PREFUSION RSV F PROTEINS AND THEIR USE

• US 20140271699A1
• Filed: 03/12/2014
• Published: 09/18/2014
• Est. Priority Date: 03/13/2013
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Kwong, Peter, Boyington, Jeffrey, Graham, Barney, McLellan, Jason, Georgiev, Ivelin Stefanov, Joyce, Michael Gordon, Kanekiyo, Masaru, Pancera, Marie, Soto, Cinque, Srivatsan, Sanjay, Zhang, Baoshan
• Granted Patent: US 9,738,689 B2
• Time in Patent Office: Days
• US Class Current: 424/187.1
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C12N 2760/18522 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2760/18534 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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.

Claims

1. An isolated immunogen, comprising:

a recombinant RSV F protein or fragment thereof comprising at least one amino acid substitution compared to a native RSV F protein that stabilizes the recombinant RSV F protein in a prefusion conformation that specifically binds to a RSV F prefusion specific antibody, wherein;

the immunogen specifically binds to the antibody after incubation at 20°

C. in phosphate buffered saline at physiological pH for at least 24 hours in the absence of the antibody, and wherein the antibody does not specifically bind to a RSV F protein in a post-fusion conformation.

2. The immunogen of claim 1, wherein the immunogen is at least 90% homogeneous and specifically binds to the prefusion antibody after the incubation at 20°

C. in phosphate buffered saline at physiological pH for at least 24 hours in the absence of the antibody.

3. The immunogen of claim 1, wherein the recombinant RSV F protein comprises an antigenic site Ø

comprising residues 62-69 and 196-209 of SEQ ID NO;

370, that specifically binds a D25 antibody, or an AM22 antibody, or both.

4. The immunogen of claim 1, wherein the amino acid sequence of the recombinant RSV F protein is at least 90% identical to the amino acid sequence of the native RSV F protein.

5. The immunogen of claim 1, wherein the native RSV F protein is from a human subtype A or subtype B, or bovine RSV.

6. The immunogen of claim 1, wherein the recombinant RSV F protein

(a) comprises an F₁ polypeptide;

(b) comprises an F₂ polypeptide;

(c) does not comprise a pep27 polypeptide or portion thereof;

or

(d) a combination of two or more of (a)-(c).

7. The immunogen of claim 6, wherein the F₂ and F₁ polypeptides comprise RSV F positions 26-109 and 137-513, respectively.

8. The immunogen of claim 1, wherein the recombinant RSV F protein is a single chain RSV F protein.

9. The immunogen of claim 1, wherein the recombinant RSV F protein is stabilized in the prefusion conformation by a cavity-filling amino acid substitution, a non-natural disulfide bond between a pair of cysteines, or both.

10. The immunogen of claim 9, wherein the pair of cysteines comprises a first cysteine and a second cysteine, each comprising a Cα

carbon and a Cβ

carbon, and wherein;

(A) the first cysteine is introduced by amino acid substitution onto one of RSV F positions 137-216 or 461-513, and the second cysteine is introduced by amino acid substitution onto one of RSV F positions 26-61, 77-97, or 271-460; and

(B) the Cα

carbon of the position of the first cysteine is from 2.0-8.0 angstroms from the Cα

carbon of the position of the second cysteine, and/or the Cβ

carbon of the position of the first cysteine is from 2.0-5.5 angstroms from the Cβ

carbon of the position of the second cysteine, in the three-dimensional structure set forth by the structural coordinates provided in Table 1.

11. The immunogen of claim 9, wherein the pair of cysteines comprises a first cysteine and a second cysteine, each comprising a Cα

carbon and a Cβ

carbon, and wherein;

(A) the first cysteine and the second cysteine are introduced by amino acid substitution onto RSV F positions 137-216 or RSV F positions 461-513;

or the first cysteine is introduced by amino acid substitution onto RSV F positions 137-216, and the second cysteine is introduced by amino acid substitution onto RSV F positions 461-513; and

(B) the Cα

carbon of the position of the first cysteine is from 2.0-8.0 angstroms from the Cα

carbon of the position of the second cysteine, and/or the Cβ

carbon of the position of the first cysteine is from 2.0-5.5 angstroms from the Cβ

carbon of the position of the second cysteine, in the three-dimensional structure set forth by the structural coordinates provided in Table 1.

