Prefusion RSV F proteins and their use
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.
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 at least 80% identical thereto; or
a pyruvate dehydrogenase synthase subunit comprising the amino acid sequence set forth as residues 516-757 of SEQ ID NO: 349, or an amino acid sequence at least 80% identical thereto.
25. The immunogen of claim 23 , wherein the protein nanoparticle subunit comprises a ferritin nanoparticle subunit and the recombinant RSV F protein linked to the ferritin nanoparticle subunit comprises or consists of:
the amino acid sequence set forth as residues 26-105 and 137-679 of any one of SEQ ID NOs: 377-382 (A, B, bovine-DSCav1, and DS S190F ferritin particles), or an amino acid sequence at least 80% identical thereto that comprises S155C, S290C, S190F, and V207L substitutions or S155C, S290C, and S190F substitutions; and
wherein the recombinant RSV F protein linked to the ferritin nanoparticle subunit forms a ferritin nanoparticle that specifically binds to the prefusion-specific antibody.
26. The immunogen of claim 23 , wherein the recombinant RSV F protein linked to the ferritin nanoparticle subunit comprises the amino acid sequence set forth as one of:
residues 28-664 of SEQ ID NO: 1429 (BZGJ9 DS-Cav1 Longlink Ferritin);
residues 28-663 of SEQ ID NO: 1430 (BZGJ9-9 DS-Cav1 Longlink Ferritin); or
residues 28-665 of SEQ ID NO: 1431 (BZGJ9-10 DS-Cav1 Longlink Ferritin); or
an amino acid sequence at least 80% identical thereto that comprises S155C, S290C, S190F, and V207L substitutions.
27. The immunogen of claim 1 , wherein the immunogen forms a homogeneous population of immunogens when incubated in aqueous solution, wherein at least 70% of the immunogens incubated in the solution specifically bind to the prefusion-specific antibody after:
(a) incubation for one hour in 350 mM NaCl pH 7.0, at 50° C.;
(b) incubation for one hour in 350 mM NaCl pH 3.5, at 25° C.;
(c) incubation for one hour in 350 mM NaCl pH 10, at 25° C.;
(d) incubation for one hour in 10 mM osmolarity, pH 7.0, at 25° C.;
(e) incubation for one hour in 3000 mM osmolarity, pH 7.0, at 25° C.; or
(f) ten freeze-thaw cycles in 350 mM NaCl pH 7.0; or
(g) a combination of two or more of (a)-(f); wherein
the immunogen is incubated in the solution in the absence of the prefusion-specific antibody.
28. A virus-like particle comprising the immunogen of claim 1 .
29. A protein nanoparticle comprising the immunogen of claim 1 .
30. An immunogenic composition comprising an effective amount of the immunogen of claim 1 ; and a pharmaceutically acceptable carrier.
31. The immunogenic composition of claim 30 , further comprising:
(a) an adjuvant;
(b) an adjuvant that promotes a Th1 immune response;
(c) an adjuvant comprising alum, an oil-in water composition, MF59, AS01, AS03, AS04, MPL, QS21, a CpG oligonucleotide, a TLR7 agonist, a TLR4 agonist, a TLR3 agonist, or a combination of two or more thereof; or
(d) two or more of (a)-(c).
32. The immunogenic composition of claim 30 , comprising a mixture of recombinant RSV F proteins or fragments thereof based on RSV F protein subtype A and B.
33. A method for generating an immune response to RSV F in a subject, comprising administering to the subject an effective amount of the immunogenic composition of claim 30 to generate the immune response.
34. The method of claim 33 , comprising a prime-boost administration of the immunogenic composition.
35. A method for detecting or isolating an RSV F binding antibody in a subject, comprising:
providing an effective amount of the immunogen of claim 1 ;
contacting a biological sample from the subject with the recombinant RSV F protein or the protein nanoparticle under conditions sufficient to form an immune complex between the recombinant RSV F protein or the protein nanoparticle and the RSV F binding antibody; and
detecting the immune complex, thereby detecting or isolating the RSV F binding antibody in the subject.
36. The method of claim 33 , wherein the subject is at risk of or has an RSV infection, a human RSV subtype A infection, a human RSV subtype B infection, or a bovine RSV infection.
37. The method of claim 33 , wherein the subject is a human or a veterinary subject; less than one year old; more than 65 years old; pregnant; a female of child bearing age; a school age child; 2-5 years old; 6-12 months old; or less than six months old.
