Novel Recombinant Prefusion RSV F Proteins And Uses Thereof

Abstract

The present invention provides immunogens comprising a recombinant Respiratory Syncytial Virus (RSV) F protein stabilized in a prefusion conformation and nucleic acids encoding such immunogens. In particular the present invention provides polypeptides, polynucleotides, compositions, and uses thereof for eliciting an immune response to bovine respiratory syncytial virus (bRSV). 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.

Claims

1. An immunogen comprising a recombinant RSV F protein or a fragment thereof specifically binding to an RSV F prefusion-specific antibody, wherein the recombinant RSV F protein or the fragment thereof comprises an F1 polypeptide and an F2 polypeptide of any RSV F protein characterized by the following substitutions at amino acid positions corresponding to the following amino acid positions in SEQ ID NO: 1 as a reference sequence: (i) S155C and S290C substitutions, which form a non-natural disulfide bond; (ii) a substitution at one or both of positions S190 and V207 by amino acids selected from the group consisting of F, L, W, Y, H, and M; and (iii) a pair of substitutions forming a non-natural disulfide bond selected from the group consisting of the following substitution pairs: Q98C and Q361C, A149C and Y458C, N183GC and N428C, N88C and N254C, E92C and N254C, and S238C and Q279C; and wherein the recombinant RSV F protein or the fragment thereof does not comprise a pep27 polypeptide.

2. The immunogen according to claim 1, wherein the F1 polypeptide and the F2 polypeptide of the recombinant RSV F protein or of the fragment thereof share at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with the F1 polypeptide and the F2 polypeptide, respectively, of a native bovine RSV F protein.

3. The immunogen according to claim 2, wherein the native bovine RSV F protein comprises an amino acid sequence according to any of SEQ ID NOs: 1-9.

4. The immunogen according to claim 2 or 3, wherein the native bovine RSV F protein consists of an amino acid sequence according to SEQ ID NO: 1.

5. The immunogen according to any of claims 1-4, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide and an F1 polypeptide comprising amino acid sequences at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% identical to amino acids 26-103 and 145-310, respectively, of SEQ ID NO: 1.

6. The immunogen according to claim 5, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide and an F1 polypeptide comprising amino acid sequences at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% identical to amino acids 26-103 and 145-513, respectively, of SEQ ID NO: 1.

7. The immunogen according to claim 6, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide and an F1 polypeptide comprising amino acid sequences at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% identical to amino acids 26-103 and 145-529, respectively, of SEQ ID NO: 1.

8. The immunogen according to claim 7, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide and an F1 polypeptide comprising amino acid sequences at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% identical to amino acids 26-103 and 145-551, respectively, of SEQ ID NO: 1.

9. The immunogen according to any of claims 1-8, wherein (i) the F2 polypeptide comprises or consists of 8-79 residues of bovine RSV F positions 26-105 and (ii) the F1 polypeptides comprises or consists of 14-365 residues of bovine RSV F positions 145-513; and wherein the bovine RSV F positions preferably correspond to the amino acid sequence of a reference F0 polypeptide according to SEQ ID NO: 1.

10. The immunogen according to any of claims 1-9, wherein the recombinant RSV F protein, or the fragment thereof, comprises: (i) an antigenic site that specifically binds to the RSV F prefusion-specific antibody, wherein the antigenic site comprises residues 62-69 and 196-209 of a native bovine RSV F protein sequence set forth in any one of SEQ ID NOs: 1-9; (ii) an epitope recognized by AM14 antibody, wherein the epitope recognized by AM14 antibody comprises at least residues L160, N183, N426, R429, H514 and H515 of a native bovine RSV F protein sequence set forth in any one of SEQ ID NOs: 1-9; and/or (iii) an epitope recognized by MPE8 antibody, wherein the epitope recognized by MPE8 antibody comprises at least residues T50, D310, L305, G307, and I309 of a native bovine RSV F protein sequence set forth in any one of SEQ ID NOs: 1-9.

11. The immunogen according to any of claims 1-9, wherein the F2 and F1 polypeptides comprise RSV F positions 62-69 and 196-209, preferably of any of SEQ ID NOs 1-9, more preferably of SEQ ID NO: 1.

12. The immunogen according to any of claims 1-11, comprising the cavity-filling amino acid substitution comprising one of: S190F; S190L; S190W; S190Y; S190H; S190M; S190F and V207L; S190F and V207F; S190F and V207W; S190L and V207L; S190L and V207F; S190L and V207W; S190W and V207L; S190W and V207F; S190W and V207W; S190Y and V207L; S190Y and V207F; S190Y and V207W; S190H and V207L; S190H and V207F; S190H and V207W; S190M and V207L; or S190M and V207F; S190M and V207W.

13. The immunogen according to claim 12, wherein the recombinant RSV F protein or the fragment thereof comprises S190F and/or V207L substitutions compared to the native bovine RSV F protein.

14. The immunogen according to any of claims 1-13, wherein the recombinant RSV F protein or the fragment thereof comprises (i) Q98C and Q361C substitutions, (ii) A149C and Y458C substitutions, and/or (iii) N183GC and N428C substitutions.

15. The immunogen according to claim 14, wherein the recombinant RSV F protein or the fragment thereof comprises (i) Q98C and Q361C substitutions, and/or (ii) A149C and Y458C substitutions.

16. The immunogen according to any of claims 1-15, wherein the recombinant RSV F protein or the fragment thereof is a single chain RSV F protein or a single chain RSV F protein fragment.

17. The immunogen according to any of claims 1-16, wherein the RSV F prefusion-specific antibody, to which the immunogen specifically binds to, is D25, MPE8 and/or AM14.

18. The immunogen according to any of claims 1-17, wherein the recombinant RSV F protein or the fragment thereof does not comprise a fusion peptide or a fragment thereof.

19. The immunogen according to claim 18, wherein the recombinant RSV F protein or the fragment thereof does not comprise amino acids 106-144 or 104-144 of the native bovine RSV F protein.

20. The immunogen according to claim 19, wherein the recombinant RSV F protein or the fragment thereof does not comprise amino acids 106-144 or 104-144 of SEQ ID NO: 1.

21. The immunogen according to any of claims 1-20, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-103 of SEQ ID NO: 31; and an F1 polypeptide comprising or consisting of amino acids 106-474 of SEQ ID NO: 31.

22. The immunogen according to any of claims 1-20, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 32; and an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 32.

23. The immunogen according to any of claims 1-20, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 31-108 of SEQ ID NO: 33; and an F1 polypeptide comprising or consisting of amino acids 111-479 of SEQ ID NO: 33.

24. The immunogen according to any of claims 1-20, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 63; and an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 63.

25. The immunogen according to any of claims 1-20, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 64; and an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 64.

26. The immunogen according to any of claims 1-20, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 65; and an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 65.

27. The immunogen according to any of claims 1-26, wherein the F2 polypeptide and the F1 polypeptide are linked by a heterologous peptide linker or are directly linked.

28. The immunogen according to claim 27, wherein the heterologous peptide linker comprises the amino acid sequence set forth as one of SEQ ID NOs: 10-26, or is a G, S, GG, GS, SG, GGG, or GSG linker.

29. The immunogen according to claim 28, wherein the F2 polypeptide and the F1 polypeptide are linked by a GS-linker.

30. The immunogen according to any of claims 1-29, wherein position 103 or 105 of the F2 polypeptide is linked to position 1 of the F1 polypeptide by a Gly-Ser linker.

31. The immunogen according to any of the previous claims, comprising a multimer of the recombinant RSV F protein or of the fragment thereof.

32. The immunogen according to any of claims 1-31, wherein the recombinant RSV F protein is linked to a trimerization domain.

33. The immunogen according to claim 32, wherein the C-terminus of the F1 polypeptide of the recombinant RSV F protein or of the fragment thereof is directly or indirectly linked to the trimerization domain.

34. The immunogen according to claim 32 or 33, wherein the trimerization domain is a foldon domain.

35. The immunogen according to any of claims 32-34, wherein the trimerization domain comprises or consists of an amino acid sequence according to any of SEQ ID NOs: 27-29.

36. The immunogen according to any of claims 32-35, wherein the immunogen comprises a protease cleavage site between the F1 polypeptide and the trimerization domain.

37. The immunogen according to any of claims 32-36, wherein the immunogen comprises a transmembrane domain between the F1 polypeptide and the trimerization domain.

38. The immunogen according to claim 37, wherein the immunogen comprises a transmembrane domain and a protease cleavage site between the F1 polypeptide and the trimerization domain, in particular a transmembrane domain between the protease cleavage site and the trimerization domain.

39. The immunogen according to any of claims 1-38, wherein the immunogen comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-103 of SEQ ID NO: 31; an F1 polypeptide comprising or consisting of amino acids 106-474 of SEQ ID NO: 31; and a foldon domain comprising or consisting of amino acids 475-513 of SEQ ID NO: 31, which is preferably directly linked to the C-terminus of the F1 polypeptide.

