# US 10,246,686 B2 · Influenza virus replication for vaccine development > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,686 B2 - Document ID: US10246686B2 - Status: Granted - Kind: B2 - Application number: 15/865,364 - Filed: 2018-01-09 - Published: 2019-01-31 - Granted: 2019-04-02 - Earliest claimed: 2015-07-06 - Assignees: Wisconsin Alumni Research Foundation (WARF) - Applicants: Wisconsin Alumni Research Foundation (WARF) - Inventors: Yoshihiro Kawaoka; Gabriele Neumann; Jihui Ping - Web page: https://patentgrep.com/patent/US10246686B2 ## CPC - 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 39/145: Orthomyxoviridae, e.g. influenza virus - C07K 14/005: from viruses - C12N 15/86: Viral vectors - C12N 2760/16111: Influenzavirus A, i.e. influenza A virus - 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 2760/16143: viral genome or elements thereof as genetic vector - C12N 2760/16151: Methods of production or purification of viral material - 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) - 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) ## Abstract The invention provides a composition useful to prepare high titer influenza viruses, e.g., in the absence of helper virus, which includes internal genes from an influenza virus vaccine strain or isolate, e.g., one that is safe in humans, for instance, one that does not result in significant disease, that confer enhanced growth in cells in culture, such as MDCK cells, or in eggs. ## Claims ### Claim 1 An isolated recombinant, reassortant influenza virus having PA, PB1, PB2, NP, NS, and M viral segments from a first influenza vaccine virus isolate, a heterologous, recombinant or chimeric influenza virus NA viral segment, and a heterologous, recombinant or chimeric HA viral segment, wherein the PB1 viral segment encodes a polypeptide having an alanine at residue 62, a glycine at residue 261, an arginine at residue 361, an arginine at residue 621, a serine at residue 654 or a glycine at residue 81 in F2, or a combination thereof, which PB1 viral segment encodes a PB1 having at least 85% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:2; or wherein the PB2 viral segment encodes a polypeptide having a lysine at residue 391 and having at least 85% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:3; or wherein the HA viral segment encodes a HA having an aspartic acid at position 136, a glutamic acid at position 162 or position 449, a leucine at position 179, a valine at position 182, an isoleucine at any of positions 184, 252 or 476, or any combination thereof; or wherein the NA viral segment encodes a NA having a serine at position 55 or a valine at position 265, or a combination thereof; and/or wherein the NS viral segment encodes a NS1 polypeptide having a lysine at position 118, and having at least 85% amino acid sequence identity to a NS1 polypeptide encoded by SEQ ID NO:6. ### Claim 2 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 wherein the PB2 viral segment encodes a PB2 polypeptide having a lysine at residue 391 and having at least 90% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:3. ### Claim 3 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 wherein the PB1 viral segment encodes a PB1 having at least 90% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:2. ### Claim 4 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 wherein the NS viral segment encodes a NS1 polypeptide having at least 90% amino acid sequence identity to a NS1 polypeptide encoded by SEQ ID NO:6. ### Claim 5 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 wherein the HA is H1, H3 or H5. ### Claim 6 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 wherein the NA is N1, N2, N3, N7, or N9. ### Claim 7 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 wherein at least one of the PA, PB1, PB2, NP, NS, and M viral segments comprise: a PB1 having the amino acid sequence encoded by SEQ ID NO:2 or PB1 with at least 95% amino acid sequence identity to the PB1 encoded by SEQ ID NO:2; a PB2 having the amino acid sequence encoded by SEQ ID NO:3 or PB2 with at least 95% amino acid sequence identity to the PB2 encoded by SEQ ID NO:3; a PA having the amino acid sequence encoded by SEQ ID NO:1 or PA with at least 95% amino acid sequence identity to the PA encoded by SEQ ID NO: 1; a NP having the amino acid sequence encoded by SEQ ID NO:4 or NP with at least 95% amino acid sequence identity to the NP encoded by SEQ ID NO:4; a M having the amino acid sequence encoded by SEQ ID NO:5 or M with at least 95% amino acid sequence identity to the M encoded by SEQ ID NO:5; or a NS having the amino acid sequence encoded by SEQ ID NO:6 or NS with at least 95% amino acid sequence identity to the NS encoded by SEQ ID NO:6 or wherein at least one of the PA, PB1, PB2, NP, NS, and M viral segments comprise: a PB1 having the amino acid sequence encoded by SEQ ID NO: 10 or PB1 with at least 95% amino acid sequence identity to the PB1 