# US 11,807,872 B2 · Recombinant influenza viruses with stabilized HA for replication in eggs

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- Number: US 11,807,872 B2
- Document ID: US11807872B2
- Status: Granted
- Kind: B2
- Application number: 17/004,583
- Filed: 2020-08-27
- Published: 2021-04-08
- Granted: 2023-11-07
- Earliest claimed: 2019-08-27
- 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/US11807872B2

## CPC

- 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 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 39/12: Viral antigens
- A61P 31/16: for influenza or rhinoviruses
- 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
- 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
- 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)
- 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

## 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 or decrease the need for HA binding to cells.

## Claims

### Claim 1

An isolated recombinant influenza virus comprising a selected neuraminidase (NA) viral segment encoding a plurality of selected residues in NA,
wherein the selected NA viral segment encodes a NA having an isoleucine (I) at residue 148, and wherein the plurality of selected residues in the NA includes when the NA does not encode an aspartic acid (D) at position 151, does not encode an asparagine (N) at position 245, does not encode a threonine (T) at position 329, does not encode a lysine (K) at position 344, does not encode a glycine (G) at position 346, does not encode a histidine (H) at residue 347, or does not encode a threonine at position 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs, has reduced sialidase activity, enhanced binding to a2-3 sialosides, or has a reduction in hemagglutinin (HA) mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine or lysine at residue 148, encodes an aspartic acid at residue 151, encodes an asparagine at residue 245, encodes a threonine at residue 329, encodes a lysine at residue 344, encodes a glycine at residue 346, encodes a histidine at residue 347, and encodes a threonine at position 369, wherein the numbering is based on N2 of SEQ ID NO:53.

### Claim 2 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the selected NA segment encodes glutamic acid (E) at residue 151, serine (S), threonine, glycine, alanine (A), leucine or isoleucine at residue 245, serine, glycine, alanine, leucine or isoleucine residue 329, glutamic acid, aspartic acid or histidine at residue 344, valine, leucine, isoleucine, threonine or serine at reside 346, glycine, alanine, valine, leucine, isoleucine or threonine at residue 347, or lysine, histidine, aspartic acid or glutamic acid at residue 369.

### Claim 3 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 151 is E, N or Q or position 344 is E, D or H.

### Claim 4 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 245 is S, T, I, L, A, V, or G.

### Claim 5 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 347 is G, Q, S, T, Y, C or W.

### Claim 6 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 369 is K, H, R, E, P, or D.

### Claim 7 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 329 is serine, valine, alanine, glycine, isoleucine or leucine.

### Claim 8 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 346 is V, S, T, A, I, or L.

### Claim 9 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 329 is S, I, L, A, W, Y, P, V, or G, the residue at position 344 is E, H, D, N or Q, the residue at position 346 is V, S, T, I, L, A, W, Y, or P, the residue at position 347 is G, Q, S, T, Y, C or W, or the residue at position 369 is K, H, R, E, P, or D.

### Claim 10 (depends on 1)

The isolated recombinant influenza virus of claim 1 which is a reassortant.

### Claim 11 (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:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74, or has at least 90% amino acid sequence identity to a NA encoded by any one of SEQ ID Nos. 55-59.

### Claim 12 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the NA viral segment encodes a N2, N3, N7, or N9 or wherein HA is H1, H2, H3, H5, H7, or H9.

### Claim 13 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein polymerase A (PA), polymerase B1 (PB1), polymerase B2 (PB2), nucleoprotein (NP), matrix (M), and non-structural (NS) viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode 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 14 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein PB2 has I, A, L, or G at residue 147 based on the numbering of a corresponding polypeptide encoded by SEQ ID NO:26.

### Claim 15 (depends on 1)

The isolated recombinant influenza virus of claim 1 which has one or more of PB2-I504V, PB1-M40L/G180W, PA-R401K, NP-I116L, and NS1-A30P/R118K based on the numbering of a corresponding polypeptide encoded by one of SEQ ID Nos. 24-27 or 29.

### Claim 16 (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 17 (depends on 1)

The isolated recombinant influenza virus of claim 1 wherein the residue at position 245 is S, the residue at position 346 is V, the residues at position 347 is G, or the residue at position 369 is K.