# US 11,510,974 B2 · Mutant virus, preparation method therefor and application thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,510,974 B2
- Document ID: US11510974B2
- Status: Granted
- Kind: B2
- Application number: 16/066,283
- Filed: 2016-08-02
- Published: 2020-08-27
- Granted: 2022-11-29
- Earliest claimed: 2015-12-31
- Assignees: Peking University
- Applicants: Peking University
- Inventors: Demin Zhou; Longlong Si; Xueying Zhou; Ziwei Zhang; Huan Xu; Zhenyu Tian; Chuanling Zhang; Sulong Xiao; Qing Xia; Lihe Zhang
- Web page: https://patentgrep.com/patent/US11510974B2

## CPC

- A61K 39/12: Viral antigens
- C07K 14/005: from viruses
- A61K 35/76: Viruses; Subviral particles; Bacteriophages
- A61P 31/16: for influenza or rhinoviruses
- C12N 5/10: Cells modified by introduction of foreign genetic material
- 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 9/93: {Ligases (6)}
- 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 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16162: by genetic engineering
- C12Y 601/01026: Pyrrolysine-tRNAPyl ligase (6.1.1.26)
- 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)
- 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)
- C12Y: ENZYMES

## Abstract

The present invention relates to a mutated virus. Said virus can be an influenza virus of human or other animal origin. The present invention also relates to a method for preparing the mutated virus, the method comprising introducing UAG codons into positions upstream of the stop codons per se of one or more genes of a viral genome by reverse genetic techniques. The present invention further relates to uses of the mutated virus, for example, as a live attenuated vaccine, or in replication of controllable and safe virus models, and the like.

## Claims

### Claim 1

A pharmaceutical composition comprising a mutated influenza virus, which is characterized in that a coding nucleic acid of at least one protein of the virus comprises one or more UAG codons at a position located one or more codons upstream of a natural, endogenous stop codon of the coding nucleic acid; wherein the at least one protein of the virus is selected from PA, PB1, PB2, or NP protein, and the at least one protein in the virus comprises an unnatural amino acid at the position corresponding to the one or more UAG codons, wherein the influenza virus comprises UAG codon(s) at positions of codon(s) of nucleic acid(s) encoding R266 of PA protein by reference to a corresponding amino acid encoded by a nucleic acid as set forth in SEQ ID NO:4, R52 of PB1 protein by reference to a corresponding amino acid encoded by a nucleic acid as set forth in SEQ ID NO:3, K33 of PB2 protein by reference to a corresponding amino acid encoded by a nucleic acid as set forth in SEQ ID NO:2, and/or D101 of NP protein by reference to a corresponding amino acid encoded by a nucleic acid as set forth in SEQ ID NO:6, wherein the composition is an active influenza virus vaccine, wherein the vaccine is capable of eliciting an immune response.

### Claim 2 (depends on 1)

The mutated virus according to claim 1,
wherein the unnatural amino acid is selected from Lys-diazirine shown in formula
Lys-azido shown in formula (II)
or at least one other unnatural amino acid.

### Claim 3 (depends on 1)

The mutated virus according to claim 1, wherein UAG stop codons of said natural, endogenous stop codons of the virus are mutated to provide UAA stop codons.

### Claim 4 (depends on 1)

The mutated virus according to claim 1 wherein,
the amino acid sequence of nonmutated PB2 is identical to the amino acid sequence encoded by the nucleic acid as set forth in SEQ ID NO: 2,
the amino acid sequence of nonmutated PB1 is identical to the amino acid sequence encoded by the nucleic acid as set forth in SEQ ID NO: 3,
the amino acid sequence of nonmutated PA is identical to the amino acid sequence encoded by the nucleic acid as set forth in SEQ ID NO: 4, or
the amino acid sequence of nonmutated NP is identical to the amino acid sequence encoded by the nucleic acid as set forth in SEQ ID NO: 6.

### Claim 5 (depends on 4)

The mutated virus according to claim 4, wherein the mutated virus comprises UAG codons at positions of codons coding for R266 of PA protein by reference to a corresponding amino acid encoded by the nucleic acid as set forth in SEQ ID NO:4, R52 of PB1 protein by reference to a corresponding amino acid encoded by the nucleic acid as set forth in SEQ ID NO:3, K33 of PB2 protein by reference to a corresponding amino acid encoded by the nucleic acid as set forth in SEQ ID NO:2 and D101 of NP protein by reference to a corresponding amino acid encoded by the nucleic acid as set forth in SEQ ID NO:6.

### Claim 6 (depends on 1)

The mutated virus according to claim 1, wherein the influenza virus is of human or other animal origin, and is an influenza A, B or C virus.