS5a, and NS5b regions of the HCV polyprotein are derived from JFH1 and the core, E1, E2, and p7 regions, and the N-terminal portion of NS2 are derived from a second HCV isolate. The second isolate may be the same or a different subtype than JFH1. Preferably, the second isolate is a different subtype than JFH1. The non-JFH1 strain or isolate can be of HCV type 1a, 1b, 2a, 2b, 3a, 3b, 4, 5, or 6. In certain embodiments, the non-JFH1 strain or isolate is H77C, Con1, NZ1, or 452. The construct also contains a 5′ HCV untranslated region, or a functional portion thereof, and a 3′ HCV untranslated region, or a functional portion thereof, flanking the two cistrons. Exemplary HCV bicistronic genomic constructs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS:2-5.
In certain embodiments, the HCV NS2 gene is a chimeric gene, encoding an amino portion of NS2 from the same HCV strain or isolate as encoding the E1, E2, and P7 proteins and a carboxy portion of NS2 from JFH1. Suitable junction sites between the amino and carboxy portions of the NS2 gene are described in Pietschmann et al. (Proc. Natl. Acad. Sci. U.S.A. (2006) 103:7408-7413; herein incorporated by reference). In certain embodiments, the junction between the amino and carboxy portions of NS2 occurs at a residue selected from the group consisting of 871-877.
In certain embodiments, the recombinant HCV bicistronic genomic construct comprises a reporter gene encoding a firefly luciferase, Renilla luciferase, or, green fluorescent protein.
In one embodiment, the recombinant HCV bicistronic genomic construct comprises an encephalomyocarditis virus (EMCV) IRES.
In another aspect, the invention includes a recombinant HCV monocistronic genomic construct comprising coding sequences for core, E1, E2, p7, NS2, NS3, NS4a, NS4b, NS5a, and NS5b regions of an HCV polyprotein. In certain embodiments, the recombinant HCV monocistronic genomic construct encodes a chimeric HCV polyprotein, wherein the C-terminal portion of NS2, and the NS3, NS4a, NS4b, NS5a, and NS5b regions of the HCV polyprotein are derived from JFH1 and the core, E1, E2, and p7 regions, and the N-terminal portion of NS2 are derived from a second HCV isolate. The second isolate may be the same or a different subtype than JFH1. Preferably, the second isolate is a different subtype than JFH1. The non-JFH1 strain or isolate can be of HCV type 1a, 1b, 2a, 2b, 3a, 3b, 4, 5, or 6. In certain embodiments, the non-JFH1 strain or isolate is H77C, Con1, NZ1, or 452. The construct also contains a 5′ HCV untranslated region, or a functional portion thereof, and a 3′ HCV untranslated region, or a functional portion thereof, on opposite ends of the construct.
In some embodiments, the HCV NS2 gene is a chimeric gene, encoding an amino portion of NS2 from the same HCV strain or type as encoding the E1, E2, and P7 proteins and a carboxy portion of NS2 from the JFH1 type. Suitable junction sites between the amino and carboxy portions of the NS2 gene are described in Pietschmann et al. (supra). In certain embodiments, the junction between the amino and carboxy portions of NS2 occurs at a residue selected from the group consisting of 871-877.
In certain embodiments, the recombinant HCV monocistronic genomic construct comprises a coding sequence for an N-terminal fragment of the JFH1 core protein linked to a reporter gene, wherein the N-terminal fragment of the JFH1 core protein comprises at least the first 12 residues and up to the first 18 residues of the JFH1 core protein, including any number of residues in between, such as residues 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, or 1-18 of the JFH1 core protein, wherein the coding sequence for the N-terminal fragment of the JFH1 core protein is linked to and translated in frame with the reporter gene. In certain embodiments, the reporter gene encodes a firefly luciferase, Renilla luciferase, or, green fluorescent protein. The recombinant HCV monocistronic genomic construct may also comprise a coding sequence for a cleavage site that allows separation of the expressed reporter gene product from the HCV polyprotein. In one embodiment, the recombinant HCV monocistronic genomic construct comprises a coding sequence for an FMDV 2A protease following the reporter gene. Additionally, the recombinant HCV monocistronic construct may further comprise a coding sequence for a ubiquitin protease cleavage site.
In certain embodiments, the invention includes a recombinant HCV monocistronic genomic construct comprising in 5′ to 3′ order:

    
    
        a) an HCV 5′ untranslated region, or a functional portion thereof;
        b) a polynucleotide comprising a coding sequence for an N-terminal fragment of the JFH1 core protein, wherein said fragment comprises at least the first 12 residues and up to the first 18 residues of the JFH1 core protein, wherein the coding sequence for the N-terminal fragment of the JFH1 core protein is linked to a reporter gene;
        c) a coding sequence for a foot-and-mouth disease virus (FMDV) 2A protease;
        d) a coding sequence for a ubiquitin protease cleavage site;
        e) a polynucleotide encoding an HCV polyprotein comprising core, E1, E2, and p7 regions from an HCV strain or isolate other than JFH1, a chimeric NS2 region comprising an N-terminal portion of NS2 from an HCV strain or isolate other than JFH1 and a C-terminal portion of NS2 from JFH1, and NS3, NS4a, NS4b, NS5a, and NS5b regions from JFH1; and
        f) an HCV 3′ untranslated region, or a functional portion thereof.
    
    


Exemplary recombinant HCV monocistronic genomic constructs comprise a polynucleotide selected from the group consisting of

    
    
        a) a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10953 of SEQ ID NO:8;
        b) a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10953 of SEQ ID NO:9;
        c) a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10971 of SEQ ID NO:10;
        d) a polynucleotide comprising the contiguous sequence from nucleotide 35 to nucleotide 9723 of SEQ ID NO:16
        e) a polynucleotide complementary to a polynucleotide of a)-d); and
        f) an RNA equivalent of a)-e).
    
    


In another aspect, the invention includes a vector comprising any of the bicistronic or monocistronic recombinant HCV genomic constructs described herein. In certain embodiments, the vector comprises a bicistronic construct comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:2-5. In other embodiments, the vector comprises a monocistronic construct comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:6-10 and SEQ ID NO:16. In certain embodiments, the vector further comprises a promoter operably linked to the genomic construct that is capable of generating an RNA transcript from a DNA template. In one embodiment, the promoter is a T7 promoter.
In another aspect, the invention includes an HCV genomic RNA transcript comprising any of the bicistronic or monocistronic recombinant HCV genomic constructs described herein, wherein transfection of a suitable host cell with the HCV genomic RNA transcript is capable of producing infectious HCV particles.
In another aspect, the invention includes a tissue culture system for production of infectious HCV particles comprising a cell capable of supporting HCV replication transfected with a recombinant HCV genomic RNA transcript of the invention.
In certain embodiments, the tissue culture system comprises a cell capable of supporting HCV replication derived from human tissue. In certain embodiments, the tissue culture system comprises a hepatocellular carcinoma cell. In certain embodiments, the tissue culture system comprises a cell capable of supporting HCV replication from a cell line selected from the group consisting of Huh7, Huh7.5, Siena 8, and T7-11. In certain embodiments, the cell capable of supporting HCV replication is interferon-cured. In one preferred embodiment, the cell capable of supporting HCV replication is a hepatocyte or a hepatocyte-derived cell.
In certain embodiments, the tissue culture system further comprises a polynucleotide encoding a double stranded RNA binding protein that acts as an interferon antagonist or RNA silencing suppressor. The double stranded RNA binding protein can be an influenza virus NS1 protein. In certain embodiments, the NS1 polypeptide comprises the amino acid sequence of SEQ ID NO 15 or a functional fragment thereof that increases the yield of viral particles. In certain embodiments, the polynucleotide encoding the RNA silencing suppressor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:11-14.
In certain embodiments, the tissue culture system further comprises a polynucleotide encoding a CD81 polypeptide to facilitate viral entry into host cells.
In certain embodiments, replication of the HCV genomic RNA transcript is maintained in the tissue culture system for at least 10 days after the genomic RNA transcript is introduced into a cell capable of supporting HCV replication. In certain embodiments, replication of the HCV genomic RNA transcript is maintained in the tissue culture system for at least 20 days after the genomic RNA transcript is introduced into a cell capable of supporting HCV replication. In certain embodiments, replication of the HCV genomic RNA transcript is maintained in the tissue culture system for at least 30 days after the genomic RNA transcript is introduced into a cell capable of supporting HCV replication. In some preferred embodiments, the cell capable of supporting HCV replication is a hepatocyte or hepatocyte-derived cell.
In certain embodiments, infectivity of virus produced from the HCV genomic construct is maintained in the tissue culture system for at least 9 days after the genomic RNA transcript is introduced into a cell capable of supporting HCV infection. In certain embodiments, infectivity of virus produced from the HCV genomic RNA transcript is maintained in the tissue culture system for at least 15 days after the genomic RNA transcript is introduced into a cell capable of supporting HCV infection.
In another aspect, the invention includes a method for producing infectious HCV particles in tissue culture, comprising transfecting a cell capable of supporting HCV infection with a recombinant HCV genomic RNA transcript of the invention and growing the cell in culture media under conditions suitable for replication of the HCV genomic RNA and secretion of infectious viral particles into the culture media.
In certain embodiments, the method further comprises the step of, prior to the transfection with the HCV genomic transcript, transfecting the cell capable of supporting HCV replication with a polynucleotide encoding an RNA silencing suppressor. The RNA silencing suppressor can be an influenza virus NS1 protein. In certain embodiments, the cell is transfected with a polynucleotide encoding an NS1 polypeptide comprising the amino acid sequence of SEQ ID NO 15 or a functional fragment thereof that increases the yield of viral particles. In certain embodiments, the polynucleotide encoding the RNA silencing suppressor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:11-14.
In certain embodiments, the cell capable of supporting HCV replication is derived from human tissue. In certain embodiments, the cell capable of supporting HCV replication is a hepatocellular carcinoma cell. In some embodiments, the cell is a hepatocyte cell. In certain embodiments, the hepatocyte cell is from a cell line selected from the group consisting of Huh7, Huh7.5, Huh7C, Siena 8, and T7-11. In certain embodiments, the hepatocyte cell is interferon-cured.
In another aspect, the invention includes a chimeric hepatitis C virus produced in a tissue culture system by any of the methods described herein, wherein the recombinant HCV genomic construct comprises a genomic sequence selected from the group of genomic sequences in SEQ ID NOS:3-5, SEQ ID NOS:8-10 and 16, the sequences of which include genomic HCV constructs and plasmid sequences.
In another aspect, the invention includes a kit comprising a cell capable of supporting HCV replication and any of the recombinant HCV genomic constructs described herein (i.e., RNA transcripts for infection and/or DNA vectors for production of viral RNA by in vitro transcription). In certain embodiments, the kit comprises a cell that is transfected with the recombinant HCV genomic construct. The kit may further comprise a polynucleotide encoding an RNA silencing suppressor. In certain embodiments, the kit comprises a cell that is transfected with the polynucleotide encoding the RNA silencing suppressor. In certain embodiments, the polynucleotide encodes an NS1 polypeptide comprising the amino acid sequence of SEQ ID NO 15 or a functional fragment thereof that increases the yield of viral particles. In certain embodiments, the polynucleotide encoding the RNA silencing suppressor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:11-14.
In certain embodiments, the cell capable of supporting HCV infection is a hepatocyte cell and in yet further embodiments, the hepatocyte is derived from human tissue. In certain embodiments, the hepatocyte cell is a hepatocellular carcinoma cell. In certain embodiments, the hepatocyte cell is from a cell line selected from the group consisting of Huh7, Huh7.5, Huh7C, Siena 8, and T7-11. In certain embodiments, the hepatocyte cell is interferon-cured.
In another aspect, the invention includes a method of identifying a compound having anti-HCV activity, the method comprising:

    
    
        a) treating an HCV tissue culture system described herein with a compound;
        b) comparing at least one of HCV replication, expression of an HCV protein, or viral infectivity in said culture system treated with said compound to said tissue culture system not treated with said compound, wherein a reduction in the level of HCV replication, expression of HCV proteins, or viral infectivity in the tissue culture system treated with said compound is indicative of a compound with anti-HCV activity.
    