12. The immunogen of claim 9, wherein the non-natural disulfide bond comprises

an intra-protomer disulfide bond between RSV F positions 155 and 290;

151 and 288;

137 and 337;

397 and 487;

138 and 353;

60 and 194;

33 and 469;

54 and 154;

59 and 192;

or;

an inter-protomer disulfide bond between RSV F positions 400 and 489;

144 and 406;

153 and 461;

149 and 458;

143 and 404;

399 and 494;

146 and 407;

402 and 141;

183 and 428, and the recombinant RSV F protein comprises a G insertion between positions 182/183;

183 and 428, and the recombinant RSV F protein comprises a C insertion between positions 427/428;

145 and 460, and the recombinant RSV F protein comprises a AA insertion between positions 146/147;

183 and 423, and the recombinant RSV F protein comprises a AAA insertion between positions 182/183;

or 330 and 430, and the recombinant RSV F protein comprises a CAA insertion between positions 329/330;

the intra-protomer disulfide bond between RSV F positions 155 and 290, and wherein the recombinant RSV F protein further comprises a non-natural disulfide bond between RSV F positions;

141 and 402;

146 and 460, and a G insertion between positions 460/461;

330 and 493, and a C insertion between positions 329/330;

183 and 428, and a G insertion between positions 182/183;

or 183 and 428, and a C insertion between positions 427/428.

13. The immunogen of claim 12, wherein the recombinant RSV F protein comprises:

the intra-protomer disulfide bond, and one or more of the following sets of substitutions;

S155C and S290C;

G151C and I288C;

F137C and T337C;

T397C and E487C;

L138C and P353C;

E60C and D194C;

Y33C and V469C;

T54C and V154C;

I59C and V192C;

or

the inter-protomer disulfide bond, and one or more of the following sets of substitutions;

T400C and D489C;

V144C and V406C;

A153C and K461C;

A149C and Y458C;

G143C and S404C;

K399C and Q494C;

S146C and I407C;

or V402C and L141C;

S155C, S290C, L141C, and V402C;

S155C, S290C;

N183C and N428C, and a G insertion between positions 182/183;

N183C and N427G, and a C insertion between positions 427/428;

S145C and 460C; and

an AA insertion between positions 146/147;

N183C and K423C, and an AAA insertion between positions 182/183;

A329C and S430C, and a CAA insertion between positions 329/330;

or

the intra-protomer disulfide bond between RSV F positions 155 and 290 and the additional non-natural disulfide bond, S155C and S290C substitutions, and one or more of the following sets of amino acid substitutions;

S146C, and N460C, and a G insertion between positions 460/461;

N183C, and N428C; and

a G insertion between positions 182/183;

or N183C, and N427G; and

a C insertion between positions 427/428.

14. The immunogen of claim 13, wherein the recombinant RSV F protein comprises

an F₁ polypeptide comprising the amino acid sequence set forth as residues 137-513 of one of SEQ ID NOs:

185, 189, 201, 202, 205, 207, 209, 213, 244, 245, 247, 257-262, 264-275, 277-282, 284, 296-299, 302, 303, 338-340;

or

an F₂ polypeptide and an F₁ polypeptide comprising the amino acid sequences set forth as residues 26-109 and 137-513, respectively, of one of SEQ ID NOs;

190, 211, 212, 243, 246, 263, 276, 283, 285.

15. The immunogen of claim 9, wherein the cavity filling amino acid substitution comprises cavity filling amino acid substitutions at RSV F positions 190 and 207, 207 and 220, 296 and 190, 220 and 153, 203, 83 and 260, 58 and 298, 87 and 90, or a combination thereof.

16. The immunogen of claim 15, wherein the recombinant RSV F protein comprises one or more cavity-filling amino acid substitutions comprising 190F and 207L substitutions, 207L and 220L substitutions, 296F and 190F substitutions, 220L and 153W substitutions, a 203W substitution, 83W and 260W substitutions, 58W and 298L substitutions, 87F and 90L substitutions, or a combination thereof.

