# US 11,078,239 B2 · Prefusion PIV F immunogens and their use

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

- Number: US 11,078,239 B2
- Document ID: US11078239B2
- Status: Granted
- Kind: B2
- Application number: 16/344,772
- Filed: 2017-10-25
- Published: 2020-02-13
- Granted: 2021-08-03
- Earliest claimed: 2016-10-25
- Assignees: The United States of America, as represented by the Secretary, Dept. of Health and Human Services; Institute for Research in Biomedicine
- Applicants: The United States of America, as represented by the Secretary, Dept. of Health and Human Services; Institute for Research in Biomedicine
- Inventors: Baoshan Zhang; Guillaume Stewart-Jones; Tongqing Zhou; John Mascola; Kai Xu; Yongping Yang; Paul Thomas; Gwo-Yu Chuang; Li Ou; Peter Kwong; Yaroslav Tsybovsky; Wing-Pui Kong; Aliaksandr Druz; Davide Corti; Antonio Lanzavecchia
- Web page: https://patentgrep.com/patent/US11078239B2

## CPC

- A61K 39/12: Viral antigens
- C07K 14/005: from viruses
- A61K 39/155: Paramyxoviridae, e.g. parainfluenza virus
- A61P 31/14: for RNA 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)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/50: containing protease site
- C12N 2760/00034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18611: Respirovirus, e.g. Bovine, human parainfluenza 1,3
- C12N 2760/18634: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18711: Rubulavirus, e.g. mumps virus, parainfluenza 2,4
- C12N 2760/18734: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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)

## Abstract

Embodiments of a recombinant human Parainfluenza Virus (hPIV) F ectodomain trimer stabilized in a prefusion conformation are provided. Also disclosed are nucleic acids encoding the hPIV F ectodomain trimer and methods of producing the hPIV F ectodomain trimer. Methods for inducing an immune response in a subject are also disclosed. In some embodiments, the method can be a method for treating or inhibiting a hPIV infection in a subject by administering a effective amount of the recombinant hPIV F ectodomain trimer to the subject.

## Claims

### Claim 1

An immunogen, comprising:
a recombinant human parainfluenza virus hPIV3 F ectodomain trimer comprising protomers comprising one or more amino acid substitutions that stabilize the hPIV3 ectodomain trimer in a prefusion conformation, wherein the one or more amino acid substitutions comprise
463V and/or 474Y cavity filling substitutions; and/or
one or more of the following sets of substitutions to form a disulfide bond to stabilize the hPIV3 ectodomain trimer in a prefusion conformation:
162C and 168C;
213C and 230C;
170C and 242C;
216C and 221C;
85C and 222C; and
172C and 238C; and
wherein amino acid substitution numbering is according to a reference hPIV3 F sequence set forth as SEQ ID NO: 9.

### Claim 2 (depends on 1)

The immunogen of claim 1, wherein the one or more amino acid substitutions comprise:
the 463V and/or 474Y cavity filling substitutions; and
the one or more of the following sets of substitutions to form a disulfide bond to stabilize the hPIV3 ectodomain trimer in a prefusion conformation:
162C and 168C;
170C and 242C;
213C and 230C;
216C and 221C;
85C and 222C; and
172C and 238C.

### Claim 3 (depends on 1)

The immunogen of claim 1, wherein the one or more amino acid substitutions comprise:
162C and 168C substitutions to form a disulfide bond, 213C and 230C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions;
162C and 168C substitutions to form a disulfide bond, 216C and 221C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions;
162C and 168C substitutions to form a disulfide bond, 85C and 222C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions;
213C and 230C substitutions to form a disulfide bond, 170C and 242C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions;
213C and 230C substitutions to form a disulfide bond, 216C and 221C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions;
213C and 230C substitutions to form a disulfide bond, 85C and 222C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions; or
172C and 238C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions.

### Claim 4 (depends on 1)

The immunogen of claim 1, wherein the 162C, 170C, 168C, 213C, 230C, 216C, 221C, 85C, 222C, 216C, 221C, 242C, 172C, 238C, 463V, and 474Y substitutions are Q162C, V170C, L168C, I213C, G230C, D216C, L221C, G85C, Q222C, D216C, L221C, I242C, I172C, N238C, A463V, and I474Y substitutions, respectively.

