# US 10,500,266 B2 · Influenza virus reassortment

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,500,266 B2
- Document ID: US10500266B2
- Status: Granted
- Kind: B2
- Application number: 15/621,270
- Filed: 2017-06-13
- Published: 2017-11-16
- Granted: 2019-12-10
- Earliest claimed: 2012-12-03
- Assignees: Seqirus UK Limited; Synthetic Genomics, Inc.
- Applicants: Seqirus UK Limited; Synthetic Genomics, Inc.
- Inventors: Peter Mason; Philip Ralph Dormitzer; Heidi Trusheim; Pirada Suphaphiphat
- Web page: https://patentgrep.com/patent/US10500266B2

## CPC

- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61K 39/12: Viral antigens
- A61P 31/16: for influenza or rhinoviruses
- 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 2039/5252: {inactivated (killed)}
- C12N 2760/16121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- 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 2760/16152: relating to complementing cells and packaging systems for producing virus or viral particles
- 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

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

## Claims

### Claim 1

A method of preparing a reassortant influenza A virus comprising:
an HA segment, an NA segment and backbone segments PA, PBI, PB2, NP, NS and M, wherein the backbone segments are from two donor strains, wherein the PB1 segment is from the A/California/07/09 influenza strain and all other backbone segments are from the PR8-X influenza strain, further wherein the PA segment has the sequence of SEQ ID NO: 1, the PB2 segment has the sequence of SEQ ID NO: 3, the NP segment has the sequence of SEQ ID NO: 4, the M segment has the sequence of SEQ ID NO: 5, and the NS segment has the sequence of SEQ ID NO: 6,
wherein the method comprises:
(i) introducing into a culture host at least one expression construct which encode(s) the viral segments required to produce the reassortant influenza A virus; and
(ii) culturing the culture host in order to produce the reassortant influenza A virus.

### Claim 2 (depends on 1)

The method of claim 1, wherein the at least one expression construct comprises at least one sequence having at least 90% identity or 100% identity with the sequences of SEQ ID NOs: 9 and 11 to 14.

### Claim 3 (depends on 1)

The method of claim 1, wherein the HA segment is from an H1 influenza virus.

### Claim 4 (depends on 1)

A method for producing influenza viruses comprising: (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 optionally (c) purifying the virus produced in step (b).

### Claim 5 (depends on 4)

A method of preparing a vaccine, comprising: (a) preparing a virus by the method of claim 4 and (b) preparing a vaccine from the virus.

### Claim 6 (depends on 4)

The method of claim 4, wherein the culture host is an embryonated hen egg.

### Claim 7 (depends on 4)

The method of claim 4, wherein the culture host is a mammalian cell, optionally an MDCK, Vero or PerC6 cell.

### Claim 8 (depends on 7)

The method of claim 7, wherein the cell grows adherently or in suspension.

### Claim 9 (depends on 5)

The method of claim 5, wherein step (b) involves inactivating the virus.

### Claim 10 (depends on 5)

The method of claim 5, wherein the vaccine is a whole virion vaccine, a split virion vaccine, a surface antigen vaccine, or a virosomal vaccine.

### Claim 11 (depends on 5)

The method of claim 5, wherein the vaccine contains less than 10 ng of residual host cell DNA per dose.

### Claim 12 (depends on 5)

The method of claim 5, wherein at least one of the influenza strains is of the H1, H2, H5, H7 or H9 subtype.

### Claim 13

A method for producing influenza viruses, comprising infecting a culture host with a reassortant influenza A virus comprising an HA segment, an NA segment and backbone segments PA, PB1, PB2, NP, NS and M, wherein the backbone segments are from two donor strains, further wherein at least one backbone segment is from the A/California/07/09 influenza strain and:
a) the PB2 segment comprises a lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm;
b) the PB2 segment comprises an asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm;
c) the PA genome segment comprises a lysine in the position corresponding to amino acid 327 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm;
d) the PA segment comprises an aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm;
e) the PA segment comprises an aspartic acid in the position corresponding to amino acid 675 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm;
f) the NP segment comprises a threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4 using a pairwise alignment algorithm; or
g) the NP segment comprises an asparagine in the position corresponding to amino acid 375 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4, using a pairwise alignment algorithm.

### Claim 14 (depends on 13)

The method of claim 13, comprising culturing the host to produce the virus and, optionally, purifying the virus.

### Claim 15 (depends on 14)

The method of claim 14, wherein the culture host is selected from: an embryonated hen egg, a mammalian cell, optionally an MDCK, a Vero cell or a PerC6 cell.

### Claim 16 (depends on 14)

A method of preparing a vaccine, comprising: (a) preparing a virus by the method of claim 14 and (b) preparing a vaccine from the virus.

### Claim 17 (depends on 13)

A method for producing influenza viruses, comprising infecting a culture host with the reassortant influenza virus of claim 13, wherein:
a) the PB2 segment comprises a lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 and asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm;
b) the PA segment comprises a lysine in the position corresponding to amino acid 327; aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 and aspartic acid in the position corresponding to amino acid 675 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm;
c) the NP genome segment comprises a threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 and asparagine in the position corresponding to amino acid 375 when aligned to SEQ ID NO: 4, using a pairwise alignment algorithm; or
d) the influenza A strain is a H1 strain.

### Claim 18 (depends on 17)

The method of claim 17, comprising culturing the host to produce the virus and, optionally, purifying the virus.

### Claim 19 (depends on 18)

The method of claim 18, wherein the culture host is selected from: an embryonated hen egg, a mammalian cell, optionally an MDCK, a Vero cell or a PerC6 cell.

### Claim 20 (depends on 18)

A method of preparing a vaccine, comprising: (a) preparing a virus by the method of claim 18 and (b) preparing a vaccine from the virus.

### Claim 21 (depends on 7)

The method of claim 7, wherein the MDCK cell is MDCK 33016 (DSM ACC2219).

### Claim 22 (depends on 15)

The method of claim 15, wherein the MDCK cell is MDCK 33016 (DSM ACC2219).

### Claim 23 (depends on 19)

The method of claim 19, wherein the MDCK cell is MDCK 33016 (DSM ACC2219).