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WO2004005524A1 - Hepatitis c virus particle formation - Google Patents

Hepatitis c virus particle formation Download PDF

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Publication number
WO2004005524A1
WO2004005524A1 PCT/US2003/020409 US0320409W WO2004005524A1 WO 2004005524 A1 WO2004005524 A1 WO 2004005524A1 US 0320409 W US0320409 W US 0320409W WO 2004005524 A1 WO2004005524 A1 WO 2004005524A1
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WIPO (PCT)
Prior art keywords
hcv
particles
nucleic acid
sequence
recombinant nucleic
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PCT/US2003/020409
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French (fr)
Inventor
Steve W. Ludmerer
Osvaldo A. Flores
Don Graham
Bohdan S. WOLANSKI
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Merck & Co., Inc.
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Publication of WO2004005524A1 publication Critical patent/WO2004005524A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24223—Virus like particles [VLP]

Definitions

  • HCV Hepatitis C virus
  • HCV-infected patients have been found to produce virions having a diameter of about 45-50 nm. (Bosman, et al, 1998. Res. Virol, 149, 311-314.) Virions isolated from a natural infection typically are coated with antibodies and Hpids, making structural determinations difficult and imprecise.
  • Virus-like particles have been observed upon infection with HCV in some cells lines such as TOFE, HPBMalO-2, HPBALL and Daudi. (Serafino, et al, 1997. Res. Virol. 148, 153-159, Shimizu, et al, 1996. Hepatology 23, 205-209.) The morphology of the particles varied among experimental protocols, and the yields of particles were low.
  • the HCV genome consists of a single strand RNA about 9.5 kb in length, encoding a precursor polyprotein about 3000 amino acids. (Choo, et al, 1989. Science 244, 362-364, Choo, et al, 1989. Science 244, 359-362, Takamizawa, et al, 1991. J. Virol. 65, 1105-1113.)
  • the HCV polyprotein contains the viral proteins in the order: C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
  • HCV structural proteins can be expressed in cultured cells from recombinant nucleic acid encoding the proteins.
  • HCV Core proteins with a molecular weight of 21 kDa (p21) and 23 kDa (p23) were observed in CHO cells using a vaccinia virus expression system encoding HCV structural proteins.
  • the p21 Core protein was suggested to be a component of native virions. (Yasui, et al, 1998.
  • the non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the virus replication machinery and are released from the polyprotein.
  • a zinc-dependent protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3.
  • RNA stimulated NTPase and helicase activities are located in the C-terminal domain of NS3.
  • NS4A provides a cofactor for NS3 protease activity.
  • NS5A is a highly phosphorylated protein conferring interferon resistance.
  • De Francesco, et al 2000. Semin Liver Dis., 20(1), 69-83, Pawlotsky, 1999. J. Viral Hepat. Suppl. 1, 47-48.
  • NS5B provides an RNA-dependent RNA polymerase.
  • the present invention features methods for producing HCV particles and measuring the ability of a compound to inhibit HCV particle formation or function.
  • Preferred methods involve the production of HCV particles in Vero cells using recombinant nucleic acid encoding for at least HCV C-E1-E2-P7-NS2.
  • Reference to "HCV particles” indicates virus-like particles that contain HCV structural proteins and are observable under electron microscopy. The particle may or may not contain HCV non-structural proteins.
  • a first aspect of the present application features a method of making HCV particles.
  • the method can be performed by incubating Vero cells containing a recombinant nucleic acid that comprises an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence under conditions suitable for producing HCV particles.
  • Conditions suitable for producing HCV particles are environmental conditions compatible with HCV structural protein expression from recombinant nucleic acid and HCV particle formation. Suitable environmental conditions provide the proper temperature and growth medium. Suitable conditions for Vero cells include those illustrated in the Example Section provided below.
  • Another aspect of the present invention features a method for measuring the ability of a compound to inhibit HCV particle formation.
  • the method comprises the steps of: (a) combining a compound, and Vero cells containing a recombinant nucleic acid comprising an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence, under conditions suitable for producing HCV particles; and (b) measuring HCV particle formation.
  • Inhibition of HCV particle formation can be measured qualitatively or quantitatively. Reference to inhibition indicates a detectable reduction in particle formation.
  • SEQ LD NO: 4 provides an example of an adenovector nucleic acid containing an expression cassette encoding HCV C-E1-E2- P7-NS2.
  • Figure 1 illustrates the construction of adenovector constructs containing an HCV structural region.
  • Figures 2A and 2B provide a comparison of Ade:NS2 and Ade:P7 Core and E2 expression.
  • Vero cells were infected with either Ade:NS2 or Ade:P7 for 2 days, cells harvested, and expression of Core ( Figure 2A) and E2 ( Figure 2B) evaluated with antibodies.
  • Lane 1 uninfected; lane 2, Ade5 at moi of 10 4 ; lane 3, Ade:NS2 at moi of 10 2 ; lane 4, Ade:NS2 at moi of 10 3 ; lane 5, Ade:NS2 at moi of 10 4 ; lane 6, Ade:P7 at moi of 10 2 ; lane 7, Ade:P7 at moi of 10 3 ; and lane 8, Ade:P7 at moi of 10 4 .
  • Viral inputs are given in terms of viral genomes; infectivity typically is 1- 2 % of these values.
  • FIG 3 shows that Ade:NS2 and Ade:P7 produce distinct molecular weight forms of Core.
  • Ade:NS2 produces predominantly the 21 kD form which has been suggested to be a component of native virions. (Yasui, et al, 1998. Journal of Virology 72, 6048-6055.)
  • Figures 4A, 4B and 4C illustrate shell particles and budding produced by Ade:P7 infection of Vero cells.
  • Figure 4A shows shell-like budding produced by Ad:P7 infection of Vero cells.
  • Figure 4B shows shell particles produced in Vero cells upon Ade:P7 infection.
  • Figures 4C shows shell budding and particles within the endoplasmic reticulum (ER) of Vero cells.
  • ER endoplasmic reticulum
  • Figures 5A, 5B and 5C illustrate electron-dense particles in Vero cells upon Ade:NS2 infection.
  • Figure 5A shows electron-dense particles produced in Vero cells upon Ade:NS2 infection.
  • Figure 5B shows budding and free electron-dense particles with the ER lumen.
  • Figure 5C shows abundant production of electron-dense particles by Ade:NS2.
  • Figures 6A and 6B illustrate immuno-electron microscopy (immunoEM) labeling of electron-dense particles.
  • Figure 6 A shows immunoEM labeling of electron-dense particles with gold-labeled (spherical particles, 10 nanometers diameter) secondary antibodies to ⁇ Core antibodies.
  • Figure 6B shows immunoEM labeling of electron-dense particles with gold-labeled secondary antibodies to an ⁇ E2 monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the present invention features methods for obtaining HCV particles and measuring the ability of a compound to inhibit HCV particle formation or function.
  • HCV particles are produced employing Vero cells and recombinant nucleic acid encoding for at least HCV C-E1-E2-P7-NS2.
  • Vero cells offer advantages such as growing well in culture; the capability to produce large amounts of protein from an exogenous gene; and containing mammalian protein secretion, glycosylation, and modification enzymes that may be important in assembling viral envelope proteins.
  • Other cell lines that meet these criteria are also candidates for supporting cell-based assembly of HCV particles. The cell line need not be hepatic in origin to support particle assembly. As described in the Example Section below, expressing HCV C-El-
  • E2-P7-NS2 resulted predominately in the p21 Core protein.
  • P21 formation correlated with a high level production of electron-dense particles. These particles have a uniform size and morphology, and a hexagonal outline is frequently seen. The particles appear to have a diffuse outer layer, possibly indicating the presence of lipids.
  • HCV inhibitory compounds can decrease replication of HCV.
  • Targets for HCV inhibitory compounds include HCV particle assembly, HCV particle secretion, and HCV particle infection.
