# US 2019/0169577 A1 · Respiratory syncytial virus having altered ns1 protein function and related materials and methods

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- Number: US 2019/0169577 A1
- Document ID: US20190169577A1
- Status: Published application
- Kind: A1
- Application number: 15/752,548
- Filed: 2016-08-12
- Published: 2019-06-06
- Earliest claimed: 2015-08-14
- Assignees: University of South Florida; Research Instiute At Nationwide Children's Hospital
- Applicants: University of South Florida; Research Instiute At Nationwide Children's Hospital
- Inventors: Mark Edward PEEPLES; Michael Nan-hao TENG; Octavio RAMILO; Maria Asuncion MEJIAS; Emilio FLANO
- Web page: https://patentgrep.com/patent/US20190169577A1

## 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
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 14/005: from viruses
- C12N 15/09: Recombinant DNA-technology
- A61K 39/12: Viral antigens
- A61K 2039/5254: {avirulent or attenuated}
- C12N 2760/18521: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- 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: 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

Embodiments disclosed herein provide compositions, methods, and uses for respiratory syncytial viruses (RSV) and immunogenic compositions thereof. Certain embodiments provide RSV having a mutated NS1 protein, where the mutation causes the uncoupling of the NS1 protein's replication and type I interferon (IFN) antagonist functions. In some embodiments, this uncoupling can produce virions capable of inducing a strong, long-lasting innate immune response while maintaining its ability to replicate in vitro. Also provided are methods for amplifying RSV in host cells, wherein amplified RSV has mutated NS1 protein in which the protein's replication and IFN antagonistic functions are uncoupled. In certain embodiments, the amplified RSV having mutated NS1 protein is formulated into immunogenic compositions, including vaccines. Other embodiments provide methods for inducing an effective immune response against RSV infection in a subject.

## Claims

### Claim 1

A mutant respiratory syncytial virus (RSV) comprising a mutated nonstructural protein 1 (NS1) comprising an amino acid deletion mutation selected from the group consisting of NΔ10; CΔ10; Δ6-15; Δ6-18; Δ8-18; Δ8-20; NΔ10; Δ6-10,12,15-19; NΔ5; Δ2-5; Δ2-7; Δ5-7; Δ6-10; Δ11-15; Δ11-13,18-20; Δ9; Δ11; Δ12; Δ13; Δ7,8; Δ9-11; and Δ9, 12 of an RSV NS1 protein having at least 95% sequence identity with SEQ ID NO: 1.

### Claim 2 (depends on 1)

The mutant RSV of claim 1, wherein the amino acid deletion mutation uncouples a replication function of NS1 from an interferon (IFN) antagonistic property of NS1.

### Claim 3 (depends on 2)

The mutant RSV of claim 2, wherein the mutant RSV comprises a trait of increased replication compared to an RSV lacking an NS1 protein (RSV ΔNS1) and reduced IFN antagonism relative to an RSV comprising a wild-type NS1 protein.

### Claim 4 (depends on 1)

The mutant RSV of claim 1, further comprising at least one substitution mutation in the 20 amino-terminal amino acids (aa 1-20) or 10 carboxy-terminal amino acids of the RSV NS1 protein.

### Claim 5 (depends on 1)

The mutant RSV of claim 1, wherein the deletion mutation is selected from the group consisting of NΔ10; NΔ11-20; Δ6-10; Δ16-20; Δ11; and Δ9,12.

### Claim 6 (depends on 1)

The mutant RSV of claim 1, wherein the mutant RSV is an attenuated RSV.

### Claim 7 (depends on 6)

An immunogenic composition against RSV comprising the attenuated RSV according to claim 6 and a pharmaceutically acceptable excipient.

### Claim 8 (depends on 1)

A nucleic acid sequence encoding the mutant RSV of claim 1.