    


In one embodiment, the compound having anti-HCV activity can be a compound that inhibits HCV infectivity. In one embodiment, the compound that inhibits HCV infectivity is an antibody which in some embodiments can be a neutralizing antibody.
In another embodiment, the compound having anti-HCV activity can be a compound that inhibits HCV replication.
These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts a schematic representation of recombinant wild-type and chimeric JFH1 genomic bicistronic constructs. Bicistronic constructs included a first cistron encoding the first 17 amino acids of the JFH1 core protein linked to a luciferase (Luc) reporter gene for monitoring RNA replication, an encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES), and a second cistron encoding a wild-type or chimeric JFH1 polyprotein. Chimeric constructs contained NS3 to NS5b coding regions from HCV type 2a strain JFH1, core to p7 coding regions from a different HCV isolate (construct LucHJ1: core to p7 region from HCV type 1a strain H77C, construct LucCJ1: core to p7 region from HCV type 1b strain Con1, and construct LucNJ1: core to p7 region from HCV type 3a strain NZ1), and a chimeric NS2 coding region with an intergenotypic junction at residue 871 of NS2 for the LucCJ1 and LucHJ1 constructs and at residue 877 of NS2 for the LucNJ1 construct.
FIG. 2 depicts a schematic representation of recombinant wild-type and chimeric JFH1 monocistronic constructs. Monocistronic constructs included a region encoding the first 12 amino acids of the JFH1 core protein linked to a green fluorescent protein (GFP) or a Renilla luciferase (RLuc) reporter gene for monitoring RNA replication, a coding sequence for a foot-and-mouth disease virus (FMDV) 2A protease, a coding sequence for a ubiquitin protease cleavage site, and a wild-type or chimeric JFH1 polyprotein coding region. Chimeric constructs contained NS3 to NS5b coding regions from HCV type 2a strain JFH1, core to p7 coding regions from a different HCV isolate (RLF2AUbHJ1 construct: core to p7 region from HCV type 1a strain H77C, RLF2AUbCJ1 construct: core to p7 region from HCV type 1b strain Con1, and RLF2AUbNJ1 construct: core to p7 region from HCV type 3a strain NZ1), and a chimeric NS2 coding region with an intergenotypic junction at residue 871 of NS2 for the RLF2AubCJ1 and RLF2AUbHJ1 constructs and at residue 877 of NS2 for the RLF2AUbNJ1 construct.
FIG. 3 compares the replication efficiencies of monocistronic RLF2AUbJFH-1 and bicistronic LucJFH1 wild-type JFH1 genomic constructs in Huh7 cells. Intracellular RNA levels were measured based on luciferase luminescence as a function of time after electroporation (eV) of the Huh7 cells.
FIG. 4 shows the JFH1 infectivity titer in Huh7 cells transfected with the monocistronic RLF2AUbJFH-1 construct as a function of time after electroporation.
FIG. 5 shows the level of RNA replication of the monocistronic RLF2AUbJFH-1 construct in Huh7 cells as a function of time after infection with recombinant virus. Naive Huh7 cells were inoculated with virus from the culture supernatant of cells transfected with the RLF2AUbJFH-1 construct, as described in Example 6. Intracellular JFH1 RNA was measured by RLuc assay for 24 days after infection of Huh7 cells.
FIG. 6 compares the infectivity of wild-type JFH1 virus produced by the monocistronic construct RLF2AUbJFH-1 and chimeric viruses produced by the monocistronic constructs RLF2AUbHJ1, RLF2AUbCJ1, and RLF2AUbNJ1.
FIG. 7 compares the RNA replication efficiencies of the RLF2AUbJFH-1, RLF2AUbHJ1, RLF2AUbCJ1, and RLF2AUbNJ1 monocistronic constructs in Huh7 cells.
FIG. 8 compares RNA replication efficiencies of JFH-1 construct RLF2AUbJFH1 in the three cell lines, Siena 8, T7-11, and Huh7. Cells were transfected with either 1 μg or 4 μg of the construct.
FIG. 9 depicts a schematic representation of NS1 of influenza. The MI-length NS1 protein contains a double-stranded RNA (dsRNA) binding domain, which inhibits the interferon-induced dsRNA-dependent protein kinase PKR and RNA interference (RNAi), and an effector domain, which functions in inhibiting pre-mRNA splicing and nuclear export of mRNA. The dsRNA binding domain and the effector domain reside at amino acid residues 1-82 and 138-147, respectively. In addition, the NS1 protein comprises two nuclear localization signals (NLS1 and NLS2), a cleavage and polyadenylation specificity factor (CPSF) binding site, and a polyadenylation binding protein II (PABII) binding site. Four RSS constructs are shown, including the full-length wild-type (wt) NS1 (residues 1-230); NS82, a truncated NS1 protein containing the dsRNA binding domain (residues 1-82); NS180, a longer truncated NS1 protein containing the dsRNA binding domain and the effector domain; and a mutant NS1 protein defective in dsRNA binding containing a deletion of residues 50-84.
FIG. 10 shows the effects of the full-length wild type and NS82 constructs on replication of the monocistronic RLF2AUbJFH-1 construct in Huh7 cells.
FIG. 11 shows the effects of the full-length wild-type and NS82 constructs on infectivity of virus produced from the monocistronic RLF2AUbJFH-1 construct in Huh7 cells.
FIG. 12 shows a schematic representation of the genetic organization of the JFH1 viral genome, including the restriction enzyme digest sites used to make the genomic constructs of the invention.
FIG. 13 shows a schematic representation of how the chimeric HCV genomic constructs were produced, including the restriction enzyme sites used to replace portions of the JFH1 genome with that of a second HCV isolate.
FIG. 14 shows a schematic representation of the viral neutralization assay performed with the HCV tissue culture system.
FIG. 15 depicts a model of HCV cell attachment and entry, showing the interactions of the HCV E1/E2 complex on the viral envelope with SRB1 and CD81 on cells during viral infection.
FIG. 16 shows JFH1 infectivity in Huh7.5 cells measured by indirect fluorescent antibody assay (IFA).
FIG. 17 depicts a schematic representation of recombinant wild-type and chimeric JFH1 monocistronic constructs, including RLF2AUbJFH1 (RJI): core to NS5b from HCV type 2a strain JFH1, RLF2AUbHJ1 (RHJ1): core to p7 region and N-terminal portion of NS2 from HCV type 1a strain H77C, RLF2AUbCJ1 (RCJ1) construct: core to p7 region and N-terminal portion of NS2 from HCV type 1b strain Con1, and RLF2AUb452JFH1 (R45211) construct: core to p7 region and N-terminal portion of NS2 from HCV type 3a strain 452, and a chimeric NS2 coding region with an intergenotypic junction at residue 871 in the RHJ1, RCJ1, and R452J1 constructs.
FIG. 18 shows the effects of CD81 on the infectivity of JFH1 in Huh7 and Huh7.5 cells.
FIG. 19 shows neutralization antibody titers for HCV types 2a, 1b, and 1a, as measured with the HCV tissue culture system.
FIG. 20 compares protective immunity against HCV type 1a in chimpanzees vaccinated with E1E2. The percent inhibition of HCV type 1a, neutralizing antibody titer, CD81 blocking antibody titer, and anti-E1E2 antibody titer were measured using a cell culture assay.
FIG. 21 shows cross-neutralization of HCV types 1a and 2a in chimpanzees, as measured using a cell culture assay.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 is the sequence of a pUC-18 derived plasmid containing the JFH1 genome.
SEQ ID NO:2 is the sequence of a pUC derived plasmid containing the JFH1genome further comprising a luciferase gene.
SEQ ID NO:3 is the sequence of a pUC derived plasmid containing the JFH1/H77C chimeric genome further comprising the luciferase gene in a bicistronic configuration.
SEQ ID NO:4 is the sequence of a pUC derived plasmid containing the JFH1/Con1 chimeric genome further comprising the luciferase gene in a bicistronic configuration
SEQ ID NO:5 is the sequence of a pUC derived plasmid containing the JFH1/NZ1 chimeric genome further comprising the luciferase gene in a bicistronic configuration
SEQ ID NO:6 is the sequence of a pUC derived plasmid containing the JFH1 genome further comprising the luciferase gene in a monocistronic configuration
SEQ ID NO:7 is the sequence of a pUC derived plasmid containing the JFH1 genome further comprising the green fluorescent protein gene in a monocistronic configuration
SEQ ID NO:8 is the sequence of a pUC derived plasmid containing the JFH1/H77C chimeric genomic construct further comprising the luciferase gene in a monocistronic configuration. The chimeric HCV genomic construct comprises from about nucleotide 7 to about nucleotide 10953 of SEQ ID NO:8
SEQ ID NO:9 is the sequence of a pUC derived plasmid containing the JFH1/Con1 chimeric genome further comprising the luciferase gene in a monocistronic configuration. The chimeric HCV genomic construct comprises from about nucleotide 7 to about nucleotide 10953 of SEQ ID NO:9
SEQ ID NO:10 is the sequence of a pUC derived plasmid containing the JFH1/NZ1 chimeric genome further comprising the luciferase gene in a monocistronic configuration. The chimeric HCV genomic construct comprises from about nucleotide 7 to about nucleotide 10971 of SEQ ID NO:10.
SEQ ID NO:11 is the sequence of the influenza NS1 gene inserted into a pCMV plasmid.
SEQ ID NO:12 is the sequence of a portion of the influenza NS1 gene inserted into a pCMV plasmid.
SEQ ID NO:13 is the sequence of a portion of the influenza NS1 gene inserted into a pCMV plasmid.
SEQ ID NO:14 is the sequence of the influenza NS1 gene inserted into a pCMV plasmid wherein an internal portion of the ns1 gene is deleted.
SEQ ID NO:15 is the amino acid sequence of NS1 from influenza.
SEQ ID NO:16 is the sequence of a pUC derived plasmid containing the JFH1/452 chimeric genome further comprising the luciferase gene in a monocistronic configuration. The chimeric HCV genomic construct comprises from about nucleotide 35 to about nucleotide 9723 of SEQ ID NO:16.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Fundamental Virology, 3rd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Cotowick and N. Kaplan eds., Academic Press, Inc.).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.
The following amino acid abbreviations are used throughout the text:

    
    
        Alanine: Ala (A) Arginine: Arg (R)
        Asparagine: Asn (N) Aspartic acid: Asp (D)
        Cysteine: Cys (C) Glutamine: Gln (O)
        Glutamic acid: Glu (E) Glycine: Gly (G)
        Histidine: His (H) Isoleucine: Ile (I)
        Leucine: Leu (L) Lysine: Lys (K)
        Methionine: Met (M) Phenylalanine: Phe (F)
        Proline: Pro (P) Serine: Ser (S)
        Threonine: Thr (T) Tryptophan: Trp (W)
        Tyrosine: Tyr (Y) Valine: Val (V)
    
    


