Prefusion RSV F proteins and their use

Patent metadata

Field	Value
Publication number	US-9738689-B2
Application number	US-201414207372-A
Country	US
Kind code	B2
Filing date	Mar 12, 2014
Priority date	Mar 13, 2013
Publication date	Aug 22, 2017
Grant date	Aug 22, 2017

Abstract

Disclosed are immunogens including a recombinant RSV F protein stabilized in a prefusion conformation. Also disclosed are nucleic acids encoding the immunogens and methods of producing the immunogens. 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 immunogen 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 intra-promoter disulfide bond between cysteine residues introduced by S155C and S290C amino acid substitutions.

2. The immunogen of claim 1 , wherein the RSV F protein is a human subtype A or subtype B, or bovine RSV F protein comprising the non-natural intra-protomer disulfide bond.

3. The immunogen of claim 1 , wherein the recombinant RSV F protein comprises a F 2 polypeptide and a F 1 polypeptide comprising or consisting of RSV F positions 26-109 and 137-513, respectively.

4. The immunogen of claim 1 , wherein the recombinant RSV F protein comprises a F2 polypeptide and a F1 polypeptide, wherein a C-terminal residue of the F2 polypeptide is linked to an N-terminal residue of the F1 polypeptide by a heterologous peptide linker.

5. The immunogen of claim 1 ,
wherein the recombinant RSV F protein further comprises a non-natural disulfide bond between cysteine residues introduced by amino acid substitutions at RSV F positions 183 and 428, and a G insertion between positions 182/183 or a C insertion between positions 427/428.

6. The immunogen of claim 5 , wherein the recombinant RSV F protein comprises:
the intra-protomer disulfide bond between S155C and S290C substitutions, and one of the following sets of amino acid substitutions: N183GC and N428C; or N183C and K427GC.

7. The immunogen of claim 1 , wherein the recombinant RSV F protein comprises an F 1 polypeptide comprising the amino acid sequence set forth as residues 137-513 of one of SEQ ID NOs: 185 or 298-299.

8. The immunogen of claim 1 , wherein the C-terminus of the recombinant RSV F protein is linked to a trimerization domain.

9. The immunogen of claim 8 , wherein the trimerization domain is a Foldon domain.

10. The immunogen of claim 9 , wherein the recombinant RSV F protein comprises the amino acid sequences set forth as positions 26-109 and 137-544 of any one of SEQ ID NOs: 185 or 298-299.

11. The immunogen of claim 1 , wherein the C-terminus of recombinant RSV F protein is linked to a ferritin domain, an encapsulin domain, a Sulfur Oxygenase Reductase (SOR) domain, a lumazine synthase domain, or a pyruvate dehydrogenase domain.

12. The immunogen of claim 1 , wherein the C-terminus of recombinant RSV F protein is linked to a transmembrane domain.

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

14. A protein nanoparticle comprising the immunogen of claim 1 .

15. The protein nanoparticle of claim 14 , wherein the protein nanoparticle is a ferritin nanoparticle, an encapsulin nanoparticle, a Sulfur Oxygenase Reductase (SOR) nanoparticle, a lumazine synthase nanoparticle, or a pyruvate dehydrogenase nanoparticle.

16. A nucleic acid molecule encoding the immunogen of claim 1 .

17. A vector comprising the nucleic acid molecule of claim 16 .

18. An isolated host cell comprising the vector of claim 17 .

19. An immunogenic composition comprising an effective amount of the immunogen of claim 1 , and a pharmaceutically acceptable carrier.

20. The immunogenic composition of claim 19 , further comprising an adjuvant.

21. The immunogenic composition of claim 20 , wherein the adjuvant promotes a Th1 immune response.

22. The immunogen of claim 1 , wherein the RSV F protein further comprises one or more additional amino acid substitutions.

23. The immunogen of claim 22 , wherein the recombinant RSV F protein is soluble and comprises an F1 ectodomain comprising a C-terminal residue linked to a trimerization domain.

24. The immunogen of claim 23 , wherein the trimerization domain is a foldon domain.

25. The immunogen of claim 22 , wherein the recombinant RSV F protein comprises a F1 ectodomain comprising a C-terminal residue linked to a transmembrane domain.

26. A nucleic acid molecule encoding the recombinant RSV F protein of claim 22 .

27. A nucleic acid molecule encoding the recombinant RSV F protein of claim 23 .

28. A nucleic acid molecule encoding the recombinant RSV F protein of claim 24 .

29. A nucleic acid molecule encoding the recombinant RSV F protein of claim 25 .

30. A vector comprising the nucleic acid molecule of claim 26 .

31. A vector comprising the nucleic acid molecule of claim 27 .

32. A vector comprising the nucleic acid molecule of claim 28 .

33. A vector comprising the nucleic acid molecule of claim 29 .

34. The immunogen of claim 22 , wherein the RSV F protein is a human subtype A or subtype B, or bovine RSV F protein comprising the non-natural disulfide bond and the one or more additional amino acid substitutions.

35. The immunogen of claim 22 , wherein the recombinant RSV F protein comprises a F 2 polypeptide and a F 1 polypeptide comprising or consisting of RSV F positions 26-109 and 137-513, respectively.

36. The immunogen of claim 22 , wherein the recombinant RSV F protein comprises a F2 polypeptide and a F1 polypeptide, wherein a C-terminal residue of the F2 polypeptide is linked to an N-terminal residue of the F1 polypeptide by a heterologous peptide linker.

37. A virus-like particle comprising the immunogen of claim 22 .

38. A protein nanoparticle comprising the immunogen of claim 22 .

39. An isolated host cell comprising the vector of claim 30 .

40. An immunogenic composition comprising an effective amount of the immunogen of claim 22 , and a pharmaceutically acceptable carrier.

41. The immunogen of claim 22 , wherein the recombinant RSV F protein comprises an antigenic site Ø comprising RSV F positions 62-69 and 196-209 that specifically binds to a D25 antibody or an AM22 antibody after incubation at 20° C. in phosphate buffered saline at physiological pH for at least 24 hours in the absence of the D25 antibody or the AM22 antibody.

42. The immunogen of claim 1 , wherein the recombinant RSV F protein comprises an antigenic site Ø comprising RSV F positions 62-69 and 196-209 that specifically binds to a D25 antibody or an AM22 antibody after incubation at 20° C. in phosphate buffered saline at physiological pH for at least 24 hours in the absence of the D25 antibody or the AM22 antibody.

Assignees

• Us Health

Inventors

• Kwong Peter
• Graham Barney
• Mclellan Jason
• Chen Man
• Joyce Michael Gordon
• Zhang Baoshan
• Zhou Tongqing

Classifications

• C12N2760/18534

  Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein · CPC title

• C07K2319/21

  containing a His-tag · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C07K2319/40

  containing a tag for immunodetection, or an epitope for immunisation · CPC title