### Claim 9 (depends on 16)

A method for producing an immunogenic composition against respiratory syncytial virus (RSV) comprising:
providing a cell culture of host cells;
inoculating the cell culture of host cells with the mutant RSV of claim 16;
incubating the cell culture of host cells with the mutant RSV;
harvesting RSV following the incubation step; and
formulating the harvested RSV into an immunogenic composition against RSV.

### Claim 10 (depends on 9)

The method of claim 9, further comprising purifying the harvested RSV.

### Claim 11 (depends on 9)

The method of claim 9, wherein formulating comprises combining the harvested RSV with a pharmaceutically acceptable carrier, vehicle, or excipient, and adjuvant, or a combination thereof.

### Claim 12 (depends on 9)

The method of claim 9, wherein the immunogenic composition against RSV comprises a pharmaceutically acceptable carrier, vehicle, or excipient, an adjuvant, or a combination thereof.

### Claim 13 (depends on 9)

An immunogenic composition against RSV produced by the method of claim 9.

### Claim 14 (depends on 7)

A method for inducing an effective immune response against respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject an immunologically effective dose of an immunogenic composition against RSV according to claim 7.

### Claim 15 (depends on 14)

The method of claim 14, wherein the subject is human

### Claim 16 (depends on 14)

The method of claim 14, wherein the subject is a human infant or child.

### Claim 17 (depends on 14)

The method of claim 14, wherein the immunogenic composition against RSV is administered via an administration route comprising intranasal administration; subcutaneous administration; intramuscular administration; intradermal administration; and oral administration.

### Claim 18 (depends on 14)

The method of claim 14, further comprising administering at least one subsequent immunologically effective dose of the immunogenic composition against RSV.

### Claim 19

A mutant respiratory syncytial virus (RSV) comprising a mutated nonstructural protein 1 (NS1) having one or more amino acid mutations that uncouples the NS1 proteins replication function from its IFN antagonistic function.

### Claim 20 (depends on 19)

The mutant RSV of claim 19, wherein the one or more amino acid mutations is a deletion of 10 or more amino acids of a first 20 amino acids of an amino-terminus of an NS1 protein having at least 95% sequence identity with SEQ ID NO: 1, a deletion of all 10 amino acids of a carboxy-terminus of an NS1 protein having at least 95% sequence identity with SEQ ID NO: 1, or a deletion of 1 to 5 amino acids in the first 20 amino acids of the amino-terminus of the NS1 protein having at least 95% sequence identity with SEQ ID NO: 1.

### Claim 21 (depends on 20)

The mutant RSV of claim 20, wherein the deletion mutation at the amino terminus is of consecutive amino acids or non-consecutive amino acids.

### Claim 22 (depends on 19)

The mutant RSV of claim 19, where the one or more amino acid mutations comprises a deletion mutation selected from the group consisting of NΔ10; CΔ10; Δ6-15; Δ6-18; Δ8-18; Δ8-20; Δ6-10,12,15-19; NΔ5; Δ2-5; Δ2-7; Δ5-7; Δ6-10; Δ11-15; Δ11-13,18-20; Δ9; Δ11; Δ12; Δ13; Δ7,8; Δ9-11; and Δ9,12 relative to an RSV NS1 protein having at least 95% sequence identity with SEQ ID NO: 1.

### Claim 23 (depends on 19)

The mutant RSV of claim 19, wherein the deletion mutation is selected from the group consisting of NΔ10; NΔ11-20; Δ6-10; Δ16-20; Δ11; and Δ9,12.

### Claim 24 (depends on 19)

The mutant RSV of claim 19, wherein the RSV exhibits increased replication relative to an RSV lacking an NS1 protein (RSV ΔNS1) and reduced IFN antagonism relative to an RSV comprising a wild-type NS1 protein.

### Claim 25 (depends on 19)

The mutant RSV of claim 19, wherein the RSV is attenuated.

### Claim 26 (depends on 25)

An immunogenic composition against RSV comprising the attenuated RSV according to claim 25 and a pharmaceutically acceptable excipient.