HK1156355B - Nucleic acid containing chimeric gene derived from hepatitis type-c virus - Google Patents

Description

Nucleic acid comprising chimeric gene derived from hepatitis c virus

Technical Field

The present invention relates to a chimeric hepatitis c virus particle containing a nucleic acid derived from a chimeric gene of hepatitis c virus, a JFH-1 strain and a strain other than the JFH-1 strain (preferably a strain belonging to genotype 1a, 1b or 2a), a vector for producing the virus particle, and a cell producing the virus particle.

The present invention also relates to a method for screening an anti-HCV agent using the viral particle, a vaccine obtained by inactivating or attenuating the viral particle, and an anti-hepatitis C virus antibody that recognizes the viral particle as an antigen.

Background

Hepatitis C virus (hereinafter referred to as "HCV") was discovered and identified as a causative virus of non-A non-B hepatitis by Choo et al in 1989 (non-patent document 1). HCV infection is a major cause of persistent infection following chronic hepatitis and is converted to cirrhosis and liver cancer, and it has been reported that about 1 hundred million and 7 million HCV-infected persons exist in the world and about 200 million HCV-infected persons exist in japan. The main infection route of HCV is blood infection, and since the screening of blood for transfusion is available, although new infected people in our country are rapidly declining, many virus carriers are still considered to exist.

Current therapies are primarily the administration of pegylated interferon, or a combination of pegylated interferon and the antiviral drug ribavirin. HCV has been classified into 6 genotypes so far, mainly genotypes 1b and 2a in japan. In particular, with regard to genotype 1b, the reality is: even when interferon and ribavirin are administered, the virus cannot be completely removed from the body, and the therapeutic effect is insufficient. In view of the above, development of a novel antiviral agent or vaccine for the purpose of preventing the onset of disease or eliminating viruses has been desired.

When HCV therapeutic agents are developed, there are no effective animals that reflect viral infection other than chimpanzees, and no effective in vitro virus culture systems, which have been obstacles. In recent years, an HCV replicon capable of evaluating the replication of HCV-RNA has been developed (non-patent document 2), and has been significantly advanced as a system for screening HCV inhibitors involved in the inhibition of viral replication.

HCV is a single-stranded RNA virus with a positive strand of about 9.6kb in genome length, which is cleaved by protease after translation, and has genes encoding precursor proteins of 10 viral proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS 5B). The replicon system is a system in which the translation region of the structural protein of HCV is recombined into a drug-resistant gene, and then IRES of EMCV (encephalomyocarditis virus) is inserted downstream thereof, and RNA replication is confirmed in cells into which the recombinant RNA is introduced. However, even when the full-length genomic RNA containing the structural protein of HCV is introduced into cells, release of viral particles into the culture medium is not observed (non-patent document 3).

Recently, Wakita et al discovered the HCV strain JFH1 belonging to genotype 2a isolated from a severe hepatitis patient, and it was clarified that it is released as infectious virus particles into a culture solution of Huh-7 cells (liver cancer cell line) (patent document 1 and non-patent document 4). This in vitro culture system for infectious HCV particles is expected to be a useful screening tool in the development of anti-HCV drugs and an effective method for producing HCV vaccines, and studies on the production of HCV particles in the in vitro culture system have been conducted, and it has been found that the HCV genome capable of producing viral particles is a chimeric HCV of other than the JFH-1 strain and the JFH-1 strain. This chimeric HCV can be produced by recombining the structural genes of the JFH-1 genome, i.e., the core, the E1, E2 and p7 protein-encoding portions, with the structural genes of other HCV strains.

As chimeric HCV between an HCV strain other than the JFH-1 strain and the JFH-1 strain, there are known: a chimeric HCV comprising the J6CF strain (genotype 2a) and the JFH-1 strain (non-patent document 5), a chimeric HCV comprising the H77 strain (genotype 1a) and the JFH-1 strain (patent document 2 and non-patent document 6), and a chimeric HCV comprising the S52 strain (genotype 3a) and the JFH-1 strain (non-patent document 7).

Non-patent document 8 shows: the virus yield of the chimeric HCV comprising the structural gene of J6CF and the non-structural gene of JFH-1 was the highest, while the yields of infectious HCV particles of the chimeric HCV of genotype 1b, i.e., Con1 strain and JFH-1 strain, were 1/10 or less. As a chimeric HCV of another genotype 1b strain and JFH-1 strain, patent document 3 describes: however, there has been no elucidation of productivity of infectious HCV particles in culture supernatants by creating a genome (full-length genomic replicon RNA) in which a region encoding a structural protein of the TH strain is recombined with the JFH-1 genome and a drug-resistant gene is inserted upstream of the encoding gene, and introducing the resulting drug-resistant strain into Huh-7 cells to obtain a drug-resistant strain.

In view of the above circumstances, there is a need for development of a method for producing HCV particles that can produce infectious HCV particles having a genotype 1b structure with a high yield, which is less effective for conventional therapies, and can be cultured in a persistent infection system.

Patent document 1: international publication WO05080575A1

Patent document 2: international publication WO06096459A2

Patent document 3: international publication WO06022422A1

Non-patent document 1: choo, QL et al, Science, 244: 359-362, 1989

Non-patent document 2: lohmann, v. et al, science, 285: 110-113, 1999

Non-patent document 3: pietschmann, t, et al, j.virol, 76: 4008-4021, 2002

Non-patent document 4: wakita, t. et al, nat.med., 11: 791-796, 2005

Non-patent document 5: lindenbach, b.d. et al, Science, 309: 623-626, 2005

Non-patent document 6: MinKyung, y, et al, j.virol., 81: 629-638, 2007

Non-patent document 7: gottwein, JM et al, Gastroenterology 133: 1614-1626, 2007

Non-patent document 8: pietschmann, t, et al, proc.natl.acad.sci.u.s.a., 103: 7408-7413, 2006

Disclosure of Invention

Problems to be solved by the invention

The invention aims to: provided are a method for efficiently producing HCV particles having structural proteins of HCV strains other than the JFH-1 strain belonging to genotypes 1a, 1b, or 2a, and a vaccine or the like using the produced HCV particles.

Means for solving the problems

The present inventors have intensively studied to solve the above problems, and have studied the productivity of HCV particles in cell culture, and found adaptive mutation (adaptive mutation) occurring when HCV grows. It is clear that: by introducing such an adaptive mutation, the productivity of HCV particles can be significantly improved as compared with the wild type before mutation introduction, and HCV particles having a structural protein of an HCV strain belonging to genotype 1a, 1b or 2a can be produced by a persistent infection system, thereby completing the present invention.

That is, the present invention relates to the following (1) to (22).

(1) A nucleic acid comprising a hepatitis C virus-derived chimeric gene in which regions encoding a core protein, an E1 protein, an E2 protein and a p7 protein derived from a hepatitis C virus strain other than the JFH-1 strain, a NS2 protein derived from the JFH-1 strain or a hepatitis C virus strain other than the JFH-1 strain, or a chimeric NS2 protein derived from the NS2 protein derived from the JFH-1 strain and a NS2 protein derived from a hepatitis C virus strain other than the JFH-1 strain, and a NS3 protein, a NS4A protein, a NS4B protein, a NS5A protein and a NS5B protein derived from the JFH-1 strain are arranged in this order from the 5 'side to the 3' side,

the proline residue at position 328 is substituted with an amino acid residue other than a proline residue, based on the number of amino acid residues at the N-terminus of the core protein.

(2) The nucleic acid molecule according to the above (1), wherein the 5 'side of the region encoding the core protein contains a 5' untranslated region of the JFH-1 strain, and the 3 'side of the region encoding the NS5B protein contains a 3' untranslated region of the JFH-1 strain.

(3) The nucleic acid according to (1) or (2), wherein the hepatitis C virus strain other than the JFH-1 strain is a strain belonging to genotype 1a, 1b or 2 a.

(4) The nucleic acid molecule according to any one of (1) to (3) above, wherein the hepatitis C virus strain other than the JFH-1 strain is selected from the group consisting of a TH strain, a Con1 strain, a J1 strain and derivatives thereof.

(5) The nucleic acid molecule according to any one of (1) to (4) above, wherein the amino acid residue other than the proline residue is selected from the group consisting of Ala, Leu, Ile, Val, Thr and Ser.

(6) The nucleic acid according to any one of (1) to (5) above, wherein the nucleic acid is a nucleic acid comprising any one of SEQ ID NOs of the sequence Listing: 1 or a nucleotide sequence having more than 90% homology with the nucleotide sequence, or a DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence having a homology of 90% or more with the nucleotide sequence.

