# US 11,371,024 B2 · Attenuation of human respiratory syncytial virus by genome scale codon-pair deoptimization

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- Number: US 11,371,024 B2
- Document ID: US11371024B2
- Status: Granted
- Kind: B2
- Application number: 15/924,012
- Filed: 2018-03-16
- Published: 2018-07-26
- Granted: 2022-06-28
- Earliest claimed: 2013-02-08
- Assignees: The United States of America, as represented by the Secretary, Dept. of Health and Human Services; The Research Foundation for The State University of New York
- Applicants: The USA, as represented by the Secretary, Dept. of Health and Human Services; The Research Foundation for the State University of New York
- Inventors: Peter L. Collins; Cyril Le Nouën; Linda G. Brock; Ursula J. Buchholz; Joshua Marc DiNapoli; Steffen Mueller; Eckard Wimmer
- Web page: https://patentgrep.com/patent/US11371024B2

## 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 39/155: Paramyxoviridae, e.g. parainfluenza virus
- C07K 14/005: from viruses
- A61K 2039/5254: {avirulent or attenuated}
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/55: {characterised by the host/recipient, e.g. newborn with maternal antibodies}
- C12N 2760/00021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/00034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/00062: by genetic engineering
- 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 2760/18561: Methods of inactivation or attenuation
- C12N 2760/18562: by genetic engineering
- 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

Described herein are RSV polynucleotide sequences that make use of multiple codons that are containing silent nucleotide substitutions engineered in multiple locations in the genome, wherein the substitutions introduce a numerous synonymous codons into the genome. Due to the large number of defects involved, the attenuated viruses disclosed herein provide a means of producing attenuated, live vaccines against RSV.

## Claims

### Claim 1

A recombinant polynucleotide comprising a recombinant nucleotide sequence encoding respiratory syncytial virus (RSV) proteins NS1, NS2, G, F, or L or a combination thereof,
wherein the codon pair bias of each recombinant nucleotide sequence encoding the RSV protein is reduced by at least 0.05 compared to a wild type RSV subgroup A virus for a corresponding nucleotide sequence encoding the RSV protein,
wherein a recombinant nucleotide sequence encoding RSV protein NS1 has about 75% to about 95% identity to the wild type RSV subgroup A virus for the corresponding nucleotide sequence encoding RSV protein NS1,
wherein a recombinant nucleotide sequence encoding RSV protein NS2 has about 75% to about 95% identity to the wild type RSV subgroup A virus for the corresponding nucleotide sequence encoding RSV protein NS2,
wherein a recombinant nucleotide sequence encoding RSV protein G has about 65% to about 85% identity to the wild type RSV subgroup A virus for the corresponding nucleotide sequence encoding RSV protein G,
wherein a recombinant nucleotide sequence encoding RSV protein F has about 65% to about 85% identity to the wild type RSV subgroup A virus for the corresponding nucleotide sequence encoding RSV protein F,
wherein a recombinant nucleotide sequence encoding RSV protein L has about 70% to about 90% identity to the wild type RSV subgroup A virus for the corresponding nucleotide sequence encoding RSV protein L,
and
wherein the recombinant nucleotide sequence encodes a RSV amino acid sequence that is the same as the corresponding wild type RSV subgroup A virus amino acid sequence, or a RSV amino acid sequence with up to four amino acid substitutions, additions or deletions compared to the corresponding wild type RSV subgroup A virus amino acid sequence.

### Claim 2 (depends on 1)

A recombinant RSV genome or antigenome comprising the recombinant polynucleotide of claim 1, said recombinant RSV genome or antigenome comprising the genome encoded by pRSV_Min A, pRSV_Min B, pRSV_Min L, pRSV_Min FLC, pRSV_Min L_N, pRSV_Min L_p, pRSV_Min L_M21, pRSV_Min L_NP, pRSV_Min L_NM21, pRSV_Min L_PM21, or pRSV_Min L_NPM21.

### Claim 3 (depends on 1)

A recombinant RSV genome or antigenome comprising the recombinant polynucleotide of claim 1, said recombinant RSV genome or antigenome further comprising a nucleotide change in one or more of the following:
a. a change in the codon encoding amino acid 136 of the N protein,
b. a change in the codon encoding amino acid 114 of the P protein, or
c. a change in the codon encoding amino acid 88 of the M2-1 protein.

### Claim 4 (depends on 3)

The recombinant RSV genome or antigenome of claim 3, wherein any one of said nucleotide changes causes an amino acid other than:
a. lysine to be encoded at position 136 of the N protein,
b. glutamic acid to be encoded at position 114 of the P protein, or
c. asparagine to be encoded at position 88 of the M2-1 protein.

### Claim 5 (depends on 4)

The recombinant RSV genome or antigenome of claim 4, wherein arginine is encoded at position 136 of the N protein, valine is encoded at position 114 of the P protein, and/or lysine is encoded at position 88 of the M2-1 protein.

### Claim 6 (depends on 2)

A method of producing a recombinant RSV, comprising expressing the genome or antigenome of claim 2 in a cell.

### Claim 7 (depends on 6)

A method of producing an immune response to a viral protein, comprising administering the recombinant RSV produced by the method of claim 6 to an animal.

### Claim 8 (depends on 7)

The method of claim 7, wherein the RSV is administered via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application.

### Claim 9 (depends on 7)

The method of claim 7, wherein the animal is a mammal.

### Claim 10 (depends on 9)

The method of claim 9, wherein the mammal is a human.

### Claim 11 (depends on 6)

A recombinant RSV produced by the method of claim 6, wherein said virus is attenuated.

### Claim 12 (depends on 11)

The recombinant RSV of claim 11, wherein the genome or antigenome is further modified to include a previously characterized RSV mutation or deletion.

### Claim 13 (depends on 6)

A vaccine composition comprising the recombinant RSV produced by the method of claim 6.

### Claim 14 (depends on 1)

The recombinant polynucleotide of claim 1, wherein the codon pair bias of the recombinant nucleotide encoding each RSV protein is reduced by at least 0.1 compared to the corresponding wild type RSV subgroup A virus nucleotide sequence encoding the RSV protein.

### Claim 15 (depends on 1)

The recombinant polynucleotide of claim 1, wherein the codon pair bias of the recombinant nucleotide encoding each RSV protein is reduced by at least 0.2 compared to the corresponding wild type RSV subgroup A virus nucleotide sequence encoding the RSV protein.

### Claim 16 (depends on 1)

The recombinant polynucleotide of claim 1, wherein the codon pair bias is calculated relative to the human genome.

### Claim 17 (depends on 1)

A recombinant RSV genome or antigenome comprising the recombinant polynucleotide of claim 1.