### Claim 5 (depends on 1)

The immunogen of claim 1, wherein the protomers further comprise a mutation to inhibit cleavage of a F2/F1 protease cleavage site.

### Claim 6 (depends on 5)

The immunogen of claim 5, wherein the mutation to inhibit cleavage of a F2/F1 protease cleavage site comprises a K108E substitution.

### Claim 7 (depends on 1)

The immunogen of claim 1, wherein an N-terminal residue of the protomers is one of hPIV3 residues 15-25 and a C-terminal residue of the protomers is one of hPIV3 F residues 475-493, wherein the residue numbering is according to the reference hPIV3 F sequence set forth as SEQ ID NO: 9.

### Claim 8 (depends on 1)

The immunogen of claim 1, wherein the protomers comprise or consist essentially of hPIV3 residues 19-484 and comprise the one or more amino acid substitutions to stabilize the trimer in the prefusion conformation and the mutation to inhibit cleavage of the F2/F1 protease cleavage site, wherein the residue numbering is according to the reference hPIV3 F sequence set forth as SEQ ID NO: 9.

### Claim 9 (depends on 1)

The immunogen of claim 1, wherein the protomers comprise or consist essentially of the amino acid sequence set forth as residues 1-463 of any one of SEQ ID NOs: 10-26 or 39-50, or an amino acid sequence at least 90% identical thereto.

### Claim 10 (depends on 1)

The immunogen of claim 1, wherein a C-terminal residue of the protomers in the ectodomain is linked to a trimerization domain by a peptide linker, or is directly linked to the trimerization domain.

### Claim 11 (depends on 10)

The immunogen of claim 10, wherein the trimerization domain is a GCN4 trimerization domain.

### Claim 12 (depends on 11)

The immunogen of claim 11, wherein the protomers comprise or consist essentially of the amino acid sequence set forth as any one of SEQ ID NOS: 10-26 or 39-50, or an amino acid sequence at least 90% identical thereto.

### Claim 13 (depends on 1)

The immunogen of claim 1, wherein the protomers further comprise one or more additional amino acid substitutions.

### Claim 14 (depends on 1)

The immunogen of claim 1, wherein the recombinant hPIV3 F ectodomain trimer specifically binds to a hPIV3 F prefusion specific antibody.

### Claim 15 (depends on 1)

The immunogen of claim 1, wherein the recombinant hPIV3 F ectodomain trimer is soluble.

### Claim 16 (depends on 1)

The immunogen of claim 1, wherein the C-terminal residue of the protomers in the F ectodomain is linked to a transmembrane domain by a peptide linker, or is directly linked to the transmembrane domain.

### Claim 17 (depends on 16)

The immunogen of claim 16, wherein the protomers comprise the amino acid sequence set forth as any one of SEQ ID NOs: 51-65 or 75-80, or an amino acid sequence at least 90% identical thereto.

### Claim 18 (depends on 1)

The immunogen of claim 1, wherein the C-terminal residue of the protomers in the F ectodomain is linked to a protein nanoparticle subunit by a peptide linker, or is directly linked to the protein nanoparticle subunit.

### Claim 19 (depends on 18)

A protein nanoparticle, comprising the immunogen of claim 18.

### Claim 20 (depends on 1)

A virus-like particle comprising the immunogen of claim 1.

### Claim 21 (depends on 1)

An immunogenic composition comprising the immunogen of claim 1.

### Claim 22 (depends on 1)

A method of inducing an immune response to hPIV3 F protein in a subject, comprising administering to the subject an effective amount of the immunogen of claim 1 to generate the immune response.

### Claim 23 (depends on 22)

The method of claim 22, wherein the immune response inhibits an hPIV3 infection.

### Claim 24 (depends on 1)

The immunogen of claim 1, wherein:
the one or more amino acid substitutions comprise 162C and 168C substitutions to form a disulfide bond, 213C and 230C substitutions to form a disulfide bond, and 463V and 474Y cavity filling substitutions; and
a C-terminal residue of protomers in the ectodomain is linked to a GCN4 trimerization domain by a peptide linker, or is directly linked to the trimerization domain.