(7) The nucleic acid according to any one of (1) to (5) above, wherein the nucleic acid is a nucleic acid comprising any one of SEQ ID NOs of the sequence Listing: 2 or a nucleotide sequence having more than 90% homology with the nucleotide sequence, or a DNA comprising the nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence having 90% or more homology with the nucleotide sequence.

(8) A vector comprising the nucleic acid according to any one of (1) to (7) above.

(9) A chimeric hepatitis C virus particle comprising the nucleic acid according to any one of (1) to (7) above as a viral genome.

(10) A cell that produces the chimeric hepatitis C virus particle described in (9) above.

(11) The cell according to (10) above, wherein the cell is Huh-7 or a derivative thereof.

(12) A method for screening an anti-hepatitis c virus substance, which comprises: culturing the cells of the following (a) or (b) in the presence of a test substance, and then detecting replicon RNA or viral particles derived from the above-mentioned nucleic acid in the resultant culture:

(a) the cell according to (10) or (11) above, or

(b) The chimeric hepatitis C virus particle and the hepatitis C virus-sensitive cell according to (9) above.

(13) A hepatitis C virus vaccine comprising the chimeric hepatitis C virus particle according to (9) above.

(14) The hepatitis C virus vaccine according to (13) above, wherein the chimeric hepatitis C virus particles are inactivated or attenuated.

(15) An anti-hepatitis C virus antibody that recognizes the chimeric hepatitis C virus particle described in (9) above as an antigen.

(16) The nucleic acid according to (4) above, wherein the hepatitis C virus strain other than the JFH-1 strain is a TH strain or a derivative thereof.

(17) The nucleic acid according to the above (5), wherein the amino acid residue other than the proline residue is Ala or Thr.

(18) A method for producing a chimeric hepatitis C virus particle, comprising the steps of:

culturing the cell of (10) or (11) above; and

a step of recovering the chimeric hepatitis C virus particle described in (9) above.

(19) A method for producing a hepatitis c virus vaccine, comprising the steps of:

a step of preparing an inactivated or attenuated chimeric hepatitis C virus particle by inactivating or attenuating the chimeric hepatitis C virus particle described in (9) above; and

preparing the inactivated or attenuated chimeric hepatitis C virus particles into a preparation in the form of a hepatitis C virus vaccine.

(20) A method for producing an anti-hepatitis C virus antibody, which comprises the steps of:

a step of immunizing an animal (excluding human) with the chimeric hepatitis C virus particle of (9) above, which may or may not be inactivated or attenuated.

(21) The production method according to (20) above, wherein the anti-hepatitis C virus antibody is a polyclonal or monoclonal antibody.

(22) The method according to (20) above, wherein the anti-hepatitis C virus antibody is a humanized antibody.

Effects of the invention

The nucleic acid containing a chimeric gene derived from hepatitis c virus of the present invention can be used to produce chimeric HCV particles having significantly higher productivity than wild-type HCV particles. Furthermore, the chimeric HCV particles of the present invention have the following advantages compared to wild-type HCV particles: has remarkably high productivity and high infectivity for cells, and therefore, has high utility value as a vaccine for prevention or treatment of HCV and as a tool for inducing anti-HCV antibodies.

The present specification includes the contents described in the specification and/or drawings of japanese patent application No. 2008-116193, which is the priority basis of the present application.

Drawings

FIG. 1 shows a method for preparing pTH/JFH1 plasmid. In the sequence of pJFH1, a nucleotide sequence encoding the 1 st to 846 nd amino acid residues with the N-terminal amino acid residue of the core protein amino acid sequence of JFH-1 as the 1 st position is recombined with a nucleotide sequence encoding the 1 st to 843 rd amino acid residues with the N-terminal amino acid residue of the core protein amino acid sequence of strain TH as the 1 st position.

FIG. 2 shows the change in the concentration of HCV core protein in the culture supernatant measured for each subculture after introducing RNA synthesized by pTH/JFH1 into Huh-7 cells and repeating subculture. The core protein in the culture supernatant decreased until day 23, but increased thereafter, and showed a constant high value after day 34.

FIG. 3A shows a method for preparing pTH/JFH1(PA) plasmid.

FIG. 3B shows a method for preparing pTH/JFH1(PT) plasmid.

FIG. 4 shows the change in the concentration of HCV core protein in the culture supernatant measured for each subculture after introducing RNA synthesized from pTH/JFH1, pTH/JFH1(PA) and pTH/JFH1(PT) into Huh-7 cells and repeating subculture. As in FIG. 2, TH/JFH-1 decreased until day 29 after introduction, but increased thereafter. On the other hand, in TH/JFH-1(PA) and TH/JFH-1(PT), high core protein was observed in the culture supernatant from the initial stage after introduction, and the values were maintained at high levels in all cases as compared with TH/JFH-1.

FIG. 5A shows the change in the concentration of HCV core protein in the culture supernatant up to 96 hours after the introduction of RNA synthesized by pTH/JFH1 and pTH/JFH1(PA) into Huh-7 cells. The yield of the nucleoprotein was higher in the culture supernatant of TH/JFH-1(PA) than in TH/JFH-1, 48 hours after RNA introduction.

FIG. 5B shows the infectious titer when each culture supernatant obtained in FIG. 5A was inoculated into uninfected Huh-7 cells. The infection titer was higher in the culture supernatant of TH/JFH-1(PA) than in TH/JFH-1 from 48 hours after the introduction of RNA.

FIG. 6 is a table showing the HCV core protein concentration of the culture supernatant obtained in the experiment shown in FIG. 5, the titer of infection with Huh-7 cells, and the intracellular HCV core protein concentration at that time. The ratio of the core protein after 96 hours to the intracellular core protein after 4 hours indicates the degree of autonomous replication of TH/JFH-1 and TH/JFH-1(PA) RNA in the cells. The HCV core secretion efficiency is an index of the secretion efficiency of HCV particles into the culture supernatant, and the efficiency is calculated by dividing (the amount of core protein in the culture supernatant after 96 hours) by (the amount of core protein in the culture supernatant after 96 hours + the amount of core protein in the cell after 96 hours). In cells into which TH/JFH-1(PA) RNA has been introduced, the secretion efficiency of HCV particles into the culture supernatant is higher than that of TH/JFH-1.

Detailed Description

The nucleic acid of the present invention is characterized by a chimeric gene of HCV comprising a nucleotide sequence coding for a nonstructural protein of a JFH-1 strain and a nucleotide sequence coding for a structural protein of an HCV strain other than the JFH-1 strain, and is characterized in that: contains a nucleotide sequence coding for the E1 protein with specific amino acid mutations.

Specifically, the nucleic acid of the present invention relates to the following (1) and (2):

(1) a chimeric gene derived from a hepatitis C virus, wherein the chimeric gene is formed by arranging regions encoding a core protein, an E1 protein, an E2 protein and a p7 protein derived from a hepatitis C virus strain other than the JFH-1 strain, a chimeric NS2 protein derived from the JFH-1 strain or the NS2 protein derived from the hepatitis C virus strain other than the JFH-1 strain or the NS2 protein derived from the JFH-1 strain and a NS2 protein derived from a hepatitis C virus strain other than the JFH-1 strain, and a NS3 protein, a NS4A protein, a NS4B protein, a NS5A protein and a NS5B protein derived from the JFH-1 strain in this order from the 5 'side to the 3' side;

(2) contains a nucleotide sequence encoding a complex protein (precursor protein) in which the amino acid residue at position 328 (or at position 137 when the amino acid residue at the N-terminus of the E1 protein is the 1 st position) is substituted from a proline residue with an amino acid residue other than a proline residue, when the methionine residue which is the N-terminal amino acid residue of the core protein is the 1 st position.

In the nucleic acid of the present invention, the NS2 protein may be derived from the JFH-1 strain, or may be derived from an HCV strain other than the JFH-1 strain, or may be a chimeric protein comprising a part of the NS2 protein derived from an HCV strain other than the JFH-1 strain and the remainder of the NS2 protein derived from the JFH-1 strain, and in this case, the chimeric protein has the same function as the wild-type NS2 protein, and for example, when a part of the NS2 protein derived from an HCV strain other than the JFH-1 strain comprises an amino acid sequence from the N-terminus to the 33-terminus of the NS2 protein, the remainder of the NS2 protein derived from the JFH-1 strain comprises an amino acid sequence from the 34-to the C-terminus.

Examples of the chimeric gene derived from hepatitis c virus include: for example, a polypeptide comprising SEQ ID NO: 1 or SEQ ID NO: 2, or a DNA comprising nucleotides 341 to 9433 of the sequence listing or a DNA comprising SEQ ID NO: 3 or SEQ ID NO: 4 from nucleotide 341 to nucleotide 9433.

