# US 7,465,456 B2 · Multi plasmid system for the production of influenza virus

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- Number: US 7,465,456 B2
- Document ID: US7465456B2
- Status: Granted
- Kind: B2
- Application number: 11/133,345
- Filed: 2005-05-20
- Published: 2005-12-01
- Granted: 2008-12-16
- Earliest claimed: 2004-06-12
- Assignees: MedImmune, LLC
- Inventors: Erich Hoffmann; Hong Jin; Bin Lu; Gregory Duke; George Kemble; Zhongying Chen
- Web page: https://patentgrep.com/patent/US7465456B2

## CPC

- C12N 15/86: Viral vectors
- A61P 31/16: for influenza or rhinoviruses
- A61P 37/04: Immunostimulants
- 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)
- C12N 15/85: for animal cells
- A61K 2039/5254: {avirulent or attenuated}
- C12N 2760/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- 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 2760/16162: by genetic engineering
- C12N 2760/16222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- 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 2760/16262: by genetic engineering
- C12N 2840/20: translation of more than one cistron
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

Vectors and methods for the production of influenza viruses suitable as recombinant influenza vaccines in cell culture are provided. Bi-directional expression vectors for use in a multi-plasmid influenza virus expression system are provided. Additionally, the invention provides methods of producing influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells (e.g., Vero and/or MDCK) and further provides influenza viruses with enhanced replication characteristics. A method of producing a cold adapted (ca) influenza virus that replicates efficiently at, e.g., 25° C. (and immunogenic compositions comprising the same) is also provided.

## Claims

### Claim 1

A reassortant influenza virus comprising:
i) an HA protein comprising a leucine at position 183 and an alanine at position 226; or
ii) an HA protein comprising a valine at position 186 and an isoleucine at position 226; or
iii) an HA protein comprising a valine at position 186 and an isoleucine at position 226 and an NA protein comprising a glutamate at position 119 and a glutamine at position 136;
wherein the HA protein is of the H3 type.

### Claim 2 (depends on 1)

An immunogenic composition comprising the reassortant influenza virus of claim 1.

### Claim 3 (depends on 1)

A method of making the reassortant influenza virus of claim 1, comprising:
(a) introducing at least one mutation in an influenza HA and/or NA gene, wherein the at least one mutation results in:
(i) an HA polypeptide comprising a leucine at position 183 and an alanine at position 226; or
(ii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226; or
(iii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226 and an NA comprising a glutamate at position 119 and a glutamine at position 136; wherein the HA protein is of the H3 type;
(b) introducing a plurality of vectors into a population of host cells, wherein the plurality of vectors corresponds to an influenza virus genome, wherein at least one of the plurality of vectors comprises an influenza HA and/or NA gene comprising the at least one mutation recited in (a), and wherein the population of host cells is capable of supporting replication of influenza virus;
(c) culturing the population of host cells; and
(d) recovering the reassortant influenza virus.

### Claim 4 (depends on 1)

A method of making the reassortant influenza virus of claim 1, comprising:
(a) introducing a plurality of vectors into a population of host cells, wherein the plurality of vectors corresponds to an influenza virus genome, wherein at least one of the plurality of vectors encodes:
(i) an HA polypeptide comprising a leucine at position 183 and an alanine at position 226; or
(ii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226; or
(iii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226 and an NA comprising a glutamate at position 119 and a glutamine at position 136; wherein the HA protein is of the H3 type;
and wherein the population of host cells is capable of supporting replication of influenza virus;
(b) culturing the population of host cells; and
(c) recovering the reassortant influenza virus.

### Claim 5 (depends on 2)

A method of stimulating an immune response, comprising:
administering the immunogenic composition of claim 2.

### Claim 6 (depends on 5)

The method of claim 5, wherein the immunogenic composition is administered intranasally.

### Claim 7 (depends on 5)

The method of claim 5, wherein the immunogenic composition is administered by subcutaneous injection.

### Claim 8 (depends on 5)

The method of claim 5 wherein the immunogenic composition is administered by intramuscular injection.

### Claim 9 (depends on 1)

The reassortant influenza virus of claim 1, wherein the virus is a 6:2 reassortant.

### Claim 10 (depends on 1)

The reassortant influenza virus of claim 1, wherein the virus is a 7:1 reassortant.