Prefusion RSV F proteins and their use

Patent metadata

Field	Value
Publication number	US-10017543-B2
Application number	US-201414776651-A
Country	US
Kind code	B2
Filing date	Mar 13, 2014
Priority date	Mar 13, 2013
Publication date	Jul 10, 2018
Grant date	Jul 10, 2018

Abstract

Disclosed are Respiratory Syncytial Virus (RSV) antigens including a recombinant RSV F protein stabilized in a prefusion conformation. Also disclosed are nucleic acids encoding the antigens and methods of producing the antigens. Methods for generating an immune response in a subject are also disclosed. In some embodiments, the method is a method for treating or preventing a RSV infection in a subject by administering a therapeutically effective amount of the antigen to the subject.

First claim

We claim:

1. An isolated immunogen, comprising:
a recombinant RSV F protein stabilized in a prefusion conformation by a non-natural disulfide bond between cysteine substitutions at position 155 and 290, and a cavity filling F, L, W, Y, H, or M substitution at position 190, position 207, or positions 190 and 207.

2. The immunogen of claim 1 , wherein the prefusion conformation of the recombinant RSV F protein or fragment thereof comprises an antigenic site Ø comprising residues 62-69 and 196-209 of a native RSV F protein sequence set forth as one of SEQ ID NOs: 1-184.

3. The immunogen of claim 1 , wherein the immunogen specifically binds to a D25, a AM22, a 5C4, or a MPE8 prefusion-specific antibody.

4. The immunogen of claim 1 , wherein the RSV F protein is a RSV A, RSV B, or bovine RSV F protein.

5. The immunogen of claim 1 , wherein the recombinant RSV F protein or fragment thereof comprises a F 1 polypeptide and a F 2 polypeptide, and optionally does not comprise a pep27 polypeptide or portion thereof.

6. The immunogen of claim 5 , wherein the C-terminal residue of the F 2 polypeptide and the N-terminal residue of the F 1 polypeptide, respectively, comprise RSV F positions 97 and 137; 97 and 145; 97 and 150; 102 and 144; 102 and 145; 102 and 146; 102 and 147; 103 and 144; 103 and 145; 103 and 146; 103 and 147; 104 and 144; 104 and 145; 104 and 146; 104 and 147; 105 and 144; 105 and 145; 105 and 146; 105 and 147; or 105 and 150.

7. The immunogen of claim 5 , wherein the F 2 and F 1 polypeptides respectively comprise or consist of RSV F positions: 26-109 and 137-513; 26-107 and 137-513; 26-107 and 145-513; 26-105 and 137-513; 26-105 and 145-513; 26-103 and 145-513; 26-109 and 137-529; 26-107 and 137-529; 26-107 and 145-529; 26-105 and 137-529; 26-105 and 145-529; 26-103 and 145-529; 46-103 and 147-310; 46-104 and 146-310; 50-96 and 149-306; 51-103 and 146-307; 51-103 and 139-307; 50-105 and 146-306; or 53-97 and 148 to one of 305-320.

8. The immunogen of claim 5 , wherein the F 1 and F 2 polypeptides comprise amino acid sequences at least 80% identical to the corresponding sequences of a native RSV F protein sequence set forth as any one of SEQ ID NOs: 1-184, wherein the RSV F protein is stabilized in the prefusion conformation by the non-natural disulfide bond between cysteine substitutions at position 155 and 290, and the cavity filling F, L, W, Y, H, or M substitution at position 190, position 207, or positions 190 and 207.

9. The immunogen of claim 5 , wherein the recombinant RSV F protein is a single chain RSV F protein and the F 2 and F 1 polypeptides are linked by a heterologous peptide linker, or are directly linked.

10. The immunogen of claim 9 , wherein
position 105 of the F 2 polypeptide is linked to position 145 of the F 1 polypeptide by a Gly-Ser linker; or
position 103 of the F 2 polypeptide is directly linked to position 145 of the F 1 polypeptide.

11. The immunogen of claim 9 , wherein the heterologous peptide linker comprises the amino acid sequence set forth as one of SEQ ID NOs: 356-365 or 1443-1453, or is a G, S, GG, GS, SG, GGG, or GSG linker.

12. The immunogen of claim 1 , wherein the recombinant RSV F protein comprises S155C, S290C, S190F, and V207L substitutions.