In an embodiment of the present invention, the nucleic acid of the present invention may further comprise a 5 'untranslated region of the JFH-1 strain on the 5' side of the region encoding the core protein, and a 3 'untranslated region of the JFH-1 strain on the 3' side of the region encoding the NS5B protein.

In an embodiment of the present invention, the HCV strain other than the JFH-1 strain is a strain belonging to genotype 1a, 1b or 2 a. Strains belonging to genotype 1b include: such as strain TH, strain Con1, strain J1 and derivatives thereof. Strains belonging to genotype 1a include: such as strain H77. Strains belonging to genotype 2a include: for example, strain J6 CF. Preferred strains are the above-exemplified strains belonging to genotype 1b, and more preferably TH strain or a derivative thereof. In the present invention, the 328 th amino acid residue must be mutated to an amino acid residue other than a proline residue from the number of N-terminal amino acid residues of the core protein derived from the exemplified strain.

In another embodiment of the invention, the other amino acid residues than proline residues are, for example, Ala, Leu, Ile, Val, Thr or Ser, preferably Ala or Thr.

In an embodiment of the present invention, the nucleic acid is a nucleic acid comprising SEQ ID NO: 1, or a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 in sequence listing. Alternatively, in yet another embodiment, the nucleic acid is a nucleic acid comprising SEQ ID NO: 2, or a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4. These nucleic acids are chimeric nucleic acids from the JFH-1 strain and TH strain, SEQ ID NOs: 1 or SEQ ID NO: 3 comprises a codon in which the 328 th amino acid residue is Ala (nucleotides 1322 to 1324 th) from the N-terminal amino acid residue number of the core protein, and SEQ ID NO: 2 or SEQ ID NO: 4 comprises the same nucleotide sequence as the above-mentioned core protein except for the codon in which the 328 th amino acid residue is Thr (nucleotide 1322 th to 1324 th nucleotides) from the N-terminal amino acid residue number of the core protein.

Furthermore, the polypeptide represented by SEQ ID NO: 1 (sequence from the N-terminal of the core to the C-terminal of NS5B) is shown in SEQ ID NO: 6, consisting of a sequence corresponding to SEQ ID NO: 2 (the sequence from the N-terminus of the core to the C-terminus of NS5B) is shown in SEQ id no: 7.

the above nucleotide sequence of the present invention may consist of a nucleotide sequence identical to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4 has a homology of 90% or more, preferably 95% or more, and more preferably 98 to 99% or more, but in this case, the nucleotide sequence is represented by SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4 encodes an amino acid residue other than proline from nucleotides 1322 to 1324 in the number of nucleotides at the 5' -end of the nucleotide sequence (see above).

In addition, the above amino acid sequence of the present invention may consist of a sequence identical to SEQ ID NO: 5 or SEQ ID NO: 6 has a homology of 90% or more, preferably 95% or more, and more preferably 98 to 99% or more, in which case the 328 th amino acid residue encodes an amino acid residue other than a proline residue from the number of amino acid residues at the N-terminus of the core protein (see above).

The reason for this is that: like HCV, which is known as an RNA virus, exists in several genotypes, the structural, non-structural and/or (5 'or 3') untranslated regions are susceptible to sudden mutations.

In the present invention, the "328 th amino acid residue from the number of amino acid residues at the N-terminus of the core protein" refers to a residue sequence represented by SEQ ID NO: 5. SEQ ID NO: 6 or SEQ ID NO: 7 (sequence from the N-terminus of the core to the C-terminus of NS5B) of HCV, which is aligned with the amino acid sequence from the N-terminus of the core to the C-terminus of NS5B of the sequence listing SEQ ID NO: 5. SEQ ID NO: 6 or SEQ ID NO: 7 is aligned to the amino acid residue at the same position as the amino acid residue at position 328. In the present invention, "the proline residue at position 328 is substituted with an amino acid residue other than a proline residue, based on the number of amino acid residues at the N-terminus of the core protein" refers to the above-mentioned sequence listing and the amino acid residues shown in SEQ ID NOs: 5 at position 328 is an amino acid residue other than proline residue, compared with the amino acid residue at the same position in the predetermined amino acid sequence.

As used herein, "% homology" between two sequences means a function of the number of positions shared by the nucleotide or amino acid sequences, and the number of homologous positions as a percentage of the total number of positions when the two sequences are aligned with or without gaps introduced. "% homology" can be determined by using the mathematical algorithms BLASTN, BLASTX, Gapped BLAST and the like (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873. 5877, 1993; Altschul et al, Nucleic Acids Res., 25: 3389. 3402, 1997, etc.).

The present invention provides a vector or a chimeric HCV particle comprising the above nucleic acid. The chimeric HCV particle has the following characteristics compared with a wild type: can be produced efficiently in a cell culture system and has high infectivity. The effects of high efficiency production and high infectivity are due to: the result shown in FIGS. 4 and 5 reveals that the 328 th amino acid residue is mutated to an amino acid residue other than Pro (preferably Ala or Thr) from the N-terminal number of amino acid residues of the core protein.

In the production of the nucleic acid, vector and chimeric HCV particle of the present invention, conventional techniques in molecular biology, virology, and the like, which are within the technical scope of the art, can be used. Such techniques are described in academic documents, patent documents, professional books, and the like, and include, for example: sambrook et al, Molecular Cloning: a Laboratory Manual (3 rd edition, 2001, CSHL PRESS); mahy et al, Virology: a practical prophach (1985, IRL PRESS); ausubel et al, Current Protocols in Molecular Biology (3 rd edition, 1995, John Wiley & Sons); U.S. Pat. No.4,683,202 (Cetus Corporation; PCR method), etc.

In order to produce the nucleic acid or infectious HCV particle of the present invention, a target sequence portion can be amplified by performing Polymerase Chain Reaction (PCR) using a forward primer and a reverse primer designed from the sequence of cDNA using a vector obtained by cloning cDNA derived from genomic RNA of HCV strains other than the JFH-1 strain or the JFH-1 strain as a template. Specifically, as shown in fig. 1 or 3, a plurality of different PCR products having a repetitive sequence with each other are synthesized, and these PCR products are mixed and used as a template to perform PCR using a forward primer containing the 5 'end of the target nucleic acid and a reverse primer containing the 5' end of the complementary strand of the nucleic acid, whereby the target nucleic acid can be amplified. Each end of the synthesized nucleic acid was cleaved with a restriction enzyme, and ligated to a plasmid pJFH1(Wakita, T. et al, Nat. Med., 11: 791-796, 2005; International publication WO2004/104198) cleaved with the same enzyme. Basic techniques for such manipulation are also described in, for example, WO04104198a1, WO06022422a1, Wakita, t. et al, nat. med., 11: 791-: 623, 626, 2005.

The PCR reaction includes, but is not limited to, the following operating conditions: in the presence of template, primer, dNTPs, heat-resistant polymerase and Mg²⁺In the presence of a buffer, the step comprising 94 to 98 ℃ for about 10 to 60 seconds, 55 to 58 ℃ for about 10 to 60 seconds, and 72 ℃ for about 30 to 60 seconds is performed for 20 to 40 cycles as 1 cycle.

The HCV genome is typically an RNA comprising a 5 'untranslated region, a core protein coding region, an E1 protein coding region, an E2 protein coding region, a p7 protein coding region, an NS2 protein coding region, an NS3 protein coding region, an NS4A protein coding region, an NS4B protein coding region, an NS5A protein coding region, an NS5B protein coding region, and a 3' untranslated region. On the other hand, the nucleic acid of the present invention capable of producing infectious HCV particles is composed of viral genomic RNA of 2 or more HCV strains or DNA encoding the RNA.

In the nucleic acid of the present invention, for example, the 5 'untranslated region, the region encoding a part of the NS2 protein, the NS3 protein coding region, the NS4A protein coding region, the NS4B protein coding region, the NS5A protein coding region, the NS5B protein coding region and the 3' untranslated region are derived from the JFH-1 strain, while the core protein coding region, the E1 protein coding region, the E2 protein coding region, the p7 protein coding region and the region encoding the remainder of the NS2 protein are derived from HCV strains other than the JFH-1 strain. The 5' -untranslated region may be derived from an HCV strain other than the JFH-1 strain.

Alternatively, in another example of the chimeric nucleic acid of the present invention, the 5 'untranslated region, the core protein coding region, the E1 protein coding region, the E2 protein coding region, the p7 protein coding region and the NS2 protein coding region are derived from an HCV strain other than the JFH-1 strain, while the NS3 protein coding region, the NS4A protein coding region, the NS4B protein coding region, the NS5A protein coding region, the NS5B protein coding region and the 3' untranslated region are derived from the JFH-1 strain.