  • Compounds inhibiting HCV replication have research and therapeutic applications. Research applications include the study of HCV morphogenesis. Therapeutic applications include using those compounds having appropriate pharmacological properties such as efficacy and lack of unacceptable toxicity to treat or inhibit onset of HCV in a patient.
  • HCV particles can be produced using a recombinant nucleic acid that comprises an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence.
  • the expression cassette contains elements driving expression of the HCV structural genes.
  • references to "recombinant nucleic acid” indicates the presence of two or more nucleic acid regions not naturally associated with each other. Recombined regions can be present within the expression cassette and in other parts of the recombinant nucleic acid.
  • the employed HCV C-E1-E2-P7-NS2 sequence can be based on a naturally occurring sequence or a functional derivative thereof able to produce particles that are recognized by an antibody targeting a naturally occurring Core or El region. Examples of naturally occurring HCV isolates and artificial HCV sequences are well known in the art.
  • HCV isolates can be classified into the following six major genotypes comprising one or more subtypes: HCV-l/(la,lb,lc), HCV-2/(2a,2b,2c), HCV- 3/(3a,3b,10a), HCV-4/(4a), HCV-5/(5a) and HCV-6/(6a,6b,7b,8b,9a,l la).
  • HCV-BK HCV-J
  • HCV-N HCV-H
  • HCV-H HCV-H
  • Gen. Virol, 1341-1347 An example of an artificial HCV sequence well known in the art is a consensus HCV lb sequence designated conl, derived from naturally occuring patient isolates. (Lohman, et al, Science 285, 110-113, 1999; Gen-Bank Accession Number
  • RNA codons as follows:
  • the HCV C-E1-E2-P7-NS2 sequence encodes a polypeptide comprising or consisting of SEQ ID NO: 3; and comprises or consists of SEQ ID NO: 2.
  • a gene expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence also contains elements needed for polypeptide expression and may contain additional sequences. Additional sequences, if present, should not prevent the expression of C-E1-E2-P7-NS2 and particle formation, and may be chosen to serve a useful purpose.
  • additional sequences examples include additional HCV sequences, sequences encoding a reporter protein and sequences that can serve as a target for complementary probe hybridization.
  • a reporter protein nucleic acid sequence encodes a protein that can produce a detectable signal.
  • reporter proteins include luciferase, beta- lactamase, secretory alkaline phosphatase, beta-glucuronidase, and green fluorescent protein and its derivatives.
  • Regulatory elements present in a gene expression cassette generally include: (a) a promoter transcriptionally coupled to a nucleotide sequence encoding the polypeptide, (b) a 5' ribosome binding site functionally coupled to the nucleotide sequence, (c) a terminator joined to the 3' end of the nucleotide sequence, and (d) a 3' polyadenylation signal functionally coupled to the nucleotide sequence. Additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing may also be present.
  • references to "functionally coupled” indicates the ability to mediate an effect on the nucleotide sequence. Functionally coupled does not require that the coupled sequences be adjacent to each other.
  • a 3' polyadenylation signal functionally coupled to the nucleotide sequence facilitates cleavage and polyadenylation of the transcribed RNA.
  • a 5' ribosome binding site functionally coupled to the nucleotide sequence facilitates ribosome binding of the transcribed RNA.
  • Promoters are genetic elements that are recognized by an RNA polymerase and mediate transcription of downstream regions.
  • Preferred promoters are strong promoters that provide for increased levels of transcription. Examples of strong promoters are the immediate early human cytomegalovirus (CMN) promoter, and CMV promoter with intron A. (Chapman, et al, 1991. Nucl Acids Res.
  • promoters include naturally occurring promoters such as the EF1 alpha promoter, the murine CMV promoter, Rous sarcoma virus promoter, SV40 early/late promoters, the ⁇ -actin promoter, and artificial promoters such as a synthetic muscle specific promoter and a chimeric muscle-specific/CMV promoter.
  • naturally occurring promoters such as the EF1 alpha promoter, the murine CMV promoter, Rous sarcoma virus promoter, SV40 early/late promoters, the ⁇ -actin promoter, and artificial promoters such as a synthetic muscle specific promoter and a chimeric muscle-specific/CMV promoter.
  • the ribosome binding site is located at or near the initiation codon.
  • ribosome binding sites include CCACC AUGG, CCGCC AUGG, and ACCAUGG, where AUG is the initiation codon. (Kozak, 1986. Cell 44, 283-292.)
  • Another example of a ribosome binding site is GCCACCAUGG.
  • the polyadenylation signal is responsible for cleaving the transcribed R ⁇ A and the addition of a poly (A) tail to the R ⁇ A.
  • the polyadenylation signal in higher eukaryotes contains an AAUAAA sequence about 11-30 nucleotides from the polyadenylation addition site. The AAUAAA sequence is involved in signaling R ⁇ A cleavage. (Lewin, Genes IN, Oxford University Press, ⁇ Y, 1990.)
  • the poly (A) tail is important for mR ⁇ A processing.
  • Polyadenylation signals that can be used as part of a gene expression cassette include the minimal rabbit ⁇ -globin polyadenylation signal and the bovine growth hormone polyadenylation signal (BGH). (Xu, et al, 2001. Gene 272, 149-156, Post, et al, U.S. Patent U. S. 5,122,458.) Additional examples include the Synthetic Polyadenylation Signal (SPA) and SN40 polyadenylation signal.
  • the SPA sequence is as follows: AAUAAAAGAUCUUUAUUUUCAUUAGAUCUGUGUGUG UUGGUUUUUGUGUG.
  • Examples of additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing that may be present include an enhancer, a leader sequence and an operator.
  • An enhancer region increases transcription. Examples of enhancer regions include the CMN enhancer and the SV40 enhancer. (Hitt, et al, 1995. Methods in Molecular Genetics 7,13-30, Xu, et al, 2001. Gene 27, 149-156.)
  • An enhancer region can be associated with a promoter.
  • a leader sequence is an amino acid region on a polypeptide that directs the polypeptide into the proteasome. Nucleic acid encoding the leader sequence is 5' of a structural gene and is transcribed along the structural gene.
  • An example of a leader sequences is tPA.
  • An operator sequence can be used to regulate gene expression.
  • the Tet operator sequence can be used to repress gene expression.
  • Both the expression cassette and other parts of an expression vector affect protein expression.
  • suitable expression vectors drive sufficient expression of HCV structural proteins to achieve particle formation.
  • vectors that can be employed to drive high levels of protein production include adenovectors, retroviral vectors, alpha virus vectors, and plasmid vectors.
  • Expression vectors can be introduced into cells, such as Vero cells, using different techniques. Examples of such techniques include RNA/DNA transfections and viral infections. (Molecular Cloning: A Laboratory Manual. 1989. Sambrook, et al, Cold Spring Harbor Press; Mackenzie, et al, 2001. J. Virol. 75, 10787-10799.)
  • the expression vector is an adenovector.
  • An adenovector is a recombinant adenovirus containing heterologous nucleic acid.
  • the adenovirus has a double-stranded linear genome with inverted terminal repeats at both ends. During viral replication, the genome is packaged inside a viral capsid to form a virion. The virus enters its target cell through viral attachment followed by internalization. (Hitt, et al, 1997. Advances in Pharmacology 40, 137- 206.)
  • Adenovectors can be based on different adenovirus serotypes such as those found in humans or animals.
  • animal adeno viruses include bovine, porcine, chimp, murine, canine, and avian (CELO).
  • Adenovectors based on human serotypes include those based on Group B, C, D or E serotypes.
  • human adenovirus Group B, C, D, or E serotypes include types 2 ("Ad2"), 4 ("Ad4"), 5 ("Ad5"), 6 (“Ad6"), 24 (“Ad24"), and 35 (“Ad35").
  • Adenovectors can contain regions from a single adenovirus or from two or more adenovirus.