encoded by SEQ ID NO: 10; a PB2 having the amino acid sequence encoded by SEQ ID NO: 11 or PB2 with at least 95% amino acid sequence identity to the PB2 encoded by SEQ ID NO: 11; a PA having the amino acid sequence encoded by SEQ ID NO: 12 or PA with at least 95% amino acid sequence identity to the PA encoded by SEQ ID NO: 12; a NP having the amino acid sequence encoded by SEQ ID NO: 13 or NP with at least 95% amino acid sequence identity to the NP encoded by SEQ ID NO: 13; a M having the amino acid sequence encoded by SEQ ID NO:14 or M with at least 95% amino acid sequence identity to the M encoded by SEQ ID NO: 14; or a NS having the amino acid sequence encoded by SEQ ID NO: 15 or NS with at least 95% amino acid sequence identity to the NS encoded by SEQ ID NO: 15. ### Claim 8 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 further comprising wherein at least one of the PA, PB1, PB2, NP, NS, and M viral segments has a C to U promoter mutation. ### Claim 9 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 further comprising 142N, 225C, 356R, or 550L in PA, one or more of 112G (PB1-F2-R81G), 247H, 507V, or 644A in PB1, one or more of 202L, 323L or 504V in PB2, one or more of 74K, 112L, 116L, 417D, or 442A in NP, 97A and/or 100H in M1, and/or 55E and/or 140Q in NS1, or combinations thereof; further comprising at least one of 202L and/or 323L in PB2, 247H in PB1 or 74K in NP and optionally at least one of 142N in PA1, 55K in NS1 or 97A and/or 100H in M1; further comprising 202L and/or 323L in PB2, 247H in PB1, has 74K in NP, 202L and/or 323L in PB2 and 247H in PB1, or 202L and/or 323L in PB2, 247H in PB1, and 74K in NP; or further comprising 40I, 40L, 112G (PB1-F2-R81G), 180W, 247H, 507V, or 644A in PB1, 202L and/or 323L in PB2, 74K, 112L, 116L, 377N, 417D, or 422L in NP, 30P, 118K, 161T or 140Q in NS1, 142N, 225C, 356R, 401K, or 550L in PA. ### Claim 10 (depends on 1) The isolated recombinant, reassortant influenza virus of claim 1 further comprising 40I, 40L, 112G (PB1-F2-R81G), 180W, 247H, 507V, or 644A in PB1 and 202L and/or 323L in PB2; 40I, 40L, 112G (PB1-F2-R81G), 180W, 247H, 507V, or 644A in PB1, 202L and/or 323L in PB2 and 74K, 112L, 116L, 377N, 417D, or 422L in NP; 40I, 40L, 112G (PB1-F2-R81G), 180W, 247H, 507V, or 644A in PB1, has 202L and/or 323L in PB2, 74K, 112L, 116L, 377N, 417D, or 422L in NP, and has 30P, 118K, 161T or 140Q in NS1; 40I, 40L, 112G (PB1-F2-R81G), 180W, 247H, 507V, or 644A in PB1, 202L and/or 323L in PB2, 74K, 112L, 116L, 377N, 417D, or 422L in NP, 30P, 118K, 161T or 140Q in NS1, and 142N, 225C, 356R, 401K, or 550L in PA; or 40I, 40L, 112G, 180W, 247H, 507V, or 644A in PB1, 202L and/or 323L in PB2, 74K, 112L, I16L, 377N, 417D, or 422L in NP, 30P, 118K, 161T or 140Q in NS1, and 142N, 225C, 356R, 401K, or 550L in PA. ### Claim 11 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 of NA has sequences for a heterologous, recombinant or chimeric NA, and wherein the HA DNA in the vector for vRNA production of HA has sequences for a heterologous, recombinant or chimeric HA, wherein the PB1 DNA encodes a PB1 polypeptide having an alanine at residue 62, a glycine at residue 261, an arginine at residue 361, an arginine at residue 621, a serine at residue 654 or a glycine at residue 81 in F2, or a combination thereof, having at least 85% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:2; or wherein the PB2 DNA encodes a polypeptide having a lysine at residue 391 and having at least 85% amino acid sequence identity to a polypeptide encoded by SEQ ID NO:3; or wherein the HA DNA encodes a HA having an aspartic acid at position 136, a glutamic acid at position 162 or 449, a leucine at position 179, a valine at position 182, an isoleucine at position 184, 252 or 476, or any combination thereof; or wherein the NA DNA encodes a NA having a serine at position 55 or a valine at position 265, or a combination thereof; and/or wherein the NS DNA encodes a NS1 polypeptide having a lysine at position 118 and having at least 85% amino acid sequence identity to a NS1 polypeptide encoded by SEQ ID NO:6; or a combination thereof; 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 M1, a 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 12 (depends on 11) The method of claim 11 wherein the cell is an avian cell or a mammalian cell. ### Claim 13 (depends on 11) The method of claim 11 wherein the cell is a Vero cell, a human cell or a MDCK cell. ### Claim 14 (depends on 11) The method of claim 11 wherein the PB1, PB2, PA, NP, NS, and M DNAs in the vectors for vRNA productions have a sequence that corresponds to one that encodes a polypeptide having at least 95% amino acid sequence identity to a corresponding polypeptide encoded by SEQ ID NOs: 1-6 or 10-15. ### Claim 15 (depends on 11) The method of claim 11 further comprising isolating the virus. ### Claim 16 (depends on 11) The method of claim 11 wherein at least one of PA, PB1, or PB2 viral segments has a C to U promoter mutation.