Alternatively, in still another example of the nucleic acid of the present invention, the 5 'untranslated region is derived from the JFH-1 strain, while the core protein coding region, the E1 protein coding region, the E2 protein coding region, the p7 protein coding region and a portion of the region coding for the NS2 protein are derived from the TH strain, and the region coding for the remainder of the NS2 protein, the NS3 protein coding region, the NS4A protein coding region, the NS4B protein coding region, the NS5A protein coding region, the NS5B protein coding region and the 3' untranslated region are derived from the JFH-1 strain, but the core protein coding region, the E1 protein coding region, the E2 protein coding region and the p7 protein coding region are not limited to the chimeric nucleic acid as long as they are derived from the TH strain.

Hereinafter, the vector, infectious HCV particle, cell producing HCV particle, and use of HCV particle of the present invention will be described in detail.

(1) Production of the Carrier

The genome of Hepatitis C Virus (HCV) is a single-stranded RNA with a full-length positive strand of about 9600 nucleotides. The genomic RNA contains a 5 'untranslated region (also referred to as 5' NTR or 5 'UTR), a translated region composed of a structural region and an unstructured region, and a 3' untranslated region (also referred to as 3 'NTR or 3' UTR). In its structural region, the structural proteins of HCV are encoded; whereas in the nonstructural region, a plurality of nonstructural proteins are encoded.

Such structural proteins (core, E1, E2 and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) of HCV are translated from the translation region in the form of a series of polyprotein complex proteins (precursor proteins), and then released and produced in infected cells by limited decomposition with a protease. Of these structural and non-structural proteins (i.e., viral proteins of HCV), Core (Core) is known as the Core protein, and E1 and E2 are envelope proteins. The nonstructural proteins are proteins involved in virus self-replication, and NS2 has metalloprotease activity, NS3 has serine protease activity (one third of the N-terminal side) and helicase activity (two thirds of the C-terminal side). Further, there are reports that: NS4A is a cofactor for the protease activity against NS3, while NS5B has RNA-dependent RNA polymerase activity.

HCV is coated with a capsule called an envelope. The envelope contains components from the host cell membrane and proteins from the virus. The proteins constituting the envelope of HCV include envelope protein 1 (referred to as E1), envelope protein 2 (referred to as E2), and p 7. In particular, E1 and E2 have a transmembrane region at their C-terminus, via which they are anchored to the HCV membrane. Thus, the E1 and E2 proteins of HCV face the outside, and HCV infects cells by adhering to them via E1 and/or E2.

According to a systematic analysis method using nucleotide sequences of HCV strains, HCV is classified into 6 types, genotype 1 to genotype 6, and each of the above types is further classified into several subtypes. Furthermore, with respect to multiple genotypes of HCV, the nucleotide sequence of the entire genome thereof has also been determined (Simmonds, P. et al, Hepatology, 10: 1321-. Specifically, as an HCV strain of genotype 1a, the H77 strain (GenBank accession number AF 011751); as the genotype 1b type HCV strains, the J1 strain (GenBank accession No. D89815), the Con1 strain (GenBank accession No. AJ238799, also referred to as Con-1 strain, Con1 strain in some cases), and the TH strain (Wakita, T. et al, J.biol.chem., 269, 14205-minus 14210, 1994, Japanese patent laid-open No. 2004-minus 179) are known; as HCV strains of genotype 2a, there are known the JFH-1 strain (GenBank accession No. AB047639, also referred to as JFH1 strain in some cases), the J6CF strain (GenBank accession No. AF177036), JCH-1(GenBank accession No. AB047640), JCH-2(GenBank accession No. AB047641), JCH-3(GenBank accession No. AB047642), JCH-4(GenBank accession No. AB047643), JCH-5(GenBank accession No. AB047644) and JCH-6(GenBank accession No. AB 047645). Also, HC-J8 strain (GenBank accession number D01221) and the like are known as HCV strains of genotype 2 b; known HCV strains of genotype 3a include NZL1 strain (GenBank accession No. D17763), S52 strain (GenBank accession no); as the HCV strain of genotype 3b, Tr-Kj (GenBank accession number D49374) and the like are known, and as the HCV strain of genotype 4a, ED43(GenBank accession number) and the like are known. For other strains, a list of GenBank accession numbers has been reported (Tokita, T. et al, J.Gen.Virol., 79: 1847-.

The genomic nucleotide sequences of the JFH-1 strain and HCV strains other than JFH-1 strain described in the present invention can be obtained from the above-mentioned documents or GenBank, and HCV strains other than JFH-1 strain can be selected from the above-mentioned genotypes, but preferred genotypes are those belonging to 1a, 1b or 2 a.

The chimeric HCV gene can be prepared as follows: a chimeric HCV gene was prepared by performing PCR using a vector obtained by cloning cDNA of each HCV genomic RNA as a template and synthetic DNA as primers to amplify and join essential regions of each HCV gene.

Furthermore, the chimeric HCV gene cDNA is ligated to an appropriate restriction site downstream of a promoter such as T7 promoter of plasmid pJFH1(Wakita, T. et al., nat. Med., 11: 791-796, 2005, International publication WO2004/104198) to prepare a vector for synthesizing HCV genomic RNA. When RNA transcribed from this vector is introduced into cells such as Huh-7, replication and assembly of the virus occur, and infectious HCV particles can be produced.

(2) Preparation of HCV particles

Chimeric HCV particles can be prepared by synthesizing RNA from HCV cDNA cloned under the control of a promoter, and introducing the RNA into cells.

That is, the chimeric HCV particle can be produced by a method including the steps of: a step of culturing cells producing the HCV particle; and a step of recovering the HCV particle. Among them, cells producing HCV particles can be obtained by infecting HCV-sensitive cells (i.e., cells that allow the formation of HCV particles) with the chimeric HCV particles of the present invention.

The promoter is not limited, and examples thereof include: t7 promoter, SP6 promoter, T3 promoter, etc., but T7 promoter is preferred.

RNA is prepared in vitro using a nucleic acid obtained by cloning HCV cDNA under the control of a T7 promoter as a template, and this can be performed using, for example, MEGAscript T7 kit (Ambion).

The cells into which RNA is introduced may be any cells that allow HCV particles to be formed, and examples thereof include: huh-7 cells, HepG2 cells, IMY-N9 cells, HeLa cells, 293T cells, or derivatives thereof, and further preferably: huh-7 cells, Huh7.5 cells and Huh7.5.1 cells as derivatives thereof, and the like. In addition, there can be enumerated: cells obtained by expressing the CD81 gene and/or Claudin-1 gene in Huh-7 cells, HepG2 cells, IMY-N9 cells, HeLa cells, 293 cells or 293T cells (Lindenbach, B.D. et al, Science, 309: 623-626, 2005; Evans, M.J. et al, Nature, 446: 801-50805, 2007; Akazawa, D. et al, J.Virol, 81: 5036-5045, 2007).

Examples of the method for introducing RNA include: calcium phosphate coprecipitation method, DEAE dextran method, lipofection method, microinjection method, and electroporation method, preferably lipofection method and electroporation method, and more preferably electroporation method.

In addition, when cDNA is introduced, HCV cDNA can be expressed in a system using an RNA polymerase I promoter and terminator (WO27037428A 1).

The virus particle-producing ability of the cell can be detected using an antibody against, for example, the core protein, the E1 protein, or the E2 protein constituting the HCV virus particles released into the culture solution. Furthermore, the presence of HCV viral particles can also be indirectly detected by amplifying HCV genomic RNA contained in HCV viral particles in a culture medium by RT-PCR using specific primers and detecting the amplified RNA.

Whether or not the produced virus has an infectious ability can be judged as follows: the determination can be made by culturing cells into which HCV RNA has been introduced, and contacting the resulting supernatant with HCV permissive cells (e.g., Huh-7 or a derivative thereof), for example, by immunostaining the cells with an anti-core antibody after 48 hours to count the number of infected cells, or by subjecting the cell extract to electrophoresis in SDS-polyacrylamide gel and detecting the core protein by Western blotting.

(3) Obtaining a particle-producing cell line

The study showed that: for efficient replication of the HCV genome, it is necessary to generate mutations in the nucleotide sequence of the genome (Lohmann, V.et al, J.Virol., 75: 1437-1449, 2001), and mutations that enhance replication are called adaptive mutations (adaptive mutation). By subculturing the cells into which the HCV genomic RNA has been introduced, which have been prepared in (2), a cell line that continuously produces HCV particles can be obtained. By continuing the culture in this manner, adaptive mutations occur in the HCV genome, and HCV particle production may be significantly improved.