1. DEFINITIONS
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an HCV genomic RNA transcript” includes a mixture of two or more such transcripts, and the like. Similarly, transfection of a cell with an HCV genomic RNA transcript is meant to include the situation where a plurality of cells are transfected with a plurality of genomic RNA transcripts.
The term “hepatitis C virus” (HCV) refers to an agent causative of Non-A, Non-B Hepatitis (NANBH). The nucleic acid sequence and putative amino acid sequence of HCV is described in U.S. Pat. Nos. 5,856,437 and 5,350,671. The disease caused by HCV is called hepatitis C, formerly called NANBH. The term HCV, as used herein, denotes a viral species of which pathenogenic strains cause NANBH, as well as attenuated strains or defective interfering particles derived therefrom.
HCV is a member of the viral family flaviviridae. The morphology and composition of Flavivirus particles are known, and are discussed in Reed et al., Curr. Stud. Hematol. Blood Transfus. (1998), 62:1-37; HEPATITIS C VIRUSES IN FIELDS VIROLOGY (B. N. Fields, D. M. Knipe, P. M. Howley, eds.) (3d ed. 1996). It has recently been found that portions of the HCV genome are also homologous to pestiviruses. Generally, with respect to morphology, Flaviviruses contain a central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of about 40-50 nm. Their cores are about 25-30 nm in diameter. Along the outer surface of the virion envelope are projections that are about 5-10 nm long with terminal knobs about 2 nm in diameter.
The HCV genome is comprised of RNA. It is known that RNA containing viruses have relatively high rates of spontaneous mutation. Therefore, there can be multiple strains, which can be virulent or avirulent, within the HCV class or species. The ORF of HCV, including the translation spans of the core, non-structural, and envelope proteins, is shown in U.S. Pat. Nos. 5,856,437 and 5,350,671.
The term “HCV genomic construct” refers to a nucleic acid, either RNA or DNA, comprising an entire HCV genome, including coding sequences for all of the structural and nonstructural proteins of HCV. A DNA vector comprising an HCV genomic construct can be used for production of HCV RNA by in vitro transcription. An RNA transcript comprising an HCV genomic construct can be used to transfect cells to produce infectious HCV viral particles.
The term “HCV genomic RNA transcript” refers to an RNA transcript comprising an HCV genomic construct that is capable of replicating and producing infectious HCV viral particles when present in a cell. HCV genomic RNA transcripts as described herein are introduced into hepatocyte cells according to the present invention. The cells can additionally contain additional viral replicons and or plasmids containing or expressing additional nucleic acids, genes or proteins. One or more other additional genes and/or regulatory regions can also optionally be present and expressed in a cell transfected with an HCV genomic RNA transcript. Additional genes or proteins can be provided on the HCV genomic RNA transcript or can be stably or transiently transfected into the host cell genome using various techniques well known in the art. An additional gene or protein can be used e.g., as a marker for cell survival. Furthermore, an additional gene or protein may provide an accessory function that enhances or enables HCV replication, expression and/or virus or virus like particle production (e.g., NS1, CD81).
As used herein, the term “additional gene” is meant to encompass a gene or other nucleic acid that expresses a protein or provides a regulatory function in the cell. The additional gene can be heterologous to HCV and the host cell or may be from HCV or the host cell genome. Thus, as used herein, the term can mean a host cell gene that is already present in the host cell genome, but is provided as an additional gene either as an episomal element or integrated into a site on the host cell genome distinct from (and in addition to) its normal location.
It should be understood that the terms “genomic construct”, “HCV genomic RNA transcript”, and “genetic element” encompass the sense strand of the viral RNA or any complementary sequence (e.g. a cDNA reverse transcript or complementary RNA) that can be converted into the sense strand (the viral genomic RNA) and can be translated into the viral proteins required for replication. The cDNA genomic constructs can comprise double stranded DNA as well known in the art. Those of skill in the art will understand that alternate constructs may be used depending upon the desired goal, e.g. cloning part or all of a viral genome, expressing an RNA viral transcript from a double strand cDNA template and generating a viral product.
It should be further understood that the description of the exemplary genomic constructs is referred to in terms of sequences comprising “about” with reference to precise borders, since the borders of the genomic sequences or other inserts can readily be modified or altered without departing from the spirit of the invention. Thus, an HCV genomic construct can comprise sequences of additional nucleotides, depending upon the well known artifacts of cloning using selected restriction enzyme sites.
A “neutralizing antibody” as used herein is meant to refer to an antibody that prevents or reduces the ability of HCV to infect host cells. The term antibody is meant to include the singular and plural and can also refer to a monoclonal or polyclonal antibody preparation, single chain antibodies, antibody Fab and F(ab)2 fragments and chimeric and humanized antibodies as known in the art.
The terms “polypeptide” and “protein” refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” are used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and these terms will be used interchangeably. Thus, these terms include, for example, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′ phosphoramidates, 2′-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide. In particular, DNA is deoxyribonucleic acid.
A polynucleotide “derived from” a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
“Recombinant host cells”, “host cells,” “cells”, “cell lines,” “cell cultures”, and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of coding sequence can be determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence.
Typical “control elements,” include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), and translation termination sequences.
“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. Expression is meant to include the transcription any one or more of transcription of an of mRNA or viral RNA from a DNA or RNA template and can further include translation of a protein from an mRNA template The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
“Encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
The term “transfection” is used to refer to the uptake of foreign DNA by a cell. A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.
The terms “effective amount” or “pharmaceutically effective amount” of a composition or agent, as provided herein, refer to a nontoxic but sufficient amount of the composition to provide the desired response, such as suppression of HCV RNA replication, expression, and/or viral infectivity, and optionally, a corresponding therapeutic effect. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
As used herein, a “biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.
2. MODES OF CARRYING OUT THE INVENTION
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
A. HCV Tissue Culture System
The present invention provides a tissue culture system for production of infectious HCV viral particles. The tissue culture system includes hepatocyte cells containing a recombinant HCV genomic RNA transcript comprising a reporter gene. The cells are transfected with an HCV genomic RNA transcript that supports the expression of viral and non-viral proteins including a reporter gene (e.g., a firefly luciferase, Renilla luciferase, or green fluorescent protein gene), linked to the replication and or expression of the HCV genome. After introducing the HCV genomic RNA transcript into hepatocyte cells, the reporter gene allows HCV RNA replication, expression, or viral particle production to be measured. According to various embodiments, human-derived cells that are adapted for growth and/or stability of the HCV genomic RNA transcript may be used. The HCV tissue culture system may be used to produce virus (e.g., for immunogenic compositions such as vaccines) or to evaluate potential activity of HCV anti-viral compounds, including determination of the presence or absence of neutralizing antibody.
Reporter gene activity can be measured at various steps in order to monitor various viral activities. For example, cells are transfected with a genomic RNA transcript comprising a reporter gene, then grown, and lysed. The amount of reporter activity in cell lysates is assayed and provides a measure of the level of viral replication occurring inside the transfected cell population.
Alternatively or in addition to the above assay, a first population of naïve cells capable of supporting HCV may be transfected, grown for a time period, and then the supernatant containing secreted virus particles is harvested. The supernatant is then used to infect a second population of naïve cells which support HCV replication. The infected naïve cells are then grown and lysed and reporter activity is measured. This measurement will be an indication of the infectivity of the virus used to infect the first cell population.
Some advantages of the tissue culture system are the following: it does not require the use of animal models; the recombinant HCV genomic RNA transcripts replicate efficiently in cultured cells and produce infectious viral particles; and all steps of the HCV virus life cycle that may be targeted for antiviral intervention are reproduced. The tissue culture system thus provides a convenient and cost-effective means for producing virus and screening and detecting potential anti-HCV therapeutic agents.
The cells used in the HCV tissue culture system may be any cells capable of replicating an HCV genomic RNA transcript and producing infectious virus, but are preferably immortalized human-derived cells, and in one embodiment are human hepatoma Huh7 cells or Huh7 derived cells. In some embodiments, the derived cells have been grown with a sub-genomic replicon, treated with interferon alpha to cure the cells of the replicon, and then Selected for an increased resistance to interferon alpha.
The HCV genomic constructs of the invention express the full HCV genome as a polyprotein, which includes the HCV structural proteins core, E1, and E2, and the HCV non-structural proteins p7, NS2, NS3, NS4a, NS4b, NS5a, and NS5b. In certain embodiments, a reporter gene is contained within the HCV genomic construct and is replicated when the construct is replicated. The presence of a reporter gene allows reporter activity to be measured for monitoring RNA replication and viral infectivity in the tissue culture system. In certain embodiments the reporter is firefly luciferase, Renilla luciferase, or green fluorescent protein.
The reporter gene may be co-expressed with the genes encoding the HCV proteins, either as a mono-cistronic, bicistronic or tricistronic RNA. In the bi-cistronic configuration, one or more genes within the genomic construct is under the translational control of a separate genetic element that does not regulate expression of the other genes also present in the construct. An additional gene or genes may be expressed (1) with the HCV protein(s), (2) with the reporter gene or (3) under the control of yet another separate translational control element contained in the genomic construct (i.e., the “tricistronic” configuration). Furthermore, the HCV proteins can be expressed together as an HCV polyprotein or may be under the expression of separate genetic elements as described above for the reporter gene and any additional gene(s). Alternatively, replication can be detected without protein expression of the reporter gene, for example, by assaying for the relative presence or absence of a nucleic acid that encodes reporter specific nucleic acid sequences.
In one preferred embodiment, the invention provides a monocistronic genomic HCV replicon having a luciferase gene encoded at the 5′ region of the HCV polyprotein, and with a cleavage site between the HCV polyprotein and the reporter gene.
Genetic elements that regulate translation of genes are well known to the skilled artisan and include, e.g., an internal ribosome entry site (IRES). Suitable IRES sequences are known from poliovirus, encephalo-myocarditis virus (EMCV), and from HCV itself. In certain embodiments, the HCV genomic construct comprises an internal ribosome entry site (IRES). FIG. 1 shows exemplary bicistronic genomic constructs (described also in SEQ ID NOS:1-5) comprising an EMCV IRES. These bicistronic genomic constructs comprise a luciferase reporter gene (Luc) and wild-type JFH1 or chimeric virus genomes, as described in Example 2. The EMCV sequence is upstream of and drives expression of the HCV polyprotein. The EMCV IRES directs translation of the core to NS5B region that is flanked at the 3′ end by the 3′ non-coding region (NCR).
When the reporter gene contained in the HCV genomic construct is provided in the monocistronic configuration, it is expressed as part of the polyprotein produced by the genomic construct. The reporter gene can be cleaved from the polyprotein by providing cleavage recognition sites at both boundaries of the reporter gene product (or at one boundary if the reporter gene product is at the amino or carboxyl terminus of the polyprotein). The cleavage sites are recognized by a protease that specifically cleaves at the sites (e.g., the HCV protease). Alternatively, a CHYSEL sequence (cis-acting hydrolase element), such as the foot-and-mouth disease virus (FMDV) 2A sequence can be used to release the reporter gene product from the HCV polyprotein (Felipe (2004) Genetic Vaccines and Therapy 2:13). In another alternative, the reporter gene can be constructed without cleavage site(s) and can thus be expressed as a fusion protein fused to one of the HCV proteins. In a genomic construct, when the reporter gene is expressed as part of a fusion protein, the reporter gene can be linked to a coding sequence for a structural protein (i.e., core or envelope), or fragment thereof. In one embodiment, a coding sequence for an N-terminal fragment of the JFH1 core protein comprising at least the first 12 residues and up to the first 18 residues of the JFH1 core protein, including any number of residues in between, such as residues 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, or 1-18 of the JFH1 core protein, is linked to the reporter gene. It is not necessary that the reporter gene always must express a protein. In some embodiments, the reporter can be detected by detecting the relative presence or absence of a nucleic acid sequence specific for the reporter.
