# US 7,968,101 B2 · Recombinant influenza vectors with tandem transcription units

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- Number: US 7,968,101 B2
- Document ID: US7968101B2
- Status: Granted
- Kind: B2
- Application number: 11/283,498
- Filed: 2005-11-18
- Published: 2006-07-27
- Granted: 2011-06-28
- Earliest claimed: 2004-11-19
- Assignees: Wisconsin Alumni Research Foundation
- Inventors: Yoshihiro Kawaoka; Gabriele Neumann
- Web page: https://patentgrep.com/patent/US7968101B2

## CPC

- 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 39/12: Viral antigens
- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61K 48/0091: {Purification or manufacturing processes for gene therapy compositions}
- C12N 15/86: Viral vectors
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 2039/525: {Virus}
- A61K 2039/5252: {inactivated (killed)}
- A61K 2039/5254: {avirulent or attenuated}
- A61K 2039/5256: {expressing foreign proteins}
- C12N 2760/16043: viral genome or elements thereof as genetic vector
- C12N 2760/16051: Methods of production or purification of viral material
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16143: viral genome or elements thereof as genetic vector
- C12N 2760/16151: Methods of production or purification of viral material
- C12N 2760/16161: Methods of inactivation or attenuation
- C12N 2760/16234: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16243: viral genome or elements thereof as genetic vector
- C12N 2760/16251: Methods of production or purification of viral material
- C12N 2760/16261: Methods of inactivation or attenuation
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention provides a composition useful to prepare influenza viruses, e.g., in the absence of helper virus, using vectors which include tandem transcription cassettes containing PolI and/or PolII promoters.

## Claims

### Claim 1

A composition comprising:
a plasmid which includes transcription cassettes for vRNA production comprising a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence;
a plasmid which includes transcription cassettes for vRNA production comprising a transcription cassette comprising a PolI promoter operably linked to an influenza virus NA cDNA linked to a PolI transcription termination sequence and a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence;
a plasmid which includes a transcription cassette for mRNA production comprising a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus NP linked to a PolII transcription termination sequence; and
a plasmid which includes transcription cassettes for mRNA production comprising a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PA linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB1 linked to a PolII transcription termination sequence, and a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB2 linked to a PolII transcription termination sequence, wherein transfection of a cell with the four plasmids yields influenza virus, and wherein transfection of a culture of Vero cells with the four plasmids yields influenza virus titers of at least 2×104 TCID50/mL.

### Claim 2 (depends on 1)

The composition of claim 1 wherein one of the plasmids for vRNA production or for mRNA production or a different plasmid further comprises a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to 3′ influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.

### Claim 3 (depends on 2)

The composition of claim 2 wherein the transcription cassette comprising the DNA of interest is on a different plasmid.

### Claim 4 (depends on 2)

The composition of claim 2 wherein the transcription cassette comprising the DNA of interest is on one of the plasmids for vRNA production or for mRNA production.

### Claim 5 (depends on 4)

The composition of claim 4 wherein the transcription cassette comprising the DNA of interest is on one of the plasmids for vRNA production.

### Claim 6 (depends on 4)

The composition of claim 4 wherein the transcription cassette comprising the DNA of interest is on one of the plasmids for mRNA production.

### Claim 7

A composition, comprising:
a plasmid which includes transcription cassettes for vRNA production comprising a transcription cassette comprising a PolII promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence, and wherein transfection of a cell with the plasmid yields influenza virus, and wherein transfection of a culture of Vero cells with the plasmid yields influenza virus titers of at least 1×102 TCID50/mL.

### Claim 8 (depends on 7)

The composition of claim 7 further comprising a plasmid having one transcription cassette for mRNA production and another plasmid having three transcription cassettes for mRNA production, wherein the cassettes for mRNA production encode PA, PB1, PB2 and NP.

### Claim 9 (depends on 7)

The composition of claim 7 further comprising four plasmids for mRNA production encoding one of PA, PB1, PB2 or NP.

### Claim 10 (depends on 7)

The composition of claim 7 further comprising a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to 3′ influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.

### Claim 11 (depends on 10)

The composition of claim 10 wherein the transcription cassette comprising the DNA of interest is on a different plasmid than the transcription cassettes for vRNA production.

### Claim 12 (depends on 10)

The composition of claim 10 wherein the transcription cassette comprising the DNA of interest is on a plasmid having one of the transcription cassettes for vRNA production.

### Claim 13 (depends on 1, 7)

The composition of claim 1 or 7 wherein the HA is a type A HA.

### Claim 14 (depends on 1, 7)

The composition of claim 1 or 7 wherein the HA is a type B HA.

### Claim 15 (depends on 1, 2, 7, 10)

A method to prepare influenza virus, comprising: contacting a cell with the composition of claim 1, 2, 7 or 10 in an amount effective to yield infectious influenza virus.

### Claim 16

A method to prepare influenza virus, comprising contacting a cell with a plasmid which includes transcription cassettes for vRNA production comprising a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence and a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence; a plasmid which includes transcription cassettes for vRNA production comprising a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NA cDNA linked to a PolI transcription termination sequence; a plasmid which includes a transcription cassette for mRNA production comprising a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus NP linked to a PolII transcription termination sequence; and a plasmid which includes transcription cassettes for mRNA production comprising a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PA linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB1 linked to a PolII transcription termination sequence, and a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB2 linked to a PolII transcription termination sequence, wherein the cell produces titers of at least 1×102 TCID50/mL.

### Claim 17 (depends on 16)

The method of claim 16 further comprising a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to 3′ influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.

### Claim 18 (depends on 16, 17)

The method of claims 16 or 17 further comprising isolating the virus.

### Claim 19

A plasmid for influenza virus production comprising: a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus HA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence.

### Claim 20

A plasmid for influenza vRNA production comprising: a transcription cassette comprising a PolI promoter operably linked to an influenza virus PA cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB1 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus PB2 cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus NP cDNA linked to a PolI transcription termination sequence, a transcription cassette comprising a PolI promoter operably linked to an influenza virus M cDNA linked to a PolI transcription termination sequence, and a transcription cassette comprising a PolI promoter operably linked to an influenza virus NS cDNA linked to a PolI transcription termination sequence, wherein the plasmid does not include transcription cassettes for vRNA production of HA and NA that if they were present in the plasmid would yield infectious virus upon transfection of a cell with the plasmid.

### Claim 21

A plasmid for mRNA production comprising a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PA linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB1 linked to a PolII transcription termination sequence, a transcription cassette comprising a PolII promoter operably linked to a DNA coding region for influenza virus PB2 linked to a PolII transcription termination sequence, wherein the plasmid does not encode NP.

### Claim 22 (depends on 19)

A method to prepare influenza virus, comprising:
contacting a cell with the plasmid of claim 19 in an amount effective to yield infectious influenza virus.

### Claim 23 (depends on 19)

The plasmid of claim 19 wherein transfection of a Vero cell with the plasmid yields influenza virus titers of at least 1×102 TCID50/mL.

### Claim 24 (depends on 19)

The plasmid of claim 19 wherein transfection of a HEK293T cell with the plasmid yields influenza virus titers of at least 1×102 TCID50/mL.

### Claim 25 (depends on 20)

The plasmid of claim 20 wherein transfection of a Vero cell with the plasmid and transcription cassettes for vRNA production of HA and NA yields influenza virus titers of at least 1×102 TCID50/mL.

### Claim 26 (depends on 20)

The plasmid of claim 20 wherein transfection of a HEK293T cell with the plasmid and transcription cassettes for vRNA production of HA and NA yields influenza virus titers of at least 1×102 TCID50/mL.