40. The immunogen according to any of claims 1-38, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 32; an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 32; and a foldon domain comprising or consisting of amino acids 477-515 of SEQ ID NO: 32, which is preferably directly linked to the C-terminus of the F1 polypeptide.

41. The immunogen according to any of claims 1-38, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 31-108 of SEQ ID NO: 33; an F1 polypeptide comprising or consisting of amino acids 111-479 of SEQ ID NO: 33; and a foldon domain comprising or consisting of amino acids 480-518 of SEQ ID NO: 33, which is preferably directly linked to the C-terminus of the F1 polypeptide.

42. The immunogen according to any of claims 1-38, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 63; an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 63; and a foldon domain comprising or consisting of amino acids 477-515 of SEQ ID NO: 63, which is preferably directly linked to the C-terminus of the F1 polypeptide.

43. The immunogen according to any of claims 1-38, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 64; an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 64; and a foldon domain comprising or consisting of amino acids 477-515 of SEQ ID NO: 64, which is preferably directly linked to the C-terminus of the F1 polypeptide.

44. The immunogen according to any of claims 1-38, wherein the recombinant RSV F protein or the fragment thereof comprises or consists of an F2 polypeptide comprising or consisting of amino acids 26-105 of SEQ ID NO: 65; an F1 polypeptide comprising or consisting of amino acids 108-476 of SEQ ID NO: 65; and a foldon domain comprising or consisting of amino acids 477-515 of SEQ ID NO: 65, which is preferably directly linked to the C-terminus of the F1 polypeptide.

45. The immunogen according to any of claims 1-39, wherein the immunogen comprises or consists of amino acids 26-474 of SEQ ID NO: 31.

46. The immunogen according to claim 45, wherein the immunogen comprises or consists of amino acids 26-513 of SEQ ID NO: 31.

47. The immunogen according to claim 45, wherein the immunogen comprises or consists of amino acids 1-474 of SEQ ID NO: 31.

48. The immunogen according to claim 45, wherein the immunogen comprises or consists of amino acids 1-513 of SEQ ID NO: 31.

49. The immunogen according to any of claims 1-39 and 45-48, wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 31.

50. The immunogen according to any of claims 1-38 and 40, wherein the immunogen comprises or consists of amino acids 26-476 of SEQ ID NO: 32.

51. The immunogen according to claim 50, wherein the immunogen comprises or consists of amino acids 26-515 of SEQ ID NO: 32.

52. The immunogen according to claim 50, wherein the immunogen comprises or consists of amino acids 1-476 of SEQ ID NO: 32.

53. The immunogen according to claim 50, wherein the immunogen comprises or consists of amino acids 1-515 of SEQ ID NO: 32.

54. The immunogen according to any of claims 1-38, 40, and 50-53, wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 32.

55. The immunogen according to any of claims 1-38 and 41, wherein the immunogen comprises or consists of amino acids 31-479 of SEQ ID NO: 33.

56. The immunogen according to claim 55, wherein the immunogen comprises or consists of amino acids 31-518 of SEQ ID NO: 33.

57. The immunogen according to claim 55, wherein the immunogen comprises or consists of amino acids 1-479 of SEQ ID NO: 33.

58. The immunogen according to claim 55, wherein the immunogen comprises or consists of amino acids 1-518 of SEQ ID NO: 33.

59. The immunogen according to any of claims 1-38, 41, and 55-58, wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 33.

60. The immunogen according to any of claims 1-38 and 42, wherein the immunogen comprises or consists of amino acids 26-476 of SEQ ID NO: 63.

61. The immunogen according to claim 60, wherein the immunogen comprises or consists of amino acids 26-515 of SEQ ID NO: 63.

62. The immunogen according to claim 60, wherein the immunogen comprises or consists of amino acids 1-476 of SEQ ID NO: 63.

63. The immunogen according to claim 60, wherein the immunogen comprises or consists of amino acids 1-515 of SEQ ID NO: 63.

64. The immunogen according to any of claims 1-38, 42, and 60-63, wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 63.

65. The immunogen according to any of claims 1-38 and 43, wherein the immunogen comprises or consists of amino acids 26-476 of SEQ ID NO: 64.

66. The immunogen according to claim 65, wherein the immunogen comprises or consists of amino acids 26-515 of SEQ ID NO: 64.

67. The immunogen according to claim 65, wherein the immunogen comprises or consists of amino acids 1-476 of SEQ ID NO: 64.

68. The immunogen according to claim 65, wherein the immunogen comprises or consists of amino acids 1-515 of SEQ ID NO: 64.

69. The immunogen according to any of claims 1-38, 43, and 65-68, wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 64.

70. The immunogen according to any of claims 1-38 and 44, wherein the immunogen comprises or consists of amino acids 26-476 of SEQ ID NO: 65.

71. The immunogen according to claim 70, wherein the immunogen comprises or consists of amino acids 26-515 of SEQ ID NO: 65.

72. The immunogen according to claim 70, wherein the immunogen comprises or consists of amino acids 1-476 of SEQ ID NO: 65.

73. The immunogen according to claim 70, wherein the immunogen comprises or consists of amino acids 1-515 of SEQ ID NO: 65.

74. The immunogen according to any of claims 1-38, 44, and 70-73, wherein the immunogen comprises or consists of an amino acid sequence according to SEQ ID NO: 65.

75. The immunogen according to any of claims 1-74, wherein the recombinant RSV F protein or the fragment thereof forms a trimer in phosphate buffered saline at a physiological pH.

76. The immunogen according to any of claims 1-75, wherein the immunogen comprises a purification tag, in particular a His-tag and/or a Strep-tag.

77. A virus-like particle comprising the immunogen according to any of claims 1-76.

78. A protein nanoparticle comprising the immunogen according to any of claims 1-76.

79. The protein nanoparticle according to claim 78, 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.

80. The immunogen, the virus-like particle, or the protein nanoparticle according to any of claims 1-79, wherein the antibodies D25, MPE8 and/or AM14 specifically bind to the immunogen, the virus-like particle, or the protein nanoparticle, preferably with a K.sub.d of 1 M or less.

81. A nucleic acid molecule comprising a polynucleotide encoding the immunogen, the virus-like particle, or protein nanoparticle according to any one of claims 1-80.

82. The nucleic acid molecule according to claim 81, wherein the polynucleotide encodes a precursor protein of the immunogen or protein nanoparticle.

83. The nucleic acid molecule according to claim 82, wherein the precursor protein comprises, from N- to C-terminus, a signal peptide, a F2 polypeptide, and a F1 polypeptide.

84. The nucleic acid molecule according to claim 83, wherein the precursor protein comprises, from N- to C-terminus, a signal peptide, a F2 polypeptide, a F1 polypeptide, and a trimerization domain.

85. The nucleic acid molecule according to any one of claims 81-84, wherein the nucleic acid molecule is codon optimized for expression in a bovine cell.

86. The nucleic acid molecule according to any one of claims 81-85, operably linked to a promoter.

87. A vector comprising the nucleic acid molecule according to any one of claims 81-86.

88. The vector according to claim 87, wherein the vector is a viral vector.

89. The vector according to claim 88, wherein the vector is a bovine parainfluenza virus vector, a human parainfluenza virus vector, a Newcastle disease virus vector, a Sendai virus vector, a measles virus vector, an attenuated RSV vector, a paramyxovirus vector, an adenovirus vector, an alphavirus vector, a Venezuelan equine encephalitis vector, a Semliki Forest virus vector, a Sindbis virus vector, an adeno-associated virus vector, a poxvirus vector, a rhabdovirus vector, a vesicular stomatitis virus vector, a picornovirus vector, or a herpes virus vector.

90. The nucleic acid molecule or the vector according to any one of claims 81-89, comprising the nucleotide sequence as set forth in any of SEQ ID NOs: 56-62.

91. An isolated host cell comprising the nucleic acid molecule or the vector according to any one of claims 81-90.

92. An immunogenic composition comprising (i) the immunogen according to any one of claims 1-76 and 80; (ii) the virus-like particle according to claim 77 or 80; (iii) the protein nanoparticle according to any one of claims 78-80; (iv) the nucleic acid molecule according to any one of claims 81-86 and 90; (v) the vector according to any one of claims 87-90; or (vi) the host cell according to claim 91; and a pharmaceutically acceptable carrier.

93. The immunogenic composition according to claim 92, further comprising an adjuvant.

94. The immunogenic composition according to claim 93, wherein the adjuvant is alum, an oil-in water composition, MF59, ASOI, AS03, ASO4, MPL, QS21, a CpG oligonucleotide, a TLR7 agonist, a TLR4 agonist, a TLR3 agonist, or a combination of two or more thereof.