  • the adenovector is based on Ad5.
  • Ad5 is described by Chroboczek, et al, 1992. J. Virology 186, 280-285.
  • the adenovector is a first generation adenovector.
  • First generation adenovectors for expressing a gene expression cassette contain the expression cassette in an recombinant adenovirus genome containing deletions in at least El.
  • First generation adenovectors for expressing at least a C-E1-E2-P7- NS2 polypeptide may contain an El, or an El and E3 deleted recombinant adenovirus genome.
  • the deletions in El, or El and E3, are sufficiently large to accommodate the gene expression cassette.
  • the size of the gene expression cassette will vary depending upon nucleic acid present in addition to nucleic acid encoding for C-E1-E2-P7-NS2.
  • a deletion in El is sufficient.
  • deletions in E3 may also be provided.
  • Adenovectors do not need to have their El, or El and E3 regions, completely removed. Rather, a sufficient amount of the El region is removed to render the vector replication incompetent in the absence of the El proteins being supplied in trans; and the El deletion or the combination of the El and E3 deletions are sufficiently large enough to accommodate a gene expression cassette.
  • El deletions can be obtained starting at about base pair 342 going up to about base pair 3523 of Ad5, or a corresponding region from other adenoviruses.
  • the deleted region may be produced by, for example, removing a region from about base pair 450 to about base pair 3511 of Ad5, or a corresponding region from other adenoviruses. Larger El region deletions starting at about base pair 341 remove elements that facilitate virus packaging.
  • E3 deletions can be obtained starting at about base pair 27865 to about base pair 30995 of Ad5, or the corresponding region of other adenovectors.
  • the deleted region may be produced by, for example, removing a region from about base pair 28134 up to about base pair 30817 of Ad5, or the corresponding region of other adenovectors.
  • the combination of the gene expression cassette and deletions to adenovirus genome should provide an overall size not exceeding about 105% of the wild type adenovirus genome.
  • the size of the recombinant adenovirus genome containing the gene expression cassette is about 85% to about 105% the size of the wild type adenovirus genome.
  • 5 kb can be inserted into an adenovirus genome with an El deletion, and approximately 7,500 kb can be inserted with an El and E3 deletion. Without any deletion, the Ad5 genome is 35,935 base pairs.
  • Replication of first generation adenovectors can be performed by supplying the El gene products in trans.
  • the El gene product can be supplied in trans, for example, by using cell lines that have been transformed with the adenovirus El region.
  • Examples of cells and cells lines transformed with the adenovirus El region are HEK 293 cells, 911 cells, PERC.6TM cells, and transfected primary human aminocytes cells. (Graham, et al, 1977. Journal of Virology 36, 59-72, Schiedner, et al., 2000. Human Gene Therapy 11, 2105-2116, Fallaux, et al, 1998. Human Gene Therapy 9, 1909-1917, Bout, et al, U.S. Patent No. 6,033,908.)
  • An expression cassette should be inserted into a recombinant adenovirus genome in the region corresponding to the deleted El region or, if present, possibly the deleted E3 region.
  • the expression cassette can have a parallel or anti- parallel orientation. In a parallel orientation the transcription direction of the inserted gene is the same direction as the deleted El gene. In an anti-parallel orientation transcription the opposite strand serves as a template and the transcription direction is in the opposite direction.
  • the recombinant nucleic acid consists of SEQ LO NO: 4.
  • HCV inhibitory compounds can be identified and evaluated using assays suitable for producing HCV particles and by measuring the ability of a compound to inhibit HCV particle formation. Control experiments can be performed in parallel at that same time the affect of a test compound is being measured, or can be performed at a different time to obtain a baseline level of particle formation.
  • Techniques for measuring particle formation in a system capable of producing such particles include visual determination and the use of antibodies recognizing Core and E2. Visual determination can be performed using electron microscopy. Additional Use of HCV Particles
  • HCV particles include one or more of the following: (a) serving as a source of immunogenetic material which can be used to obtain HCV antibodies recognizing a native particle form; and (b) and serving as a source of HCV particles for evaluating HCV particle function and infectivity, and evaluating the ability of a compound to inhibit particle function or infectivity.
  • HCV particles can be obtained from cultured cells based on techniques to fractionate HCV infected sera.
  • techniques to fractionate HCV infected sera include: (a) obtaining a highly enriched fraction of electron-dense particles by mechanical disruption of cells using a dounce homogenizer; (b) a low-speed spin to remove cellular debris; and (c) density centrifugation on a sucrose or cesium chloride cushion to obtain purified particles.
  • Purified HCV particles can used to infect a HCV susceptible host cell and HCV inhibitory compounds can be identified or evaluated based on their ability to inhibit particle formation or infectivity.
  • Assays measuring the ability of a compound to inhibit infective particle formation involve evaluating the effect of the compound prior to particle assembly or expression of the capsid and envelop proteins.
  • Assays measuring the ability of a compound to inhibit particle infectivity employ HCV particles that were purified, and measure the ability of a compound to inhibit infection of a HCV susceptible host cell. Examples of HCV susceptible host cells include Huh7, HepG2, HPBALL, and Daudi cells.
  • This example describes the production of adenovector constructs containing an expression cassette encoding HCV BK C-E1-E2-P7 (SEQ ID NO: 1) or C-E1-E2-P7-NS2 (SEQ ID NO: 2).
  • the resulting adenovectors were designated Ade:P7 and Ade:NS2 (SEQ ID NO: 4).
  • Expression of the HCV transgene in these vectors was driven by the human cytomegalovirus promoter.
  • Ade:P7 and Ade:NS2 adenovectors are based on serotype 5, with a deletion of the El a gene.
  • the promoter for the Ade:P7 vector contains a tetracycline repressor element to prevent or limit expression of the transgene during viral rescue.
  • the vectors can be propagated in tissue culture if transcription factor El a is provided in trans.
  • the overall adenovector construction strategy involved the production of an HCV structural region expression cassette in a shuttle vector, transfer of the expression cassette to an adenovirus genome plasmid, and viral rescue ( Figure 1).
  • the HCV structural region was cloned into a shuttle vector where expression was driven by the human cytomegalo virus promoter, followed by a stop codon and the bovine growth hormone polyadenylation signal.
  • the expression cassette is flanked by Adenovirus Serotype 5 sequences on both ends to enable homologous recombination.
  • the shuttle vector was mixed with a second plasmid containing the Adenovirus Serotype 5 ⁇ E1 genome to produce a recombinant adenovirus expression construct. Both vectors were linearized to facilitate recombination and transformed into bacterial strain BJ5183. (Hanahan, 1983 J. Moi Biol. 166, 557-580.) Recombination resulting in the production of an adenovirus genome plasmid was confirmed by restriction mapping. The expression cassette and flanking regions were sequenced to verify that recombination did not produce deletions.
  • the adenovirus vector was rescued from the adenovirus genome plasmid by digesting with Pad and transfecting either PerC.6 cells (Ade:NS2) or TREX cells (Ade:P7).
  • the TREX cells contain the tetracycline repressor, which prevents expression of the transgene during rescue and packaging.
  • Recombinant virus was prepped by two rounds of cesium chloride banding. The genetic structure was verified through restriction mapping of packaged genomic DNA.
  • Vero cells were infected with either Ade:P7 or Ade:NS2 for 2 days at different multiplicity of infections. Vero cells were approximately 25 % confluent at the time of infection. Cells were grown at 37 ° C, at 5 % CO 2 , in Minimal Essential Medium Alpha Medium, supplemented with 10 % Fetal Bovine Serum, and 1 X Penn/Strep and GlutaMax. All medium reagents were purchased from Invitrogen. The cells were harvested to analyze for protein expression using the following procedure.
