# US 8,772,022 B2 · Hepatitis C virus expressing reporter tagged NS5A protein

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- Number: US 8,772,022 B2
- Document ID: US8772022B2
- Status: Granted
- Kind: B2
- Application number: 13/121,565
- Filed: 2009-10-05
- Published: 2012-01-05
- Granted: 2014-07-08
- Earliest claimed: 2008-10-03
- Assignees: Hvidovre Hospital; Kobenhavns Universitet
- Applicants: Judith M. Gottwein; Troels Kasper Hoyer Scheel; Jens Bukh; Jannick Prento; Tanja Bertelsen Jensen; Jacob Bo Lademann; Yiping Li
- Inventors: Judith M. Gottwein; Troels Kasper Hoyer Scheel; Jens Bukh; Jannick Prento; Tanja Bertelsen Jensen; Jacob Bo Lademann; Yiping Li
- Web page: https://patentgrep.com/patent/US8772022B2

## CPC

- C12N 15/86: Viral vectors
- C07K 14/005: from viruses
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 2039/525: {Virus}
- C12N 2770/24222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2770/24243: viral genome or elements thereof as genetic vector
- C12N 2770/24251: Methods of production or purification of viral material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A were developed. A deletion upstream of the inserted reporter gene sequence conferred favorable growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. JFH1-based intergenotypic recombinants with genotype specific homotypic 5′UTR, or heterotypic 5′UTR (either of genotype 1a (strain H77) or of genotype 3a (strain S52)) were also developed. The present inventors additionally developed J6/JFH1 recombinants with the 5′UTR of genotypes 1-6. These recombinants with different 5′UTRs are a useful to study the function of the 5′UTR in a genotype specific manner.

## Claims

### Claim 1

A chimeric nucleic acid molecule comprising the structural genes Core, E1, and E2, the p7 gene, and the non-structural gene NS2 of any one of a genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, or 7a Hepatitis C virus (HCV) and the non-structural genes NS3, NS4A, NS4B, NS5A and NS5B from the HCV genotype 2a JFH1 strain, wherein a reporter gene is inserted in domain III of the non-structural gene NS5A, wherein said NS5A gene encodes a non-structural NS5A protein having a 40 amino acid deletion of amino acid positions 250 to 289 or having a 25 amino acid deletion of amino acid positions 266-290, and wherein said nucleic acid encodes an intragenotypic or intergenotypic recombinant HCV genome that is infective and genetically stable in cell cultures.

### Claim 2 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the genotype 1a is of the strain H77, TN or DH6, wherein the genotype 1b is of the strain J4, DH1, or DH5, wherein the genotype 2a is of the strain J6 or JFH1, wherein the genotype 2b is of the strain J8, wherein the genotype 3a is of the strain S52 or DBN, wherein the genotype 4a is of the strain ED43, wherein the genotype 5a is of the strain SA13, wherein the genotype 6a is of the strain HK6a, wherein the genotype 7a is of the strain QC69.

### Claim 3 (depends on 1)

The nucleic acid molecule of claim 1, wherein the reporter gene is an EGFP reporter gene.

### Claim 4 (depends on 1)

The nucleic acid molecule of claim 1, which encodes human hepatitis C virus of strain selected from the group consisting of:
HCV genotype 1a/JFH1-hRLucΔ, HCV genotype 1b JFH1-hRlucΔ, HCV genotype 2a/JFH1-hRlucΔ, HCV genotype 2b/JFH1-hRlucΔ, HCV genotype 3a/JFH1-hRlucΔ, HCV genotype 4a/JFH1-hRlucΔ, HCV genotype 4b/JFH1-hRlucΔ, HCV genotype 5a/JFH1-hRlucΔ, HCV genotype 6a/JFH1-hRlucΔ, and HCV genotype 7a/JFH1-hRlucΔ,
wherein HCV genotype 1a/JFH1-hRLucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 130,
wherein HCV genotype 1b/JFH1-hRLucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 131,
wherein HCV genotype 2a/JFH1-hRlucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 20,
wherein HCV genotype 2b/JFH1-hRlucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 103,
wherein HCV genotype 3a/JFH1-hRlucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 132,
wherein HCV genotype 4a/JFH1-hRlucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 133,
wherein HCV genotype 5a/JFH1-hRlucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 134,
wherein HCV genotype 6a/JFH1-hRlucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 135, and
wherein HCV genotype 7a/JFH1-hRlucΔ encodes the amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 136.

### Claim 5 (depends on 4)

The nucleic acid molecule according to claim 4, which encodes human hepatitis C virus of strain selected from the group consisting of:
HCV genotype 1a/JFH1-hRLucΔ, HCV genotype 1b/JFH1-hRlucΔ, HCV genotype 2a/JFH1-hRlucΔ, HCV genotype 2b/JFH1-hRlucΔ, HCV genotype 3a/JFH1-hRlucΔ, HCV genotype 4a/JFH1-hRlucΔ, HCV genotype 4b/JFH1-hRlucΔ, HCV genotype 5a/JFH1-hRlucΔ, HCV genotype 6a/JFH1-hRlucΔ, and HCV genotype 7a/JFH1-hRlucΔ,
wherein HCV genotype 1a/JFH1-hRLucΔ encodes the nucleic acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 122;
wherein HCV genotype 1b/JFH1-hRlucΔ (1b/2a) encodes the nucleic acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 123;
wherein HCV genotype 2a/JFH1-hRlucΔ encodes the nucleic acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 10,
wherein HCV genotype 2b/JFH1-hRlucΔ encodes the nucleic acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 83,
wherein HCV genotype 3a/JFH1-hRlucΔ encodes the nucleic acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 124,
wherein HCV genotype 4a/JFH1-hRlucΔ the nucleic acid sequence acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 125,
wherein HCV genotype 5a/JFH1-hRlucΔ the nucleic acid sequence acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 126,
wherein HCV genotype 6a/JFH1-hRlucΔ the nucleic acid sequence acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 127, and
wherein HCV genotype 7a/JFH1-hRlucΔ the nucleic acid sequence acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 128.

### Claim 6 (depends on 2)

The nucleic acid molecule according to claim 2, wherein the reporter gene is an EGFP reporter gene.

### Claim 7 (depends on 1)

The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule encodes a Hepatitis C virus that is capable of infectivity in vivo.

### Claim 8 (depends on 1)

The nucleic acid molecule according to claim 1, wherein said molecule comprises one or more adaptive mutations in Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A or NS5B singly or in combination.

### Claim 9 (depends on 1)

The nucleic acid molecule according to claim 1, wherein said molecule is capable of generating a HCV RNA titer not represented by input of said molecule itself of 104 IU/ml or above following transfection and/or subsequent viral passage.

### Claim 10 (depends on 1)

The nucleic acid molecule according to claim 1 wherein said molecule is capable of generating a HCV infectivity titer of 102 TCID50/ml (50% tissue culture infectious doses)/ml or above following transfection and/or subsequent viral passage.

### Claim 11 (depends on 1)

A method for producing a cell which replicates HCV RNA and produces a virus particle comprising introducing a nucleic acid molecule according to claim 1 into a cell.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the cell is Huh7.5.

### Claim 13 (depends on 1)

An isolated cell comprising the nucleic acid molecule of claim 1.

### Claim 14 (depends on 1)

A hepatitis C virus particle comprising the nucleic acid molecule of claim 1.