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Hepatitis C virus vaccine composition

A technology of hepatitis C virus and vaccine composition, which is applied in the direction of virus, virus peptide, drug combination, etc., can solve the problem of HCV infection inhibitory activity of undisclosed antibodies, and achieve the effect of preventing virus infection

Inactive Publication Date: 2012-07-18
TORAY IND INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Non-Patent Document 12 discloses that by mixing HCV core protein, NS3 protein, NS4 protein, and NS5 protein as an antigen, and mixing Alum and CpG-ODN as an adjuvant, antibodies against the core protein, NS3 protein, NS4 protein, and NS5 protein The amount of production increased, but similar to Patent Documents 1 and 2, the HCV infection inhibitory activity of the antibody was not disclosed

Method used

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  • Hepatitis C virus vaccine composition
  • Hepatitis C virus vaccine composition
  • Hepatitis C virus vaccine composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Preparation of J6 / JFH1-HCV particles

[0127] The cDNA (genomic cDNA) obtained by reverse transcription of the entire genomic RNA region of the hepatitis C virus (HCV) JFH1 strain (genotype 2a) isolated from patients with fulminant hepatitis was cloned into the downstream of the T7 RNA promoter sequence of the pUC19 plasmid, and the obtained The plasmid DNA pJFH1 (Wakita, T. et al., Nat.Med., 11 (2005) 791-796 pages and International Publication WO 2004 / 104198) was digested with EcoR I, and further partially digested with Bcl I to prepare the excised A plasmid DNA fragment of a fragment (about 2840 bp) from the EcoR I site to the original BclI site was purified.

[0128] On the other hand, genomic cDNA from HCV J6CF strain (GenBank accession number AF177036, Yanagi, M., et al., Virology 262:250-263 (1999)) was cloned into the downstream of the T7 RNA promoter sequence of pUC19 plasmid, and The obtained plasmid DNA, i.e. pJ6CF (International Publication WO 2006 / 022422),...

Embodiment 2

[0135] Purification of J6 / JFH1-HCV particles

[0136] The J6 / JFH1-HCV particles produced in Example 1 were purified by the following 3 steps.

[0137] 1) Concentration and diafiltration

[0138] Using a hollow fiber cartridge (Hollow Fiber Cartridge) (GE Healthcare company: 500kDa cut-off, model UFP-500-C-8A, hereinafter referred to as "hollow fiber"), the culture supernatant containing HCV particles obtained in Example 1 above was concentrated to 60 times.

[0139] 2) Density gradient ultracentrifugation

[0140] Add 3 mL of cooled TNE buffer (10 mM Tris-HCl (pH7.4), 150 mM NaCl, 1 mM EDTA) containing 60% sucrose to an Ultra-clear 25×89 mm centrifuge tube (Beckman Coulter Company: catalog number 344058), covering 7 mL of TNE buffer containing 20% ​​sucrose. Another 25 mL sample was overlaid on TNE buffer containing 20% ​​sucrose. Using SW-28 (Beckman Coulter Co.), ultracentrifugation was performed at 28,000 rpm at 4° C. for 4 hours.

[0141] A 25G injection needle (Teru...

Embodiment 3

[0145] Inactivation of HCV particles

[0146] The concentrated hepatitis C virus obtained in steps 1) to 3) of Example 2 was inactivated by ultraviolet irradiation. GL-15 manufactured by Toshiba Corporation was used as a line source of ultraviolet rays. Specifically, the purified infection titers were 1 × 10 6 The ffu / mL solution containing hepatitis C virus particles (JFH1 strain) was placed in a silicon-coated polyethylene microcentrifuge tube (manufactured by Assist), and placed in a place where the irradiation intensity of ultraviolet light was 20 mW / cm 2 distance, irradiate UV-C for 5 minutes.

[0147] After treatment, the hepatitis C virus particles were serially diluted 50 times, 250 times, 1250 times, 6250 times, 31250 times, 156250 times and 781250 times in Dulbecco's modified Eagle medium (DMEM).

[0148] The day before, press 1×10 4 Cells / well were placed in a 96-well poly-L-lysine coated plate (manufactured by Corning; Corning 96 Well Clear Flat Bottom Poly-L-l...

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Abstract

Disclosed is an effective HCV vaccine composition, which is developed as a result of the finding of an optimum combination of an HCV antigen capable of inducing an antibody having an inhibitory activity on the infection by an HCV and an adjuvant. Specifically disclosed is a hepatitis C virus vaccine composition which comprises: inactivated virus particles produced by inactivating infectious hepatitis C virus particles that are produced from hepatitis C virus genome containing sequences respectively encoding NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein derived from hepatitis C virus strain JFH1; a non-methylated CpG-containing oligonucleotide represented by SEQ ID NO:5 shown in the Sequence Listing; and aluminum hydroxide.

Description

technical field [0001] The present invention relates to hepatitis C virus vaccine compositions. Background technique [0002] Hepatitis C virus (hepatitis C virus; hereinafter sometimes abbreviated as "HCV") was discovered as the main causative virus of non-A non-B hepatitis (Non-Patent Document 1). HCV is an RNA virus having a single-stranded positive-strand RNA of about 9.6 kb as a genome, which is cleaved into 10 viral proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) precursor proteins. Among them, core, E1, E2 and p7 proteins are classified as structural proteins, and NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins are classified as non-structural proteins. HCV is also classified into more than 10 genotypes (such as 1a, 1b, 2a, 2b, 3a, and 3b) according to the difference in the genome nucleotide sequence (non-patent literature 2-4), which can be detected by HCV antibody, HCV Core antigen detection or nucleic acid amplification detection to determine genot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/29A61K9/16A61K39/39A61K47/02A61K47/34A61P1/16A61P31/12A61P37/04C07K14/18C07K19/00C12N7/04C12N15/09
CPCC12N2770/24223A61K2039/55561A61K2039/55505A61K2039/5252C12N2770/24263A61K39/29C12N7/00C12N2770/24222C12N2770/24234C07K2319/30A61K2039/5258A61K2039/545A61K2039/57A61K39/12A61P1/16A61P31/12A61P31/14A61P37/04
Inventor 胁田隆字森山正树赤泽大辅中村纪子
Owner TORAY IND INC
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