# US 10,119,124 B2 · Influenza M2 protein mutant viruses as live influenza attenuated vaccines > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,124 B2 - Document ID: US10119124B2 - Status: Granted - Kind: B2 - Application number: 15/292,595 - Filed: 2016-10-13 - Published: 2017-04-06 - Granted: 2018-11-06 - Earliest claimed: 2007-06-18 - Assignees: Wisconsin Alumni Research Foundation (WARF) - Applicants: Wisconsin Alumni Research Foundation (WARF) - Inventors: Tokiko Watanabe; Yoshihiro Kawaoka - Web page: https://patentgrep.com/patent/US10119124B2 ## 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 39/12: Viral antigens - A61K 39/145: Orthomyxoviridae, e.g. influenza virus - C07K 14/005: from viruses - A61K 2039/5254: {avirulent or attenuated} - A61K 2039/543: {intranasal} - C07K 16/108: Orthomyxoviridae (F), e.g. influenza virus - C12N 2760/00: ssRNA viruses negative-sense - C12N 2760/16011: Orthomyxoviridae - 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 2760/16121: 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: 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 A method to prepare recombinant influenza viruses comprising a mutant M2 protein which has a deletion of two or more residues in the cytoplasmic tail and is attenuated in vivo, is provided, as well the resulting virus and vaccines with the virus. ## Claims ### Claim 1 An immunogenic composition comprising an isolated, live, in vivo attenuated recombinant influenza virus comprising a PA viral gene segment, a PB1 viral gene segment, a PB2 viral gene segment, a HA viral gene segment, a NA viral gene segment, a NP viral gene segment, a M viral gene segment having coding sequences for M1 and M2, and a NS viral gene segment having coding sequences for NS1 and NS2, wherein the M gene segment comprises a mutant M2 protein gene for a M2 protein which has a deletion consisting of 2 to 21 residues of the C-terminus of the cytoplasmic tail which M2 protein is encoded by the mutant M2 protein gene, wherein the deletion attenuates the replication of the recombinant virus in vivo relative to the corresponding influenza virus without the deletion, wherein the amount of the virus in the composition is about 0.1 micrograms to 200 micrograms of influenza virus hemagglutinin (HA) per influenza virus isolate or about 103 to 107 PFU per influenza virus isolate. ### Claim 2 (depends on 1) The immunogenic composition of claim 1, wherein the recombinant virus comprises influenza A HA. ### Claim 3 (depends on 2) The immunogenic composition of claim 2, wherein the recombinant virus comprises H5 HA. ### Claim 4 (depends on 2) The immunogenic composition of claim 2, wherein the HA is not H1 or H3 HA. ### Claim 5 (depends on 1) The immunogenic composition of claim 1, wherein the HA in the recombinant virus is modified at the HA cleavage site so that in vitro plaque formation occurs only in the presence of trypsin relative to an unmodified HA cleavage site where in vitro plaque formation occurs in the absence of trypsin. ### Claim 6 (depends on 1) The immunogenic composition of claim 1, wherein the recombinant virus further comprises an additional attenuating mutation. ### Claim 7 (depends on 1) The immunogenic composition of claim 1, further comprising at least one different influenza virus. ### Claim 8 (depends on 1) The immunogenic composition of claim 1, wherein 2 up to 7 C-terminal residues of the cytoplasmic tail of M2 are deleted. ### Claim 9 (depends on 1) The immunogenic composition of claim 1, wherein 9 up to 21 C-terminal residues of the cytoplasmic tail of M2 are deleted. ### Claim 10 (depends on 1) The immunogenic composition of claim 1, further comprising a pharmaceutically acceptable carrier. ### Claim 11 (depends on 1) The immunogenic composition of claim 1, wherein the mutant M2 protein gene has a mutation that codes for a deletion of 5 to 15 residues from the C-terminus of the M2 protein. ### Claim 12 (depends on 11) The immunogenic composition of claim 11, wherein the recombinant influenza virus has a HA gene segment for a H5 HA with an HA cleavage site of an avirulent influenza virus. ### Claim 13 (depends on 1) A method to raise an immune response against influenza virus in a vertebrate, comprising: contacting the vertebrate with the immunogenic composition of claim 1. ### Claim 14 (depends on 13) The method of claim 13, wherein the vertebrate is an avian or a mammal. ### Claim 15 (depends on 14) The method of claim 14, wherein the mammal is a human. ### Claim 16 (depends on 13) The method of claim 13, wherein 2 up to 7 C-terminal residues of the cytoplasmic tail of M2 are deleted. ### Claim 17 (depends on 13) The method of claim 13, wherein 9 up to 21 C-terminal residues of the cytoplasmic tail of M2 are deleted. ### Claim 18 (depends on 13) The method of claim 13, wherein the recombinant virus further comprises an additional attenuating mutation. ### Claim 19 (depends on 13) The method of claim 13, wherein the recombinant virus comprises H5 HA. ### Claim 20 (depends on 13) The method of claim 13, wherein the HA of the recombinant virus is not H1 or H3 HA.