# US 11,390,651 B2 · Vaccine candidates for human respiratory syncytial virus (RSV) having attenuated phenotypes

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- Number: US 11,390,651 B2
- Document ID: US11390651B2
- Status: Granted
- Kind: B2
- Application number: 17/008,025
- Filed: 2020-08-31
- Published: 2021-06-24
- Granted: 2022-07-19
- Earliest claimed: 2016-09-23
- Assignees: The USA, Secretary, DHHS; Codagenix Inc.
- Applicants: The USA, AS Represented By the Secretary, Dept. of Health and Human Services; Codagenix, Inc.
- Inventors: Cyril Lenouen; Ursula J. Buchholz; Peter L. Collins; Steffen Mueller
- Web page: https://patentgrep.com/patent/US11390651B2

## CPC

- C07K 14/08: RNA viruses
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/0043: {Nose}
- A61K 39/155: Paramyxoviridae, e.g. parainfluenza virus
- A61P 31/20: for DNA viruses
- C12N 7/04: Inactivation or attenuation; Producing viral sub-units
- 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
- 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)
- 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)
- 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)

## Abstract

Reported herein are presumptively de-attenuating mutations that are useful, either individually or in combinations that may include other known mutations, in producing recombinant strains of human respiratory syncytial virus (RSV) exhibiting attenuation phenotypes. Also described herein is a novel RSV construct, Min_L-NPM2-1(N88K)L, which exhibits an attenuated phenotype, is stable and is as immunogenic as wild type RSV. The recombinant RSV strains described here are suitable for use as live-attenuated RSV vaccines. Exemplary vaccine candidates are described. Also provided are polynucleotide sequences capable of encoding the described viruses, as well as methods for producing and using the viruses.

## Claims

### Claim 1

An isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by a mutation in the L ORF at a position corresponding to T1166 of the L protein in SEQ ID NO:11 and further modified by at least two mutations selected from the group consisting of:
i. a mutation in the M2-1 ORF at a position corresponding to N88 or A73 of the M2-1 protein in SEQ ID NO:9;
ii. a mutation in the N ORF at a position corresponding to K136 of the N protein in SEQ ID NO:3;
iii. a mutation in the P ORF at a position corresponding to E114 of the P protein in SEQ ID NO:4; and
iv. combinations thereof.

### Claim 2 (depends on 1)

The isolated polynucleotide molecule of claim 1, wherein the RSV genome or antigenome is further modified by the following mutations:
i. a mutation in the M2-1 ORF at a position corresponding to N88 of the M2-1 protein in SEQ ID NO:9;
ii. a mutation in the N ORF at a position corresponding to K136 of the N protein in SEQ ID NO:3; and
iii. a mutation in the P ORF at a position corresponding to E114 of the P protein in SEQ ID NO:4.

### Claim 3 (depends on 1)

The isolated polynucleotide molecule of claim 1, wherein the RSV genome or antigenome is further modified by the following mutations:
i. a mutation in the M2-1 ORF at a position corresponding to A73 of the M2-1 protein in SEQ ID NO:9;
ii. a mutation in the N ORF at a position corresponding to K136 of the N protein in SEQ ID NO:3; and
iii. a mutation in the P ORF at a position corresponding to E114 of the P protein in SEQ ID NO:4.

### Claim 4 (depends on 1)

The isolated polynucleotide molecule of claim 1, wherein the RSV genome or antigenome is modified by corresponding mutations selected from the group consisting of (1) T1166I in the L protein, N88K in the M2-1 protein, K136R in the N protein and E114V in the P protein; and (2) T1166I in the L protein, A73S in the M2-1 protein, K136R in the N protein and E114V in the P protein.

### Claim 5 (depends on 4)

The isolated polynucleotide molecule of claim 4, wherein the RSV genome or antigenome comprises a deletion in at least one of the proteins selected from M2-2, NS1 and NS2.

### Claim 6 (depends on 4)

The isolated polynucleotide molecule of claim 4, wherein the RSV genome or antigenome is codon-pair deoptimized.

### Claim 7 (depends on 4)

The isolated polynucleotide molecule of claim 4, wherein the L ORF of the RSV genome or antigenome is codon-pair deoptimized.

### Claim 8 (depends on 1)

A vector comprising the isolated polynucleotide molecule of claim 1.

### Claim 9 (depends on 1)

A cell comprising the isolated polynucleotide of claim 1.

### Claim 10 (depends on 1)

A pharmaceutical composition comprising an immunologically effective amount of the recombinant RSV variant encoded by the isolated polynucleotide molecule of claim 1.

### Claim 11 (depends on 10)

A method of vaccinating a subject against RSV comprising administering the pharmaceutical composition of claim 10.

### Claim 12 (depends on 10)

A method of inducing an immune response comprising administering the pharmaceutical composition of claim 10.

### Claim 13 (depends on 11)

The method of claim 11, wherein the pharmaceutical composition is administered intranasally via injection, aerosol delivery, nasal spray or nasal droplets.

### Claim 14 (depends on 4)

A live attenuated RSV vaccine comprising the recombinant RSV variant encoded by the isolated polynucleotide of claim 4.

### Claim 15 (depends on 14)

A pharmaceutical composition comprising the RSV vaccine of claim 14.

### Claim 16 (depends on 4)

A method of making a live attenuated RSV vaccine comprising expressing the isolated polynucleotide molecule of claim 4.

### Claim 17

An isolated polynucleotide molecule that is at least about 95% identical to the nucleotide sequence of SEQ ID NO:14.

### Claim 18 (depends on 17)

The isolated polynucleotide molecule of claim 17, comprising the nucleotide sequence of SEQ ID NO:14.

### Claim 19 (depends on 18)

A vector comprising the isolated polynucleotide molecule of claim 18.

### Claim 20 (depends on 18)

A cell comprising the isolated polynucleotide of claim 18.

### Claim 21 (depends on 18)

A pharmaceutical composition comprising an immunologically effective amount of the recombinant RSV variant encoded by the isolated polynucleotide molecule of claim 18.

### Claim 22 (depends on 21)

A method of vaccinating a subject against RSV comprising administering the pharmaceutical composition of claim 21.

### Claim 23 (depends on 21)

A method of inducing an immune response comprising administering the pharmaceutical composition of claim 21.

### Claim 24 (depends on 22)

The method of claim 22, wherein the pharmaceutical composition is administered intranasally.

### Claim 25 (depends on 24)

The method of claim 24, wherein the pharmaceutical composition is administered via injection, aerosol delivery, nasal spray or nasal droplets.

### Claim 26 (depends on 18)

A live attenuated RSV vaccine comprising the recombinant RSV variant encoded by the isolated polynucleotide of claim 18.

### Claim 27 (depends on 26)

A pharmaceutical composition comprising the RSV vaccine of claim 26.

### Claim 28 (depends on 18)

A method of making a live attenuated RSV vaccine comprising expressing the isolated polynucleotide molecule of claim 18.