Typical examples of using this phenomenon are: a method for selecting a mutant having improved virus productivity by introducing genomic RNA of chimeric HCV into cells. Examples of such mutations are: permissive mutations in chimeric HCV particles of strain H77 and JFH-1 (MinKyung Y et al, JVirol, 81: 629-638, 2007). Mutations were allowed to occur randomly depending on the strain, the design (construct) of the chimeric HCV genome, and the experimental conditions. Therefore, this mutation also does not have suitable certainty in genotype 1b, so it is necessary to perform an experiment for each desired construct to obtain a permissive mutant.

The replication ability and the HCV particle productivity are greatly changed by mutation of 1 amino acid residue, and the mutation varies depending on the genotype of HCV, the kind of cell used in culture, and experiments. In addition, since mutations in nucleic acids required for mutation of 1 amino acid residue cannot be detected by hybridization techniques, it is necessary to determine the gene sequence of HCV in order to detect these mutations.

Therefore, by isolating HCV genomic RNA from such cells and determining the nucleotide sequence thereof, it is possible to specify an HCV genomic sequence that enables high-yielding HCV particles.

In order to confirm whether these mutations are related to HCV replication ability or HCV particle productivity, it is necessary to introduce mutations into the original HCV genome to confirm whether HCV replication ability or HCV particle productivity has been reproduced. When a mutation is introduced into the original HCV genome, a PCR method or a commercially available mutation introduction kit (for example, KOD-Plus-mutagenesis kit manufactured by Toyobo Co., Ltd.) may be used.

Whether or not the mutation is specific to the HCV genome to be used or effective for other HCV genomes can be confirmed by introducing a mutation into the HCV genome without a mutation again.

In the present invention, it was confirmed that when the N-terminal amino acid residue of the core protein of TH/JFH-1 (example 1 described later) is the 1 st position, the 328 TH amino acid residue (or, when the N-terminal amino acid residue of the E1 protein is the 1 st position, the 137 TH amino acid residue), i.e., the proline residue, was mutated to the alanine residue or the threonine residue. Furthermore, it also implies: the 328 TH amino acid residue from the N-terminal amino acid residue of the core protein is common not only to the TH strain but also to Con1 strain of genotype 1b (GenBank accession No. AJ238799), J1 strain (GenBank accession No. D89815), H77 strain of genotype 1a (GenBank accession No. AF011751), JFH-1 strain of genotype 2a (GenBank accession No. AB047639) and J6CF strain (GenBank accession No. AF177036), and mutation of a proline residue into another amino acid residue, preferably an alanine residue or a threonine residue, is effective not only for the TH strain but also for any HCV strain having a proline residue as the 328 TH amino acid residue from the N-terminal amino acid residue of the core protein.

(4) Use of HCV particles

HCV particles are suitable for use as vaccines, and for use as antigens for the production of anti-HCV antibodies.

Specifically, HCV particles can also be used as a vaccine as they are, but they can also be attenuated or inactivated by a method known in the art. Inactivation of viruses can be achieved by adding and mixing an inactivating agent such as formalin, β -propiolactone, glutaraldehyde, or the like, for example, to a virus suspension, and allowing it to react with the viruses (Appaiahgari, M.B. & Vrati, s., Vaccine, 22: 3669-3675, 2004). It is also considered that an attenuated vaccine can be obtained by infecting cultured animal cells or animals (excluding humans) with chimeric HCV particles and repeating subculture to extremely reduce pathogenicity, or by genetically modifying a region involved in the proliferation or infectivity of HCV, for example, the core-NS 5B region, to be negative (negative), thereby reducing pathogenicity.

Thus, the HCV vaccine of the present invention can be produced by a method comprising the steps of: a step of preparing an inactivated or attenuated chimeric HCV particle by inactivating or attenuating the chimeric HCV particle of the present invention; and a step of preparing the inactivated or attenuated chimeric HCV particle into a preparation in the form of HCV vaccine.

The vaccine of the present invention can be formulated, for example, in any administration form of solution or suspension. Alternatively, they may be prepared in solid form suitable for dissolution or suspension in a liquid (e.g., lyophilized products) so that they may be reconstituted prior to use. Alternatively, such solids or preparations may be opalescent in the presence of a pharmaceutically acceptable surfactant, or may be encapsulated into liposomes.

The active immunogenic ingredients, such as HCV particles, can often be mixed with excipients that are pharmaceutically acceptable and suitable for the active ingredient. Suitable excipients include: such as water, physiological saline, glucose, glycerol, ethanol, and the like, and mixtures thereof.

Also, the vaccine may contain minor amounts of adjuvants (e.g., wetting or emulsifying agents), pH buffering agents, and/or adjuvants to enhance the efficacy of the vaccine, as desired.

Examples of adjuvants that can act are not limited, and include aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-N-muramyl-L-alanyl-D-isoglutamine (also known as CGP11637, nor-MDP), N-acetyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (also known as CGP19835A, MTP-PE), and RIBI. Among them, RIBI may contain 3 components extracted from bacteria, namely monophosphoryl lipid a, trehalose dimycolate and cell wall skeleton (HPL + TDM + CWS), in a 2% squalene/Tween (registered trademark) 80 emulsion.

The efficacy of an adjuvant can be determined by administering to a mammal a vaccine composed of HCV particles and measuring the amount of antibody produced.

The vaccines of the present invention are typically administered by parenteral administration, such as by injection, for example, subcutaneous or intramuscular injection. Other formulations suitable for other modes of administration include suppositories, and oral prescriptions used in some cases.

If necessary, 1 or more compounds having adjuvant activity may be added to the HCV vaccine. Adjuvants are non-specific stimulators of the immune system. They enhance the host immune response to HCV vaccines. Specific examples of adjuvants known in the art are: freund's complete and incomplete adjuvant, vitamin E, nonionic block polymer, muramyl dipeptide, saponin, mineral oil, vegetable oil, and Carbopol (Carbopol). Adjuvants particularly suitable for mucosal use are, for example: coli (e.coli) heat Labile Toxin (LT) or Cholera Toxin (CT). Other suitable adjuvants are, for example: aluminium hydroxide, aluminium phosphate or alumina, an oily emulsion (for example an emulsion of Bayol (registered trade mark) or Marcol 52 (registered trade mark)), a saponin or a vitamin E solubiliser. Thus, in a preferred form, the vaccine of the invention contains an adjuvant.

For example, in an injection to be administered subcutaneously, intradermally, intramuscularly or intravenously, the HCV vaccine of the present invention may be administered together with other specific examples of pharmaceutically acceptable carriers or diluents, for example, stabilizers, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing substances such as bovine serum or skim milk, and buffers (e.g., phosphate buffer).

Existing binders and carriers used in suppositories may include: such as polyalkylene glycol or triglycerol. Such suppositories may be formed of mixtures containing the active ingredient in the range of 0.5% to 50%, preferably in the range of 1% to 20% by weight. Oral prescription drugs contain commonly used excipients. Examples of such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

The vaccine of the invention is prepared in the form of a solution, suspension, tablet, pill, capsule, sustained release prescription or powder containing 10-95%, preferably 25-70% by weight of the active ingredient (viral particles or a fraction thereof).

The vaccine of the present invention is administered in a method suitable for the administration form and in an amount having a prophylactic and/or therapeutic effect. Suitable amounts for administration are typically: the antigen is administered in the range of 0.01. mu.g to 100,000. mu.g per administration, depending on the patient to be treated, the antibody synthesizing ability in the immune system of the patient, and the desired degree of defense, and also depending on the administration routes such as oral, subcutaneous, intradermal, intramuscular, intravenous administration routes.

The vaccines of the present invention may be administered according to a single dosing regimen, or preferably according to a combination dosing regimen. When administered according to a combination regimen, 1-10 separate administrations are given at the time of initiation of vaccination and then another administration may be given at intervals necessary to maintain and/or enhance the immune response, for example, 2 nd administration after 1-4 months. Administration may follow several months later, as desired. The summary of administration is also determined at least in part by the necessity of the individual, depending on the judgment of the physician.

Furthermore, the vaccine containing HCV particles of the present invention may be administered together with other immune control agents (e.g., immunoglobulin).

Also, the present invention provides the following method: by administering the HCV particle vaccine of the present invention to a healthy person, an immune response to HCV is induced in the healthy person, for the prevention of a new HCV infection. The invention also provides methods for use as therapeutic vaccines: the HCV particle vaccine of the present invention is administered to a patient already infected with HCV to induce a strong immune response against HCV in vivo, thereby excluding HCV.