38. A kit comprising the immunogen of claim 1 ; and instructions for using the kit.
39. A recombinant RSV F protein stabilized in a prefusion conformation by:
a non-natural disulfide bond between cysteine residues introduced by S155C and S290C substitutions; and
a S190F cavity filling substitution and a V207L cavity filling substitution; and
wherein a C-terminal residue of an F 1 polypeptide on the recombinant RSV F protein is one of RSV F positions 513-529 and is linked to a foldon trimerization domain by a heterologous peptide linker.
40. The immunogen of claim 1 , wherein the recombinant RSV F protein further comprises one or more additional amino acid substitutions.
41. The immunogen of claim 40 , wherein the recombinant RSV F protein further comprises a non-natural disulfide bond between cysteine substitutions at positions 183 and 428 and a glycine insertion between positions 182/183.
42. The immunogen of claim 41 , wherein the recombinant RSV F protein comprises S155C, S290C, N183C, N428C, S190F, and V207L substitutions.
43. The immunogen of claim 41 , wherein the RSV F protein is a single-chain RSV F protein and wherein position 103 of an F 2 polypeptide of the RSV F protein is linked to position 145 of an F 1 polypeptide of the RSV F protein by a heterologous glycine-serine peptide linker.
44. The immunogen of claim 43 , wherein the F 2 and F 1 polypeptides comprise RSV F positions 26-103 and 145-513, respectively.
45. The immunogen of claim 41 , wherein a C-terminal residue of an F 1 polypeptide of the recombinant RSV F protein comprises one of RSV F positions 513-529 and is linked to a trimerization domain by a heterologous peptide linker.
46. The immunogen of claim 44 , wherein the trimerization domain is a foldon domain.
47. The immunogen of claim 40 , wherein the recombinant RSV F protein further comprises a second non-natural disulfide bond between cysteine substitutions at positions 149 and 458.
48. The immunogen of claim 47 , wherein the recombinant RSV F protein comprises S155C, S290C, A149C, Y458C, S190F, and V207L substitutions.
49. The immunogen of claim 47 , wherein the RSV F protein is a single-chain RSV F protein and wherein position 103 of an F 2 polypeptide of the RSV F protein is linked to position 145 of an F 1 polypeptide of the RSV F protein by a heterologous glycine-serine peptide linker.
50. The immunogen of claim 49 , wherein the F 2 and F 1 polypeptides comprise RSV F positions 26-103 and 145-513, respectively.
51. The immunogen of claim 47 , wherein a C-terminal residue of an F 1 polypeptide of the recombinant RSV F protein comprises one of RSV F positions 513-529 and is linked to a trimerization domain by a heterologous peptide linker.
52. The immunogen of claim 51 , wherein the trimerization domain is a foldon domain.
53. The immunogen of claim 12 , wherein the recombinant RSV F protein comprises the amino acid sequence set forth as residues 26-109 and 137-513, respectively, of SEQ ID NO: 371 (DS-Cav1, subtype A), or an amino acid sequence at least 80% identical thereto that comprises the S155C, S290C, S190F, and V207L substitutions.
54. The immunogen of claim 12 , wherein the recombinant RSV F protein comprises the amino acid sequence set forth as residues 26-105 and 145-513, respectively, of SEQ ID NO: 371 (DS-Cav1, subtype A), or an amino acid sequence at least 80% identical thereto that comprises the S155C, S290C, S190F, and V207L substitutions.
55. The immunogen of claim 12 , wherein the recombinant RSV F protein comprises the amino acid sequence set forth as residues 26-103 and 145-513, respectively, of SEQ ID NO: 371 (DS-Cav1, subtype A), or an amino acid sequence at least 80% identical thereto that comprises the S155C, S290C, S190F, and V207L substitutions.
56. The immunogen of claim 18 , wherein the recombinant RSV F protein linked to the trimerization domain comprises of the amino acid sequence set forth as residues 26-109 and 137-544, respectively, of SEQ ID NO: 371 (DS-Cav1, subtype A), or an amino acid sequence at least 80% identical thereto that comprises the S155C, S290C, S190F, and V207L substitutions.
57. The immunogen of claim 18 , wherein the recombinant RSV F protein linked to the trimerization domain comprises of the amino acid sequence set forth as residues 26-105 and 137-544, respectively, of SEQ ID NO: 371 (DS-Cav1, subtype A), or an amino acid sequence at least 80% identical thereto that comprises the S155C, S290C, S190F, and V207L substitutions.
58. The immunogen of claim 18 , wherein the recombinant RSV F protein linked to the trimerization domain comprises of the amino acid sequence set forth as residues 26-103 and 145-544, respectively, of SEQ ID NO: 371 (DS-Cav1, subtype A), or an amino acid sequence at least 80% identical thereto that comprises the S155C, S290C, S190F, and V207L substitutions.