95. The immunogenic composition according to claim 93 or 94, wherein the adjuvant promotes a Th1 immune response.

96. The immunogenic composition according to any of claims 92-95, further comprising a RSV F prefusion-specific antibody that specifically binds the immunogen.

97. The immunogen according to any one of claims 1-76 and 80; the virus-like particle according to claim 77 or 80; the protein nanoparticle according to any one of claims 78-80; the nucleic acid molecule according to any one of claims 81-86 and 90; the vector according to any one of claims 87-90; the host cell according to claim 91; or the immunogenic composition according to any of claims 92-96; for use in generating an immune response to RSV F in a subject, in particular in cattle.

98. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to claim 97, wherein the immune response comprises a Th1 immune response.

99. A method for generating an immune response to RSV F in a subject, comprising administering to the subject an effective amount of the immunogen according to any one of claims 1-76 and 80; the virus-like particle according to claim 77 or 80; the protein nanoparticle according to any one of claims 78-80; the nucleic acid molecule according to any one of claims 81-86 and 90; the vector according to any one of claims 87-90; the host cell according to claim 91; or the immunogenic composition according to any of claims 92-96; to generate the immune response.

100. The method of claim 99, wherein the immune response comprises a Th1 immune response.

101. The immunogen according to any one of claims 1-76 and 80; the virus-like particle according to claim 77 or 80; the protein nanoparticle according to any one of claims 78-80; the nucleic acid molecule according to any one of claims 81-86 and 90; the vector according to any one of claims 87-90; the host cell according to claim 91; or the immunogenic composition according to any of claims 92-96; for use in prevention and/or treatment of RSV infection in a subject, in particular in cattle.

102. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101, wherein the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition is administered intravenously or intramuscularly.

103. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101-102, wherein the administration comprises a prime-boost administration of the immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition.

104. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101-103, wherein the immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition is administered repeatedly.

105. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101-104, wherein a single dose comprises 1 ng-10 g of the immunogen, preferably 100 ng-5 g of the immunogen, more preferably 1-1000 g of the immunogen, even more preferably 10-100 g of the immunogen, and most preferably 50 g of the immunogen.

106. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101-105, wherein the immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition is administered in combination with an anti-RSV agent.

107. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101-106, wherein the subject is at risk of or has an RSV infection.

108. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101-107, wherein the RSV infection is bovine RSV infection.

109. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to any of claims 97-98 and 101-108, wherein the subject is a bovine subject.

110. The immunogen, the virus-like particle, the protein nanoparticle, the nucleic acid molecule, the vector, the host cell, or the immunogenic composition for use according to claim 109, wherein the subject is a calf.

111. A method for treating or preventing a RSV infection in a subject, comprising administering to the subject a therapeutically effective amount of the immunogen according to any one of claims 1-76 and 80; the virus-like particle according to claim 77 or 80; the protein nanoparticle according to any one of claims 78-80; the nucleic acid molecule according to any one of claims 81-86 and 90; the vector according to any one of claims 87-90; the host cell according to claim 91; or the immunogenic composition according to any of claims 92-96; thereby treating or preventing RSV infection in the subject.

112. The method according to any one of claims 99-100 and 111, comprising a prime-boost administration of the immunogenic composition.

113. The method according to any one of claims 99-100 and 111-112, further comprising administering to the subject a therapeutically effective amount of an anti-RSV agent.

114. A method for detecting or isolating an RSV F binding antibody in a subject, comprising: (a) providing the immunogen according to any one of claims 1-76 and 80; the virus-like particle according to claim 77 or 80; the protein nanoparticle according to any one of claims 78-80; the nucleic acid molecule according to any one of claims 81-86 and 90; the vector according to any one of claims 87-90; the host cell according to claim 91; or the immunogenic composition according to any of claims 92-96; (b) contacting a biological sample from the subject with the recombinant RSV F protein or with the fragment thereof under conditions sufficient to form an immune complex between the recombinant RSV F protein or the fragment thereof and the RSV F binding antibody; and (c) detecting the immune complex, thereby detecting or isolating the RSV F binding antibody in the subject.

115. The method claim 114, wherein the method is an in-vitro method for detecting an RSV F binding antibody in an isolated biological sample of a subject.

116. The method according to any one of claims 99-100 and 111-115, wherein the subject is at risk of or has an RSV infection.

117. The method according to any one of claims 99-100 and 111-116, wherein the RSV infection is bovine RSV infection.

118. The method according to any one of claims 99-100 and 111-117, wherein the subject is a bovine subject.

119. The method according to claim 118, wherein the subject is a calf.

120. A kit comprising (i) the immunogen according to any one of claims 1-76 and 80; (ii) the virus-like particle according to claim 77 or 80; (iii) the protein nanoparticle according to any one of claims 78-80; (iv) the nucleic acid molecule according to any one of claims 81-86 and 90; (v) the vector according to any one of claims 87-90; (vi) the host cell according to claim 91; and/or (vii) the immunogenic composition according to any of claims 92-96; and instructions for using the kit.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0359] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0360] FIG. 1 shows for Example 1 a ClustalOmega sequence alignment of hRSVF strain A2 with RSV F from nine bovine strains (corresponding to SEQ ID NOs: 1-9) as indicated. Each row covers 60 positions. Residue positions completely conserved are designated by a *, homologous residues by a : and variable residues by a space.

[0361] FIG. 2 shows for Example 1 strain names and accession numbers for human and bovine RSV F proteins.

[0362] FIG. 3 shows for Example 1 the translation of pre-F hRSV F stabilization to bRSV F. a, Structural model of a pre-F hRSV F trimer stabilized by DS-Cav1 mutations (PDB ID 4MMU). One monomer is depicted by a blue ribbon model with the four DS-Cav1 mutations shown by red stick models outlined by red squares. The other two monomers are depicted by gray surface representations. b, Sequence variation between hRSV strain A2 and eight different bRSV strains is mapped (orange surface representation) onto a blue ribbon model of one monomer of a DS-Cav1 pre-F RSV F trimer colored as in (a). c, The locations of the DS-Cav1 mutations (red), the sc linkage (green) and interprotomer disulfide stabilization mutations (green) introduced into bovine RSV F protein are indicated by boxed stick models for one RSV F monomer. In (b) and (c), the other two monomers of the trimer are shown as gray surface representations. d, Phylogenetic tree for human and bovine RSV F proteins. Names shown indicate the virus strain or isolate. GenBank accession numbers for all strains used in this study are shown in FIG. 2.

[0363] FIG. 4 shows for Example 1 the expression screen for bDS-Cav1 RSV F in seven different strains.

[0364] FIG. 5 shows for Example 1 the antigenic screening of bRSV F single chain immunogens.

[0365] FIG. 6 shows for Example 1 the antigenic screening of bRSVF single chain immunogens with interprotomer disulfides.

[0366] FIG. 7 shows for Example 1 the yields of bRSV single chain immunogens in liter-scale production.

[0367] FIG. 8 shows the antigenic and physical characterization of bRSV F glycoprotein immunogens.

[0368] FIG. 9 shows protein purification and electron microscopy analysis. a, Representative SDS PAGE gel analysis for engineered bRSV F glycoproteins. DS2-v1 (391-2 sc9 DS-Cav1 Q98C-Q361, 391-2 DS-Cav1 and 391-2 post-F. Proteins are observed to collapse to smaller molecular weight bands in the presence of a reducing agent, confirming disulfide bond formation. b, Gel filtration chromatograms of bRSV F glycoprotein variants. Variants stabilized in pre-F conformation had longer retention times than variants with post-F conformation. c, Negative stain electron microscopy of pre-fusion and post-fusion forms of bovine RSV F. Images shown here are 2D class averages of variants with measured dimensions. Dh, diameter of head; Lh, length of head; Ls, length of stalk.

[0369] FIG. 10 shows for Example 4 crystallographic data collection and refinement statistics.

[0370] FIG. 11 shows the crystal structures of pre-F-stabilized bRSV F immunogens. a, Crystal structure of bRSV F ATue51908 DS-Cav1 depicted by a C-worm representation color-coded by atomic mobility factors, with thick, red worm for flexible regions and thin, blue worm for more rigid regions. Atomic level details are shown in insets on the right with stick representations and 2Fo-Fc electron density (blue) for regions that were mutated to stabilize the pre-F conformation. The upper left inset shows a ribbon superposition of the antigenic site region of ATue51908 DS-Cav1 (lime) with the structure of hRSV F DS-Cav1 (gray; PDB ID 4MMU). b, Crystal structure of the DS2 immunogen bRSV F 391-2 DS-Cav1 sc9 Q98C-Q361C, depicted as in (a).