FIGS. 2 and 17 show exemplary monocistronic genomic constructs comprising wild-type JFH1 or chimeric viral genomes and Renilla luciferase (RLuc) or green fluorescent protein (GFP) reporter genes (see Example 3 and SEQ ID NOS:6-10 and 16). These constructs, in addition, comprise downstream of the reporter gene a sequence encoding an FMDV 2A protease followed by a sequence encoding a ubiquitin (Ub) protease cleavage site, which together permit production of the HCV polyprotein with its natural N-terminus free of extraneous amino acids. The expressed FMDV2a protease undergoes self-cleavage at the penultimate amino acid at its C-terminus, thereby separating the reporter gene product from the HCV polyprotein, but leaving the one C-terminal FMDV2a amino acid linked to the remaining polyprotein fusion. Cleavage of the ubiquitin cleavage site at its C-terminus by host endogenous ubiquitin-specific proteases releases the HCV polyprotein product with its precise native N-terminus.
In some embodiments, the genomic construct expresses a selectable marker. The selectable marker allows for the selection of cells harboring the genomic construct. In some embodiments neomycin phosphotransferase gene (Neo) for selection with G418 is used. Examples of other markers based on drug resistance include aad, ble, dhfr, hpt, nptII, aphII, gat, and pac. Other markers may allow selection using antibiotics, such as Puromycin, Zeocin or Hygromycin, but one of skill in the art will understand that any marker gene known in the art that allows for selection of cells harboring the genomic construct may be used. In full length genomic constructs a neo gene is usually not present.
In certain embodiments, the genomic construct may have one or more cell-adaptive mutations. These mutations can enhance or allow HCV replication in cells that are either non-permissive or otherwise not efficient for HCV production. In one embodiment, wherein Huh7 cells are used, the genomic construct can have the following cell-adaptive mutations: E1202G, T12801 in the NS3 region and K1846T in NS4B region (Nicole Krieger, Volker Lohmann, and Ralf Bartenschlager, Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. 2001, Journal of Virology, 75(10): 4614-4624; Volker Lohmann, Sandra Hoffmann, Ulrike Herian, Francois Penin, and Ralf Bartenschlager, Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. 2003, Journal of Virology, 77(5):3007-3019). Such cell-adaptive mutations are not necessarily required, however, as some cells can support HCV replication without having been adapted in cell culture (e.g., HCV-N or JFH-1). The tissue culture system of the invention can support replication of HCV genomic RNA transcripts with or without these mutations.
HCV genomic constructs may be derived from various viral strains and genotypes. The HCV genome can be a chimera of any types (e.g. 1, 2, 3, 4, 5, 6) or subtypes of HCV (e.g. 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1 h, 1i, 1j, 1k, 1l, 1m, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 2m, 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3k, 4a, 4c, 4d, 4e, 4f, 4g, 4h, 4k, 4l, 4m, 4n, 4o, 4p, 4q, 4r, 4s, 4t, 5a, 6a, 6b, 6d, 6f, 6g, 6h, 6i, 6j, 6k, 6l, 6m, 6n). This nomenclature is the current standard, as set out by the NIAID Hepatitis C Virus (HCV) Sequence Database [hcv.lanl.govicontent/hcv-db/classification/genotable.hind] in which previous genotypes 7-9 have been reclassified as subtypes of type 6 (Simmonds et al. (1996) J. Gen. Viral. 77:3013-3024). Thus the new classification includes previous classifications I, II, III, IV, V, VI, 4α, 4β, 7a, 7b, 7c/NGII/VII, 7d, NGI, 8a, 8b, 9a, 9b, 9c, 10a/1133 and 11a.
For example, the complete sequences for several HCV-1 isolates are reported. See, e.g., NCBI accession nos. AB016785, AB049087, AB049088, AB049089, AB049090, AB049091, AB049092, AB049093, AB049094, AB049095, AB049096, AB049097, AB049098, AB049099, AB0490100, AB0490101, AB080299, AB 119282, AB154177, AB154178, AB154179, AB154180, AB154181, AB154182, AB154183, AB154184, AB154185, AB154186, AB154187, AB154188, AB154189, AB154190, AB154191, AB154192, AB154193, AB154194, AB154195, AB154196, AB154197, AB154198, AB 154199, AB 154200, AB 154201, AB 154202, AB 154203, AB 154204, AB 154205, AB154206, AB19133, AF009606, AF011751, AF011752, AF 011753, AF054247, AF054248, AF054249, AF054250, AF139594, AF165045, AF165046, AF165047, AF165048, AF165049, AF165050, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF165063, AF165064, AF176573, AF207752, AF207753, AF207754, AF207755, AF207756, AF207757, AF207758, AF207759, AF207760, AF207761, AF207762, AF207763, AF207764, AF207765, AF207766, AF207767, AF207768, AF207769, AF207770, AF207771, AF207772, AF207773, AF207774, AF208024, AF271632, AF290978, AF313916, AF333324, AF356827, AF483269, AF511948, AF511949, AF511950, AJ000009, AJ132996, AJ132997, AJ238799, AJ238800, AJ278830, AY045702, AY051292, AY460204, AY587016, D10934, D11168, D11355, D13558, D14484, D14853, D30613, D45172, D50480, D50481, D50482, D50483, D50484, D50485, D63857, D85516, D89815, D89872, D90208, L02836, M58335, M62321, M67463, M84754, M96362, NC—004102, S62220, U01214, U16362, U45476, U89019, X61596.
Similarly, the complete sequences for several HCV-2 isolates are reported. See, e.g., NCBI accession nos. AB030907, AB031663, AB047639, AB047640, AB047641, AB047642, AB047643, AB047644, AB047645, AF169002, AF169003, AF169004, AF169005, AF177036, AF238481, AF238482, AF238483, AF238484, AF238485, AF238486, AY232730, AY232731, AY232732, AY232733, AY232734, AY232735, AY232736, AY232737, AY232738, AY232739, AY232740, AY232741, AY232742, AY232743, AY232744, AY232745, AY232746, AY232747, AY232748, AY232749, AY587845, AY746460, D00944, D10988, D50409. One suitable HCV strain for use with the invention is the genotype 2a strain JFH1 (Kato et al. (2003) J. Med. Virol. 64:334-339).
The complete sequences for several HCV-3 isolates are also reported. See, e.g., NCBI accession nos. AF046866, D17763, D28917, D49374, D63821, X76918. The complete sequences for HCV-4 isolates are also known. See, e.g., NCBI accession no. Y11604. The complete sequences for several HCV-5 isolates are known. See, e.g., NCBI accession nos. AF064490 and Y13184. The complete sequences for several HCV-6 isolates are also known. See, e.g., NCBI accession nos. AY859526, AY878650, D63822, D84262, D84263, D84264, D84265, Y12083.
Additional sequences are as follows. Isolate HCV J1.1 is described in Kubo et al. (1989) Japan. Nucl. Acids Res. 17:10367-10372; Takeuchi et al. (1990) Gene 91:287-291; Takeuchi et al. (1990) J. Gen. Virol. 71:3027-3033; and Takeuchi et al. (1990) Nucl. Acids Res. 18:4626. The complete coding sequences of two independent isolates, HCV-J and BK, are described by Kato et al., (1990) Proc. Natl. Acad. Sci. USA 87:9524-9528 and Takamizawa et al., (1991) J. Virol. 65:1105-1113, respectively. HCV-1 isolates are described by Choo et al. (1990) Brit. Med. Bull. 46:423-441; Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455 and Han et al. (1991) Proc. Natl. Acad. Sci. USA 88:1711-1715. HCV isolates HC-J1 and HC-J4 are described in Okamoto et al. (1991) Japan J. Exp. Med. 60:167-177. HCV isolates HCT 18, HCT 23, Th, HCT 27, EC1 and EC10 are described in Weiner et al. (1991) Virol. 180:842-848. HCV isolates Pt-1, HCV-K1 and HCV-K2 are described in Enomoto et al. (1990) Biochem. Biophys. Res. Commun. 170:1021-1025. HCV isolates A, C, D & E are described in Tsukiyama-Kohara et al. (1991) Virus Genes 5:243-254.
In certain embodiments, the HCV genomic construct is chimeric comprising sequences derived from more than one viral strain or genotype. Such chimeric constructs encode a chimeric HCV polyprotein. In one embodiment, the chimeric polyprotein comprises a C-terminal portion of NS2, and the NS3, NS4a, NS4b, NS5a, and NS5b regions from JFH1 and the core, E1, E2, and p7 regions, and the N-terminal portion of NS2 from a second HCV isolate. The second isolate may be the same or a different subtype than JFH1. Preferably, the second isolate is a different subtype than JFH1. In certain embodiments, the non-JFH1 strain or isolate is of HCV type 1a, 1b, 2a, 2b, 3a, 3b, 4, 5, or 6. In certain embodiments, the non-JFH1 strain or isolate is H77C, Con1, NZ1, or 452.
In certain embodiments, the HCV NS2 gene is a chimeric gene, encoding an amino portion of NS2 from the same HCV strain or isolate as encoding the E1, E2, and P7 proteins and a carboxy portion of NS2 from JFH1. Suitable junction sites between the amino and carboxy portions of the NS2 gene are described in Pietschmann et al. (Proc. Natl. Acad. Sci. U.S.A. (2006) 103:7408-7413; herein incorporated by reference). In certain embodiments, the junction between the amino and carboxy portions of NS2 occurs at a residue selected from the group consisting of 871-877.
Exemplary bicistronic and monocistronic chimeric constructs are depicted in FIGS. 1, 2, and 17. Representative recombinant HCV monocistronic genomic constructs comprise a polynucleotide selected from the group consisting of: a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10953 of SEQ ID NO:8; a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10953 of SEQ ID NO:9; a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10971 of SEQ ID NO:10; a polynucleotide comprising the contiguous sequence from nucleotide 35 to nucleotide 9723 of SEQ ID NO:16; or RNA equivalents thereof. An HCV genomic RNA transcript comprising any of these bicistronic or monocistronic recombinant HCV genomic constructs can be used for transfection of a suitable host cell to produce infectious HCV particles.
RNA from an HCV genome can be used to make a complementary DNA copy. A DNA copy can be made by reverse transcription (RT). Alternatively, a DNA copy, or fragment thereof, can be made entirely or in part by synthetic means. Once a DNA copy of the RNA genome is available, it can be converted to double stranded DNA and can then be cloned into a vector (e.g. a plasmid), and the plasmid can be used to make DNA templates from which viral RNA can be transcribed. As an alternative, the DNA may be used to generate viral RNA without using a vector, in which case a suitable promoter for a DNA-dependent RNA polymerase will be introduced upstream of the start of the viral genome. Suitable promoters include the T7, T3 and SP6 promoters, which are used in in vitro transcription (‘IVT’) reactions. DNA copies of the virus can optionally be amplified e.g. using the polymerase chain reaction (PCR).
As an alternative, viral transcripts can be made from an RNA template using an RNA-dependent RNA polymerase, such as Qβ replicase. The transcription mediated amplification (‘TMA’) reaction may be used to amplify RNA.
Plasmids containing HCV sequences may be constructed by any method well-known in the art. See, e.g., Sambrook, Fritsch, and Maniatis, Molecular cloning a laboratory manual (2nd Edition) 1989 Cold Spring Harbor Laboratory Press; Joseph Sambrook, David W. Russell, Joe Sambrook, Molecular Cloning: a Laboratory Manual, ed. Cold Spring Harbor Laboratory Press, 2000 or PCR Applications, 1999, ed. M. Innis, D. Gelfand and J. Sninsky, Academic Press. The replicon LucJFH1 shown in FIGS. 12 and 13 may be constructed by replacing the Age1-NotI fragment from pJFH1 with the Age1-Not1 fragment from Luc-JFH1. Except for the chimeric constructs containing a part of JFH1 and part of various other HCV serotype structural protein genes, the genomic constructs of the invention were synthesized artificially utilizing (phosphodiester synthesis and cloned into pUC plasmids as described in the Figures and accompanying text.
The fragment of DNA that contained the coding sequences for the FMDV 2A protease and the ubiquitin protease cleavage site was generated by artificial synthesis as part of the pLucJFH1 monocistronic plasmid.
It will readily be appreciated by one of ordinary skill in the art that various alternative cloning strategies can be employed to construct the genomic viral constructs of the invention without departing from the scope of the invention.
Restriction enzyme sites were utilized to clone various chimeric HCV genomes into pUC plasmid vectors. Following successful generation of recombinant viral genomic constructs, restriction enzyme sites were utilized to generate genomic constructs. Sequences were confirmed following cloning using standard sequencing techniques.
A wide variety of methods can be used to deliver the expression constructs to cells. Such methods include DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyornithine-mediated transfection, or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like. Other useful methods of transfection include electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery, or microinjection. See, e.g., Sambrook et al., supra, for a discussion of techniques for transforming cells of interest; and Feigner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Methods of delivering DNA using electroporation are described in, e.g., U.S. Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No. 2002/0146831; and International Publication No. WO/0045823, all of which are incorporated herein by reference in their entireties.
Reporter Genes
The inclusion of reporter genes in the recombinant genomic constructs of the present invention allows anti-HCV activity of compounds to be monitored in the tissue culture system in real-time. In addition, it is desirable that anti-HCV activity be repeatedly quantitated rather than at a single time point, and the presence of a reporter gene facilitates such observations.
Preferably, the reporter gene is easily assayed. For example, the reporter gene may give a detectable signal, such as a visible signal. The reporter gene may encode a protein which gives a visible signal itself, or which catalyses a reaction, which gives a visible, change, e.g., a fluorescent protein or an enzyme. The reporter gene may encode an enzyme such as a beta-galactosidase or a peroxidase, both of which are commonly used with colored substrates and/or products. The reporter gene may encode a fluorescent protein, such as green fluorescent protein (GFP) or a fluorescent derivative thereof such as yellow fluorescent protein (YFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP). The reporter gene may encode a luminescent protein, such as firefly or Renilla luciferase. The reporter gene may drive DNA replication in the cell or may encode a drug resistance marker. The reporter gene may encode an antigen that can be detected with an antibody that specifically binds the antigen. In addition, reporter genes that encode for secreted proteins that are measurable in media may be used. Examples of such reporter genes are genes encoding for secreted alkaline phosphatase or human growth hormone. The reporter gene may also be a unique nucleic acid sequence that can be detected utilizing nucleic acid hybridization and/or amplification techniques. The above list is exemplary and is not meant to be limiting to sources for the reporter gene, many more which can readily be envisioned by the person of ordinary skill in the art.
Reporter genes that may find use in this invention include, e.g., antigens that have a known antibody for detection (e.g., human growth hormone and antibody to human growth hormone) and enzymes that have an activity that can be detected in vitro or in vivo (e.g., alkaline phosphatase, the luciferases, green fluorescent protein and the like). Additionally, as indicated above, detection of the presence of nucleic acid encoding the reporter can also be monitored to determine replication, expression or particle production.
