# US 10,245,312 B2 · Influenza virus reassortment

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- Number: US 10,245,312 B2
- Document ID: US10245312B2
- Status: Granted
- Kind: B2
- Application number: 15/210,485
- Filed: 2016-07-14
- Published: 2017-04-06
- Granted: 2019-04-02
- Earliest claimed: 2012-03-02
- Assignees: Seqirus UK Limited
- Applicants: Novartis AG
- Inventors: Pirada Suphaphiphat; Peter Mason; Bjoern Keiner; Philip Dormitzer; Heidi Trusheim
- Web page: https://patentgrep.com/patent/US10245312B2

## CPC

- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61P 31/16: for influenza or rhinoviruses
- 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}
- A61K 2039/5252: {inactivated (killed)}
- 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 2760/16161: Methods of inactivation or attenuation
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

New influenza donor strains for the production of reassortant influenza A viruses are provided.

## Claims

### Claim 1

A non-naturally occurring reassortant influenza A virus comprising six backbone viral segments, a hemagglutinin (HA) segment and a neuraminidase (NA) segment, wherein the virus comprises backbone segments from two or more donor strains, each donor strain providing more than one backbone segment, further wherein at least one backbone viral segment comprises a nucleotide sequence (a) having at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 9-14 or SEQ ID NOs: 17-22, or (b) that encodes a viral polypeptide having at least 99% sequence identity with an amino acid sequence of a polypeptide encoded by the groups consisting of SEQ ID NOs 9-14 or SEQ ID NOs: 17-22;
wherein the reassortant influenza A virus comprises PB1 and PB2 viral segments from the same donor strain and (1) the PB1 viral segment comprises a nucleotide sequence that encodes a viral polypeptide comprising the amino acid sequence of a polypeptide encoded by SEQ ID NO: 18 and (2) the PB2 viral segment comprises a nucleotide sequence that encodes a viral polypeptide comprising the amino acid sequence of a polypeptide encoded by SEQ ID NO: 19.

### Claim 2 (depends on 1)

The reassortant influenza A virus of claim 1, wherein the virus comprises:
(a) an NS segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 22, or (ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 22; or
(b) an M segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 21, or (ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 21; or
(c) an NP segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 20, or ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 20; or
(d) a PA segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 17, or ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 17.

### Claim 3 (depends on 1)

The reassortant virus of claim 1, wherein the virus comprises:
(a) an NS segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 14, or (ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 14;
(b) an M segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 13, or (ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 13;
(c) an NP segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 20, or ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 20; and
(d) a PA segment comprising a nucleotide sequence (i) having at least 95% identity with the sequence of SEQ ID NO: 9, or ii) that encodes a viral polypeptide having at least 99% sequence identity with the amino acid sequence of a polypeptide encoded by SEQ ID NO: 9.

### Claim 4 (depends on 1)

The reassortant virus of claim 1, wherein the virus comprises:
(a) a PB1 segment comprising a nucleotide sequence (i) having the sequence of SEQ ID NO: 18, or (ii) that encodes a viral polypeptide having the amino acid sequence of a polypeptide encoded by SEQ ID NO: 18;
(b) a PB2 segment comprising a nucleotide sequence (i) having the sequence of SEQ ID NO: 19, or (ii) that encodes a viral polypeptide having the amino acid sequence of a polypeptide encoded by SEQ ID NO: 19;
(c) an NS segment comprising a nucleotide sequence (i) having the sequence of SEQ ID NO: 14, or (ii) that encodes a viral polypeptide having the amino acid sequence of a polypeptide encoded by SEQ ID NO: 14;
(d) an M segment comprising a nucleotide sequence (i) having the sequence of SEQ ID NO: 13, or (ii) that encodes a viral polypeptide having the amino acid sequence of a polypeptide encoded by SEQ ID NO: 13;
(e) an NP segment comprising a nucleotide sequence (i) having the sequence of SEQ ID NO: 20, or ii) that encodes a viral polypeptide having the amino acid sequence of a polypeptide encoded by SEQ ID NO: 20; and
(f) a PA segment comprising a nucleotide sequence (i) having the sequence of SEQ ID NO: 9, or ii) that encodes a viral polypeptide having the amino acid sequence of a polypeptide encoded by SEQ ID NO: 9.

### Claim 5 (depends on 1)

The reassortant influenza A virus of claim 1, wherein the virus comprises backbone segments from two donor strains.

### Claim 6 (depends on 5)

The reassortant influenza A virus of claim 5, wherein
(a) the first donor strain has backbone viral segments comprising nucleotide sequences (i) having at least 95% identity with the sequences of SEQ ID NOs: 17-22, or (ii) that encode polypeptides having at least 99% sequence identity with the amino acid sequences of a polypeptides encoded by SEQ ID NOs: 17-22; and
(b) the second donor strain has backbone viral segments comprising nucleotide sequences (i) having at least 95% identity with the sequences of SEQ ID NOs: 9-14, or (ii) that encode polypeptides having at least 99% sequence identity with the amino acid sequences of polypeptides encoded by SEQ ID NOs: 9-14.

### Claim 7 (depends on 1)

The reassortant influenza A virus of claim 1, wherein the virus contains the H1, H2, H3, H4, H5, H6, H8, H9, H10, H11, H12, H13, H14, H15, H16, or H7 HA subtype.

### Claim 8 (depends on 1)

The reassortant influenza A virus of claim 1, wherein the virus has the HA segment from a pandemic influenza strain.

### Claim 9 (depends on 1)

A method of preparing a reassortant influenza virus comprising the steps of:
(i) introducing into a culture host one or more expression construct(s) which encode(s) viral segments required to produce an influenza virus according to claim 1; and
(ii) culturing the culture host in order to produce reassortant virus.

### Claim 10 (depends on 9)

The method of claim 9, wherein at least one expression construct comprises a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 9-14 and 17-22.

### Claim 11 (depends on 9)

The method of claim 9, further comprising the step (iii) of purifying the reassortant virus obtained in step (ii).

### Claim 12 (depends on 1)

A method of producing influenza viruses comprising the steps of: (a) infecting a culture host with the reassortant influenza virus of claim 1; (b) culturing the host from step (a) to produce the virus; and (c) purifying the virus obtained in step (b).

### Claim 13 (depends on 9)

A method of preparing a vaccine, comprising the steps of: (a) preparing a virus by the method of claim 9 and (b) preparing a vaccine from the virus.

### Claim 14 (depends on 12)

The method of claim 12, wherein the culture host is an embryonated hen egg or a mammalian cell.

### Claim 15 (depends on 14)

The method of claim 14, wherein the culture host is a mammalian cell and the mammalian cell is an MDCK, Vero or PerC6 cell.

### Claim 16 (depends on 15)

The method of claim 15, wherein the mammalian cell grows adherently or in suspension.

### Claim 17 (depends on 16)

The method of claim 16, wherein the mammalian cell is cell line MDCK 33016 (DSM ACC2219).

### Claim 18 (depends on 13)

The method of claim 13, wherein step (b) includes inactivating the virus.

### Claim 19 (depends on 13)

The method of claim 13, wherein the vaccine is a whole virion vaccine, a split virion vaccine, a surface antigen vaccine or a virosomal vaccine.

### Claim 20 (depends on 13)

The method of claim 13, wherein the vaccine contains less than 10 ng of residual host cell DNA per dose.

### Claim 21 (depends on 1)

A vaccine composition comprising the reassortant influenza A virus of claim 1.