[0371] FIG. 12 shows serum neutralizing antibody titers elicited by engineered bRSV F pre-F trimers. Pre-F-stabilized bRSV F glycoproteins elicited geometric mean EC.sub.5s neutralization titers between 43-344 fold higher than post-F in mice and calves respectively. Schematic immunization procedures for bRSV F variants in seronegative mice (a) and calves (b). Neutralization titer from each animal is shown as an individual dot, and geometric means are indicated by black horizontal lines. Immunization groups are shape-coded. Lod, limit of detection (titer=100) is indicated with a horizontal dashed line. Vertical dotted lines separate immunogen strains in (a) and weeks post prime in (b). Serum antibody binding ELISA data is summarized in FIG. 14. P values were determined by two-tailed Mann-Whitney tests. * indicates P0.05, ** indicates P0.01, *** indicates P0.001 and **** indicates P0.0001. There are 10 mice per group for the mouse immunizations. For calf immunizations, the DS2-v1 (391-2 sc9 DS-Cav1Q98C-Q361C) and post-F (391-2 post-F) groups each contained 5 animals and the placebo group contained 4 animals.

[0372] FIG. 13 shows bRSV neutralization EC50 titers measured from week 5 mouse sera.

[0373] FIG. 14 shows immunogenicity of engineered bovine RSV F pre-F trimers. ELISA binding titers of week five sera from mice (a) and longitudinal sera samples from calves (b) immunized with bRSV F variants. titers from each animal are represented by shape-coded symbols. Solid symbols indicate sera recognition of immobilized 391-2 sc9 DS-Cav1Q98C-Q361C RSV F trimers and open symbols indicate recognition of immobilized 391-2 post-F RSV F trimers. Vertical dotted lines separate immunogen strains in (a) and weeks post prime in (b). 6 dpi, 6 days post inoculation for calf challenge study in (b). Geometric mean titers are indicated by black horizontal lines. Calf immunization groups: DS2-v1 (391-2 sc9 DS-Cav1 Q98C-Q361C), 391-2 post-F and placebo (PBS).

[0374] FIG. 15 shows biographical data for immunized calves.

[0375] FIG. 16 shows bRSV neutralization EC.sub.50 titers measured from calf sera.

[0376] FIG. 17 shows blocking of neutralizing antibody binding. ELISA plates coated with 391-2 sc9 DS-Cav1Q98C-Q361C pre-F bRSV F trimer were incubated with serial dilutions of week 6 calf sera followed by biotinylated mAbs. The serum dilution that blocked mAb binding by 80% (defined as BD80) was determined. Higher BD80 values indicate the presence of sera that specifically blocks the respective mAbs. Vertical dotted lines separate the six different biotinylated mAbs (indicated above) used for pre-F trimer detection. Blocking of mAb binding titer of serum from each animal are represented by shape-coded symbols. Calf immunization groups: DS2-v1 (391-2 sc9 DS-Cav1Q98C-Q361C), 391-2 post-F and placebo (PBS).

[0377] FIG. 18 shows the effect of vaccination on bRSV replication in the respiratory tract of calves and on pulmonary pathology. a, Peak titers of bRSV in nasal secretions. Each dot represents the virus titer from nasopharyngeal swabs obtained at day 6 post challenge. Groups of 5 calves were vaccinated with DS2-v1 (391-2 DS-Cav1 sc9 Q98C-Q361C) and Post-F (391-2 post-F), and 4 calves were vaccinated with PBS (Placebo in adjuvant). Geometric mean peak titers are indicated by black horizontal lines. b, Effect of F protein vaccination on numbers of cells in BAL, 6 days after challenge with bRSV. c, Analysis of percentage of lung with macroscopic lung lesions from photographs of lungs. d, bRSV titers in samples of tracheal epithelium (TrSc), lung wash cells (LWC), and homogenates of samples taken from the right apical (RA), right cardiac (RC) and left cardiac (LC) lobes of the lung, 6 days post-infection. Each bar represents the bRSV titer of a lung sample. Each group of five bars is from an individual calf. Titers are expressed as log.sub.10 pfu/ml or g. The limit of detection (lod) is log.sub.10 0.7 pfu/ml (a and d). Virus titers for each individual testing point are listed in FIG. 15. P values were determined by two-tailed Mann-Whitney tests. ns indicates not significant (P>0.05), * indicates P0.05, ** indicates P0.01, *** indicates P0.001 and **** indicates P0.0001

[0378] FIG. 19 shows viral titers as a measure of bRSV replication in nasopharyngeal secretion.

[0379] FIG. 20 shows viral titers as a measure of bRSV replication in the respiratory tract of calves.

[0380] FIG. 21 shows the effect of vaccination on bRSV replication in the respiratory tract of calves and clinical signs of disease. a, Clinical sore at day 6 post RSV challenge. Each dot represents the score from each animal obtained at day 6 post challenge. DS2-v1 (391-2 sc9 DS-Cav1Q98C-Q361C), Post-F (391-2 post-F), PBS (Placebo). Geometric mean scores are indicated by black horizontal lines. b, Effect of F protein vaccination on proportion of neutrophils in BAL, 6 days after challenge with bRSV. P0.0105 for calves vaccinated with pre-F compared to controls. P values were determined by two-tailed Mann-Whitney tests. * indicates P0.05, ** indicates P0.01, *** indicates P0.001 and **** indicates P0.0001.

[0381] FIG. 22 shows the clinical scores and signs of immunized calves.

[0382] FIG. 23 shows the definition of clinical scores.

[0383] FIG. 24 shows the effect of bRSV F vaccination on pulmonary pathology.

[0384] FIG. 25 shows the histology of lung sections from vaccinated calves. Haematoxylin and eosin stained lung sections (left panel 4 objective, middle panel 10 objective and right panel 20 objective) from calves vaccinated with 50 g of adjuvanted pre-F (a), post-F (b) and PBS (c), 6 days after challenge with bRSV. White scale bars represent 1000 m (left panel), 400 m (middle panel) 200 m (right panel).

EXAMPLES

[0385] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Experimental: Material and Methods

Protein Expression, Purification

[0386] RSV F variants were expressed by transient transfection of Expi293F cells using 293Fectin (Invitrogen). Cell culture supernatants were harvested five days post transfection and centrifuged at 10,000 g to remove cell debris. The supernatants were sterile-filtered, and RSV F variants were purified by nickel (Roche) and Strep-Tactin (iba) affinity chromatography followed by size-exclusion chromatography (SEC. The foldon domain was removed only when proteins were prepared for animal immunization. The C-terminal tags were removed from the variants by digestion with 2 U/ml restriction-grade thrombin (Novagen) overnight at 4 C. The bRSV F glycoprotein with purification tags removed were then purified by a second round of size-exclusion chromatography in PBS.

Expression and Purification of Antibodies and Antigen-Binding Fragments (Fabs).

[0387] Antibodies were expressed by transient co-transfection of Expi293F cells (Thermo Fisher Scientific, MA) with both heavy- and light-chain plasmids using 293 fectin (Thermo Fisher Scientific, MA). Cell supernatants were harvested after 4-5 days and passed over Protein A agarose (GE Healthcare, PA). Bound antibodies were washed with PBS and eluted with IgG elution buffer (Pierce, Ill.) into 1/10th volume of 1 M Tris-HCl pH 8.0. Fabs were generated by digesting the IgG with Lys-C or HRV3C protease, and the cleaved Fc region was removed by passing the mixture over Protein A agarose. Final purification of Fabs was performed by SEC.

Antigenic Screening of bRSV F Immunogens

[0388] Initial assessment of all constructs were performed using a 96-well microplate format for high throughput expression followed by an ELISA-based antigenic evaluation as described previously 17. Briefly, 24 h prior to transfection HEK 293T cells (Thermo Fisher Scientific, MA) were seeded in each well of a 96-well microplate at a density of 2.5105 cells/ml in expression medium (high glucose DMEM supplemented with 10% ultra-low IgG fetal bovine serum and 1-non-essential amino acids), and incubated at 37 C., 5% CO2 for 20 h. Plasmid DNA and TrueFect-Max (United BioSystems, MD) were mixed and added to the growing cells, and the 96-well plate incubated at 37 C., 5% CO2. One day post transfection, enriched medium (high glucose DMEM plus 25% ultra-low IgG fetal bovine serum, 2 nonessential amino acids, 1 glutamine) was added to each well, and the 96-well plate was returned to the incubator for continuous culture. Five days post transfection supernatants with the expressed bRSV F variants were harvested and tested by ELISA for binding to D25, MPE8 and motavizumab antibodies using Ni2+-NTA microplates.