Thus, as defined herein, detection of reporter gene “activity” includes the detection of the physical presence or chemical activity of the product of the reporter gene and also includes detection of all or part of a nucleic acid sequence that is specific for the reporter gene. The reporter signal can be detected in vivo in cell cultures or quantitated in vitro from cell lysates.
Cell Lines
Any cell line capable of supporting replication of the HCV genomic RNA transcripts may be used. Besides the Huh7 cell line, the human cancer cell line HeLa, murine hepatoma Hepa1-6, HEK293 human hepatoma cell lines HepG2 And IMY-N9 (a cell line derived from fusing human hepatocytes and HepG2 cells) are also known to support HCV replicon replication. Therefore, any of these tumor cell lines, or any other cell line that supports the replication of an HCV genomic RNA transcript may be used in the tissue culture system. In some embodiments, the cell line is derived from human cells, preferably from human hepatoma cell line Huh7.
One cell line for use with the invention is derived from a hepatocellular carcinoma, namely the human hepatoma cell line known as ‘Huh7’ (Nakabayashi et al. (1982) Cancer Res 42:3858-63). A useful Huh7-derived cell line is ‘21-5’, which supports a full length HCV replicon (Pietschmann et al. (2002) J Virol 76:4008-21). Other suitable cell lines are Huh-7.5 and Huh-7.8, which are sub-lines of Huh-7 that can support complete HCV replication in cell culture (Blight et al. (2002) J. Virol. 76:13001-14; WO2004/044182), with Huh-7.5 being preferred. Cells derived by passaging of Huh7 cells (and their derivatives) can also be used, as well as cells derived by treating Huh7 cells with α-interferon and/or γ-interferon. As described in WO2004/044182, cell lines permissive for HCV can be prepared by a process comprising (a) culturing cells infected with HCV; (b) curing the cells of HCV; and (c) identifying a sub-line of the cured cells that is permissive for HCV replication.
The use of human-derived cells for HCV replication is desirable in order to provide a more accurate model of HCV replication and response to treatment in humans. It should be noted that in some embodiments, the cells are tumor cells. Tumor cells (e.g., Huh-7, HeLa, etc.) may be more easily passaged in culture. However, the invention is not limited to tumor cells but may be practiced with any cell or cell line that supports the replication of an HCV genomic RNA transcript.
According to various embodiments, the cell line may be adapted for increased growth rate in culture or increased HCV replication or expression (for example, as measured by a bioluminescent luciferase signal where a luciferase reporter is used). Cell lines that have been adapted for increased growth rate and/or adapted for stability of the HCV genomic RNA transcript in culture may be referred to herein as adapted cell lines.
In some embodiments, cells stably harboring HCV genomic RNA transcripts may be isolated by performing a luciferase (or other reporter) assay on the cells and then expanding the cells expressing the highest luciferase (or other reporter) signal.
In embodiments wherein adaptation for growth kinetics is performed first, the cells adapted for growth kinetics may not contain the HCV genomic RNA transcript (e.g., the cells may have been “cured” of the RNA transcript during passaging or the adapted cells may be derived from cells not harboring the RNA transcript). In these cases, the cells may be transfected with the HCV genomic RNA transcript prior to performing the luciferase (or other reporter) assay.
It should be noted that while adaptation for growth and/or stability may be desirable for some studies and cell lines, the invention may also be practiced with directly derived (non-adapted) cell lines, for example, Huh7 cells transfected with an HCV genomic RNA transcript.
Media
Any media capable of supporting cell growth and replication of the HCV genomic RNA transcripts may be used. In general, the methods described in Lidenbach et al. (Science (2005) 309:623-626; herein incorporated by reference) were utilized for cell culture.
Other Factors
In addition, the tissue culture system may also include various other factors that improve viral replication or infectivity. For example, the tissue culture system may further comprise a polynucleotide encoding a double stranded RNA binding protein that acts as an interferon antagonist or RNA silencing suppressor. Viral titers can be improved by blocking host antiviral defense mechanisms that interfere with viral replication. One trigger of host defense mechanisms is viral double-stranded RNA, which is produced as an intermediate during viral replication. Double stranded RNA induces host interferon and RNA silencing antiviral responses. Suppressors of interferon and RNA silencing responses are produced by certain viruses. For example, the NS1 protein of influenza virus is a double stranded RNA binding protein that blocks activation of host interferon-induced dsRNA-dependent protein kinase PKR and RNA interference (RNAi). NS1 is required for efficient influenza virus replication and also improves HCV infectivity.
Thus, in certain embodiments, cells are transfected with a polynucleotide encoding an RNA silencing suppressor prior to transfection with HCV genomic RNA. A polynucleotide encoding NS1 from any influenza strain, or a fragment thereof that suppresses RNA silencing, can be used in the practice of the invention. The full-length NS1 protein contains a dsRNA binding domain, which inhibits PKR and RNAi, and an effector domain, which functions in inhibiting pre-mRNA splicing and nuclear export of mRNA. A representative NS1 gene comprises the sequence of SEQ ID NO:15. The dsRNA binding domain and the effector domain reside at amino acid residues 1-82 and 138-147, respectively. In addition, the NS1 protein comprises two nuclear localization signals (NLS1 and NLS2), a cleavage and polyadenylation specificity factor (CPSF) binding site, and a polyadenylation binding protein II (PABII) binding site.
In certain embodiments, a polynucleotide encoding an NS1 polypeptide comprising the amino acid sequence of SEQ ID NO 15 or a functional fragment thereof that increases the yield of viral particles is used in the practice of the invention. In certain embodiments, the polynucleotide encoding an NS1 polypeptide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:11-14.
In addition, the tissue culture system may include a polynucleotide encoding a CD81 polypeptide. Increasing the expression of the CD81 receptor on the surface of cells facilitates viral entry into cells (Pileri et al. (1998) Science 282:938-941; herein incorporated by reference). Cells can be transfected with a CD81 expression plasmid prior to transfection with HCV RNA to improve infectivity.
B. Applications
The HCV tissue culture system can be used to monitor replication, expression, or infectivity of HCV by detecting reporter gene activity from HCV genomic RNA transcripts. The HCV tissue culture system also provides a means for monitoring one or more essential steps in the life cycle of HCV by detecting or monitoring reporter gene activity present in HCV virus produced from cells containing the recombinant HCV genomic RNA transcript. The virus life cycle includes the steps required for a virus to enter into a cell, produce virus particles and exit the cell in order to infect other cells. These steps include binding to the cell surface, interacting with receptors on the surface which facilitate cell entry, uncoating the virus particle to release the viral genome, replication and expression of the viral genome, particle maturation including packaging the viral genome into a particle, egress through the cell and finally exiting the cell and the release of viral progeny. (See Fields Virology (1996) Chapter 10: Virus-Host Interactions D. Knipe).
In another aspect, the tissue culture system provides a model of HCV growth that may be used for evaluation and identification of inhibitors of HCV replication and infection, for pharmacokinetic (PK) and pharmacodynamic (PD), toxicity studies and rapid screening of anti-viral HCV candidate compounds.
In another aspect, the invention provides a method of identifying a compound having anti-HCV activity, i.e. a compound that reduces or inhibits the level of HCV replication, expression of HCV proteins, and/or viral infectivity. A compound can directly regulate replication by interfering with replication of the HCV genomic RNA transcript in the host cell or can indirectly regulate replication by interfering with expression of HCV proteins that are necessary for HCV replication in the host cell. Alternatively, as discussed below, the HCV tissue culture system provides a method of identifying compounds that have no effect on replication or expression per se, but instead reduce or inhibit virus particle formation.
Replication of an HCV genomic RNA transcript and/or viral infectivity titers can be measured by detecting reporter gene activity in cell cultures. In one embodiment, cells containing an HCV genomic RNA transcript further comprising a reporter gene, are contacted with one or more test compounds. The reporter gene is only replicated if the HCV genomic RNA transcript is replicated. As discussed herein, depending on the embodiment, expression of the reporter gene protein does not have to, but may optionally be linked to expression of the HCV polyprotein. Activity of the reporter gene contained within the HCV genomic RNA transcript in cells exposed to one or more test compounds is compared to activity of the reporter gene contained within the HCV genomic RNA transcript in cells that are not contacted with a test compound. A compound that reduces RNA replication, expression, and/or viral infectivity is a compound that may have use as a medicament for the regulation of HCV replication, expression, and/or infectivity in a human.
The HCV tissue culture system may be used to evaluate potential HCV antiviral compounds. In one embodiment, the hepatocyte cells, transfected with recombinant HCV genomic RNA transcripts, are treated with a compound known to reduce or prevent HCV replication and or particle formation and then the cells are treated with a second candidate compound to determine whether the second candidate compound has an additive or synergistic effect with the known compound in reducing or preventing HCV infection. The tissue culture system may also be used to measure replication, expression, and infectivity of HCV in culture as described herein.
Screening for Anti-Viral Compounds
The tissue culture system described herein can be used to identify compounds that have antiviral effects, for example, reduce replication, expression, or production of HCV in culture. In addition, combinations of compounds can be tested for synergistic or additive effects.
An “antiviral agent” or “antiviral compound” as used herein refers to an agent that is effective in reducing or inhibiting the formation and/or replication of a virus in a mammal. Antiviral agents as used herein are meant to include agents that act specifically on virus biology to reduce or inhibit viral replication. Antiviral agents can include agents targeted at interfering with one or more virus functions, including, but not limited to viral or host required enzymatic activity, viral binding, viral entry, viral packaging and viral infectivity. This group of compounds can include small molecules and antibodies and other biologics.
In the case of anti HCV agents, any antiviral agent that inhibits the replication or particle formation of HCV infectious virus is considered an antiviral agent and an anti-HCV virus agent. This includes agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of a virus in a mammal. Anti-HCV virus agents include, for example, ribavirin, amantadine, VX-497 (merimepodib, Vertex Pharmaceuticals), VX-498 (Vertex Pharmaceuticals), Levovirin, Viramidine, Ceplene (maxamine), XTL-001 and XTL-002 (XTL Biopharmaceuticals), MN283-Valopictitabine, Idenix). Antiviral agents can also include nucleic acids (e.g., siRNA and antisense RNA) or small molecules that may interfere with one or more enzymatic activities necessary for viral replication and packaging such as protease, helicase and polymerase functions. Anti-HCV virus agents can also include nucleic acids (e.g., siRNA and antisense RNA) or small molecules that may interfere with one or more enzymatic activities necessary for HCV replication and packaging such as protease, helicase and polymerase functions. Such nucleic acids can be provided in the form of anti-sense molecules, double stranded duplex DNA, siRNA and the like. Antiviral agents can include immunomodulatory compounds, either as non-specific or specific modulators of the immune system. Anti HCV agents can also include vaccines such as therapeutic and prophylactic vaccines against HCV and antibodies or small molecules which interfere with HCV receptor binding.
Candidate Compounds
Typical candidate compounds for use in all the screening methods of the invention include, but are not restricted to, peptides, peptoids, proteins, lipids, metals, small organic molecules, RNA aptamers, antibiotics and other known pharmaceuticals, polyamines, antibodies or antibody derivatives (e.g., antigen-binding fragments, single chain antibodies including scFvs, etc.), and combinations or derivatives thereof. Small organic molecules have a molecular weight of about more than 50 and less than about 2,500 daltons, and most preferably between about 300 and about 800 daltons. Candidate compounds may be derived from large libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from MayBridge Chemical Co. (Revillet, Cornwall, UK) or Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts may be used. Additionally, candidate compounds may be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures. Compounds can include antibodies or fragments of antibodies that retain the antibody antigen binding capacity.
Pharmacological Studies
The tissue culture system of the present invention may be used for pharmacokinetic, pharmacodynamic and toxicology studies of potential anti-viral compounds and protocols. The tissue culture system may also be used to monitor the course of HCV replication and also to determine the drug mechanism of action.
The tissue culture system may be used to identify and evaluate compounds or protocols that inhibit HCV replication. The tissue culture system may also be used to identify anti-viral compounds that may inhibit other steps in the viral life cycle (e.g., compounds that inhibit virus packaging or egress from the cell).
The tissue culture system is treated with a candidate compound or using a candidate protocol. Alternatively, the tissue culture system is treated with a combination of compounds already known or determined to be effective immunomodulators against HCV, such as, e.g., interferon-α. Anti-viral activity may be quantified and compared with a control or untreated tissue culture system, or quantified and used to determine the relative rankings of multiple candidates. The schedule of administration may be determined individually for each candidate compound. The schedule may be chosen to maintain an effective amount of test compound over a period of several days.
