# US 10,864,264 B2 · Influenza virus reassortment

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- Number: US 10,864,264 B2
- Document ID: US10864264B2
- Status: Granted
- Kind: B2
- Application number: 16/657,082
- Filed: 2019-10-18
- Published: 2020-07-09
- Granted: 2020-12-15
- Earliest claimed: 2013-03-13
- Assignees: Seqirus UK Limited; Synthetic Genomics, Inc.
- Applicants: Seqirus UK Limited; Synthetic Genomics, Inc.
- Inventors: Philip Dormitzer; Peter Mason; Pirada Suphaphiphat; Raul Gomila
- Web page: https://patentgrep.com/patent/US10864264B2

## 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)
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16234: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16251: Methods of production or purification of viral material
- C12N 2760/16252: 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 B viruses are provided.

## Claims

### Claim 1

A method of preparing a reassortant influenza B virus comprising steps of:
(i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza B virus wherein the expression construct(s) encode the HA segment from a first influenza B virus and the NP and/or PB2 segment from a second influenza B virus which is a B/Victoria/2/87-like strain; and
(ii) culturing the culture host in order to produce a reassortant influenza B virus.

### Claim 2

A method of preparing a reassortant influenza B virus comprising steps of:
(i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza B virus wherein the expression construct(s) encode the HA segment from a first influenza B virus and the NP segment from a second influenza B virus which is not B/Lee/40 or B/Ann Arbor/1/66 or B/Panama/45/90; and
(ii) culturing the culture host in order to produce a reassortant influenza B virus.

### Claim 3 (depends on 2)

The method of claim 2, wherein the NP and PB2 segments are from the second influenza B virus.

### Claim 4 (depends on 2)

The method of claim 2, wherein the second influenza B virus is a B/Victoria/2/87-like strain.

### Claim 5 (depends on 1)

The method of claim 1, wherein the PA, PB1, PB2, NP, NS and M segments are from the second influenza B virus.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reassortant influenza B virus comprises backbone segments from two or more influenza B strains.

### Claim 7 (depends on 6)

The method of claim 6, wherein at least one backbone segment is from a B/Yamagata/16/88-like strain.

### Claim 8

A method of preparing a reassortant influenza B virus comprising steps of
(i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza B virus comprising the HA segment from a B/Yamagata/16/88-like strain and at least one backbone segment from a B/Victoria/2/87-like strain; and
(ii) culturing the culture host in order to produce a reassortant influenza B virus.

### Claim 9 (depends on 8)

The method of claim 8, wherein two, three, four, five or six backbone segments are from the B/Victoria/2/87-like strain.

### Claim 10 (depends on 8)

The method of claim 8, wherein the ratio of segments from the B/Victoria/2/87-like strain and the B/Yamagata/16/88-like strain is 7:1, 6:2, 4:4, 3:5 or 1:7.

### Claim 11

A method of preparing a reassortant influenza B virus comprising steps of
(i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza B virus comprising viral segments from a B/Victoria/2/87-like strain and a B/Yamagata/16/88-like strain, wherein the ratio of segments from the B/Victoria/2/87-like strain and the B/Yamagata/16/88-like strain is 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:1; and
(ii) culturing the culture host in order to produce a reassortant influenza B virus.

### Claim 12 (depends on 10)

The method of claim 10, wherein the ratio is 7:1, 6:2, 4:4, 3:5 or 1:7.

### Claim 13 (depends on 1)

The method of claim 1, wherein the B/Victoria/2/87-like strain is B/Brisbane/60/08.

### Claim 14 (depends on 11)

The method of claim 11, wherein the B/Yamagata/16/88-like strain is B/Panama/45/90.

### Claim 15 (depends on 1)

The method of claim 1, wherein the NP segment encodes a protein which has at least 97%, at least 98%, at least 99% identity or 100% identity to the sequence of SEQ ID NO: 4.

### Claim 16 (depends on 1)

The method of claim 1, wherein the PB2 segment encodes a protein which has at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 3.

### Claim 17 (depends on 1)

The method of claim 1, wherein the NS segment encodes a protein which has at least 97%, at least 98%, at least 99% or 100% identity with the sequence of SEQ ID NO: 35 and/or wherein the M1 segment encodes a protein which has at least 97%, at least 98%, at least 99% or 100% identity with the sequence of SEQ ID NO: 34.