RSV F Antigenic Characterization

[0389] A fortBio Octet Red384 instrument was used to measure binding kinetics of RSV F variants to antibodies that target the pre-F or post-F form (D25, AM14, MPE8 and Mota). All assays were performed with agitation set to 1,000 rpm in phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA) to minimize nonspecific interactions. The final volume for all solutions was 50 l/well. Assays were performed at 30 C. in tilted black 384-well plates (Geiger Bio-One). Ni-NTA sensor tips were used to capture relevant RSV F variants. Typical capture levels for each loading step were between 1.4 and 1.5 nm, and variability within a row of eight tips did not exceed 0.1 nm for each of these steps. The nm unit is a measure of the change in the interference pattern of white light reflected from the surface of the biosensor tip compared to an internal reference. This was measured in real-time and correlated with a change in the thickness of bound molecules on the biosensor tip surface. This can also be defined as a change in response units measured in nm. Biosensor tips were equilibrated for 120 s in PBS+1% BSA prior to loading bRSV F variants. Biosensor tips were then equilibrated for 120 s in PBS+1% BSA prior to measuring association with antigen binding fragments (Fabs) in solution (0.007 M to 0.5 M) for 300 s; Fabs were then allowed to dissociate for 300-1200 s depending on the observed dissociation rate. Parallel correction to subtract systematic baseline drift was carried out by subtracting the measurements recorded for a loaded sensor incubated in PBS+1% BSA. Data analysis and curve fitting were carried out using Octet software, version 9.0. Experimental data were fitted with the binding equations describing a 1:1 interaction. Global analysis of the data sets assuming reversible binding (full dissociation) were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all of the concentrations used in each experiment.

Physical Stability of RSV F Variants

[0390] To assess the physical stability of the pre-fusion conformation of designed bRSV F glycoproteins under various stress conditions, the proteins were treated with a variety of pharmaceutically relevant stresses such as extreme pH, high temperature, low and high osmolarity, and repeated freeze/thaw cycles while at a concentration of 50 g/ml. The physical stability of treated bRSV F variants was evaluated by the preservation of antigenic site after treatment as assessed by binding of the site -specific antibody D25. In pH treatments, the bRSV F glycoprotein solution was adjusted to pH 3.5 and pH 10 with appropriate buffers and incubated at room temperature for 60 minutes and subsequently neutralized to pH 7.5. Temperature treatments were carried out by incubating the bRSV F glycoprotein solutions at 50 C. and 70 C. for 60 minutes in a PCR cycler with heated lid. In osmolarity treatments, bRSV F glycoprotein solutions originally containing 150 mM NaCl were either diluted with 2.5 mM Tris buffer (pH 7.5) to an osmolarity of 10 mM NaCl or adjusted with 4.5 M MgCl2 to a final concentration of 3.0 M MgCl2. Protein solutions were incubated for 60 minutes at room temperature and then returned to 150 mM salt by adding 5.0 M NaCl or dilution with 2.5 mM Tris buffer, respectively, and concentrated to 50 g/ml. The freeze/thaw treatment was carried out by repeatedly freezing bRSV F glycoprotein solutions in liquid nitrogen and thawing at 37 C. ten times in the presence of 10% glycerol. All bRSV F glycoproteins were diluted to 40 g/ml with PBS+1% BSA, and their ability to bind D25 Fab was measured with an Octet instrument using the protocol described above. The degree of physical stability is reported as the ratio of steady state D25-binding level before and after stress treatment.

Negative Stain Electron Microscopy

[0391] Samples were diluted to approximately 0.01 mg/ml, adsorbed to freshly glow-discharged carbon-coated grids, rinsed with several drops of buffer containing 10 mM HEPES, pH 7.0, and 150 mM KCl, and stained with 0.75% uranyl formate. Images were recorded on an FEI T20 microscope with a 2k2k Eagle CCD camera at a pixel size of 2.2 . Reference-free 2D classification and averaging were performed with EMAN2 (Tang, G., et al. EMAN2: An extensible image processing suite for electron microscopy. Journal of structural biology 157, 38-46 (2007)) and SPIDER.

Crystallization and X-Ray Data Collection of Pre-F-Stabilized bRSV F Proteins

[0392] Crystallization conditions were screened by vapor diffusion using a Mosquito crystallization robot (TTP labtech) that generated sitting drops at 20 C. by mixing 0.2 l of bRSV immunogens with 0.2 l of reservoir solution. Optimized crystals for data collection were grown by manually setting up hanging drops combining 0.5 l protein with 0.5 l of reservoir solution. ATue51908 DS-Cav1 crystals were grown in 12% (w/v) PEG 3350, and 0.1M sodium acetate pH 5.5, and 391-2 sc9 DS-Cav1Q98C Q361C crystals were grown in 0.9 M K/Na tartrate, 0.16 M Li2SO4, and 0.1 M CHES pH 9.5. Prior to data collection, ATue51908 DS-Cav1 crystals were transferred to 15% (v/v) 2R,3R-butanediol, 18% (w/v) PEG 3350, and 0.1M sodium acetate pH 5.5 and 391-2 sc9 DS-Cav1Q98C Q361C crystals were transferred to 15% (v/v) 2R,3R-butanediol, 1.3 M K/Na tartrate, 0.16 M Li2SO4, and 0.1 M CHES pH 9.5 followed by flash freezing in liquid nitrogen. X-ray diffraction data were collected at a wavelength of 1.00 at the SER-CAT beamline ID-22 (Advanced Photon Source, Argonne National Laboratory).

Structure Determination, Refinement and Analysis of Pre-F-Stabilized bRSV F

[0393] Diffraction data were integrated and scaled with the HKL2000 suite, and a molecular replacement solutions for both structures were obtained by PHASER using the pre-F RSV F structure (PDB ID: 4MMS) as a search model. Manual model building was carried out using COOT, with secondary structure elements built first. Refinement of individual coordinates, TLS parameters, and individual B-factors was performed in PHENIX. Final data collection and refinement statistics are presented in FIG. 10. All structural images were created using PyMol (The PyMol Molecular Graphics System, version 1.1; Schrdinger, LLC).

Mouse Immunizations

[0394] All mouse experiments were reviewed and approved by the Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH, under animal protocol 13-454, and all animals were housed and cared for in accordance with local, state, federal, and institute policies in an American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited facility at the NIH. Mice were randomized into groups of ten and these groups were not blinded to the investigators. As in previous experiments, hybrid female mice that were the first filial offspring of a cross between BALB/cJ females (C) and C57BL6) males (B6) (The Jackson Laboratory) known as CB6F1/J at ages 6 weeks to 12 weeks were intramuscularly injected with RSV F immunogens at week 0 and week 3. The frozen RSV F variant immunogen proteins were thawed on ice and mixed with 5-fold w/w poly I:C (Invivogen) adjuvant (i.e. 10 g RSV F, 50 g Poly I:C per animal per immunization), with injections taking place within 1 h of immunogen:adjuvant preparation. No adverse effect from immunization was observed. Blood was collected at least three days before immunization, and at week 2, week 5 and week 7 post initial immunization.

bRSV Neutralization Assays

[0395] bRSV microneutralization assay was performed using BT cells (ATCC CRL1390) and 500-1000 TCID50 (50% tissue culture infectious doses) of bRSV, strain 375 (ATCC VR1339). Briefly, immune sera were serially diluted in quadruplicates prior to mixing with 500-1000 TCID50 of bRSV for 1 hour at 37 C. in a humidified 5% CO2 atmosphere prior to addition to monolayers of BT cells seeded the day before at 8,000 cells/well. Cells were then incubated for 7 days, fixed with 70% methanol, stained with 1% crystal violet and examined at the microscope for syncytia formation and cytophatic effect (CPE). Neutralizing titer was defined as the reciprocal of the highest sera dilution at which the infectivity of bRSV was completely neutralized in 50% of the wells. Infectivity was identified by the presence of CPE and syncytia on day 7, and the titer was calculated by the Reed-Muench method.

ELISA Binding Assays

[0396] A standard ELISA was used to determine binding of immune sera to bRSV pre- and post-bRSV F proteins. Briefly, ELISA plates were coated with antigens at 5 g/ml, blocked with 1% BSA in PBS, incubated with serial dilutions of sera and washed. Bound mAbs were detected by incubation with AP-conjugated Goat Anti-Mouse adsorbed against human IgG (Southern Biotech) or goat anti-bovine IgG (Southern Biotech). Plates were then washed, substrate (4-Nitrophenyl phosphate disodium salt hexahydrate, Sigma) was added and plates were read at 405 nm. The relative titer of sera binding to respective coated antigens were determined by measuring the concentration of each serum required to achieve 50% binding relative to the maximum (ED50). The ED50 values were calculated by interpolation of binding curves fitted with a four-parameter nonlinear regression with a variable slope.