Anti-viral activity may be measured in several ways. A first method is using one or more of the reporter genes mentioned above (e.g. a luciferase assay technique). Reporter gene activity, or the amount of the reporter protein should correlate with the amount of HCV replicon replication. In addition to measuring the level of reporter protein, the efficacy of the candidate compound or protocol may be determined by measuring the level of HCV RNA or the level of viral proteins. Dose and/or time-dependent response curves (e.g., EC50, IC50) may be determined for candidate compounds and protocols.
In some embodiments, a second reporter gene may be stably or transiently transfected into the cell to allow the viability of cells to be assessed. If a test compound kills target cells then expression of the first reporter will not be seen, but because of cell death rather than because of any antiviral activity. Thus the second reporter acts as a marker for viability of cells containing the HCV genomic RNA transcript.
One suitable second reporter is a Renilla luciferase gene. Another method to determine this is to quantitate the gene copy number of an HCV gene on the HCV genomic RNA transcript, and a human gene contained in the hepatocyte cells (e.g., Huh7 or Huh7.5). Thus, by comparing the ratio of HCV gene copy number to human gene copy number in cells, it can be determined if treatment with an HCV inhibitor is inhibiting replication of the HCV genomic RNA transcript (HCV gene to human gene copy number ratio decreases) or if the compound is inhibiting human cell growth or inducing cell death in cells harboring the HCV genomic RNA transcript (HCV to human gene copy number ratio stays relatively constant). HCV gene and human gene copy number in cells is quantitated at day zero for the baseline and again at a later time or times.
Kits
The above-described tissue culture system, including the recombinant HCV genomic constructs, hepatocyte cells, and transfection reagents, can be provided in kits, with suitable instructions and other necessary reagents, in order to produce HCV virus as described above. The kit will normally contain in separate containers the combination of recombinant HCV genomic constructs (e.g., RNA transcripts for infection and/or DNA vectors for production of viral RNA by in vitro transcription) and hepatocyte cells (either already transfected or separate). Instructions (e.g., written, tape, VCR, CD-ROM, DVD, etc.) for growing the virus in tissue culture usually will be included in the kit. The kit can also contain other packaged reagents and materials (i.e., transfection reagents, polynucleotides encoding RNA silencing suppressors and/or CD81, antiviral compounds, buffers, media, and the like). Wild-type JFH1 and chimeric viruses, such as those described above, can be grown in culture using these kits.
EXPERIMENTAL
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Example 1
General Materials and Methods
Cells
Huh-7
Human hepatoma cell line Huh-7 (Cancer Res 42:3858-63) was maintained in Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Carlsbad, Calif.) containing high glucose, 10% fetal bovine serum, L-glutamine, and non-essential amino acids, at 37° C. in 5% CO2. Unless otherwise indicated, all cell culture in presence of HCV was performed as described and referenced herein.
Huh7C
Interferon-cured Huh7 (Huh7C) cells were prepared by treating Huh7 cells containing a subgenomic replicon with interferon-α at a concentration of 100 U/ml of culture for 14 days, as described previously by Kanazawa et al. (J. Virol. (2004) 78:9713-9720). Briefly, cured Huh7 cells, from which the replicon had been eliminated, were established by treating Huh7/Rep-Neo cells with 100 U of IFN-α/ml for 14 days. Clearance of replicon RNA was confirmed by reverse transcription-PCR (RT-PCR) and by the loss of resistance to G418.
T7-11
Five million Huh7 derived cells containing a subgenomic replicon in 0.2 ml of Hank's Balanced Salt Solution (HBSS) were implanted subcutaneously in female C.B17 SCID mice, which were irradiated (3 Gy) before cell implantation. A tumor emitting strong bioluminescence was excised 27 days later and the bioluminescent portion of the tumor was cultured in vitro in the presence of 0.5 mg/ml G418. Luciferase expression of G418-resistant colonies was evaluated using the Xenogen IVISTM imaging system (Xenogen Corporation, Alameda, Calif.) and one colony with high expression was expanded and cured of the subgenomic replicon to create the cell line T7-11.
Siena 8
Huh 5.2 cells carrying a subgenomic replicon (Lohmann et al. (1999) Science 285(5424):110-113) were treated with interferon alpha at 100 IU/ml for 3 weeks to cure HCV RNA and individual colony was isolated to create Siena 8 cells. Cells were maintained in the same condition described for Huh 7 cells above.
RNA Preparation
RNA transcripts were prepared using the MEGASCRIPT kit (Ambion, Austin, Tex.). RNA was extracted and purified using TRIZOL reagent following manufacturer's instructions (Invitrogen, Carlsbad, Calif.).
RNA Transfection
Subconfluent cells were trypsinized and washed once with complete DMEM and once with serum-free DMEM-F12 medium. Cell pellets were resuspended in serum-free DMEM-F12 medium at a density of 10 cells/ml. To 200 ml of the cell suspensions in an electroporation cuvette (0.2-cm gap; BTX, San Diego, Calif.) 1 to 10 mg of in vitro-transcribed RNA was added. The cells were immediately electroporated with an ECM 630 apparatus (BTX) set to 200 V and 1,000 mF. After electroporation the cell suspension was kept for 5 minutes at room temperature and then diluted into DMEM supplemented with 10% fetal bovine serum and nonessential amino acids and seeded into a 10-cm diameter petri dish.
Example 2
Recombinant JFH1 Genomic Bicistronic Constructs
Several wild-type and chimeric JFH1 genomic constructs were prepared (see FIG. 1). A recombinant wild-type JFH1 genomic construct contained within a pUC18 plasmid as shown in the sequence of SEQ ID NO:1 was prepared as described previously (Wakita et al. (2005) Nature Medicine 11:791-796; herein incorporated by reference in its entirety). This construct includes open reading frames for HCV proteins core, E1, E2, p7, NS2, NS3, NS4A, NS4b, NS5a, and NS5b, flanked by 5′ and 3′ untranslated regions, and a T7 promoter.
In addition, a bicistronic wild-type JFH1 genomic construct (LucJFH1) comprising the sequence of SEQ ID NO:2 was generated that included a first cistron encoding the first 17 amino acids of the JFH1 core protein linked to a luciferase reporter gene for monitoring viral infection and RNA replication. This bicistronic construct also included an encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) for expression of the second cistron, which encoded the wild-type JFH1 polyprotein.
A number of bicistronic chimeric constructs were also prepared encoding chimeric HCV polyproteins comprising sequences derived from JFH1 and a second HCV isolate. All bicistronic constructs contained a first cistron encoding the first 17 amino acids of the JFH1 core protein linked to a luciferase reporter gene, an EMCV IRES, and a second cistron, encoding a chimeric polyprotein comprising NS3 to NS5b regions from strain JFH1 and core to p7 regions from a second HCV isolate, linked by a chimeric NS2 region with an intergenotypic junction at residue 871 (LucCJ1 and LucHJ1) or 877 (LucNJ1) of NS2. Chimeric constructs included: (a) LucHJ1, contained in the sequence of SEQ ID NO:3, containing sequences from HCV type 2a strain JFH1 and type 1a strain (HJ1 fragment from the H77 isolate); (b) LucCJ1, contained in the sequence of SEQ ID NO:4, containing sequences from HCV type 2a strain JFH1 and type 1b strain (CJ1 fragment from the Con1 isolate); and (c) LucNJ1, contained in the sequence of SEQ ID NO:5, containing sequences from HCV type 2a strain JFH1 and type 1b strain (NJ1 fragment from the HNZ1 isolate).
Example 3
Recombinant JFH1 Genomic Monocistronic Constructs
Several monocistronic wild-type and chimeric JFH1 genomic constructs were also prepared (see FIG. 2). Monocistronic constructs included a region encoding the first 12 amino acids of the JFH1 core protein linked to a green fluorescent protein (GFP) or a Renilla luciferase (RLuc) reporter gene, a coding sequence for a foot-and-mouth disease virus (FMDV) 2A protease, a coding sequence for a ubiquitin protease cleavage site, and a wild-type JFH1 or chimeric polyprotein coding region. In some of these chimeric constructs, the polyprotein coding region comprised NS3 to NS5b sequences derived from strain JFH1 and core to p7 sequences derived from a second HCV isolate, linked by a chimeric NS2 coding region with an intergenotypic junction at residue 871 (RLF2AUba1 and RLF2AUbHJ1) or 877 (RLF2AUbNJ1) of NS2. Monocistronic constructs included plasmids encoding chimeric DNA constructs of the invention as follows: (a) RLF2AubJFH-1, as in SEQ ID NO:6, containing the wild-type JFH1 genome and an RLuc reporter gene; (b) GFPF2AUbJFH-1, comprising the sequence of SEQ ID NO:7, containing the wild-type JFH1 genome and a Green Fluorescent Protein (GFP) reporter gene and pUC plasmid sequences; (c) RLF2AUbHJ1, comprising the sequence of SEQ ID NO:8, containing sequences from HCV type 2a strain JFH1 and type 1a strain HJ1 and an RLuc reporter gene and pUC plasmid sequences, wherein the genomic construct includes from about nucleotide 7 to about nucleotide 10953 of SEQ ID NO:8; (d) RLF2AUbCJ1, comprising the sequence of SEQ ID NO:9, containing sequences from HCV type 2a strain JFH1 and type 1b strain CuI and an RLuc reporter gene and plasmid sequences, wherein the genomic construct includes from about nucleotide 7 to about nucleotide 10953 of SEQ ID NO:9; and (e) RLF2AUbNJ1, comprising the sequence of SEQ 1:13 NO:10, containing sequences from HCV type 2a strain JFH1 and type 1b strain NJ1 and an RLuc reporter gene and plasmid sequences, wherein the genomic construct includes from about nucleotide 7 to about nucleotide 10971 of SEQ ID NO:10.
Pietschmann et al. have demonstrated that bicistronic chimeric constructs containing an NS2 fusion protein with an intergenotypic junction between the first and second putative transmembrane domains of the NS2 protein yielded productive virus particles (Proc. Natl. Acad. Sci. U.S.A. (2006) 103:7408-7413).
FIGS. 12 and 13, show the restriction enzyme sites that were utilized to make the described recombinant HCV genomic constructs. In particular, FIG. 12 shows JFH-1 and Luc JFH-1, two recombinant HCV genomes that were synthesized using phosphodiester synthesis. The HCV genomic constructs were cloned into the EcoRI/XbaI restriction enzyme sites of the plasmid PUC 18. Cloning was performed in E. coli as described in the figures and accompanying text.
Referring now to FIG. 13, the JFH-1 strain and the compatible restriction enzyme fragments from each HCV genome and the HCV genome containing the luciferase gene are shown. Compatible fragments were cloned into pUC vectors and transformed into E. coli. As indicated above, the full length JFH1 and JFH1 having reporter gene genomic constructs were synthesized in vitro and cloned into the EcoRI/Xba1 site of pUC18. The chimeric constructs were cloned using conventional restriction enzyme digestion, ligation and transformation of E. coli utilizing the restrictions sites depicted in FIGS. 1-3, 12, and 13. In each of the genomic constructs shown, the genomic construct is bordered by EcoR1 and Xba1 sites. Any other internal Eco R1 or Xba1 sites can be removed without altering the expression of any corresponding products.
Example 4
Renilla Luciferase Assay
Replication of viral RNA produced from recombinant genomic RNA transcripts can be measured by using a Renilla luciferase (RLuc) assay (see, e.g., Srikantha et al. (1996) J. Bacteriol. 178:121-129; Liu et al. (1999) Gene 237:153-159; Haan et al. (2004) J. Virol. 78:6048-6054; Kiel et al. (2003) J. Biol. Chem. 278:5659-5668; herein incorporated by reference). Luminescence is measured at 480 nm with a luminometer. Light emission is integrated over 10-30 seconds. RLuc assays were used to measure intracellular HCV RNA in cell lysates and infectious viral titers in culture media. RLuc activity was measured using cell lysates to measure the level of HCV replication in cells. To measure the titer of infectious virus titer present in the culture supernatant, conditioned media from the transfected or infected cells were inoculated into naïve Huh7 cells and RLuc activity from the infected cells was measured.
Example 5
Comparison of RNA Replication Efficiencies of Monocistronic and Bicistronic Recombinant Wild-Type JFH1 Genomic RNA Transcripts
RNA replication efficiencies of monocistronic and bicistronic wild-type JFH1 genomic RNA transcripts were compared. RLF2AUbJFH-1 and LucJFH1 genomic RNA transcripts were introduced into separate cultures of Huh7 cells by electroporation. RNA levels were measured for 33 days after electroporation by quantitating luminescence from luciferase reporters.
FIG. 3 shows that the replication efficiency of the monocistronic RLF2AUbJFH-1 RNA transcript was superior to the bicistronic LucJFH1 RNA transcript. Although the replication of the bicistronic RNA transcript was comparable to the monocistronic RNA transcript initially, replication declined after 9 days, whereas the monocistronic RNA transcript maintained replication at higher levels for 33 days.
Example 6
Infectivity and RNA Replication Efficiencies of HCV Viruses Produced by the Monocistronic Recombinant Genomic RNA Transcripts
Monocistronic HCV genomic constructs were evaluated for their ability to produce infectious viral particles and to replicate viral RNA in Huh7 cells. The infectivity of virus produced by recombinant genomic RNA transcripts in transfected Huh7 cells was determined by inoculating naive Huh7 cells with virus released into the supernatants of cultures of transfected cells. Infectious viral titers and RNA replication levels were determined by RLuc assays as described in Example 4.
A. Recombinant Wild-Type JFH1 Genomic Monocistronic Construct
The recombinant wild-type JFH1 genomic monocistronic RLF2AUbJFH-1 RNA transcript was introduced into Huh7 cells by electroporation. Virus present in the culture supernatant after electroporation was then used to infect 5×104 naive Huh7 cells, and infectious viral titers were measured by RLuc assay for 28 days after electroporation. As shown in FIG. 4, the RLF2AUbJFH-1 RNA transcript produced viral particles that were secreted from Huh7 cells and infectious in naive Huh7 cells.
The level of RNA replication of RLF2AUbJFH-1 was also measured in Huh7 cells as a function of time after infection with recombinant virus. Naive Huh7 cells (5×104) were inoculated with virus from the culture supernatant of transfected cells 10 days after electroporation. Intracellular JFH1 RNA was measured by RLuc assay for 24 days after infection of Huh7 cells. As shown in FIG. 5, RNA was detectable by day 2 and persisted for 24 days.
B. Recombinant Chimeric Genomic Monocistronic Constructs
The infectivity of wild-type JFH1 virus, produced by the monocistronic construct RLF2AUbJFH-1, and chimeric viruses, produced by the monocistronic constructs RLF2AUbHJ1, RLF2AUbCJ1, and RLF2AUbNJ1, were compared. Cultures of naive Huh7 cells were separately inoculated with each virus from culture supernatants of transfected cells after electroporation with genomic RNA transcripts. Viral titers were measured by RLuc assay. FIG. 6 shows that the recombinant wild-type JFH1 virus produced from the monocistronic RLF2AUbJFH-1 RNA transcript and the chimeric virus produced from the RLF2AUbCJ1 RNA transcript produced infectious virus in Huh7 cells.