  • Cells were trypsinized for 5 minutes, resuspended in phosphate- buffered saline and washed twice in the same buffer, counted, aliquoted into samples of 5 x 10 5 cells, pelleted and stored at -80°C until use. An aliquot of cells was resuspended in 200 microliters of 2 x Laemmli buffer, boiled for 5 minutes, 4 microliters run on an SDS-PAGE minigel (BioRad), and transferred to nitrocellulose. Expression of Core and E2 was evaluated using anti-Core polyclonal or an anti-E2 monoclonal antibody.
  • Anti-Core polyclonal antibodies were raised against a bacterially expressed GST:Core(aal-124) fusion protein that were first cleared by eluting through a glutathione column.
  • An anti-E2 murine monoclonal antibody which recognizes an E2 linear epitope (amino acids 461-491) was generated against the E2 hypervariable region imotope B14 and boosted with purified E2 glycoprotein from the N strain of HCV genotype lb (Zucchelli, et. al, 2001.
  • Antibody detection was with Horseradish Peroxidase conjugated anti-rabbit or anti-mouse antibodies (Zymed Laboratories) and the ECL Detection System (Amersham Pharmacia).
  • E2 expression was dose dependent upon viral input ( Figure 2B). In contrast, Core expression appears to require a threshold viral input level, with expression either at high levels or not at all ( Figure 2A). Expression levels of Core were similar between Ade:P7 or Ade:NS2. E2 expression was greater than 10-fold higher with Ade:P7 at a similar viral input (compare Figure 2B lanes 5 to 7 and 8).
  • Ade:P7 directs expression almost exclusively of the higher molecular weight p23 form. A faint band near 16 kD was observed and attributed to a proteolytic artifact that has been described by others and considered to not be physiologically relevant. (Ravaggi, et al, 1994. J. Hepatol 20, 833-836; Ray, et al, 1995. Virus Res. 37, 209-220.)
  • Ade:NS2 produces the p21 and p23 forms of Core in Vero cells, where greater than 80 % of the total p21 and p23 expression consists of the lower molecular form.
  • One possibility that accounts for the increased production of the shorter form from Ade:NS2 is that the longer HCV polyprotein produced by Ade:NS2 folds in a way that facilitates subsequent cleavage, and NS2 expression is not directly involved in this event.
  • Example 4 Shell Particles and Budding Produced by Ade:P7 Infection of Vero Cells
  • Vero cells were infected with Ade:P7 at a genomic moi of 10 and harvested after 2 days.
  • Cells were fixed in glutaraldehyde and osmium tetroxide, dehydrated, embedded in Spurr' s resin, thin sectioned, and analyzed by EM. No particles or budding were observed in uninfected or Ade5 infected Nero cells. Some signs of distended ER and cell degeneration were observed in Ade5 infected cells (data not shown).
  • Figure 4A illustrates budding of distended ER membranes into the lumen.
  • the outer layer is very electron dense. Particle size varies from 40-80 nm.
  • Figure 4B illustrates a shell particle completely budded from the ER membrane. Protrusions or spikes appear to emanate from the surface of the particles shown in Figure 4B.
  • Figure 4C illustrates shell particles and budding of shell particles from distended ER. Protrusions or spikes appear to emanate from the surface of the particles shown in Figure 4C.
  • Example 5 Electron-Dense Particles Were Produced in Vero Cells Upon Ade: ⁇ S2 Infection
  • Vero cells were infected with Ade:NS2 at a genomic moi of 5 x 10 3 and harvested after 2 days. Cells were fixed in glutaraldehyde and osmium tetroxide, dehydrated, embedded in Spurr' s resin, thin sectioned, and analyzed by EM. No particles or budding were observed in uninfected or Ade5 infected Vero cells.
  • Figures 5A, 5B, and 5C illustrate the accumulation of electron-dense particles in different regions and association with lipid storage droplets.
  • Figure 5A illustrates the accumulation of electron-dense particles within the cell cytoplasm.
  • the electron-dense particles display a homogenous size of 45-60 nm. Many appear six- sided. In some cases a lighter hue encasing the particles was observed, possibly indicating lipid association.
  • Figure 5B illustrates electron-dense material accumulation at the ER surface membrane and apparent budding towards the ER lumen (labeled B). Whole pieces of broken ER membrane are observed within the lumen. Free particles (A) are observed within the cell cytoplasm.
  • Figure 5C illustrates abundant production of electron-dense particles associated with lipid storage droplets.
  • the electron dense particles appear to gravitate towards the lipid storage droplet, and further appear to be in the midst of merging with the droplet.
  • ImmunoEM labeling of electron-dense particles demonstrates the presence of Core and E2 at the surface of the particles. ImmunoEM labeling was performed using Vero cells infected with Ade:NS2.
  • Vero cells were infected with Ade:NS2 at a genomic moi of 5 x 10 3 and harvested after 2 days. Cells were fixed in formaldehyde/glutaraldehyde, and permeabilized with TritonX-100. The cells were then incubated with anti-Core or anti-E2 antibodies, followed by a second incubation with gold-labeled secondary antibodies (spherical particles, 10 nanometers diameter), washed, dehydrated, and embedded in Spurr' s resin.
  • gold-labeled secondary antibodies spherical particles, 10 nanometers diameter

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Abstract

The present invention features methods for producing HCV particles and measuring the ability of a compound to inhibit HCV particle formation or function. Preferred methods involve the production of HCV particles in Vero cells using recombinant nucleic acid encoding for at least HCV C-E1-E2-P7-NS2.

Description

TITLE OF THE INVENTION
HEPATITIS C VIRUS PARTICLE FORMATION
CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of U.S. Provisional
Application No. 60/393,167, filed July 2, 2002, hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION The references cited in the present application are not admitted to be prior art to the claimed invention.
It is estimated that about 3% of the world's population are infected with the Hepatitis C virus (HCV). (Wasley, et al, 2000. Semin. Liver Dis. 20, 1-16.) Exposure to HCV results in an overt acute disease in a small percentage of cases, while in most instances the virus establishes a chronic infection causing liver inflammation and slowly progresses into liver failure and cirrhosis. (Iwarson, 1994. FEMS Microbiol. Rev. 14, 201-204.) In addition, epidemiological surveys indicate an important role of HCV in the pathogenesis of hepatocellular carcinoma. (Kew, 1994. FEMS Microbiol. Rev. 14, 211-220, Alter, 1995. Blood 85, 1681-1695.) HCV-infected patients have been found to produce virions having a diameter of about 45-50 nm. (Bosman, et al, 1998. Res. Virol, 149, 311-314.) Virions isolated from a natural infection typically are coated with antibodies and Hpids, making structural determinations difficult and imprecise.
Virus-like particles have been observed upon infection with HCV in some cells lines such as TOFE, HPBMalO-2, HPBALL and Daudi. (Serafino, et al, 1997. Res. Virol. 148, 153-159, Shimizu, et al, 1996. Hepatology 23, 205-209.) The morphology of the particles varied among experimental protocols, and the yields of particles were low.
The HCV genome consists of a single strand RNA about 9.5 kb in length, encoding a precursor polyprotein about 3000 amino acids. (Choo, et al, 1989. Science 244, 362-364, Choo, et al, 1989. Science 244, 359-362, Takamizawa, et al, 1991. J. Virol. 65, 1105-1113.) The HCV polyprotein contains the viral proteins in the order: C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
Individual viral proteins are produced by proteolysis of the HCV polyprotein. Host cell proteases release the putative structural proteins C, El, E2, and P7, and create the N-terminus of NS2 at amino acid 810. (Mizushima, et al, 1994. J. Virol. 68, 2731-2734, Hijikata, et al, 1993. P.N.A.S. USA 90, 10773-10777.)