Calf Immunization

[0397] The calf experiment was performed under the regulations of the Home Office Scientific Procedures Act (1986) of the United Kingdom. The study had been reviewed and approved by the Animal and Plant Health Agency (APHA) Ethical Review Committee. Calve groups were not blinded to the investigators. Male calves were obtained from local farms and were removed from their mothers at birth to ensure that they did not receive any colostrum and transported to APHA at 1 day of age. Calves were bled on arrival at APHA and were fed 250 ml of colostrum, 48 hrs after birth, in order produce calves with little or no maternally derived bRSV-specific serum antibodies. Sera obtained before and after colostrum intake was analyzed for bRSV-specific and prefusion bRSV F protein-specific antibodies by ELISA. All but two calves were free from bRSV-specific serum antibodies. Calves were allocated to three groups of 5 to give groups matched for calf age, and the two animals with maternally derived bRSV-specific antibodies were allocated to the control group. Calves were 3 to 6 weeks old at the time of vaccination. The frozen bRSV F proteins, pre-F (DS2) and post-F (391-2 post-F) were thawed on ice and mixed with Montanide ISA71 VG (Seppic, France) in a water in oil emulsion in a ratio of 70:30 adjuvant to aqueous phase. Calves were inoculated intramuscularly with 50 g protein in a volume of 2 ml on two occasions 4 weeks apart. As controls, calves were inoculated with 2 ml PBS in ISA71 VG. Vaccinations took place within 3 h of immunogen:adjuvant preparation. Calves developed a transient fever 24 h after vaccination and no or only mild diffuse swelling at the injection sites. Calves were bled at defined time points for analysis of bRSV-specific serum antibody responses.

Calf Challenge Virus

[0398] Virulent bRSV used to challenge calves consisted of bronchoalveolar lavage (BAL) prepared from a gnotobiotic calf inoculated 6 days previously with the Snook strain of bRSV, which had been passaged on four previous occasions in gnotobiotic or specific pathogen free calves. The BAL was free from other viruses, mycoplasmas, and bacteria as assessed by inoculation of tissue culture cells, mycoplasmal or bacterial media. Virus titers were determined by plaque assay on fetal calf kidney cells.

Calf Challenge

[0399] Four weeks after the last vaccination, calves were challenged by intranasal and intratracheal administration of 10.sup.4 pfu of bRSV, Snook strain, in BAL. Following bRSV challenge, nasopharyngeal swabs were obtained daily to monitor bRSV excretion, and calves were examined daily for clinical signs of disease. The severity of disease was given a score as shown in FIG. 22. The clinical scores are defined in FIG. 23. Calves were euthanized 6 days after challenge to determine the extent of gross pneumonic consolidation and the extent of virus infection in the lower respiratory tract as described previously. Titers of virus in the trachea were determined by scraping the epithelium from a piece of trachea approximately 3 cm long into 2 ml of Hanks balanced salt solution (Sigma) containing 1% bovine serum albumin (BSA) (Sigma). The apical and cardiac lung lobes were clamped and the lungs lavaged with 1 liter of PBS to obtain bronchalveolar lavage (BAL). Cytospin preparations of BAL cells were fixed and stained with Diff Quik (Thermo Fisher Scientific) and differential cell counts made using oil immersion microscopy. Samples of lung taken from three different apical lung lobes were homogenized to give a 20% w/v suspension. Lung tissue for histology was also taken from 3 different apical lobes and fixed in 10% neutral buffered formalin, paraffin wax embedded and sections were stained with haematoxylin and eosin.

Statistical Analysis

[0400] Statistical analyses were performed using two-tailed Mann-Whitney tests with GraphPad Prism 6.0 software (La Jolla, Calif.). Differences were considered statistically significant at P0.05.

Example 1: Design and Initial Characterization of bRSV F Immunogens

[0401] The RSV F glyoprotein is conserved between bRSV and hRSV, with sequence identities of 80% (FIGS. 1 and 2, and FIG. 3 b,d) and multiple F-directed antibodies able to neutralize both hRSV and bRSV. Based on the success of the prior engineering of pre-F hRSV F trimer (McLellan, J. S., et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592-598 (2013)), bRSV F was modified to create thermostable pre-F trimers. A disulfide between residues 155 and 290 (DS) along with cavity-filling mutations S190F and V207L (Cav1) and a C-terminal T4 foldon trimerization domain (foldon) were incorporated into bRSV F from seven different strains to make bovine versions of DS-Cav1 (bDS-Cav1 s) (FIG. 3 a, b and FIG. 4), which included cleavable C-terminal His and Strep tags for purification.

[0402] Upon expression in Expi293F cells only three of the seven bDS-Cav1s (strains 391-2, ATue51908, and RB94 respectively) expressed at greater than 0.5 mg/L of culture (FIG. 4). Amino acid sequences of those three bDS-Cav1 s are shown in the following:

TABLE-US-00015 bRSV391-2DSCav1: [SEQIDNO:36] MAATAMRMIISIIFISTYMTHITLCQNITEEFYQSTCSAVSRGYLSALRT GWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVIELQSLMQNE PASFSRAKRGIPELIHYTRNSTKRFYGLMGKKRKRRFLGFLLGIGSAIAS GVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTFKVLDLKNYID KELLPKLNNHDCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTY MLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCVVKEEVIAYV VQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVS FEPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKT DISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTV SVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKIN QSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPR GSHHHHHHSAWSHPQFEK bRSVATue51908DSCav1: [SEQIDNO:37] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNITEEFYQSTCSAVSRGYL SALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQS LMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIG SAVASGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTFKVLDL KNYIDKELLPQLNNHDCRISNIETVIEFQQKNNRLLEIAREFSVNAGITT PLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCVVKEE VIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDN AGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKI MTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQV NAKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLG GLVPRGSHHHHHHSAWSHPQFEK bRSVRB94DSCav1: [SEQIDNO:38] MPMGSLQPLATLYLLGMLVASVLAAQNITEEFYQSTCSAVSRGYLSALRT GWYTSVVTIELSKIQKNVCNSTDSNVKLIKQELERYNNAVVELQSLMQNE PASSSRAKRGIPELIHYKRNSTKKFYGLMGKKRKRRFLGFLLGIGSAIAS GVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTFKVLDLKNYID KELLPKLNNHDCQISNIATVIEFQQKNNRLLEIAREFSVNAGITTPLSTY MLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCVVKEEVMAYV VQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNAKYDCKIMTSKT DISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNRGVDTV SVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKIN QSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPR GSHHHHHHSAWSHPQFEK

[0403] All three of these bDS-Cav1 s were recognized by pre-F-specific mAbs D25 (McLellan, J. S., et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113-1117 (2013)) and MPE8 (Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)) as well as by mAb motavizumab (Mota; McLellan, J. S., et al. Structural basis of respiratory syncytial virus neutralization by motavizumab. Nat Struct Mol Biol 17, 248-250 (2010)) (FIG. 4).

[0404] To enhance immunogenicity, next bDS-Cav1 thermostability was sought to be optimized. To minimize the number of designs evaluated, two RSV strains (391-2 and RB94) were selected to optimize initially, with the intent to introduce the best mutations from the final set into the third strain, ATue51908. Previous investigations (Georgiev, I. S., et al. Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env. Journal of virology 89, 5318-5329 (2015); Sharma, S. K., et al. Cleavage-Independent HIV-1 Env Trimers Engineered as Soluble Native Spike Mimetics for Vaccine Design. Cell Rep 11, 539-550 (2015); Krarup, A., et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat Commun 6, 8143 (2015); Chen, J., et al. Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation. Cell 95, 409-417 (1998)) of type 1 fusion machines have indicated that removal of the cleavage site to create sc variants can improve pre-F stability. Therefore, with a focus on that aforementioned type of stabilizations and on the introduction of interprotomer disulfide bonds (DS2), 92 variants of bDS-Cav1 were designed, all of which employed a sc topology and 32 of which contained an interprotomer disulfide (DS2 variants). Additionally, many of the 92 designs incorporated internal cavity-filling mutations, core residues from hRSV F for increased stability, and additional sites of N-linked glycosylation to mask irrelevant epitopes.

