# US 7,504,248 B2 · Production of viruses viral isolates and vaccines

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- Number: US 7,504,248 B2
- Document ID: US7504248B2
- Status: Granted
- Kind: B2
- Application number: 10/497,832
- Filed: 2002-12-09
- Published: 2005-06-09
- Granted: 2009-03-17
- Earliest claimed: 2001-12-07
- Assignees: Crucell Holland B.V.
- Inventors: Giuseppe Marzio; Maria Grazia Pau; Dirk Jan Elbertus Opstelten; Alphonsus Gerardus Cornelis Maria Uytdehaag
- Web page: https://patentgrep.com/patent/US7504248B2

## CPC

- C07K 14/005: from viruses
- A61K 39/12: Viral antigens
- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61P 31/16: for influenza or rhinoviruses
- A61P 37/04: Immunostimulants
- 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 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 2039/525: {Virus}
- C12N 2760/16021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/16022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/16121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- 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 2760/16151: Methods of production or purification of viral material
- C12N 2760/16221: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- 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
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention discloses methods for producing and/or propagating virus particles, such as influenza virus particles, that are present in a virus isolate obtained from an infected subject by contacting a host cell with a virus particle and culturing the cell under conditions conducive to propagation of the virus particle. The invention also provides a method for selective propagation of a set of virus particles, such as influenza virus particles present in an influenza isolate, which have an affinity for receptors comprising a specific glycosylation residue.

## Claims

### Claim 1

A method for producing an influenza virus particle, said method comprising the steps of:
contacting a human embryonal retina cell transformed with E1 of an adenovirus with an influenza virus particle in a culture medium under conditions conducive to infection of said cell by said influenza virus particle, wherein the influenza virus particle is from an influenza isolate obtained from a subject infected with influenza virus; and
culturing said cell under conditions conducive to propagation of said influenza virus particle, wherein said cell comprises a heterologous nucleic acid encoding an alpha2,6 sialyltransferase or a functional equivalent thereof and overexpresses the alpha2,6 sialyltransferase or functional equivalent thereof as compared to the cell that does not comprise the heterologous nucleic acid.

### Claim 2

A method for producing an influenza virus particle, the method comprising:
providing a host cell that is a human embryonal retina cell transformed with E1 of an adenovirus;
expressing a nucleic acid encoding alpha2,6 sialyltransferase in the host cell by a heterologous nucleic acid at a higher level than the expression of alpha2,6 sialyltransferase in a cell the same as the host cell, but not comprising the heterologous nucleic acid;
infecting the host cell with an influenza virus particle from an influenza isolate obtained from a subject infected with influenza virus, and
culturing the host cell under conditions that allow for propagation of the influenza virus particle.

### Claim 3 (depends on 2)

The method according to claim 2, comprising over-expressing a human alpha2,6 sialyltransferase.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the influenza isolate is obtained from at least one influenza-infected mammal.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the influenza isolate is obtained from a human or a pig.

### Claim 6 (depends on 2)

The method according to claim 2, wherein the influenza isolate is obtained from at least one bird.

### Claim 7 (depends on 2)

The method according to claim 2, wherein the host cell is a PER.C6 cell wherein the heterologous nucleic acid has been introduced.

### Claim 8 (depends on 2)

The method according to claim 2, wherein the heterologous nucleic acid is integrated into a genome of the host cell.

### Claim 9 (depends on 2)

The method according to claim 2, said method further comprising making a vaccine by inactivating the propagated influenza virus particles.

### Claim 10 (depends on 9)

The method according to claim 9, further comprising:
treating the inactivated influenza virus particles to yield antigenic parts; and isolating at least one antigenic part.

### Claim 11 (depends on 10)

The method according to claim 10, comprising isolating at least one antigenic part of a hemagglutinin or neuraminidase protein, or combinations thereof, from an influenza virus.