# US 11,305,004 B2 · Compositions and methods related to ebolavirus vaccines

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- Number: US 11,305,004 B2
- Document ID: US11305004B2
- Status: Granted
- Kind: B2
- Application number: 17/397,340
- Filed: 2021-08-09
- Published: 2022-02-10
- Granted: 2022-04-19
- Earliest claimed: 2020-08-10
- Assignees: The Scripps Research Institute
- Applicants: The Scripps Research Institute
- Inventors: Linling He; Jiang Zhu; Anshul Chaudhary; Ian Wilson
- Web page: https://patentgrep.com/patent/US11305004B2

## CPC

- A61K 39/12: Viral antigens
- C07K 14/005: from viruses
- C07K 14/473: {alpha-Glycoproteins}
- 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 2039/55505: {Inorganic adjuvants}
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- A61K 2039/55566: {Emulsions, e.g. Freund's adjuvant, MF59}
- A61K 2039/6068: {Other bacterial proteins, e.g. OMP}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2319/02: containing a signal sequence
- C07K 2319/735: containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
- C12N 2760/14122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/14134: 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides novel engineered Ebolavirus GP proteins and polypeptides, as well as scaffolded vaccine compositions that display the engineered proteins. The invention also provides methods of using such engineered Ebolavirus GP proteins and vaccine compositions in various therapeutic applications, e.g., for preventing or treating Ebolavirus infections.

## Claims

### Claim 1

An engineered Ebolavirus glycoprotein (GP) protein, comprising (a) substitution of residue W615 in heptad repeat 2 (HR2) with residue L, A, V, I or F, and (b) one or more proline substitutions in heptad repeat 1 C segment (HR1C); wherein the amino acid numbering is based on Zaire Ebolavirus GP sequence with UniProt ID Q05320 (SEQ ID NO:1).

### Claim 2 (depends on 1)

The engineered Ebolavirus GP protein of claim 1, wherein the Ebolavirus is Zaire Ebolavirus (EBOV), Sudan virus (SUDV), Taï forest virus (TAFV), Bundibugyo virus (BDBV), or Reston Ebolavirus (RESTV).

### Claim 3 (depends on 1)

The engineered Ebolavirus GP protein of claim 1, wherein the proline substitutions comprise T577P or L579P.

### Claim 4 (depends on 1)

The engineered Ebolavirus GP protein of claim 1, further comprising a truncation at the C-terminus of the membrane proximal external region (MPER).

### Claim 5 (depends on 4)

The engineered Ebolavirus GP protein of claim 4, further comprising (a) a deletion of the mucin-like domain (MLD) deleted from the GP1 subunit, and/or (b) a deletion of MPER from the GP2 subunit.

### Claim 6

An engineered Ebolavirus glycoprotein (GP) protein, comprising: (a) a truncated soluble GP of an Ebolavirus that has the mucin-like domain (MLD) deleted from the GP1 subunit and the membrane proximal external region (MPER) deleted from the GP2 subunit, and (b) substitution of residue W615 in heptad repeat 2 (HR2) with residue L, A, V, I or F; wherein the amino acid numbering is based on Zaire Ebolavirus GP sequence corresponding to UniProt ID Q05320 (SEQ ID NO:1).

### Claim 7 (depends on 6)

The engineered Ebolavirus GP protein of claim 6, further comprising one or more modifications selected from (i) an extension of HR2 at the C terminus, (ii) one or more proline substitutions in heptad repeat 1 C segment (HR1C), and (iii) one or more engineered inter-GP disulfide bonds.

### Claim 8 (depends on 6)

The engineered Ebolavirus GP protein of claim 6, wherein the truncated soluble GP comprises SEQ ID NO:2 or SEQ ID NO:3, or a variant thereof with at least 99% sequence identity.

### Claim 9 (depends on 6)

The engineered Ebolavirus GP protein of claim 6, comprising the sequence SEQ ID NO:4, or a variant thereof with at least 99% sequence identity.

### Claim 10 (depends on 6)

The engineered Ebolavirus GP protein of claim 6, further comprising an extension of HR2 at the C-terminus.

### Claim 11 (depends on 10)

The engineered Ebolavirus GP protein of claim 10, wherein the HR2 extension comprises (a) extending HR2 C terminus with a N-terminal fragment of the adjacent membrane proximal external region (MPER) of the Ebolavirus glycoprotein or (b) replacing a HR2 C-terminal fragment with a longer leucine zipper motif.