[0405] All 92 bDS-Cav1 designs were evaluated for expression and antigenic recognition by mAbs D25, MPE8 and Mota in a 96 well-microplate transient transfection format (McLellan, J. S., et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592-598 (2013)). Each design was scored by summing ELISA readings for the pre-F-specific mAbs D25 and MPE8 (FIGS. 5 and 6). The top three-scoring DS2 designs (391-2 DS-Cav1 sc9 Q98C-Q361C [SEQ ID NO: 32], RB94 DS-Cav1 sc9 A149C Y458C [SEQ ID NO: 39], and RB94 sc9 DS-Cav1 N183GC-N428C [SEQ ID NO: 40]) and the top two-scoring 391-2 and RB94 sc designs without interprotomer disulfides (sc9-10_bRSV(RB94) DS-Cav1_fd_hp2_fp2_ig1 [SEQ ID NO: 41] and 391-2-site hRSV bovsurf DS-Cav1-BZGJ9 Long [SEQ ID NO: 42]) were selected for additional evaluation (FIGS. 5 and 6). To expand the dimensions of our search for optimal immunogens, interprotomer disulfides (Q98C-Q361C, A149C-Y458C and N183GC-N428C) and sc formats (sc9 and sc9-10) from the top five designs were mixed and matched and the ATue51908 strain was added to generate additional designs for a total of nine constructs, which were expressed in 1 liter Expi293F cultures (FIG. 7). Amino acid sequences of those nine constructs are shown in the following:

TABLE-US-00016 bRSV391-2sc9-10DS-Cav1Q98C-Q361C: [SEQIDNO:31] MAATAMRMIISIIFISTYMTHITLCQNITEEFYQSTCSAVSRGYLSALRT GWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVIELQSLMCNE PASgsGSAIASGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLT FKVLDLKNYIDKELLPKLNNHDCRISNIETVIEFQQKNNRLLEIAREFSV NAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIM CVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDR GWYCDNAGSVSFFPQAETCKVCSNRVFCDTMNSLTLPTDVNLCNTDIFNT KYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGC DYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFD ASIAQVNAKINQSLAFIRRSDELLSaiggyipeaprdgqayvrkdgewvl lstflgglvprgshhhhhhsawshpqfek bRSV391-2sc9DS-Cav1Q98C-Q361C: [SEQIDNO:32] MAATAMRMIISIIFISTYMTHITLCQNITEEFYQSTCSAVSRGYLSALRT GWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVIELQSLMCNE PASFSgsGSAIASGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSV LTFKVLDLKNYIDKELLPKLNNHDCRISNIETVIEFQQKNNRLLEIAREF SVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMCVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRT DRGWYCDNAGSVSFFPQAETCKVCSNRVFCDTMNSLTLPTDVNLCNTDIF NTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSN GCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDE FDASIAQVNAKINQSLAFIRRSDELLSaiggyipeaprdgqayvrkdgew vllstflgglyprgshhhhhhsawshpqfek bRSV391-2sc9DS-Cav1A149C-Y458C: [SEQIDNO:43] MAATAMRMIISIIFISTYMTHITLCQNITEEFYQSTCSAVSRGYLSALRT GWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVIELQSLMQNE PASFSGSGSAlcSGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSV LTFKVLDLKNYIDKELLPKLNNHDCRISNIETVIEFQQKNNRLLEIAREF SVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMCVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRT DRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIE NTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSN GCDYVSNKGVDTVSVGNTLYcVNKLEGKALYIKGEPIINYYDPLVFPSDE FDASIAQVNAKINQSLAFIRRSDELLsaiggyipeaprdgqayvrkdgew vllstflgglvprgshhhhhhsawshpqfek bRSVATue51908sc9-10DS-Cav1A149C-Y458C: [SEQIDNO:33] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNITEEFYQSTCSAVSRGYL SALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQS LMQNEPASgsGSAVcSGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNG VSVLIFKVLDLKNYIDKELLPQLNNHDCRISNIETVIEFQQKNNRLLEIA REFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQ SYSIMCVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICL TRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNT DIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKT FSNGCDYVSNKGVDTVSVGNTLYcVNKLEGKALYIKGEPIINYYDPLVFP SDEFDASIAQVNAKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKD GEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK bRSVATue51908sc9-10DS-Cav1N183GC-N428C: [SEQIDNO:44] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNITEEFYQSTCSAVSRGYL SALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQS LMQNEPASgsGSAVASGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSgc GVSVLTFKVLDLKNYIDKELLPQLNNHDCRISNIETVIEFQQKNNRLLEI AREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQ QSYSIMCVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNIC LTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCN TDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKcRGIIK TFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVF PSDEFDASIAQVNAKINQSLAFIRRSDELLsaiggyipeaprdgqayvrk dgewvllstflgglvprgshhhhhhsawshpqfek bRSVRB94sc9DS-Cav1A149C-Y458C: [SEQIDNO:39] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNITEEFYQSTCSAVSRGYL SALRTGWYTSVVTIELSKIQKNVCNSTDSNVKLIKQELERYNNAVVELQS LMQNEPASSSgsGSAlcSGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLS NGVSVLTFKVLDLKNYIDKELLPKNNHDCQISNIATVIEFQQKNNRLLEI AREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQ QSYSIMCVVKEEVMAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNIC LTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCN TDIFNAKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIK TFSNGCDYVSNRGVDTVSVGNTLYcVNKLEGKALYIKGEPIINYYDPLVF PSDEFDASIAQVNAKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRK DGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK bRSVRB94sc9DS-Cav1N183GC-N428C: [SEQIDNO:40] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNITEEFYQSTCSAVSRGYL SALRTGWYTSVVTIELSKIQKNVCNSTDSNVKLIKQELERYNNAVVELQS LMQNEPASSSgsGSAIASGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLS gcGVSVLTFKVLDLKNYIDKELLPKLNNHDCQISNIATVIEFQQKNNRLL EIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIV RQQSYSIMCVVKEEVMAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSN ICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNL CNTDIFNAKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKcRGI IKTFSNGCDYVSNRGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPL VFPSDEFDASIAQVNAKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYV RKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK sc9-10_bRSV(RB94)DS-Cav1_fd_hp2_fp2_ig1: [SEQIDNO:41] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRT GWYTSVITIELSKIQKNVCNSTDSNVKLIKQELERYNNAVVELQSLMQST PATGSGSAIASGVAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLI TKVLDLKNYIDKELLPILNNHDCQISNIATVIEFQQKNNRLLEIAREFSV NAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIM CIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDR GWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNA KYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGC DYVSNRGVDTVSVGNTLYYVNKQEGKSLYIKGEPIINYYDPLVFPSDEFD ASIAQVNAKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVL LSTFLGGLVPRGSHHHHHHSAWSHPQFEK 391-2-site hRSVbovsurfDS-Cav1-BZGJ9Long: [SEQIDNO:42] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGqniteefyqstcsavsrgyl salrtgwytsvitielsKIQKNVCKSTDSKVKLIKQELERYNNAVlelql lmqstpatnngsgsaiasgVAVCKylhlegevnkiknallstnkavvsls ngvsVLTFkvldlknyidkELLPKLNNHDCRISNIEtviefqqknnrlle itrefsvnagvttpvstymltnsellslindmpitndqkklmssnvqivr qqsySIMCllkeevlayvvqlpiygvidtpcwklhtsplcttdnkegsni cltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsrtlptdvnlc ntdifntkydckimtsktdvsssvitslgaivscygktkctasnknrgii ktfsngcdyvsnkgvdtvsygntlyyvnkqegkslyvkgepiinfydplv fpsdefdasisqvnekinqslafirrsdeLLhnvnagksttGGYIPEAPR DGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK

[0406] The sc designs sc9 and sc9-10 differed only in two residues, with a GS linker replacing F.sub.2F1 cleavage and fusion residues 106-144 or 104-144, respectively. Of these nine designs, the two sc-only variants, namely bRSV 391-2 sc9 DS-Cav1Q98C-Q361C (SEQ ID NO: 32) and bRSV 391-2 sc9-10 DS-Cav1Q98C-Q361C (SEQ ID NO: 31) gave 7-9 fold higher expression yields as compared to the other variants (FIG. 7). However, size exclusion chromatography revealed that their respective sizes were compatible with aggregation. Therefore, three of the remaining five DS2 designs were chosen (each with sc topology and added interprotomer disulfides) with the highest yield (FIG. 7) for immunogenic, antigenic, physical, and structural characterizations.

[0407] Additionally, as benchmarks, the DS-Cav1 variant of each of the three strains and also the post-F form of each of the three strains (the latter created by removing the RSV F fusion loop residues 137-146), as previously described (McLellan, J. S., Yang, Y., Graham, B. S. & Kwong, P. D. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. Journal of virology 85, 7788-7796 (2011)), were used. Altogether the sc-DS2, DS-Cav1 and post-F immunogens of each of the three strains totals nine final immunogen constructs (cf. FIG. 8). Namely, the following nine immunogen constructs were used: bRSV 391-2 DSCav1 (SEQ ID NO: 36), bRSV ATue51908 DSCav1 (SEQ ID NO: 37), bRSV 391-2 DSCav1 (SEQ ID NO: 38), bRSV 391-2 sc9-10 DS-Cav1Q98C-Q361C (SEQ ID NO: 31), bRSV 391-2 sc9 DS-Cav1 Q98C-Q361C (SEQ ID NO: 32), bRSV ATue51908 sc9-10 DS-Cav1 A149C-Y458C (SEQ ID NO: 33), bRSV 391-2 postF (SEQ ID NO: 45), bRSV ATue51908 postF (SEQ ID NO: 46), and bRSV 391-2 postF (SEQ ID NO: 47).