RNA replication efficiencies of the wild-type JFH1 and chimeric genomic monocistronic constructs were also compared. RLF2AUbJFH-1, RLF2AUbHJ1, RLF2AUbCJ1, and RLF2AUbNJ1 RNA transcripts were introduced into separate cultures of Huh7 cells by electroporation. Cultures of the Huh7 cells were split three days and twelve days after electroporation, and RNA levels were measured by RLuc assay for 21 days after electroporation. A replication incompetent JFH-1 mutant (JFH1/GND mutant previously described by Wakita et al., supra) was used as a negative control for comparison.
As shown in FIG. 7, all of the recombinant constructs replicated in Huh7 cells and continued to replicate after culture dilutions. The replication efficiencies of the chimeric RLF2AUbCJ1 and RLF2AUbNJ1 constructs were nearly as high as the wild-type RLF2AUbJFH-1 construct. The chimeric RLF2AUbHJ1 construct, however, showed significantly lower replication levels than the other recombinant constructs.
Example 7
RNA Replication Efficiencies of HCV Viruses in Different Cell Lines
The RNA replication efficiencies of JFH-1 in the three cell lines, Siena 8, Huh7C, and Huh7 were compared (FIG. 8). 5×106 cells were transfected with either 1 μg or 4 μg of the genomic RNA transcript RLF2aUbJFH-1. Rluc was measured 3 days after electroporation in cell lysates.
Example 8
Influenza Virus NS1 Constructs for RNA Silencing Suppression
JFH1 is a type 2a virus that replicates efficiently and produces infectious virus in cell culture. Infectivity titers of JFH1 in Huh7.5 cells are limited to about 104/ml, and titers of chimeric viruses, comprising genotype's other than 2a, are significantly lower (about 103/ml). Viral titers can be improved by blocking host antiviral defense mechanisms that interfere with viral replication.
One trigger of host defense mechanisms is viral double-stranded RNA, which is produced as an intermediate during viral replication. Double stranded RNA induces host interferon and RNA silencing antiviral responses. Suppressors of interferon and RNA silencing responses are produced by certain viruses. For example, the NS1 protein of influenza virus is a double stranded RNA binding protein that blocks activation of host interferon-induced dsRNA-dependent protein kinase PKR and RNA interference (RNAi). NS1 is required for efficient influenza virus replication. The ability of NS1 to also improve HCV infectivity titers was therefore tested.
Several RNA silencing suppressors comprising full-length and truncated forms of NS1 were constructed (FIG. 9). A representative NS1 gene can be found at GenBank accession number AAA43536. Other influenza strains available, or fragments thereof, can also be used in the practice of the invention. The full-length NS1 protein contains a dsRNA binding domain, which inhibits PKR and RNAi, and an effector domain, which functions in inhibiting pre-mRNA splicing and nuclear export of mRNA. The dsRNA binding domain and the effector domain reside at amino acid residues 1-82 and 138-147, respectively. In addition, the NS1 protein comprises two nuclear localization signals (VLSI and NLS2), a cleavage and polyadenylation specificity factor (CPSF) binding site, and a polyadenylation binding protein II (PABII) binding site.
Four constructs were prepared, including (i) the full-length wild-type (wt) NS1 (residues 1-230), comprising about nucleotide 2033 to about nucleotide 2723 of SEQ ID NO: 11; (ii) NS82, a truncated NS1 protein containing the dsRNA binding domain (residues 1-82 of Seq ID No. 15); (iii) NS180, a longer truncated NS1 protein containing the dsRNA binding domain and the effector domain, comprising residues 1-180 of SEQ ID NO:15; and (iv) a mutant NS1 protein defective in dsRNA binding containing a deletion of residues 50-84 of SEQ ID NO:15. Constructs were each cloned into a pCMV vector for expression and the constructs are represented by SEQ ID NOS:11-14.
Example 9
Effects of Influenza Virus NS1 and NS82 on Replication of JFH1
The effects of NS1 and NS82 expression on JFH1 replication in Huh7 cells were tested. Interferon-cured Huh7 cells were transfected with pCMV-NS1 or pCMV-NS82, followed by electroporation with monocistronic JFH1 RNA. The level of JFH1 RNA replication in the electroporated cells was measured by (RLuc) assay on cell lysates. As shown in FIG. 10, the level of JFH1 replication peaked on day 3 after electroporation. The level of replication in NS1 transfected cells was about 3-fold higher than that of mock-transfected cells. High levels of JFH1 replication were maintained for up to 5 days after electroporation in cells transfected with NS82, whereas replication levels decreased in cells transfected with wild-type NS1.
Example 10
Effects of Influenza Virus NS1 and NS82 on Infectivity of JFH1
JFH1 was produced from interferon-cured Huh7 cells by first transfecting cells with pCMV-NS1 (SEQ ID NO:11) or pCMV-NS82 (SEQ ID NO:12) and then electroporating cells with the monocistronic RLF2AUbJFH-1 RNA. Virus replication in electroporated cells was measured. Virus present in the culture supernatant was then used to infect naive Huh7 cells, and the level of infectious virus titer was measured by RLuc assay. As shown in FIG. 11, expression of NS82 (transfected with 1.8 μg of pCMV-NS82) prior to JFH1 electroporation of IFN-cured cells increased the infectious JFH1 titer by about 8-fold. The NS1 gene was cloned into the pCMV plasmid as an Asc1-Not1 fragment.
Example 11
Effects of CD81 on Replication and Infectivity of JFH1 in Interferon Cured Huh7 and Naive Huh7 Cells
The effects of NS1 on JFH1 replication and infectivity were compared in interferon cured Huh7 cells (Huh7C) and naïve Huh7 cells. Expression of a portion of the NS1 coding region prior to viral RNA transfection resulted in a significant increase in infectious JFH1 titers in Huh7C cells compared to naïve Huh7 cells. Huh7C cells were more permissive for JHF1 replication compared to naïve Huh7 cells, but the infectivity of JFH1 in Huh7C cells was severely compromised.
The CD81 level on the Huh7C cell surface was about 1/10 that of Huh7 cells. In order to verify the role of CD81 and improve JFH1 infectivity, a variety of cell lines derived from Huh7 were transfected with a CD81 expression plasmid (Pileri et al. (1998) Science 282:938-941; herein incorporated by reference). CD81 was required for infection and improved JFH1 infectivity, but over-expression of CD81 beyond a threshold level inhibited JFH1 replication. Huh7C was optimal for supporting replication but was suboptimal for the infectivity assay. Huh7 was optimal for infection.
Example 12
Assays Using HCV Produced in Cell Culture
Culture Medium
Huh7.5 cells were grown in a culture medium containing 1 liter of Dulbecco's Modification of Eagle's Medium (DMEM) with 4.5 g/L glucose and L-glutamine without sodium pyruvate (Cellgro, Catalog No. 10-017-CN), 10 ml of penicillin-streptomycin solution (10,000 I.U. penicillin/ml and 10,000 μg streptomycin/ml; Cellgro, Catalog No. 30-002-CI), 10 ml of nonessential amino acids (Cellgro, Catalog No. 25-025-CI) and 100 ml of undialized fetal bovine serum (FBS).
Infection and Neutralization
Day 0:
8×104 to 10×104 Huh7.5 cells (for luciferase assay) or 5×104 Huh7.5 cells (for indirect fluorescent antibody assay) were added per well to a 24-well plate.
Day 1:
The Huh7.5 cells were infected with HCV virus. The viral medium was warmed to 37° C. before adding to the wells. The culture medium in the wells was replaced with about 200 μl of the viral medium per well, and the 24-well plate was then placed in a CO2 incubator and incubated overnight.
For studies of antibody neutralization, anti-HCV antibodies or serum containing anti-HCV antibodies were diluted as appropriate and added to the viral medium. The mixture was incubated at 37° C. for one hour and added to the cells at 200 μl per well. The cells were incubated in a CO2 incubator for five hours (for RJ1 or JFH1) or overnight (for RCJ1 or HJ1). In some cases, the viral medium was removed (e.g., if RJ1 was used) and washed with fresh culture medium twice and replaced with 500 μl of fresh culture medium per well.
Day 2:
The viral medium was removed and washed twice and replaced with 500 μl of fresh culture medium.
Day 3:
The medium was replaced with fresh culture medium
Day 4:
Luciferase and WA assays were performed on HCV produced in cell culture (HCVcc). See FIG. 14 for a schematic diagram of assaying viral neutralization of HCV infected hepatocytes grown in cell culture.
Luciferase Assay
Luciferase assays were performed using the Renilla luciferase assay system (Promega, Catalog No. E2820). The 24-well plate was washed twice with 500 μl phosphate buffered saline (PBS) per well, and the PBS was removed as completely as possible. 200 μl of 1× lysis buffer (Promega) was added per well, and the plate was shaken at room temperature for over one hour. 100 μl aliquots of luciferase assay buffer (Promega) mixed with the Renilla luciferase assay substrate (Promega) were added to luminometer cuvettes. 50 μl of lysate was added to the 100 μl of assay buffer in each cuvette and mixed. Luciferase activity was measured with a MOONLIGHT 3010 luminometer.
Indirect Fluorescent Antibody Assay
Indirect fluorescent antibody (IFA) assays were performed using the CYTOFIX/CYTOPERM kit for fixation and permeabilization of cells and immunofluorescent staining (BD Biosciences, Catalog No. 554714). The plate was washed twice with 500 μl PBS per well. The PBS was removed and replaced with 1× cytofixation/permeabilization solution (BD Biosciences) for overnight fixation. The plate was washed three times with PBS or 1× cytowash/permeabilization solution (BD Biosciences). 1× cytowash/permeabilization solution containing a blocking reagent was added and incubated at room temperature for about 30 minutes to 1 hour. The blocking reagent was removed and 150-200 μl of the primary antibody (rabbit anti-NS5A diluted by 1:1000 in 1× cytowash/permeablization solution) was added per well. The plate was incubated at room temperature for 1 hour and washed three times with 1× cytowash/permeabilization solution (BD Biosciences) before adding the secondary antibody (ALEXA FLUOR 488-conjugated goat-anti-rabbit antibody diluted 1:1000 in 1× cytowash/permeabilization solution). The plate was incubated at room temperature for about one hour and then washed twice with PBS. The PBS was removed and the wells were partially air dried and sealed. The plate was stored at −20° C. until observation under a fluorescence microscope.
Example 13
IFA Assay of HCV Infection in Huh7.5 Cells
The IFA assay was performed on Huh7.5 cells infected with JFH-1 as described in Example 12. Huh7.5 cells were transfected with either 2 μg or 4 μg of the RLF2AUbJFH-1 RNA genomic transcript by electroporation. Cells were fixed and immunostained with anti-NS5a antibody at either day 3 or 16 after infection with JFH1. Viral titers were measured for 25 days after electroporation. As shown in FIG. 16, the synthetic JFH1 genome replicated in Huh7.5 cells and secreted infectious viral particles at high viral titers. By day 16 after electroporation, about 90% of the Huh7.5 cells were infected with virus.
Example 14
Effects of CD81 on Infectivity of JFH1 in Huh7 and Huh7.5 Cells
The effect of CD81 on JFH1 infectivity was compared in Huh7 and Huh7.5 cells. CD81 is expected to improve infectivity by increasing the expression of the CD81 receptor on the surface of cells, which facilitates viral entry into cells. Huh7 and Huh7.5 cells were transfected with a CD81 expression plasmid (Pileri et al., supra) prior to viral RNA transfection. As shown in FIG. 18, both Huh7 and Huh7.5 cells showed significant increases in infectious JFH1 titers in the presence of CD81 compared to naive cells.
Example 15
Selection of Anti-HCV Antibodies Using Cell Culture System
The HCV cell culture system was used to select neutralizing antibodies against HCV. HCV viruses were produced from RHJ1 (type 1a H77C:type 2a JFH1 chimera), RCJ1 (type 1b con1:type 2a JFH1 chimera), and RJ1 (type 2a JFH1) RNA genomic transcripts (see FIG. 17) in cell culture. Anti-HCV human monoclonal antibodies, HCE6 or S3.1, were tested in neutralization assays as described previously in Example 12. The antibodies were mixed with viral medium prior to infection and added to cells at concentrations of 50, 10, 2, 0.5, or 0.1 μg/ml. As shown in FIG. 19, the HCE6 monoclonal antibody showed superior neutralization of HCV compared to S3.1 for all of the HCV viruses tested, approaching nearly 100% HCV neutralization at the highest antibody concentrations (i.e., 10 and 50 μg/ml) for the JFH1 and the type 1a H77C:type 2a JFH1 chimera and about 90% neutralization at the highest antibody concentration (i.e., 50 μg/ml) for the type 1b con1:type 2a JFH1 chimera.
Example 16
Correlation Between Antibody Neutralization in Cell Culture Assay and Protection Against HCV Infection in an Animal Model
Plasma samples were collected from four chimpanzees (Pan troglodytes; L357, L534, L559, and L470) vaccinated with HCV-1 E1 and E2 proteins, as described previously (Proc. Natl. Acad. Sci. U.S.A. (1994) 91:1294-1298, herein incorporated by reference). Three of the vaccinated chimpanzees, L357, L534, and L559 were completely protected from subsequent HCV infection. One of the chimpanzees, L470, showed partial protection, that is, vaccination significantly retarded infection and disease development. A plasma sample, collected from a fifth unvaccinated chimpanzee, was used as a control.
Plasma samples were tested for the presence of neutralizing antibodies by using the cell culture assay described in Example 12. In FIG. 20, results are shown for neutralization of the type 1a H77C:type 2a JFH1 chimeric virus grown in cell culture. Plasma from the protected chimpanzees, L357, L534, and L559 neutralized 80% to 88% of the virus, whereas plasma from the partially protected chimpanzee, L470, neutralized 46% of the virus, and neutralization was undetectable for the control. These results indicate that viral neutralization in the cell culture assay correlates well with actual protection from HCV infection. For comparison, the results of previous work on the neutralizing titers of the same chimp plasma as assayed against an HCV type 4a pseudoparticle (HCVpp), CD81 blocking antibody titers, and anti-E1E2 antibody titers are also shown.
In FIG. 21, results are shown for neutralization of the type 2a JFH1 virus grown in cell culture. Plasma from the protected chimpanzees, L357, L534, and L559 neutralized 53% to 69% of the virus, whereas plasma from the partially protected chimpanzee, L470, neutralized 28% of the virus. Thus, neutralizing titers are lower against the type 2a virus than the type 1a virus. The results of the cell culture assay correlate with cross-protection observed for HCV types 1a and 2a.
Thus, wild-type JFH1 and chimeric monocistronic and bicistronic genomic constructs, and methods of using the constructs for production of HCV in tissue culture are described. In addition, a neutralization assay using HCV grown in cell culture is described. Neutralization titers measured by this assay have been found to correlate well with actual protection from HCV infection in vaccinated animals. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined herein. All references cited herein are hereby incorporate by reference in their entirety.

