# US 11,197,926 B2 · Recombinant influenza viruses with stabilized HA for replication in eggs

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- Number: US 11,197,926 B2
- Document ID: US11197926B2
- Status: Granted
- Kind: B2
- Application number: 16/170,321
- Filed: 2018-10-25
- Published: 2019-06-06
- Granted: 2021-12-14
- Earliest claimed: 2017-10-25
- Assignees: Wisconsin Alumni Research Foundation (WARF); The University of Tokyo
- Applicants: Wisconsin Alumni Research Foundation (WARF); The University of Tokyo
- Inventors: Yoshihiro Kawaoka; Shinya Yamada; Shiho Chiba
- Web page: https://patentgrep.com/patent/US11197926B2

## CPC

- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/0043: {Nose}
- 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)
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- 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 2760/16034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16051: Methods of production or purification of viral material
- C12Y 302/01018: Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
- 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)
- C12Y: ENZYMES

## Abstract

Modified influenza virus neuraminidases are described herein that improve viral replication, thus improving the yield of vaccine viruses. Expression of such modified neuraminidases by influenza virus may also stabilize co-expressed hemagglutinins so that the hemagglutinins do not undergo mutation.

## Claims

### Claim 1

An isolated recombinant influenza virus comprising a selected NA viral segment encoding at least three selected residues in NA, wherein the residues are selected from residue A, I, G, or L at position 32; the residue N or Q at position 147: residue K, R or H at position 148: residue E, N or Q at position 151: residue S, T, I, L, A, N, W, Y, P, V, or G at position 245; residue D or E at position 329: residue S, T, P, Y, W, A, N, I, L, or V at position 346: residue G, Q, S, T, Y, C or W at position 347,
or any combination thereof, wherein the numbering is relative to SEQ ID NO:3, wherein the recombinant influenza virus has enhanced replication in avian eggs or has a reduction in HA mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine at residue 32, does not have a deletion of residues 46 to 50, encodes an aspartic acid at position 147, encodes a threonine at residue 148, encodes an aspartic acid at residue 151, encodes an asparagine at residue 245, encodes an asparagine at residue 329, encodes a glycine at residue 346, encodes a histidine at residue 347, or any combination thereof.

### Claim 2 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the NA viral segment encodes a NA that has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:54.

### Claim 3 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the NA viral segment encodes a N2, N3, N7, or N9.

### Claim 4 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 32 is A; the residue at position 147 is N; the residue at position 148 is K; the residue at position 151 is E; the residue at position 245 is S; the residue at position 329 is D; the residue at position 346 is V; the residue at position 347 is Q; or any combination thereof.

### Claim 5 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 147 is N or Q, the residue at position 329 is D or E, the residue at position 347 is G or Q, or any combination thereof.

### Claim 6 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 148 is K, R or H, the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, or V, or any combination thereof.

### Claim 7 (depends on 1)

The isolated recombinant influenza virus of claim 1 which comprises PA, PB1, PB2, NP, M, and NS viral segments having at least 85% nucleic acid sequence identity to SEQ ID NOS: 24 to 29 or 39 to 44 or encoding a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NOS: 24 to 29 or 39 to 44.

### Claim 8

A method to prepare influenza virus, comprising: contacting a cell with:
a vector for vRNA production comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to an influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1, PB2, PA, NP, NS, and M DNAs in the vectors for vRNA production are from one or more influenza vaccine virus isolates, wherein the NA DNA in the vector for vRNA production encodes a NA having at least three residues selected from residue A, I, G, L at position 32: the residue N or Q at position 147; residue K, R or H at position 148; residue E, N or Q at position 151; residue S, T, I, L, A, N, W, Y, P, V, or G at position 245; residue D or E at position 329; residue S, T, P, Y, W, A, N, I, L, or V at position 346; residue G, Q, S, T, Y, C or W at position 347, or any combination thereof, wherein the numbering for NA residues is relative to SEQ ID NO:3; and
a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; in an amount effective to yield infectious influenza virus.

### Claim 9 (depends on 8)

The method of claim 8 wherein the NA has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:54.

### Claim 10 (depends on 8)

The method of claim 8 wherein the residue at position 147 is N; the residue at position 329 is D; the residue at position 347 is Q; the residue at position 151 is E; the residue at position 148 is K; or the residue at position 245 is S.

### Claim 11 (depends on 8)

The method of claim 8 wherein the virus comprises PA, PB1, PB2, NP, M, and NS viral segments having at least 85% nucleic acid sequence identity to SEQ ID NOS: 24 to 29 or 39 to 44 or encoding a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID NOS: 24 to 29 or 39 to 44.

### Claim 12 (depends on 8)

An isolated virus prepared by the method of claim 8.

### Claim 13 (depends on 1)

A method of immunizing an avian or a mammal, comprising: administering to the avian or the mammal a composition having an effective amount of the virus of claim 1.

### Claim 14 (depends on 13)

The method of claim 13 wherein the composition comprises at least one other different influenza virus.

### Claim 15 (depends on 13)

The method of claim 13 wherein the mammal is a human.

### Claim 16 (depends on 13)

The method of claim 13 wherein the composition is administered intranasally.

### Claim 17 (depends on 13)

The method of claim 13 wherein the composition is administered via injection.

### Claim 18 (depends on 1)

A method comprising passaging the virus of claim 1 in eggs.