17. The immunogen of claim 16, wherein the cavity-filling amino acid substitutions comprise S190F and V207L substitutions, V207L and V220L substitutions, V296F and S190F substitutions, V220L and A153W substitutions, a L203W substitution, L83W and L260W substitutions, T58W and A298L substitutions, K87F and V90L substitutions, or a combination thereof.

18. The immunogen of claim 17, wherein the recombinant RSV F protein comprises

an F₁ polypeptide comprising positions 137-513 of one of SEQ ID NOs:

191, 193, 196, 197, or 248;

an F₂ polypeptide and an F₁ polypeptide comprising residues 26-109 and 137-513, respectively, of one of SEQ ID NOs;

192, 195 or 194.

19. The immunogen of claim 1, wherein the recombinant RSV F protein further comprises a non-natural disulfide bond to stabilize the membrane proximal domain of the RSV F protein, wherein the non-natural disulfide bond to stabilize the membrane proximal domain is between RSV positions 512 and 513;

486 and 487;

486 and 487, and the recombinant RSV F protein comprises a P insertion between positions 486/487;

330 and 493, and the recombinant RSV F protein comprises a C insertion between positions 329/330;

or 330 and 493, and the recombinant RSV F protein comprises a C insertion between positions 329/330 and a G insertion between positions 492/493, wherein the amino acid positions correspond to positions of the reference F₀ polypeptide set forth as SEQ ID NO;

124.

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

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

22. The immunogen of claim 21, 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, 189-197, 201-202, 205, 207, 209, 211-213, 243-248, 257-285, 296-299, or 302-303.

23. 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.

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

25. The immunogen of claim 1, wherein

(a) the prefusion specific antibody specifically binds to the immunogen after the immunogen is incubated at 20 degrees Celsius in phosphate buffered saline at pH 7.4 for at least 24 hours in the absence of the prefusion specific antibody;

(b) at least 90% of the recombinant RSV F proteins in the homogeneous population are stabilized in the prefusion conformation.

(c) at least 90% of the recombinant RSV F proteins in the homogeneous population are specifically bound by a RSV F protein prefusion specific antibody;

or

(d) a combination of two or more of (a)-(c).

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

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

28. The protein nanoparticle of claim 26, 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.

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

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

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

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

33. The immunogenic composition of claim 32, further comprising an adjuvant.

34. The immunogenic composition of claim 33, wherein the adjuvant promotes a Th1 immune response.

35. A method for generating an immune response to RSV F protein in a subject, comprising administering an effective amount of the immunogenic composition of claim 32 to the subject to generate the immune response.

36. The method of claim 35, wherein the immune response comprises a Th1 immune response.

37. The method of claim 35, comprising a prime-boost administration of the immunogenic composition.

38. The method of claim 35, wherein the subject is a human subject or cattle or sheep.

39. The method of claim 35, wherein the subject is

less than one year old and immunogenic composition comprises an AS01 adjuvant;

or

more than 65 years old and the immunogenic composition comprises an AS04 adjuvant.

40. The method of claim 35, wherein the subject is pregnant.

41. The method of claim 40, wherein the immunogenic composition comprises an adjuvant and wherein the adjuvant comprises Alum.

42. A method for treating or preventing a RSV infection in a subject, comprising administering to the subject a therapeutically effective amount of the immunogenic composition of claim 32, thereby treating or preventing RSV infection in the subject.

43. A kit comprising the immunogen of claim 1 and instructions for using the kit.

45. The epitope scaffold protein of claim 43, comprising RSV F₂ positions 62-69, and RSV F₁ positions 196-209, wherein the positions correspond to positions of the reference F₀ polypeptide set forth as SEQ ID NO:

124.

47. The recombinant RSV F protein of claim 45, wherein the recombinant RSV F protein is a single chain RSV F protein.

44. An epitope-scaffold protein, comprising a heterologous scaffold protein linked to a polypeptide comprising a RSV F protein prefusion epitope, wherein monoclonal antibody D25 or AM22 specifically binds to the epitope-scaffold protein.

46. A recombinant RSV F protein comprising cysteine residues at positions 155 and 290, wherein the RSV F protein induces an immune response to RSV F protein in a subject.