13. The immunogen of claim 1 , wherein the recombinant RSV F protein comprises or consists of
(A) the amino acid sequence set forth as residues 26-109 and 137-513, respectively, or 26-103 and 145-513, respectively, or 26-105 and 145-513, respectively, of
(i) SEQ ID NO: 372 (DSCav1, subtype B), 373 (DSCav1, bovine), or an amino acid sequence at least 80% identical thereto that comprises S155C, S290C, S190F, and V207L substitutions; or
(ii) one of SEQ ID NOs: 374 (DS S190F, subtype A), 375 (DS, S190F, subtype B), or 376 (DS, S190F, bovine); or an amino acid sequence at least 80% identical thereto that comprises S155C, S290C, and S190F substitutions; or

(B) the amino acid sequence set forth as one of:
residues 26-476 of SEQ ID NO: 669 (BZGJ9 DS-Cav1);
residues 26-472 of SEQ ID NO: 706 (BZGJ9-9 DS-Cav1);
residues 26-474 of SEQ ID NO: 707 (BZGJ9-10 DS-Cav1); or
an amino acid sequence at least 80% identical thereto that comprises the S155C, S290C, S190F, and V207L substitutions.

14. The immunogen of claim 1 , wherein the F 1 polypeptide comprises an RSV α10 helix comprising from RSV position 492 to one of positions 510-529, and wherein the α10 helix comprises at least two cysteine substitutions that form a non-natural inter-protomer disulfide bond.

15. The immunogen of claim 14 , wherein positions 512-524 of the F 1 polypeptide comprise the amino acid sequence set forth as CCHNVNAGKSTTN (residues 512-524 of SEQ ID NO: 844) or CCHNVNACCSTTN (residues 512-524 of SEQ ID NO: 849); or wherein positions 512-529 of the F 1 polypeptide comprise the amino acid sequence set forth as CCHNVNACCSTTNICCTT (residues 512-529 of SEQ ID NO: 853).

16. The immunogen of claim 1 , wherein the recombinant RSV F protein is linked to a scaffold protein.

17. The immunogen of claim 1 , comprising a multimer of the recombinant RSV F protein or fragment thereof.

18. The immunogen of claim 1 , wherein the recombinant RSV F protein or fragment thereof is linked to a trimerization domain.

19. The immunogen of claim 18 , wherein the C-terminus of the F 1 polypeptide of the recombinant RSV F protein is linked to the trimerization domain; and
optionally further comprising a protease cleavage site between the F 1 polypeptide and the trimerization domain.

20. The immunogen of claim 18 , wherein the trimerization domain is a Foldon domain.

21. The immunogen of claim 18 , wherein the recombinant RSV F protein linked to the trimerization domain comprises or consists of the amino acid sequence set forth as residues 26-109 and 137-544, or residues 26-103 and 145-544, of one of:
SEQ ID NO: 372 (DSCav1, subtype B), or 373 (DSCav1, bovine), or an amino acid sequence at least 80% identical thereto that comprises S155C, S290C, S190F, and V207L substitutions;
SEQ ID NOs: 374 (DS S190F, subtype A), 375 (DS, S190F, subtype B), or 376 (DS, S190F, bovine); or
an amino acid sequence at least 80% identical thereto that comprises S155C, S290C, and S 190F substitutions.

22. The immunogen of claim 18 , wherein the recombinant RSV F protein linked to the trimerization domain comprises or consists of the sequence of the recombinant RSV F protein linked to the trimerization domain set forth as SEQ ID NO: 669 (BZGJ9 DSCav1), SEQ ID NO: 706 (BZGJ9-9 DSCav1), or SEQ ID NO: 707 (BZGJ9-10 DSCav1); or an amino acid sequence at least 80% identical thereto.

23. The immunogen of claim 1 , wherein the recombinant RSV F protein or fragment thereof is linked to a protein nanoparticle subunit.

24. The immunogen of claim 23 , wherein the protein nanoparticle subunit comprises:
a ferritin nanoparticle subunit comprising the amino acid sequence set forth as residues 517-679 of SEQ ID NO: 350, or an amino acid sequence at least 80% identical thereto, and optionally includes a C31S, C31A or C31V substitution in the ferritin polypeptide;
a SOR subunit comprising the amino acid sequence set forth as residues 516-825 of SEQ ID NO: 344 or SEQ ID NO: 345, or an amino acid sequence at least 80% identical thereto;
a lumazine synthase subunit comprising the amino acid sequence set forth as residues 517-670 of SEQ ID NO: 346 or SEQ ID NO: 348, or residues 517-669 of SEQ ID NO: 347, or an amino acid sequence a

Assignees

• Us Health

Inventors

• Kwong Peter D
• Graham Barney S
• Mclellan Jason S
• Boyington Jeffrey
• Chen Lei
• Chen Man
• Chuang Gwo-Yu
• Georgiev Ivelin Stefanov
• Gorman Jason
• Joyce Michael Gordon
• Kanekiyo Masaru
• Ofek Gilad
• Pancera Marie
• Sastry Mallika
• Soto Cinque
• Srivatsan Sanjay
• Stewart-Jones Guillaume
• Yang Yongping
• Zhang Baoshan
• Zhou Tongqing

Classifications

• A61P31/14

  for RNA viruses · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C12N2710/10343

  viral genome or elements thereof as genetic vector · CPC title

• C07K2319/735

  containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title