# US 10,307,476 B2 · Genetically stable live attenuated respiratory syncytial virus vaccine and its production

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- Number: US 10,307,476 B2
- Document ID: US10307476B2
- Status: Granted
- Kind: B2
- Application number: 15/455,438
- Filed: 2017-03-10
- Published: 2017-07-06
- Granted: 2019-06-04
- Earliest claimed: 2012-04-13
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The United States of America, AS Represented By the Secretary, Department of Health and Human Services
- Inventors: Peter L. Collins; Cindy L. Luongo; Ursula J. Buchholz; Brian R. Murphy
- Web page: https://patentgrep.com/patent/US10307476B2

## CPC

- A61K 39/155: Paramyxoviridae, e.g. parainfluenza virus
- A61K 39/12: Viral antigens
- 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 2039/5254: {avirulent or attenuated}
- C12N 2760/18521: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/18522: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/18534: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18562: by genetic engineering
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are recombinant respiratory syncytial viruses that contain mutations that make the disclosed viruses attractive vaccine candidates. The viruses disclosed contain attenuating mutations designed to have increased genetic and phenotypic stability. Desired combinations of these mutations can be made to achieve desired levels of attenation. Exemplary vaccine candidates are described. Also provided are polynucleotides capable of encoding the described viruses, as wells as methods for producing the viruses and methods of use.

## Claims

### Claim 1

A recombinant infectious respiratory syncytial virus comprising a large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N) and a M2-1 protein and a genome or antigenome having a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein, and optionally comprises nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH).

### Claim 2 (depends on 1)

The recombinant infectious respiratory syncytial virus of claim 1, wherein the virus is attenuated.

### Claim 3 (depends on 1)

The recombinant infectious respiratory syncytial virus of claim 1, wherein the viral genome or antigenome further comprises a mutation of the codon that encodes the tyrosine at position 1321, or a corresponding position, of the L protein.

### Claim 4 (depends on 1)

The recombinant infectious respiratory syncytial virus of claim 1, wherein the viral genome or antigenome further comprises a deletion of the NS2 gene.

### Claim 5 (depends on 1)

The recombinant infectious respiratory syncytial virus of claim 1, wherein the viral genome or antigenome further comprises one or more of the following mutations:
the L protein mutation of 1321K, 1321E, 1321P, 1321G, 1321K(AAA), 1321E(GAA), 1321P(CCT), 1321G(GGA), or 1321G(GGT);
the L protein mutation Q831L;
V267I in the N protein, E218A in the F protein, T523 I in the F protein, C319Y in the L protein, and H1690Y in the L protein;
a T to C substitution at the ninth nucleotide position of the gene-start signal of the M2 gene, which corresponds to nucleotide 7606 of a positive sense complement of the genome of RSV strain A2; and
deletion of the SH gene.

### Claim 6 (depends on 1)

The recombinant infectious respiratory syncytial virus of claim 1, wherein the viral genome or antigenome further comprises:
a mutation of the codon that encodes the tyrosine at position 1321, or a corresponding position, of the L protein, wherein the mutation of the codon is an AAA mutation.

### Claim 7 (depends on 1)

The recombinant infectious respiratory syncytial virus of claim 1, wherein the viral genome or antigenome further comprises:
a mutation of the codon that encodes the tyrosine at position 1321, or a corresponding position, of the L protein, wherein the mutation of the codon is an AAA mutation;
V267I in the N protein, E218A and T523I in the F protein, and C319Y and H1690Y in the L protein; and
a deletion of the SH gene.

### Claim 8 (depends on 1)

The recombinant infectious respiratory syncytial virus of claim 1, wherein the viral genome or antigenome further comprises a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine.

### Claim 9 (depends on 8)

The recombinant infectious respiratory syncytial virus of claim 8, wherein the mutation of L protein amino acid sequence residue 1314 is encoded by the codon CTG.

### Claim 10 (depends on 8)

The recombinant infectious respiratory syncytial virus of claim 8, wherein the virus is attenuated.

### Claim 11 (depends on 8)

The recombinant infectious respiratory syncytial virus of claim 8, wherein the viral genome or antigenome further comprises a mutation of the codon that encodes a tyrosine at position 1321, or a corresponding position, of the L protein.

### Claim 12 (depends on 8)

The recombinant infectious respiratory syncytial virus of claim 8, wherein the viral genome or antigenome further comprises a deletion of the NS2 gene.

### Claim 13 (depends on 8)

The recombinant infectious respiratory syncytial virus of claim 8, wherein the viral genome or antigenome further comprises one or more of the following mutations:
the L protein mutation of 1321K, 1321E, 1321P, 1321G, 1321K(AAA), 1321E(GAA), 1321P(CCT), 1321G(GGA), or 1321G(GGT);
the L protein mutation Q831L;
V267I in the N protein, E218A in the F protein, T523I in the F protein, C319Y in the L protein, and H1690Y in the L protein;
a T to C substitution at the ninth nucleotide position of the gene-start signal of the M2 gene, which corresponds to nucleotide 7606 of a positive sense complement of the genome of RSV strain A2; and
deletion of the SH gene.

### Claim 14 (depends on 1)

A method of producing an immune response to a viral protein, comprising administering the respiratory syncytial virus of claim 1 to an animal.

### Claim 15 (depends on 14)

The method of claim 14, wherein the respiratory syncytial virus is administered via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application.

### Claim 16 (depends on 14)

The method of claim 14, wherein the animal is a mammal.

### Claim 17 (depends on 16)

The method of claim 16, wherein the mammal is a human.

### Claim 18

A recombinant infectious respiratory syncytial virus comprising a large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), a M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), a M2-2 protein, and a small hydrophobic protein (SH), and a genome or antigenome having:
a deletion of the codon that encodes the serine at position 1313, or a corresponding position, of the L protein,
a mutation of amino acid sequence residue 1314, or a corresponding position, of the L protein, wherein the mutation of L protein amino acid sequence residue 1314 is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon set forth as CTG; and
a deletion of the NS2 gene.

### Claim 19 (depends on 18)

A method of producing an immune response to a viral protein, comprising administering the respiratory syncytial virus of claim 18 to an animal.

### Claim 20 (depends on 19)

The method of claim 19, wherein the respiratory syncytial virus is administered via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application.

### Claim 21 (depends on 19)

The method of claim 19, wherein the animal is a mammal.

### Claim 22 (depends on 21)

The method of claim 21, wherein the mammal is a human.