HCV structural proteins can be expressed in cultured cells from recombinant nucleic acid encoding the proteins. HCV Core proteins with a molecular weight of 21 kDa (p21) and 23 kDa (p23) were observed in CHO cells using a vaccinia virus expression system encoding HCV structural proteins. (Yasui, et al, 1998. Journal of Virology 72, 6048-6055.) The p21 Core protein was suggested to be a component of native virions. (Yasui, et al, 1998. Journal of Virology 72, 6048- 6055.) The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the virus replication machinery and are released from the polyprotein. A zinc-dependent protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3. (Grakoui, et al, 1993. J. Virol 67, 1385-1395, Hijikata, et al, 1993. P.N.A.S. USA 90, 10773-10777.) A distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4ANS4B, NS4B NS5A and NS5A/NS5B junctions. (Barthenschlager, et al, 1993. J. Virol 67, 3835-3844, Grakoui, et al, 1993. Proc. Natl Acad. Sci. USA 90, 10583-10587, Tomei, et al, 1993. J. Virol. 67, 4017-4026.) RNA stimulated NTPase and helicase activities are located in the C-terminal domain of NS3.
NS4A provides a cofactor for NS3 protease activity. (Failla, et al, J. Virol. 1994. 68, 3753-3760, De Francesco, et al, U.S. Patent No. 5,739,002.) NS5A is a highly phosphorylated protein conferring interferon resistance. (De Francesco, et al, 2000. Semin Liver Dis., 20(1), 69-83, Pawlotsky, 1999. J. Viral Hepat. Suppl. 1, 47-48.)
NS5B provides an RNA-dependent RNA polymerase. (De Francesco, et al, International Publication Number WO 96/37619, Behrens, et al, 1996. EMBO 15, 12-22, Lohmann, et al, 1998. Virology 249, 108-118.)
SUMMARY OF THE INVENTION
The present invention features methods for producing HCV particles and measuring the ability of a compound to inhibit HCV particle formation or function. Preferred methods involve the production of HCV particles in Vero cells using recombinant nucleic acid encoding for at least HCV C-E1-E2-P7-NS2. Reference to "HCV particles" indicates virus-like particles that contain HCV structural proteins and are observable under electron microscopy. The particle may or may not contain HCV non-structural proteins.
Thus, a first aspect of the present application features a method of making HCV particles. The method can be performed by incubating Vero cells containing a recombinant nucleic acid that comprises an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence under conditions suitable for producing HCV particles.
Conditions suitable for producing HCV particles are environmental conditions compatible with HCV structural protein expression from recombinant nucleic acid and HCV particle formation. Suitable environmental conditions provide the proper temperature and growth medium. Suitable conditions for Vero cells include those illustrated in the Example Section provided below.
Another aspect of the present invention features a method for measuring the ability of a compound to inhibit HCV particle formation. The method comprises the steps of: (a) combining a compound, and Vero cells containing a recombinant nucleic acid comprising an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence, under conditions suitable for producing HCV particles; and (b) measuring HCV particle formation. Inhibition of HCV particle formation can be measured qualitatively or quantitatively. Reference to inhibition indicates a detectable reduction in particle formation.
Another aspect of the present invention describes a recombinant nucleic acid consisting of SEQ ID NO: 4. SEQ LD NO: 4 provides an example of an adenovector nucleic acid containing an expression cassette encoding HCV C-E1-E2- P7-NS2.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the construction of adenovector constructs containing an HCV structural region.
Figures 2A and 2B provide a comparison of Ade:NS2 and Ade:P7 Core and E2 expression. Vero cells were infected with either Ade:NS2 or Ade:P7 for 2 days, cells harvested, and expression of Core (Figure 2A) and E2 (Figure 2B) evaluated with antibodies. Lane 1, uninfected; lane 2, Ade5 at moi of 104; lane 3, Ade:NS2 at moi of 102; lane 4, Ade:NS2 at moi of 103; lane 5, Ade:NS2 at moi of 104; lane 6, Ade:P7 at moi of 102; lane 7, Ade:P7 at moi of 103; and lane 8, Ade:P7 at moi of 104. Viral inputs are given in terms of viral genomes; infectivity typically is 1- 2 % of these values.
Figure 3 shows that Ade:NS2 and Ade:P7 produce distinct molecular weight forms of Core. Ade:NS2 produces predominantly the 21 kD form which has been suggested to be a component of native virions. (Yasui, et al, 1998. Journal of Virology 72, 6048-6055.)
Figures 4A, 4B and 4C illustrate shell particles and budding produced by Ade:P7 infection of Vero cells. Figure 4A shows shell-like budding produced by Ad:P7 infection of Vero cells. Figure 4B shows shell particles produced in Vero cells upon Ade:P7 infection. Figures 4C shows shell budding and particles within the endoplasmic reticulum (ER) of Vero cells.
Figures 5A, 5B and 5C illustrate electron-dense particles in Vero cells upon Ade:NS2 infection. Figure 5A shows electron-dense particles produced in Vero cells upon Ade:NS2 infection. Figure 5B shows budding and free electron-dense particles with the ER lumen. Figure 5C shows abundant production of electron-dense particles by Ade:NS2.
Figures 6A and 6B illustrate immuno-electron microscopy (immunoEM) labeling of electron-dense particles. Figure 6 A shows immunoEM labeling of electron-dense particles with gold-labeled (spherical particles, 10 nanometers diameter) secondary antibodies to αCore antibodies. Figure 6B shows immunoEM labeling of electron-dense particles with gold-labeled secondary antibodies to an αE2 monoclonal antibody (mAb).
DETAILED DESCRIPTION OF THE INVENTION
The present invention features methods for obtaining HCV particles and measuring the ability of a compound to inhibit HCV particle formation or function. Preferably, HCV particles are produced employing Vero cells and recombinant nucleic acid encoding for at least HCV C-E1-E2-P7-NS2.
For HCV particle morphogenesis, Vero cells offer advantages such as growing well in culture; the capability to produce large amounts of protein from an exogenous gene; and containing mammalian protein secretion, glycosylation, and modification enzymes that may be important in assembling viral envelope proteins. Other cell lines that meet these criteria are also candidates for supporting cell-based assembly of HCV particles. The cell line need not be hepatic in origin to support particle assembly. As described in the Example Section below, expressing HCV C-El-
E2-P7-NS2 resulted predominately in the p21 Core protein. P21 formation correlated with a high level production of electron-dense particles. These particles have a uniform size and morphology, and a hexagonal outline is frequently seen. The particles appear to have a diffuse outer layer, possibly indicating the presence of lipids.
Methods for producing HCV particles have a variety of different uses including being used as a source to obtain HCV particles, being used to study HCV particle morphogenesis, and being used to obtain HCV inhibitory compounds. HCV inhibitory compounds can decrease replication of HCV. Targets for HCV inhibitory compounds include HCV particle assembly, HCV particle secretion, and HCV particle infection.
Compounds inhibiting HCV replication have research and therapeutic applications. Research applications include the study of HCV morphogenesis. Therapeutic applications include using those compounds having appropriate pharmacological properties such as efficacy and lack of unacceptable toxicity to treat or inhibit onset of HCV in a patient.
Expression Cassettes
HCV particles can be produced using a recombinant nucleic acid that comprises an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence. The expression cassette contains elements driving expression of the HCV structural genes.
Reference to "recombinant nucleic acid" indicates the presence of two or more nucleic acid regions not naturally associated with each other. Recombined regions can be present within the expression cassette and in other parts of the recombinant nucleic acid.
The employed HCV C-E1-E2-P7-NS2 sequence can be based on a naturally occurring sequence or a functional derivative thereof able to produce particles that are recognized by an antibody targeting a naturally occurring Core or El region. Examples of naturally occurring HCV isolates and artificial HCV sequences are well known in the art.
HCV isolates can be classified into the following six major genotypes comprising one or more subtypes: HCV-l/(la,lb,lc), HCV-2/(2a,2b,2c), HCV- 3/(3a,3b,10a), HCV-4/(4a), HCV-5/(5a) and HCV-6/(6a,6b,7b,8b,9a,l la).