### Claim 12 (depends on 10)

The engineered Ebolavirus GP protein of claim 10, wherein the HR2 extension comprises (a) extending the HR2 C terminus from residue 632 to residue 637 in MPER, (b) extending the HR2 C terminus from residue 632 to residues 643 in MPER, or (c) replacing residues 617-632 with a GCN4 leucine zipper sequence shown in SEQ ID NO:34.

### Claim 13 (depends on 10)

The engineered Ebolavirus GP protein of claim 10, wherein the W615 substitution comprises W615L substitution or P612G/W615F double mutation.

### Claim 14 (depends on 10)

The engineered Ebolavirus GP protein of claim 10, comprising (a) W615L substitution and (b) extension of the HR2 C terminus from residue 632 to residue 637 in MPER of the Ebolavirus GP.

### Claim 15 (depends on 10)

The engineered Ebolavirus GP protein of claim 10, comprising (a) P612G/W615F double mutation in HR2 and (b) replacement of residues 617-632 with a GCN4 leucine zipper sequence shown in SEQ ID NO:34.

### Claim 16 (depends on 10)

The engineered Ebolavirus GP protein of claim 10, comprising an amino acid sequence as set forth in any one of SEQ ID NOs:5-8, or a variant thereof with at least 99% sequence identity.

### Claim 17 (depends on 10)

The engineered Ebolavirus GP protein of claim 10, further comprising a C-terminal trimerization motif.

### Claim 18 (depends on 17)

The engineered Ebolavirus GP protein of claim 17, wherein the C-terminal trimerization motif comprises SEQ ID NO:29 or SEQ ID NO:30, or a variant thereof with at least 99% sequence identity.

### Claim 19 (depends on 6)

The engineered Ebolavirus GP protein of claim 6, further comprising one or more proline substitutions in the HR1C segment.

### Claim 20 (depends on 19)

The engineered Ebolavirus GP protein of claim 19, comprising an amino acid sequence as set forth in any one of SEQ ID NOs:9-17 and 20, or a variant thereof with at least 99% sequence identity.

### Claim 21 (depends on 19)

The engineered Ebolavirus GP protein of claim 19, comprising (a) W615L substitution, (b) proline substitution T577P or L579P, and (c) HR2 extension from residue 632 to residue 637 in MPER.

### Claim 22 (depends on 19)

The engineered Ebolavirus GP protein of claim 19, further comprising a C-terminal trimerization motif.

### Claim 23 (depends on 6)

The engineered Ebolavirus GP protein of claim 6, further comprising an engineered disulfide bond between two neighboring GP protomers.

### Claim 24 (depends on 23)

The engineered Ebolavirus GP protein of claim 23, wherein the engineered disulfide bond is engineered between residues G91/A575 (SS2), F153/Y534 (SS1), T520/A575 (SS3), G157/I532 (SS4), D522/A575 (SS5) or K56/G599 (SS6).

### Claim 25 (depends on 6)

A pharmaceutical composition, comprising the engineered Ebolavirus GP protein of claim 6, and a pharmaceutically acceptable carrier.

### Claim 26 (depends on 6)

A vaccine composition, comprising an engineered Ebolavirus GP protein of claim 6 that is displayed on the surface of a self-assembling nanoparticle.

### Claim 27 (depends on 26)

The vaccine composition of claim 26, comprising (1) a polypeptide sequence containing from N terminus to C terminus (a) the engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence ferritin, (b) the engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence E2p, or (c) the engineered Ebolavirus GP protein, linker sequence (G4S)2 (SEQ ID NO:42), nanoparticle sequence I3-01v9; wherein the engineered Ebolavirus GP protein comprises W615L substitution, proline substitution T577P, and HR2 extension from residue 632 to residue 637 in MPER.

### Claim 28 (depends on 27)

The vaccine composition of claim 27, further comprising a locking domain and/or a T-cell epitope that is fused to the C-terminus of the nanoparticle subunit sequence.

### Claim 29 (depends on 28)

The vaccine composition of claim 28, comprising (1) a polypeptide sequence containing from N-terminus to C-terminus (a) the engineered Ebolavirus GP protein shown in SEQ ID NO:17, nanoparticle subunit sequence shown in SEQ ID NO:36 (E2p), locking domain shown in SEQ ID NO:39 (LD4), and T cell epitope shown in SEQ ID NO:31 or (b) the engineered Ebolavirus GP protein shown in SEQ ID NO:17, nanoparticle sequence shown in SEQ ID NO:37 (I3-01v9), locking domain shown in SEQ ID NO:40 (LD7), or (2) a variant of the polypeptide sequence with at least 99% sequence identity.