## Claims

1. A recombinant hepatitis C virus (HCV) monocistronic genomic construct comprising in 5′ to 3′ order:
a) an HCV 5′ untranslated region, or a functional portion thereof;
b) a polynucleotide comprising a coding sequence for an N-terminal fragment of the JFH1 core protein, wherein said fragment comprises at least the first 12 residues and up to the first 18 residues of the JFH1 core protein, wherein the coding sequence for the N-terminal fragment of the JFH1 core protein is linked to a reporter gene;
c) a coding sequence for a foot-and-mouth disease virus (FMDV) 2A protease;
d) a coding sequence for a ubiquitin protease cleavage site;
e) a polynucleotide encoding an HCV polyprotein comprising core, E1, E2, and p7 regions from an HCV strain or isolate other than JFH1, a chimeric NS2 region comprising an N-terminal portion of NS2 from an HCV strain or isolate other than JFH1 and a C-terminal portion of NS2 from JFH1, and NS3, NS4a, NS4b, NS5a, and NS5b regions from JFH1; and
f) an HCV 3′ untranslated region, or a functional portion thereof.

2. The recombinant HCV genomic construct of claim 1, comprising a polynucleotide selected from the group consisting of:
a) a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10953 of SEQ ID NO:8;
b) a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10953 of SEQ ID NO:9;
c) a polynucleotide comprising the contiguous sequence from nucleotide 7 to nucleotide 10971 of SEQ ID NO:10;
d) a polynucleotide comprising the contiguous sequence from nucleotide 35 to nucleotide 9723 of SEQ ID NO:16
e) a polynucleotide complementary to a polynucleotide of a)-d); and
f) an RNA equivalent of a)-e).

3. The recombinant HCV genomic construct of claim 1, wherein the HCV strain or isolate other than JFH1 is selected from the group consisting of HCV 1a, 1b, 2a, 2b, 3a, 3b, 4, 5, and 6.

4. The recombinant HCV genomic construct of claim 3, wherein the HCV strain or isolate is H77C, Con1, NZ1, or 452.

5. The recombinant HCV genomic construct of claim 1, wherein the reporter gene encodes a firefly luciferase, Renilla luciferase, or, green fluorescent protein.

6. A vector comprising the recombinant HCV genomic construct of claim 1.

7. The vector of claim 6, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:8-10 and SEQ ID NO:16.

8. The vector of claim 6, further comprising a T7 promoter, wherein the T7 promoter is operably linked to the polynucleotide encoding an HCV polyprotein.

9. An HCV genomic RNA transcript comprising the recombinant HCV genomic construct of claim 1.

10. A tissue culture system for production of infectious HCV particles comprising a cell capable of supporting HCV infection transfected with the HCV genomic RNA transcript of claim 9.

11. The tissue culture system of claim 10, wherein the cell is a hepatocyte.

12. The tissue culture system of claim 10, further comprising a polynucleotide encoding an RNA silencing suppressor.

13. The tissue culture system of claim 12, wherein the RNA silencing suppressor is an influenza virus NS1 polypeptide or a functional fragment thereof that increases the yield of viral particles.

14. The tissue culture system of claim 13, wherein the NS1 polypeptide comprises the amino acid sequence of SEQ ID NO 15 or a functional fragment thereof that increases the yield of viral particles.

15. The tissue culture system of claim 13, wherein the polynucleotide encoding the RNA silencing suppressor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:11-14.

16. The tissue culture system of claim 10, further comprising a polynucleotide encoding a CD81 polypeptide.

17. The tissue culture system of claim 11, wherein the hepatocyte cell is derived from human tissue.

18. The tissue culture system of claim 17, wherein the hepatocyte cell is a hepatocellular carcinoma cell.

19. The tissue culture system of claim 17, wherein the hepatocyte cell is from a cell line selected from the group consisting of Huh7, Huh7.5, Siena 8, Huh7C and T7-11.

20. The tissue culture system of claim 11, wherein the hepatocyte cell is interferon-cured.

21. The tissue culture system of claim 11, wherein replication of the HCV genomic RNA transcript is maintained for at least 10 days after the HCV genomic RNA transcript is introduced into the cell.

22. The tissue culture system of claim 21, wherein replication of the HCV genomic RNA transcript is maintained for at least 20 days after the HCV genomic RNA transcript is introduced into the cell.

23. The tissue culture system of claim 22, wherein replication of the HCV genomic RNA transcript is maintained for at least 30 days after the HCV genomic RNA transcript is introduced into the cell.

24. The tissue culture system of claim 11, wherein infectivity of virus produced from the HCV genomic RNA transcript is maintained for at least 9 days after the HCV genomic RNA transcript is introduced into the cell.

25. The tissue culture system of claim 24, wherein infectivity of virus produced from the HCV genomic RNA transcript is maintained for at least 15 days after the HCV genomic RNA transcript is introduced into the cell.

26. A method for producing HCV in tissue culture, comprising transfecting a cell with the HCV genomic RNA transcript of claim 9 and growing said cell in culture media under conditions suitable for replication of the construct and secretion of infectious viral particles into said culture media.

27. The method of claim 26, further comprising transfecting said cell with a polynucleotide encoding an RNA silencing suppressor prior to transfecting the cell with the HCV genomic RNA transcript.

28. The method of claim 27, wherein the RNA silencing suppressor is an influenza virus NS1 polypeptide or a functional fragment thereof that increases the yield of viral particles.

29. The method of claim 28, wherein the NS1 polypeptide comprises the amino acid sequence of SEQ ID NO 15 or a functional fragment thereof that increases the yield of viral particles.

30. The method of claim 28, wherein the polynucleotide encoding the RNA silencing suppressor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:11-14.

31. The method of claim 26, further comprising a polynucleotide encoding a CD81 polypeptide.

32. The method of claim 26, wherein the cell is a hepatocyte cell is derived from human tissue.

33. The method of claim 32, wherein the hepatocyte cell is a hepatocellular carcinoma cell.

34. The method of claim 32, wherein the hepatocyte cell is from a cell line selected from the group consisting of Huh7, Huh7.5, Siena 8, Huh7C and T7-11.

35. The method of claim 32, wherein the hepatocyte cell is interferon-cured.

36. A chimeric hepatitis C virus produced by the method of claim 26.

37. A chimeric hepatitis C virus comprising the HCV genomic RNA transcript of claim 9.

38. A kit comprising a cell capable of supporting HCV infection and the vector of claim 7.

39. A kit comprising a cell capable of supporting HCV infection and the HCV genomic RNA transcript of claim 9.

40. A method of identifying a compound having anti-HCV activity, the method comprising:
a) treating the tissue culture system of claim 10 with a compound;
b) comparing HCV replication, expression of HCV proteins, or viral infectivity in said culture system treated with said compound to said tissue culture system not treated with said compound, wherein a reduction in the level of HCV replication, expression of HCV proteins, or viral infectivity in the tissue culture system treated with said compound is indicative of a compound with anti-HCV activity.

41. A method of identifying an antibody having anti-HCV activity, the method comprising:
a) treating the tissue culture system of claim 10 with the antibody;
b) comparing HCV replication, expression of HCV proteins, or viral infectivity in said culture system treated with said compound to said tissue culture system not treated with the antibody, wherein a reduction in the level of HCV replication, expression of HCV proteins, or viral infectivity in the tissue culture system treated with said compound is indicative of an antibody with anti-HCV activity.

42. A method for producing HCV in tissue culture, comprising transfecting a cell with the HCV genomic RNA transcript of claim 9 and growing said cell in culture media under conditions suitable for replication of the HCV genomic RNA transcript and secretion of infectious viral particles into said culture media, wherein the transfected cell expresses CD81 and an influenza NS1 polypeptide.

43. The method of claim 42, wherein the NS1 polypeptide comprises the amino acid sequence of SEQ ID NO 15 or a functional fragment thereof that increases the yield of viral particles.

44. The method of claim 42, wherein the transfected cell comprises a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:11-14.

45. The method of claim 42, wherein the cell is a hepatocyte cell and is derived from human tissue.

46. The method of claim 45, wherein the hepatocyte cell is a hepatocellular carcinoma cell.

47. The method of claim 45, wherein the hepatocyte cell is from a cell line selected from the group consisting of Huh7, Huh7.5, Siena 8, Huh7C and T7-11.

48. The method of claim 45, wherein the hepatocyte cell is interferon-cured.