(Simmonds, J. Gen. Virol, 693-712, 2001.) Examples of particular HCV sequences such as HCV-BK, HCV-J, HCV-N, HCV-H, have been deposited in GenBank and described in various publications. (See, for example, Chamberlain, et al, 1997 J.
Gen. Virol, 1341-1347.) An example of an artificial HCV sequence well known in the art is a consensus HCV lb sequence designated conl, derived from naturally occuring patient isolates. (Lohman, et al, Science 285, 110-113, 1999; Gen-Bank Accession Number
AJ238799.)
Artificial HCV encoding sequences can also be produced based on a naturally occurring amino acid sequence taking into account the degeneracy of the genetic code. The degeneracy of the genetic code arises because almost all amino acids are encoded by different combinations of nucleotide triplets or "codons". The translation of a particular codon into a particular amino acid is well known in the art
(see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990). Amino acids are encoded by RNA codons as follows:
A=Ala=Alanine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=Glu=Glutamic acid: codons GAA, GAG F=Phe=Phenylalanine: codons UUC, UUU
G=Gly=Glycine: codons GGA, GGC, GGG, GGU
H=His=Histidine: codons CAC, CAU
I=Ue=Isoleucine: codons AUA, AUC, AUU
K=Lys=Lysine: codons AAA, AAG L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU M=Met=Methionine: codon AUG
N=Asn=Asparagine: codons AAC, AAU
P=Pro=Proline: codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: codons CAA, CAG R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG Y=Tyr=Tyrosine: codons UAC, UAU.
In different embodiments, the HCV C-E1-E2-P7-NS2 sequence encodes a polypeptide comprising or consisting of SEQ ID NO: 3; and comprises or consists of SEQ ID NO: 2.
A gene expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence also contains elements needed for polypeptide expression and may contain additional sequences. Additional sequences, if present, should not prevent the expression of C-E1-E2-P7-NS2 and particle formation, and may be chosen to serve a useful purpose.
Examples of additional sequences that may be present include additional HCV sequences, sequences encoding a reporter protein and sequences that can serve as a target for complementary probe hybridization.
A reporter protein nucleic acid sequence encodes a protein that can produce a detectable signal. Examples of reporter proteins include luciferase, beta- lactamase, secretory alkaline phosphatase, beta-glucuronidase, and green fluorescent protein and its derivatives. Regulatory elements present in a gene expression cassette generally include: (a) a promoter transcriptionally coupled to a nucleotide sequence encoding the polypeptide, (b) a 5' ribosome binding site functionally coupled to the nucleotide sequence, (c) a terminator joined to the 3' end of the nucleotide sequence, and (d) a 3' polyadenylation signal functionally coupled to the nucleotide sequence. Additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing may also be present.
Reference to "functionally coupled" indicates the ability to mediate an effect on the nucleotide sequence. Functionally coupled does not require that the coupled sequences be adjacent to each other. A 3' polyadenylation signal functionally coupled to the nucleotide sequence facilitates cleavage and polyadenylation of the transcribed RNA. A 5' ribosome binding site functionally coupled to the nucleotide sequence facilitates ribosome binding of the transcribed RNA.
Promoters are genetic elements that are recognized by an RNA polymerase and mediate transcription of downstream regions. Preferred promoters are strong promoters that provide for increased levels of transcription. Examples of strong promoters are the immediate early human cytomegalovirus (CMN) promoter, and CMV promoter with intron A. (Chapman, et al, 1991. Nucl Acids Res. 19, 3979- 3986.) Additional examples of promoters include naturally occurring promoters such as the EF1 alpha promoter, the murine CMV promoter, Rous sarcoma virus promoter, SV40 early/late promoters, the β-actin promoter, and artificial promoters such as a synthetic muscle specific promoter and a chimeric muscle-specific/CMV promoter. (Li, et al, 1999. Nat. Biotechnol 17, 241-245, Hagstrom, et al, 2000. Blood 95, 2536-2542.)
The ribosome binding site is located at or near the initiation codon. Examples of ribosome binding sites include CCACC AUGG, CCGCC AUGG, and ACCAUGG, where AUG is the initiation codon. (Kozak, 1986. Cell 44, 283-292.) Another example of a ribosome binding site is GCCACCAUGG.
The polyadenylation signal is responsible for cleaving the transcribed RΝA and the addition of a poly (A) tail to the RΝA. The polyadenylation signal in higher eukaryotes contains an AAUAAA sequence about 11-30 nucleotides from the polyadenylation addition site. The AAUAAA sequence is involved in signaling RΝA cleavage. (Lewin, Genes IN, Oxford University Press, ΝY, 1990.) The poly (A) tail is important for mRΝA processing.
Polyadenylation signals that can be used as part of a gene expression cassette include the minimal rabbit β -globin polyadenylation signal and the bovine growth hormone polyadenylation signal (BGH). (Xu, et al, 2001. Gene 272, 149-156, Post, et al, U.S. Patent U. S. 5,122,458.) Additional examples include the Synthetic Polyadenylation Signal (SPA) and SN40 polyadenylation signal. The SPA sequence is as follows: AAUAAAAGAUCUUUAUUUUCAUUAGAUCUGUGUG UUGGUUUUUUGUGUG.
Examples of additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing that may be present include an enhancer, a leader sequence and an operator. An enhancer region increases transcription. Examples of enhancer regions include the CMN enhancer and the SV40 enhancer. (Hitt, et al, 1995. Methods in Molecular Genetics 7,13-30, Xu, et al, 2001. Gene 27, 149-156.) An enhancer region can be associated with a promoter.
A leader sequence is an amino acid region on a polypeptide that directs the polypeptide into the proteasome. Nucleic acid encoding the leader sequence is 5' of a structural gene and is transcribed along the structural gene. An example of a leader sequences is tPA.
An operator sequence can be used to regulate gene expression. For example, the Tet operator sequence can be used to repress gene expression.
Expression Vectors
Both the expression cassette and other parts of an expression vector affect protein expression. Overall, suitable expression vectors drive sufficient expression of HCV structural proteins to achieve particle formation. Examples of vectors that can be employed to drive high levels of protein production include adenovectors, retroviral vectors, alpha virus vectors, and plasmid vectors.
Expression vectors can be introduced into cells, such as Vero cells, using different techniques. Examples of such techniques include RNA/DNA transfections and viral infections. (Molecular Cloning: A Laboratory Manual. 1989. Sambrook, et al, Cold Spring Harbor Press; Mackenzie, et al, 2001. J. Virol. 75, 10787-10799.)
In an embodiment of the present invention, the expression vector is an adenovector. An adenovector is a recombinant adenovirus containing heterologous nucleic acid. The adenovirus has a double-stranded linear genome with inverted terminal repeats at both ends. During viral replication, the genome is packaged inside a viral capsid to form a virion. The virus enters its target cell through viral attachment followed by internalization. (Hitt, et al, 1997. Advances in Pharmacology 40, 137- 206.)
Adenovectors can be based on different adenovirus serotypes such as those found in humans or animals. Examples of animal adeno viruses include bovine, porcine, chimp, murine, canine, and avian (CELO). Adenovectors based on human serotypes include those based on Group B, C, D or E serotypes. Examples of human adenovirus Group B, C, D, or E serotypes include types 2 ("Ad2"), 4 ("Ad4"), 5 ("Ad5"), 6 ("Ad6"), 24 ("Ad24"), and 35 ("Ad35"). Adenovectors can contain regions from a single adenovirus or from two or more adenovirus. In an embodiment of the present invention, the adenovector is based on Ad5. Ad5 is described by Chroboczek, et al, 1992. J. Virology 186, 280-285.
In an embodiment of the present invention, the adenovector is a first generation adenovector. First generation adenovectors for expressing a gene expression cassette contain the expression cassette in an recombinant adenovirus genome containing deletions in at least El.
First generation adenovectors for expressing at least a C-E1-E2-P7- NS2 polypeptide may contain an El, or an El and E3 deleted recombinant adenovirus genome. The deletions in El, or El and E3, are sufficiently large to accommodate the gene expression cassette.