The HCV particles of the present invention are also effective as antigens for producing antibodies. Antibodies recognizing the HCV particles of the present invention used as antigens are useful as passive immunizing agents for the prevention or treatment of HCV infection. The type of antibody is not limited, and examples thereof include: for example, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, fragments of the above antibodies (Fc, Fab, (Fab')₂Etc.), single chain antibodies (scFv, etc.), camel antibodies, multivalent antibodies (bivalent, trivalent, etc.), etc. The antibody may be of any species, such as IgG, IgE, IgM, IgD, IgA, IgY, and its class (class) includes, for example, IgG1 to IgG4, IgA1 to IgA2, and the like. Furthermore, the antibody may comprise chemical modifications such as glycosylation, pegylation, acetylation, phosphorylation, amidation, and the like.

anti-HCV antibodies can be made according to a method comprising the steps of: a step of administering the above-described chimeric HCV particle of the present invention, which may or may not be inactivated or attenuated, to an animal (excluding human), preferably a mammal or bird.

The mammals include mice, rats, rabbits, goats, sheep, horses, cattle, guinea pigs, dromedary, bactrian camels, and U.S. camels. Dromedary, bactrian and american camels are suitable for making antibodies comprising only H chains. Examples of the birds include: chicken, goose, ostrich, etc.

The antibody can be obtained by collecting serum from an animal to which the HCV particle of the present invention has been administered, and performing a known method (for example, ammonium sulfate fractionation, ion exchange chromatography, protein A or protein G binding affinity chromatography, gel filtration chromatography, etc.).

Furthermore, hybridomas that produce monoclonal antibody-producing cells can be prepared using cells or tissues (B cells, spleen cells, lymph nodes) of animals immunized with the HCV particles of the present invention and myeloma cells (mouse, rat, etc.). Methods for producing hybridomas are well known, and Antibodies: a Laboratory Manual (Cold Spring harbor Laboratory, 1988; eds. Fushan and Andon, monoclonal antibodies, protocols, lectures, 1987, etc.).

Cells producing monoclonal antibodies can be produced by cell fusion, or by other methods such as introduction of oncogene DNA or immortalization of B lymphocytes by Epstein-Barr virus infection.

Humanized antibodies or human antibodies can be prepared by using phage display method (Brinkman et al, J.Immunol. methods, 182: 41-50, 1995; Ames et al, J.Immunol. methods, 184: 177-186, 1995; International publication WO 98/46645; International publication WO 98/50433; International publication WO98/24893, etc.), or human antibody-producing mice (KM mice (キリンフア - マ Co., Abgenix/Amgen, etc.).

The monoclonal antibody, polyclonal antibody, and human antibody or humanized antibody obtained by the above methods are effective for diagnosis, treatment, and prevention of HCV.

The antibody produced using the HCV particles of the present invention is administered together with a pharmaceutically acceptable lytic agent, additive, stabilizer, buffer, and the like. The route of administration may be any route of administration, but is preferably subcutaneous, intradermal, intramuscular, more preferably intravenous.

Preferred examples of antibodies produced using the HCV particles of the present invention include: an anti-hepatitis c virus antibody that recognizes the chimeric HCV (hepatitis c virus) particles of the present invention (i.e., chimeric HCV particles containing the nucleic acid of the present invention as a viral genome) as an antigen. The anti-hepatitis c virus antibody may be an antibody produced against the chimeric HCV particle of the present invention, and regardless of the production process, it binds (reacts) not only with the chimeric HCV particle of the present invention but also with other broad hepatitis c virus particles, and may inhibit the function thereof.

The HCV particle (chimeric HCV particle) of the present invention or the cell producing the particle can be further used for screening an anti-HCV substance.

The method for screening an anti-HCV substance specifically comprises the steps of: culturing the cells of the following (a) or (b) in the presence of a test substance, and then detecting replicon RNA or viral particles derived from the nucleic acid contained in the chimeric HCV particle of the present invention in the resulting culture:

(a) a cell that produces a chimeric HCV particle; or

(b) Chimeric HCV particles and hepatitis c virus-sensitive cells.

According to the above method, an anti-HCV substance is selected as a substance which can inhibit viral infection or proliferation. In the present invention, the "replicon RNA" refers to RNA that is produced by altering the HCV viral genome and has an autonomous replication ability. In the present invention, the term "autonomous replication ability" refers to an ability to autonomously regenerate (i.e., replicate) a copy of a nucleic acid itself in a cell, like plasmid DNA. Examples of previously known subgenomic replicon RNAs include: the recombinant RNA prepared by recombining the translation region of the structural protein of HCV into a drug-resistant gene and inserting IRES of EMCV (encephalomyocarditis virus) downstream thereof was confirmed to show RNA replication in cells into which the recombinant RNA was introduced. On the other hand, the full-length genomic replicon RNA is RNA that can autonomously replicate RNA derived from the full-length genome of HCV introduced into cells, and typical examples thereof include: among the RNAs derived from the full-length genome of HCV, a recombinant RNA having a drug resistance gene (or reporter gene) and an IRES inserted between its 5' untranslated region and the gene encoding the HCV core protein. In the above method, "replicon RNA derived from the above nucleic acid" refers to replicon RNA transcribed from the nucleic acid. The hepatitis C virus-sensitive cells are not limited to the following cells, but include: the cells to which HCV-derived RNA is introduced in the above-mentioned "(2) preparation of HCV particle" include, for example, Huh-7, HepG2, IMY-N9, HeLa, 293 or 293T cells, or derivatives thereof.

Examples

The present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these examples. In the examples, the TH strain is exemplified as an HCV strain other than the JHF-1 strain, but strains other than the specific strain can be prepared in the same manner.

Example 1: construction of TH/JFH-1 plasmid

As a cDNA for HCV genomic RNA, a TH/JFH-1 chimeric cDNA was prepared, wherein 5 'UTR was JFH-1 strain of genotype 2a (GenBank accession No. AB047639, Kato, T. et al, Gastroenterology, 125: 1808-No. 1817, 2003), and N-terminal 33 amino acid residue of core protein-NS 2 protein was TH strain of genotype 1b (Wakita, T. et al, J.biol.chem., 269: 14205-No. 14210, 1994, Mordapour, D. et al, biochem.Biophys.Res.Commun.246: 920-No. 924, 1998 and International publication WO2006/022422), and N-terminal 34 amino acid residue-3' UTR of NS2 was JFH-1 strain of genotype 2 a.

The amino acid sequence of the protein coded by TH/JFH-1 is shown in SEQ ID NO: 5. the method for preparing these plasmids is shown in FIG. 1.

Specifically, pJFH1(Wakita, T. et al, Nat. Med., 11: 791-796, 2005, International publication WO2004/104198) and pTH (International publication WO2006/022422) comprising a part of the TH strain of the viral genome isolated from a hepatitis patient were used, and pJFH1 was a plasmid DNA constructed by cloning cDNA corresponding to the entire region of genomic RNA derived from the JFH-1 strain into the pUC19 plasmid.

Using pJFH1 as a template, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primer 21M13(SEQ ID NO: 8) and MS98(SEQ ID NO: 9) attached to the Phusion High-Fidelity DNA polymerase kit (FINNZYMES Co.), were added, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 45 seconds were used as 1 cycle, and 30 cycles were carried out. The resulting PCR product was designated as PCR product No. 1.

Next, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS97(SEQ ID NO: 10) and MS96(SEQ ID NO: 11), which were attached to the Phusion High-Fidelity DNA polymerase kit (FINNZYMES), were added using pTH as a template, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 45 seconds were set to 1 cycle, and 30 cycles were performed. The resulting PCR product was designated as PCR product No. 2.

Next, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS99(SEQ ID NO: 12) and MS89(SEQ ID NO: 13) attached to Phusion High-Fidelity DNA polymerase kit (FINNZYMES) were added using pJFH1 as a template, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 45 seconds were used as 1 cycle, and 30 cycles were carried out. The resulting PCR product was used as PCR product No. 3.

Each PCR product was purified on an agarose gel, and eluted with 50. mu.l of EB buffer attached thereto using QIAquick gel extraction kit (QIAGEN). Mu.l each of the DNAs of PCR product No.1, PCR product No.2 and PCR product No.3 was mixed, and using this as a template, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers 21M13(SEQ ID NO: 8) and MS89(SEQ ID NO: 13) attached to the Phusion High-Fidelity DNA polymerase kit (FINNZYMES Co.), were added, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to carry out PCR. The PCR reaction was carried out under the following conditions: each cycle was carried out for 30 cycles, with 1 cycle comprising a step of 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 2 minutes. The resulting PCR product was designated as PCR product No. 4.

pJFH1 and the purified PCR product No.4 were digested with restriction enzymes EcoRI and SpeI, and each DNA fragment was separated by agarose gel electrophoresis and purified. These 2 DNA fragments were mixed with Ligation high (New England Biolabs) and ligated. This vector was designated as pTH/JFH 1. The pTH/JFH1 encodes a DNA fragment containing a 5' untranslated region derived from the JFH-1 strain; from the core of the TH strain to the N-terminal 33 amino acid residues of the protein regions E1, E2, p7 and NS 2; a nucleotide sequence of a chimeric gene of the N-terminal 34 amino acid residues start of the NS2 protein region, NS3, NS4A, NS4B, NS5A, NS5B protein region and 3' untranslated region of the JFH-1 strain.