### Claim 18 (depends on 1)

The method of claim 1, wherein the reassortant influenza B virus comprises:
a) a PA protein which has at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to the sequence of SEQ ID NO: 1; and/or
b) a PB1 protein which has at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to the sequence of SEQ ID NO: 2; and/or
c) a M1 protein which has at least 97% identity, at least 98%, at least 99% identity or 100% identity to the sequence of SEQ ID NO: 5; and/or
d) a M2 protein which has at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to the sequence of SEQ ID NO: 6; and/or
e) a NS1 protein which has at least 97% identity, at least 98%, at least 99% identity or 100% identity to the sequence of SEQ ID NO: 7; and/or
f) a NS2 protein which has at least 97% identity, at least 98%, at least 99% identity or 100% identity to the sequence of SEQ ID NO: 8.

### Claim 19

A method of preparing a reassortant influenza B virus comprising steps of
(i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza B virus comprising:
a) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15; or
b) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 31, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34; or
c) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 31, the PB2 segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15; or
d) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 12, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 16 and the M segment of SEQ ID NO: 15, or
e) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34;
f) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 33, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or
g) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31, the PB2 segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or
h) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 33, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or
i) the PA segment of SEQ ID NO: 11, the PB1 segment of SEQ ID NO: 12, the PB2 segment of SEQ ID NO: 32, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or
j) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 12, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34, or
k) the PA segment of SEQ ID NO: 30, the PB1 segment of SEQ ID NO: 31, the PB2 segment of SEQ ID NO: 13, the NP segment of SEQ ID NO: 14, the NS segment of SEQ ID NO: 35 and the M segment of SEQ ID NO: 34; and
(ii) culturing the culture host in order to produce a reassortant influenza B virus.

### Claim 20 (depends on 1)

The method of claim 1, further comprising the step (iii) of purifying the reassortant virus obtained in step (ii).

### Claim 21 (depends on 1)

A reassortant influenza B virus obtainable by the method of claim 1.

### Claim 22 (depends on 1)

The method of claim 1, wherein the culture host is an embryonated hen egg.

### Claim 23 (depends on 1)

The method of claim 1, wherein the culture host is a cell.

### Claim 24 (depends on 23)

The method of claim 23, wherein the cell is an MDCK, Vero or PerC6 cell.

### Claim 25 (depends on 23)

The method of claim 23, wherein the cell grows adherently.

### Claim 26 (depends on 23)

The method of claim 23, wherein the cell grows in suspension.

### Claim 27 (depends on 26)

The method of claim 26, wherein the MDCK cell is cell line MDCK 33016 (DSM ACC2219).

### Claim 28 (depends on 1)

A method of preparing a vaccine, comprising the steps of (a) preparing a virus by the method of claim 1 and (b) preparing a vaccine from the virus.

### Claim 29 (depends on 21)

A method of preparing a vaccine, comprising the step of preparing a vaccine from the virus of claim 21.

### Claim 30 (depends on 28)

The method of claim 28, wherein step (b) involves inactivating the virus.

### Claim 31 (depends on 28)

The method of claim 28, wherein the vaccine is a whole virion vaccine.

### Claim 32 (depends on 28)

The method of claim 28, wherein the vaccine is a split virion vaccine.

### Claim 33 (depends on 28)

The method of claim 28, wherein the vaccine is a surface antigen vaccine.

### Claim 34 (depends on 28)

The method of claim 28, wherein the vaccine is a virosomal vaccine.

### Claim 35 (depends on 28)

The method of claim 28, wherein the vaccine contains less than 10 ng of residual host cell DNA per dose.

### Claim 36 (depends on 28)

The method of claim 28, wherein the vaccine comprises an antigen from an influenza A strain.

### Claim 37 (depends on 36)

The method of claim 36, wherein the influenza A strain is of the H1, H2, H5, H7 or H9 subtype.

### Claim 38 (depends on 28)

A vaccine obtainable by the method of claim 28.

### Claim 39 (depends on 21)

An expression system comprising one or more expression construct(s) comprising the vRNA encoding segments of an influenza B virus of claim 21.

### Claim 40 (depends on 39)

A host cell comprising the expression system of claim 39.

### Claim 41 (depends on 40)

The host cell of claim 40, wherein the host cell is a mammalian cell.

### Claim 42 (depends on 41)

The host cell of claim 41, wherein the host cell is an MDCK, Vero or PerC6 cell.