The size of the gene expression cassette will vary depending upon nucleic acid present in addition to nucleic acid encoding for C-E1-E2-P7-NS2. For first generation adenovector expressing only the C-E1-E2-P7-NS2 polypeptide, a deletion in El is sufficient. For first generation adenovectors containing additional nucleic acid, such as reporter or additional HCV regions, deletions in E3 may also be provided.
Adenovectors do not need to have their El, or El and E3 regions, completely removed. Rather, a sufficient amount of the El region is removed to render the vector replication incompetent in the absence of the El proteins being supplied in trans; and the El deletion or the combination of the El and E3 deletions are sufficiently large enough to accommodate a gene expression cassette.
El deletions can be obtained starting at about base pair 342 going up to about base pair 3523 of Ad5, or a corresponding region from other adenoviruses. The deleted region may be produced by, for example, removing a region from about base pair 450 to about base pair 3511 of Ad5, or a corresponding region from other adenoviruses. Larger El region deletions starting at about base pair 341 remove elements that facilitate virus packaging.
E3 deletions can be obtained starting at about base pair 27865 to about base pair 30995 of Ad5, or the corresponding region of other adenovectors. The deleted region may be produced by, for example, removing a region from about base pair 28134 up to about base pair 30817 of Ad5, or the corresponding region of other adenovectors.
The combination of the gene expression cassette and deletions to adenovirus genome should provide an overall size not exceeding about 105% of the wild type adenovirus genome. For example, as the recombinant adenovirus Ad5 genome increase in size above about 105% the genome becomes unstable. (Bett, et al, Journal of Virology 67:5911-5921, 1993.) Preferably, the size of the recombinant adenovirus genome containing the gene expression cassette is about 85% to about 105% the size of the wild type adenovirus genome. Approximately, 5 kb can be inserted into an adenovirus genome with an El deletion, and approximately 7,500 kb can be inserted with an El and E3 deletion. Without any deletion, the Ad5 genome is 35,935 base pairs.
Replication of first generation adenovectors can be performed by supplying the El gene products in trans. The El gene product can be supplied in trans, for example, by using cell lines that have been transformed with the adenovirus El region. Examples of cells and cells lines transformed with the adenovirus El region are HEK 293 cells, 911 cells, PERC.6™ cells, and transfected primary human aminocytes cells. (Graham, et al, 1977. Journal of Virology 36, 59-72, Schiedner, et al., 2000. Human Gene Therapy 11, 2105-2116, Fallaux, et al, 1998. Human Gene Therapy 9, 1909-1917, Bout, et al, U.S. Patent No. 6,033,908.)
An expression cassette should be inserted into a recombinant adenovirus genome in the region corresponding to the deleted El region or, if present, possibly the deleted E3 region. The expression cassette can have a parallel or anti- parallel orientation. In a parallel orientation the transcription direction of the inserted gene is the same direction as the deleted El gene. In an anti-parallel orientation transcription the opposite strand serves as a template and the transcription direction is in the opposite direction.
In an embodiment of the present invention the recombinant nucleic acid consists of SEQ LO NO: 4.
Particle Formation Assays
HCV inhibitory compounds can be identified and evaluated using assays suitable for producing HCV particles and by measuring the ability of a compound to inhibit HCV particle formation. Control experiments can be performed in parallel at that same time the affect of a test compound is being measured, or can be performed at a different time to obtain a baseline level of particle formation.
Techniques for measuring particle formation in a system capable of producing such particles include visual determination and the use of antibodies recognizing Core and E2. Visual determination can be performed using electron microscopy. Additional Use of HCV Particles
Additional uses of HCV particles include one or more of the following: (a) serving as a source of immunogenetic material which can be used to obtain HCV antibodies recognizing a native particle form; and (b) and serving as a source of HCV particles for evaluating HCV particle function and infectivity, and evaluating the ability of a compound to inhibit particle function or infectivity.
HCV particles can be obtained from cultured cells based on techniques to fractionate HCV infected sera. (E.g., Andre, et al. 2002. /. Virol. 76, 6919-6928, Maillard, et al. 2001. J. Virol. 75, 8240-8250, Prince, et al, 1997. J. Vir. Hep. 3, 11- 17.) An example of techniques that may be employed includes: (a) obtaining a highly enriched fraction of electron-dense particles by mechanical disruption of cells using a dounce homogenizer; (b) a low-speed spin to remove cellular debris; and (c) density centrifugation on a sucrose or cesium chloride cushion to obtain purified particles. Purified HCV particles can used to infect a HCV susceptible host cell and HCV inhibitory compounds can be identified or evaluated based on their ability to inhibit particle formation or infectivity. Assays measuring the ability of a compound to inhibit infective particle formation involve evaluating the effect of the compound prior to particle assembly or expression of the capsid and envelop proteins. Assays measuring the ability of a compound to inhibit particle infectivity employ HCV particles that were purified, and measure the ability of a compound to inhibit infection of a HCV susceptible host cell. Examples of HCV susceptible host cells include Huh7, HepG2, HPBALL, and Daudi cells.
EXAMPLES
Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
Example 1: Generation of Recombinant Adenovectors
This example describes the production of adenovector constructs containing an expression cassette encoding HCV BK C-E1-E2-P7 (SEQ ID NO: 1) or C-E1-E2-P7-NS2 (SEQ ID NO: 2). The resulting adenovectors were designated Ade:P7 and Ade:NS2 (SEQ ID NO: 4). Expression of the HCV transgene in these vectors was driven by the human cytomegalovirus promoter. Ade:P7 and Ade:NS2 adenovectors are based on serotype 5, with a deletion of the El a gene. The only difference between the two vectors is that the promoter for the Ade:P7 vector contains a tetracycline repressor element to prevent or limit expression of the transgene during viral rescue. The vectors can be propagated in tissue culture if transcription factor El a is provided in trans.
The overall adenovector construction strategy involved the production of an HCV structural region expression cassette in a shuttle vector, transfer of the expression cassette to an adenovirus genome plasmid, and viral rescue (Figure 1). The HCV structural region was cloned into a shuttle vector where expression was driven by the human cytomegalo virus promoter, followed by a stop codon and the bovine growth hormone polyadenylation signal. The expression cassette is flanked by Adenovirus Serotype 5 sequences on both ends to enable homologous recombination.
The shuttle vector was mixed with a second plasmid containing the Adenovirus Serotype 5 ΔE1 genome to produce a recombinant adenovirus expression construct. Both vectors were linearized to facilitate recombination and transformed into bacterial strain BJ5183. (Hanahan, 1983 J. Moi Biol. 166, 557-580.) Recombination resulting in the production of an adenovirus genome plasmid was confirmed by restriction mapping. The expression cassette and flanking regions were sequenced to verify that recombination did not produce deletions. The adenovirus vector was rescued from the adenovirus genome plasmid by digesting with Pad and transfecting either PerC.6 cells (Ade:NS2) or TREX cells (Ade:P7). The TREX cells contain the tetracycline repressor, which prevents expression of the transgene during rescue and packaging. Recombinant virus was prepped by two rounds of cesium chloride banding. The genetic structure was verified through restriction mapping of packaged genomic DNA.