Example 2: in vitro RNA synthesis and introduction thereof into cells

pTH/JFH1 was cleaved with XbaI, and phenol/chloroform extraction and ethanol precipitation were performed. Next, the XbaI-cleaved fragment was treated with mung bean nuclease to remove the remaining nucleotide sequence from the 3' -end of the XbaI recognition sequence. Then, protease K treatment, phenol/chloroform extraction and ethanol precipitation are carried out to purify the DNA fragment. Using the cleaved plasmid as a template, the reaction was carried out at 37 ℃ for 3 hours using MEGAscript T7 kit (Ambion Co.) to synthesize HCV RNA. After DNaseI treatment, the reacted synthetic RNA was extracted with acidic phenol, and then ethanol-precipitated and purified.

Will be 3X 10⁶Each of Huh7 cells and 10. mu.g of HCV RNA were suspended in 400. mu.l of Cytomix solution (120mM KCl, 0.15mM CaCl)₂、10mM K₂HPO₄/KH₂PO₄、25mMHepes、2mM EGTA、5mM MgCl₂20mM ATP and 50mM glutathione) was transferred to a 4mM cuvette (Cuvettes), and then electroporation was performed at 260V and 950. mu.F using a Gene Pulser (BioRad). Thereafter, the cells into which HCV RNA had been introduced were inoculated at 10cm²Subculture was performed in a petri dish.

Example 3: HCV particle production in TH/JFH-1 RNA-introduced cells

When passaging cells into which TH/JFH-1RNA was introduced was performed, HCV core protein contained in the culture supernatant was quantified using an HCV antigen ELISA test kit (オ - ソ), and the production of HCV particles was confirmed. As a result, the amount of HCV core in the culture supernatant decreased with time up to 23 days after the introduction, but increased in the amount of HCV core at the elapse of 26 days from the introduction, and showed a constant high yield at the elapse of 34 days (fig. 2). It is thus assumed that: TH/JFH-1RNA does not have a high virus-producing ability at the beginning when introduced into Huh7 cells, but then an adaptive mutation necessary for virus production is introduced into the viral genome, thereby imparting a high virus-producing ability to TH/JFH-1 RNA.

Example 4: analysis of HCV genomic sequence in repeatedly passaged TH/JFH-1 Virus-infected cells

In order to examine the adaptive mutation required for the TH/JFH-1 virus to have a high yield, total RNA in infected cells on day 34 from the introduction of the RNA was extracted, and the HCV genome contained therein was subjected to sequence analysis.

Total RNA was extracted using Trizol (Invitrogen) and subjected to transcription reaction to produce cDNA. The cDNA was divided into 5 DNA fragments by PCR, ligated with pGEM-T Easy vector (Promega), and transformed into E.coli DH5a to obtain colonies. Plasmids were extracted from 10 colonies using a QIAprep Mini kit (QIAGEN Co.) and the nucleotide sequence of each DNA fragment was confirmed.

As a result, proline in E1 region of TH strainCCU(P) by substitution with alanineACU(A) Or threonineGCU(T). Note that, the N-terminal amino acid residue, i.e., methionine residue, of the core protein of strain TH (Wakita, T. et al, J.biol.chem., 269(1994) 14205-14210, Mordapour et al, biochem.Biophys.Res.Commun., 246 (1998)) 920-924, and International publication WO2006/022422 corresponds to the 328 TH amino acid residue, as counted from the 1 st position; alternatively, the amino acid residue at the N-terminus of the E1 protein corresponds to the amino acid residue at position 137, starting with the 1 st position.

The amino acid sequence of TH/JFH-1(PA) is shown in SEQ ID NO: 6, the amino acid sequence of TH/JFH-1(PT) is shown in SEQ ID NO: 7.

example 5: construction of TH/JFH-1 mutant plasmid

A plasmid having the adaptive mutation required for high yield of TH/JFH-1 virus as shown in example 4 was constructed. The plasmid was prepared according to the method shown in FIG. 3.

Specifically, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS151(SEQ ID NO: 14) and MS165(SEQ ID NO: 15) attached to the Phusion High-Fidelity DNA polymerase kit (FINNZYMES Co.) were added using pTH/JFH1 as a template, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute were set to 1 cycle, and 30 cycles were carried out. The resulting PCR product was designated as PCR product No. 5.

Next, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS164(SEQ ID NO: 16) and MS156(SEQ ID NO: 17) attached to the Phusion High-Fidelity DNA polymerase kit (FINNZYMES Co.) were added using pTH/JFH1 as a template, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute were used as 1 cycle, and 30 cycles were carried out. The resulting PCR product was designated as PCR product No. 6.

Each PCR product was purified on an agarose gel, and eluted with 50. mu.l of EB buffer attached thereto using QIAquick gel extraction kit (QIAGEN). Mu.l of each of the DNAs of PCR product No.5 and PCR product No.6 was mixed, and using this as a template, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS151(SEQ ID NO: 14) and MS156(SEQ ID NO: 17) attached to Phusionhigh-Fidelity DNA polymerase kit (FINNZYMES Co.), and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute and 30 seconds were set to 1 cycle, and 30 cycles were performed. The resulting PCR product was designated as PCR product No.7 (FIG. 3A).

Next, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS151(SEQ ID NO: 14) and MS163(SEQ ID NO: 18) attached to the Phusion High-Fidelity DNA polymerase kit (FINNZYMES Co.) were added using pTH/JFH1 as a template, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute were used as 1 cycle, and 30 cycles were carried out. The resulting PCR product was designated as PCR product No. 8.

Next, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS162(SEQ ID NO: 19) and MS156(SEQ ID NO: 17) attached to the Phusion High-Fidelity DNA polymerase kit (FINNZYMES Co.) were added using pTH/JFH1 as a template, and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute were used as 1 cycle, and 30 cycles were carried out. The resulting PCR product was designated as PCR product No. 9.

Each PCR product was purified on an agarose gel, and eluted with 50. mu.l of EB buffer attached thereto using QIAquick gel extraction kit (QIAGEN). Mu.l each of the DNAs of PCR product No.8 and PCR product No.9 was mixed, and using this as a template, 10. mu.l of 5 Xbuffer, 1. mu.l of 10mM dNTP mixture, 2.5. mu.l each of 10. mu.M primers MS151(SEQ ID NO: 14) and MS156(SEQ ID NO: 17) attached to Phusionhigh-Fidelity DNA polymerase kit (FINNZYMES Co.), and finally deionized water was added to make the total amount to 49.5. mu.l. Then, 0.5. mu.l of Phusion DNA polymerase (FINNZYMES) was added thereto to conduct PCR. The PCR reaction was carried out under the following conditions: the steps comprising 98 ℃ for 10 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute and 30 seconds were used as 1 cycle, and 30 cycles were carried out. The resulting PCR product was designated as PCR product No.10 (FIG. 3B).

The pTH/JFH1 and the purified PCR product No.7 were digested with the restriction enzyme Acc65I, and the respective DNA fragments were separated by agarose gel electrophoresis and purified. These 2 DNA fragments were mixed with Ligation high (New England Biolabs) to ligate the 2 DNA fragments. This vector was designated as pTH/JFH-1 (PA). The pTH/JFH1(PA) has the following nucleotide sequence: encoding a 5' untranslated region comprising a gene derived from the JFH-1 strain; from the core of the TH strain to the N-terminal 33 amino acid residues of the protein regions E1, E2, p7 and NS 2; a nucleotide sequence of a chimeric gene comprising a NS3, NS4A, NS4B, NS5A, NS5B protein region and a 3' untranslated region, starting from the N-terminal 34 amino acid residue of the NS2 protein region of the JFH-1 strain, wherein the N-terminal amino acid residue of the core protein, i.e., the methionine residue, is the 1 st position and the 328 th amino acid residue is the alanine residue.

Next, pTH/JFH1 and the purified PCR product No.10 were digested with the restriction enzyme Acc65I, and the DNA fragments were separated by agarose gel electrophoresis and purified. These 2 DNA fragments were mixed with Ligation high (New England Biolabs) to ligate the 2 DNA fragments. This vector was designated as pTH/JFH1 (PT). The pTH/JFH1(PT) has the following nucleotide sequence: a nucleotide sequence encoding a chimeric gene comprising the 5 '-untranslated region derived from the JFH-1 strain, the core derived from the TH strain, E1, E2, p7, and the N-terminal 33 amino acid residues of the NS2 protein region of the JFH-1 strain, the N-terminal 34 amino acid residues of the NS2 protein region of the JFH-1 strain, the NS3, NS4A, NS4B, NS5A, and the NS5B protein region, and the 3' -untranslated region, wherein the methionine residue, which is the N-terminal amino acid residue of the core protein, is the 1 st amino acid residue, and the threonine residue at the 328 TH amino acid residue is the threonine residue.

pTH/JFH1(PA) was used as SEQ ID NO: 1. pTH/JFH1(PT) was used as SEQ ID NO: 2, the nucleotide sequences are shown in a sequence table.

Example 6: production of TH/JFH-1(PA) and TH/JFH-1(PT) viruses

Each plasmid prepared in example 5 was cleaved with XbaI, followed by phenol/chloroform extraction and ethanol precipitation. Next, the XbaI-cleaved fragment was treated with mung bean nuclease to remove the remaining nucleotide sequence from the 3' -end of the XbaI recognition sequence. Further, protease K treatment, phenol/chloroform extraction, ethanol precipitation, and purification of DNA fragments were carried out. Using the cleaved plasmid as a template, each HCV RNA was synthesized using MEGAscript T7 kit (Ambion).

Will be 3X 10⁶Each Huh-7 cell and 10. mu.g of HCV RNA were suspended in 400. mu.l of Cytomix solution (120mM KCl, 0.15mM CaCl)₂、10mM K₂HPO₄/KH₂PO₄、25mMHepes、2mM EGTA、5mM MgCl₂20mM ATP and 50mM glutathione) was transferred to a 4mM cuvette, and then electroporation was carried out at 260V and 950. mu.F using a Gene Pulser (BioRad). Thereafter, the cells into which HCV RNA had been introduced were inoculated at 10cm²The culture dish (2) is subjected to subculture.

When passaging was performed on each cell into which TH/JFH-1RNA, TH/JFH-1(PA) RNA (SEQ ID NO: 3) and TH/JFH-1(PT) RNA (SEQ ID NO: 4) prepared in example 2 were introduced, HCV core protein contained in the culture supernatant was quantified using an HCV antigen ELISA test kit (オ - ソ), and HCV particle production was confirmed. From the initial stage to the late stage of the culture, when the amount of HCV core contained in the culture supernatant of the cells into which the RNA not having the mutation had been introduced was compared with the amount of HCV core contained in the culture supernatant of the cells into which the RNA having the mutation had been introduced, it was revealed that the latter amount of HCV core was high (FIG. 4).

Example 7: evaluation of infectivity of mutant-introduced Virus

The infectivity of the virus produced by the cells into which TH/JFH-1(PA) RNA has been introduced was compared with that of the wild type TH/JFH-1. The changes in intracellular HCV core protein and culture supernatants up to 4, 24, 48, 72 and 96 hours after the introduction of each RNA were examined, and the infectivity of the culture supernatants was examined.

Specifically, TH/JFH-1 and TH/JFH-1(PA) RNAs were synthesized in the same manner as in example 6, except that the RNA was changed to 3X 10⁶Each Huh-7 cell and 10. mu.g of HCV RNA were suspended in 400. mu.l of Cytomix solution (120mM KCl, 0.15mM CaCl)₂、10mM K₂HPO₄/KH₂PO₄、25mM Hepes、2mM EGTA、5mM MgCl₂20mM ATP and 50mM glutathione) was transferred to a 4mM cuvette, and then electroporation was carried out at 260V and 950. mu.F using a Gene Pulser (BioRad). Thereafter, the cells into which HCV RNA had been introduced were inoculated at 10cm²The culture dishes of (4) were collected after 4, 24, 48, 72 and 96 hours, and the culture supernatants were filtered through a 0.45 μm filter (Millipore Co.), followed by quantification of HCV core protein using an HCV antigen ELISA kit (オ - ソ Co.). In addition, 10cm of the culture supernatant was removed²After the culture dish was washed with PBS, the cells were collected (り from scratch き) by using 500. mu.l of PBS and a spatula (Sumitomo ベ - クライト Co.), centrifuged, and the cells were collected. To the recovered cells, 100. mu.l of passive lysis buffer (Promega corporation) was added to prepare a lysate, and HCV core protein was quantified using an HCV antigen ELISA kit (オ - ソ corporation) in the same manner as the culture supernatant.

The culture medium was subjected to stepwise dilution, and the infectious titer of the culture supernatant was quantified as follows. Huh-7 cells were cultured at 1X 10⁴One well was inoculated in 96 wells coated with polylysineAfter culturing in a plate (Corning Co., Ltd.) for one day and night, the plate was replaced with a culture supernatant obtained by stepwise dilution with a culture medium, and the plate was further cultured for 3 days. Thereafter, the culture solution was discarded, washed 3 times with PBS, and fixed with methanol for 15 minutes. Next, each well was blocked with Block Ace (manufactured by Sumitomo Dainippon Co., Ltd.) containing 0.3% Triton X-100, and reacted with an anti-HCV core-specific antibody (clone 2H 9). Subsequently, the wells were washed with PBS and reacted with Alexa 488-labeled anti-mouse IgG antibody (Invitrogen). Thereafter, the wells were washed with PBS, the number of infectious foci in each well was observed under a fluorescence microscope (オリンパス Co.), and the infectious titer of each culture supernatant was calculated as a Foci Formation Unit (FFU).

As a result, in the cells into which TH/JFH-1(PA) RNA was introduced, the HCV core protein was secreted into the culture supernatant at a higher rate than in the wild-type TH/JFH-1, and the infection titer of the culture supernatant was also high (FIGS. 5 and 6). The results show that: the mutation of the 328 TH proline residue in TH/JFH-1(SEQ ID NO: 5) is a mutation that increases the production of HCV particles.

Industrial applicability

The HCV particles provided by the method of the present invention have high expression, high productivity and high infectivity, and are therefore suitable as a vaccine for prevention or treatment of HCV. Furthermore, the HCV particles of the present invention can also be used as a tool for inducing anti-HCV antibodies.

All publications, patents and patent applications cited in this specification are herein incorporated in their entirety by reference.

Claims (10)

1. A nucleic acid consisting of a JFH-1 strain 5 'untranslated region, a chimeric gene derived from hepatitis C virus, and a JFH-1 strain 3' untranslated region, wherein the chimeric gene is a gene encoding a core protein derived from a TH strain, an E1 protein, an E2 protein, and a p7 protein; a NS2 protein derived from the JFH-1 strain or the TH strain, or a chimeric NS2 protein of an NS2 protein derived from the JFH-1 strain and an NS2 protein derived from the TH strain; and each region of NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein derived from the JFH-1 strain in this order from the 5 'side to the 3' side,

wherein the 328 th proline residue is substituted with a threonine or alanine residue, based on the number of N-terminal amino acid residues of the core protein,

wherein said 5 'untranslated region is located on the 5' side of said core protein coding region, and said 3 'untranslated region is located on the 3' side of said NS5B protein coding region.

2. The nucleic acid according to claim 1, wherein said nucleic acid is:

consisting of SEQ ID NO: 1, and DNA consisting of the nucleotide sequence shown in the specification;

(ii) a region encoded by said 5' untranslated region, encoding SEQ ID NO: 6 amino acid sequence and the 3' untranslated region;

consisting of SEQ ID NO: 3, and RNA consisting of the nucleotide sequence shown in the specification; or

The 5' untranslated region described above, encodes SEQ ID NO: 6 amino acid sequence and the 3' untranslated region.

3. The nucleic acid of claim 1, wherein the nucleic acid is:

consisting of SEQ ID NO: 2, and 2 is DNA consisting of nucleotide sequence shown in the specification;

(ii) a region encoded by said 5' untranslated region, encoding SEQ ID NO: 7 and the 3' untranslated region;

consisting of SEQ ID NO: 4, and RNA consisting of the nucleotide sequence shown in the specification; or

The 5' untranslated region encodes the amino acid sequence of SEQ ID NO: 7 and said 3' untranslated region.

4. A vector comprising the nucleic acid of any one of claims 1 to 3.

5. A chimeric hepatitis C virus particle comprising the nucleic acid of any one of claims 1 to 3 as the viral genome.

6. A cell that produces the chimeric hepatitis C virus particle of claim 5.

7. The cell according to claim 6, wherein the cell is Huh-7 strain or a derivative thereof.

8. A hepatitis C virus vaccine comprising the chimeric hepatitis C virus particle of claim 5.

9. The hepatitis C virus vaccine according to claim 8, wherein said chimeric hepatitis C virus particle is inactivated or attenuated.

10. Use of the chimeric hepatitis c virus particle of claim 5 as an antigen for the preparation of an anti-hepatitis c virus antibody recognizing said virus particle.