Example 2: Expression of HCV Structural Proteins in Vero cells
Vero cells were infected with either Ade:P7 or Ade:NS2 for 2 days at different multiplicity of infections. Vero cells were approximately 25 % confluent at the time of infection. Cells were grown at 37 ° C, at 5 % CO2, in Minimal Essential Medium Alpha Medium, supplemented with 10 % Fetal Bovine Serum, and 1 X Penn/Strep and GlutaMax. All medium reagents were purchased from Invitrogen. The cells were harvested to analyze for protein expression using the following procedure. Cells were trypsinized for 5 minutes, resuspended in phosphate- buffered saline and washed twice in the same buffer, counted, aliquoted into samples of 5 x 105 cells, pelleted and stored at -80°C until use. An aliquot of cells was resuspended in 200 microliters of 2 x Laemmli buffer, boiled for 5 minutes, 4 microliters run on an SDS-PAGE minigel (BioRad), and transferred to nitrocellulose. Expression of Core and E2 was evaluated using anti-Core polyclonal or an anti-E2 monoclonal antibody. Anti-Core polyclonal antibodies were raised against a bacterially expressed GST:Core(aal-124) fusion protein that were first cleared by eluting through a glutathione column. An anti-E2 murine monoclonal antibody which recognizes an E2 linear epitope (amino acids 461-491) was generated against the E2 hypervariable region imotope B14 and boosted with purified E2 glycoprotein from the N strain of HCV genotype lb (Zucchelli, et. al, 2001.
Hepatology 33, 692-703). Antibody detection was with Horseradish Peroxidase conjugated anti-rabbit or anti-mouse antibodies (Zymed Laboratories) and the ECL Detection System (Amersham Pharmacia).
E2 expression was dose dependent upon viral input (Figure 2B). In contrast, Core expression appears to require a threshold viral input level, with expression either at high levels or not at all (Figure 2A). Expression levels of Core were similar between Ade:P7 or Ade:NS2. E2 expression was greater than 10-fold higher with Ade:P7 at a similar viral input (compare Figure 2B lanes 5 to 7 and 8).
Example 3: Ade:NS2 and Ade:P7 Direct Expression of Distinct Molecular Weight Forms of Core
Core produces molecular weight forms p21 and p23 in cell-based expression systems. (Liu, et. al, 1997. J. Virol. 71, 657-662; Yasui, et. al, 1998. J. Virol. 72, 6048-6055.) Ade:P7 and Ade:NS2 were found to differ in their ability to produce these different forms (Figure 3).
Ade:P7 directs expression almost exclusively of the higher molecular weight p23 form. A faint band near 16 kD was observed and attributed to a proteolytic artifact that has been described by others and considered to not be physiologically relevant. (Ravaggi, et al, 1994. J. Hepatol 20, 833-836; Ray, et al, 1995. Virus Res. 37, 209-220.)
Ade:NS2 produces the p21 and p23 forms of Core in Vero cells, where greater than 80 % of the total p21 and p23 expression consists of the lower molecular form. One possibility that accounts for the increased production of the shorter form from Ade:NS2 is that the longer HCV polyprotein produced by Ade:NS2 folds in a way that facilitates subsequent cleavage, and NS2 expression is not directly involved in this event.
Example 4: Shell Particles and Budding Produced by Ade:P7 Infection of Vero Cells Vero cells were infected with Ade:P7 at a genomic moi of 10 and harvested after 2 days. Cells were fixed in glutaraldehyde and osmium tetroxide, dehydrated, embedded in Spurr' s resin, thin sectioned, and analyzed by EM. No particles or budding were observed in uninfected or Ade5 infected Nero cells. Some signs of distended ER and cell degeneration were observed in Ade5 infected cells (data not shown).
The presence of shell particles and budding is shown in Figures 4A- 4C. Figure 4A illustrates budding of distended ER membranes into the lumen. The outer layer is very electron dense. Particle size varies from 40-80 nm.
Figure 4B illustrates a shell particle completely budded from the ER membrane. Protrusions or spikes appear to emanate from the surface of the particles shown in Figure 4B.
Figure 4C illustrates shell particles and budding of shell particles from distended ER. Protrusions or spikes appear to emanate from the surface of the particles shown in Figure 4C.
Example 5: Electron-Dense Particles Were Produced in Vero Cells Upon Ade:ΝS2 Infection
Vero cells were infected with Ade:NS2 at a genomic moi of 5 x 103 and harvested after 2 days. Cells were fixed in glutaraldehyde and osmium tetroxide, dehydrated, embedded in Spurr' s resin, thin sectioned, and analyzed by EM. No particles or budding were observed in uninfected or Ade5 infected Vero cells.
Figures 5A, 5B, and 5C, illustrate the accumulation of electron-dense particles in different regions and association with lipid storage droplets. Figure 5A illustrates the accumulation of electron-dense particles within the cell cytoplasm. The electron-dense particles display a homogenous size of 45-60 nm. Many appear six- sided. In some cases a lighter hue encasing the particles was observed, possibly indicating lipid association.
Figure 5B illustrates electron-dense material accumulation at the ER surface membrane and apparent budding towards the ER lumen (labeled B). Whole pieces of broken ER membrane are observed within the lumen. Free particles (A) are observed within the cell cytoplasm.
Figure 5C illustrates abundant production of electron-dense particles associated with lipid storage droplets. The electron dense particles appear to gravitate towards the lipid storage droplet, and further appear to be in the midst of merging with the droplet.
Example 6: ImmunoEM Labeling of Electron-Dense Particles
ImmunoEM labeling of electron-dense particles demonstrates the presence of Core and E2 at the surface of the particles. ImmunoEM labeling was performed using Vero cells infected with Ade:NS2.
Vero cells were infected with Ade:NS2 at a genomic moi of 5 x 103 and harvested after 2 days. Cells were fixed in formaldehyde/glutaraldehyde, and permeabilized with TritonX-100. The cells were then incubated with anti-Core or anti-E2 antibodies, followed by a second incubation with gold-labeled secondary antibodies (spherical particles, 10 nanometers diameter), washed, dehydrated, and embedded in Spurr' s resin.
Cells were then thin-sectioned and analyzed by EM. Anti-Core antibodies were visualized at the surface of the electron-dense particles (Figure 6A). Anti-Core labeling was found exclusively at the surface of the particles. Anti-E2 antibodies were also visualized at the surface of the electron-dense particles (Figure
6B), although anti-E2 antibodies were also observed at ER and nuclear membranes.
No cell labeling was observed with uninfected or Ade5 infected Vero cells (data not shown). These results demonstrate that Core and E2 are both contained within the electron-dense particles.
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:
1. A method of making Hepatitis C virus (HCV) particles comprising the step of incubating Vero cells containing a recombinant nucleic acid that comprises an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence under conditions suitable for producing said HCV particles.
2. The method of claim 1 , wherein said recombinant nucleic acid is an adenovector.
3. The method of claim 2, wherein said expression cassette comprises a human cytomegalovirus promoter and a bovine growth hormone polyadenylation signal functionally coupled to said HCV C-E1-E2-P7-NS2 sequence.
4. The method of claim 3, wherein said adenovector is based on
Ad5 containing deletions in the El region.
5. The method of claim 4, wherein said HCV C-E1-E2-P7-NS2 sequence consists of SEQ LO NO: 2.
6. The method of claim 1 , wherein said recombinant nucleic acid consists of SEQ ID NO: 4.
7. A method for measuring the ability of a compound to inhibit HCV particle formation comprising the steps of: a) combining said compound, and Vero cells containing a recombinant nucleic acid comprising an expression cassette encoding for at least a HCV C-E1-E2-P7-NS2 sequence, under conditions suitable for producing HCV particles; and b) measuring HCV particle formation.
8. The method of claim 7, wherein said recombinant nucleic acid is an adenovector.
9. The method of claim 8, wherein said expression cassette comprises a human cytomegalovirus promoter and a bovine growth hormone polyadenylation signal functionally coupled to said HCV C-E1-E2-P7-NS2 sequence.
10. The method of claim 9, wherein said adenovector is based on
Ad5 containing deletions in the El region.
11. The method of claim 10, wherein said HCV C-E1-E2-P7-NS2 sequence consists of SEQ ID NO: 2.
12. The method of claim 7, wherein said recombinant nucleic acid consists of SEQ LD NO: 4.
13. The method of claim 12, wherein said step (b) involves the use of immuno-electron microscopy.
14. A recombinant nucleic acid consisting of SEQ LD NO: 4.
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