TABLE-US-00017 bRSV391-2postF: [SEQIDNO:45] MAATAMRMIISIIFISTYMTHITLCQNITEEFYQSTCSAVSRGYLSALRT GWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVIELQSLMQNE PASFSRAKRGIPELIHYTRNSTKRFYGLMGKKRKRRAIASGVAVSKVLHL EGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNH DCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSL INDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGVID TPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSI GAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVN KLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSD ELLGLEVLFQGPHHHHHHHHSAWSHPQFEK bRSVATue51908postF: [SEQIDNO:46] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNITEEFYQSTCSAVSRGYL SALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQS LMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRAVASGVAVS KVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLP QVNNHDCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNS ELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPI YGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQT ETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSS VITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNT LYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAF IRRSDELLGLEVLFQGPHHHHHHHHSAWSHPQFEK bRSVRB94postF: [SEQIDNO:47] MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNITEEFYQSTCSAVSRGYL SALRTGWYTSVVTIELSKIQKNVCNSTDSNVKLIKQELERYNNAVVELQS LMQNEPASSSRAKRGIPELIHYKRNSTKKFYGLMGKKRKRRAIASGVAVS KVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLP KVNNHDCQISNIATVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNS ELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVMAYVVQLPI YGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNAKYDCKIMTSKTDISSS VITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNRGVDTVSVGNT LYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAF IRRSDELLGLEVLFQGPHHHHHHHHSAWSHPQFEK

[0408] All nine of these constructs gave expression yields of 2-5 mg/L except for ATue51908 sc9-10 DS-Cav1 A149C-Y458C (0.24 mg/L) and RB94 DS-Cav1 (0.76 mg/L) (FIG. 8), and the post-F forms consistently gave the highest expression (3-5 mg/L). After purification on nickel and Strep-Tactin affinity columns, and subsequent cleavage of C-terminal affinity tags, all nine immunogens behaved well when analyzed by size-exclusion chromatography, eluting with peaks consistent with trimer formation (FIG. 9 a, b), with post-F forms eluting at slightly larger sizes than pre-F forms due to their elongated shape (McLellan, J. S., et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113-1117 (2013); McLellan, J. S., Yang, Y., Graham, B. S. & Kwong, P. D. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. Journal of virology 85, 7788-7796 (2011)).

Example 2: Antigenic Characteristics of bRSV F Immunogens

[0409] The antigenicity of each purified immunogen was evaluated with biolayer interferometry to assess recognition by the antigenic site -directed mAb D25 (McLellan, J. S., et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113-1117 (2013)), antigenic site II-directed mAb Mota (McLellan, J. S., et al. Structural basis of respiratory syncytial virus neutralization by motavizumab. Nat Struct Mol Biol 17, 248-250 (2010)) and quaternary-specific mAbs AM14 (Gilman, M. S. A., et al. Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein. PloS pathogens 11, e1005035 (2015)) and MPE8 (Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)) (FIG. 8). The three pre-F-specific mAbs, D25, MPE8 and AM14 recognized all six immunogens containing pre-F stabilizing mutations, confirming stabilization of their pre-F conformations. Moreover, recognition by the quaternary-specific mAbs MPE8 and AM14 substantiated the formation of native-like trimers. In contrast, these mAbs did not recognize any of the post-F immunogens. As expected, Mota recognized all nine immunogens since its site II epitope is not affected by pre- and post-F conformational changes. Notably, all mAbs except for D25, recognized the pre-F immunogens with nanomolar affinity (0.2-12.8 nM) even though these mAbs were elicited by hRSV F. D25 recognized the pre-F immunogens with affinities ranging from 0.4-420 nM, suggesting that antigenic site may be adversely effected by some of the stabilizing mutations. Interestingly, the three DS-Cav1-only immunogens which also had the least number of mutations had the highest affinity for D25.

Example 3: Physical Characteristics of bRSV F Immunogens

[0410] Next, the stability of purified immunogens was assessed by subjecting them to high temperature, pH extremes, osmolarity extremes and cycles of freeze-thaw and quantifying their subsequent recognition by D25 (FIG. 8). All nine immunogens were observed to generally tolerate pH and osmolarity extremes and freeze-thaw cycles. The three DS-Cav1-only immunogens, 391-2 DS-Cav1, Atue51908 DS-Cav1 and RB94 DS-Cav1, were most susceptible to physical extremes and lost 25-60% of their D25 reactivity upon exposure to high (3M) osmolarity. Curiously, the majority of immunogens actually increased reactivity to D25 after exposure to high pH. Although none of the DS-Cav1-only immunogens were able to survive exposure to high temperature (70 C.), all three of the DS2 immunogens tolerated high temperature. Not surprisingly, all three post-F immunogens were heat resistant.

Example 4: Structural Characteristics of bRSV F Immunogens

[0411] To further confirm the pre- and post-F conformations of the engineered bRSV F immunogens, they were examined by negative stain electron microscopy (EM), followed by reference-free 2D class averaging of the images (FIG. 9c). Each of the pre-F immunogens displayed bulb-like trimer structures with a short stem-like structure at one end, whereas the post-F immunogens displayed longer and more slender structures each of which were consistent with known crystal structures of pre- and post-F hRSV F (McLellan, J. S., et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592-598 (2013); McLellan, J. S., et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113-1117 (2013); McLellan, J. S., Yang, Y., Graham, B. S. & Kwong, P. D. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. Journal of virology 85, 7788-7796 (2011); Swanson, K. A., et al. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proceedings of the National Academy of Sciences of the United States of America 108, 9619-9624 (2011)).

[0412] The crystal structures of two pre-F-stabilized bRSV F immunogens, ATue51908 DS-Cav1 (SEQ ID NO: 37) and 391-2 DS-Cav1 sc9 Q98C-Q361C (SEQ ID NO: 32) were determined to 2.65 and 3.6 resolution, respectively (FIG. 10). ATue51908 DS-Cav1 crystallized in a monoclinic lattice not previously observed with hRSV F immunogens. The overall structure of ATue51908 DS-Cav1 was similar to that of hRSV DS-Cav1 (McLellan, J. S., et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592-598 (2013)) with a root mean square deviation (rmsd) of 1.0 for 437 C atoms excluding residues 209-215 in a membrane distal loop adjacent to antigenic site (FIG. 11a). In this crystal form, the appended C-terminal foldon trimerization domain was visible in the electron density map, although its three-fold axis was misaligned by 17 degrees relative to the three-fold axis of bRSV pre-F due to crystal packing (FIG. 11a). The introduced DS and S190F mutations showed strong electron density, while V207L showed weaker but detectable electron density (FIG. 11a). The DS2 immunogen 391-2 DS-Cav1 sc9 Q98C-Q361C (SEQ ID NO: 32) crystallized in the cubic lattice commonly observed with hRSV F pre-F immunogens and like ATue51908 DS-Cav1, its structure was similar to hRSV DS-Cav1 with an rsmd of 1.1 for 435 C atoms. Although side chains for the DS-Cav1 mutations were not clearly apparent in the electron density, partly due to the 3.6- resolution of structure, the DS2 interprotomer Q98C-Q361C disulfide and nearby sc linker showed traceable electron density (FIG. 11b). Comparison of the two bRSV pre-F-stabilized structures at the backbone level revealed high structural similarity with an rmsd of 0.9 between 434 equivalent C atoms excluding the F.sub.2F.sub.1 linker region. The greatest structural differences between the hRSV and bRSV pre-F immunogens were observed in residues 206-215 at the apical loop between 4 and 5 near antigenic site (FIGS. 11a, b, left panels), which is also the region of highest sequence divergence (50% identity) between the two species (FIG. 1). These structural and sequence difference may explain the lower binding affinity of D25 to bRSV pre-F (FIG. 8) relative to hRSV pre-F (McLellan, J. S., et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592-598 (2013)).

Example 5: Immunogenic Characterization of bRSV F Immunogens in Mice

[0413] To evaluate immunogenicity, each of the nine bRSV F immunogens were used to immunize a group of 10 CB6F1/J mice. Each immunogen dose comprised 10 g of protein adjuvanted with 50 g of polyinosinic:polycytidylic acid (Polyl:C). Mice were primed and boosted intramuscularly at weeks 0 and 3 respectively. Analysis of week five sera revealed geometric mean reciprocal IC.sub.50 neutralization titers of 6,880-11,453 for pre-F immunogen-immunized mice, which were 33- to 55-fold higher (P0.0001) than the titers (geometric mean 100-210) observed for the post-F immunogen-immunized mice (FIG. 12a and FIG. 13). Neutralization titers elicited from pre-F-immunized mice were 82-110 fold greater than the calibrated protective titer of 100 (McLellan, J. S., et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592-598 (2013)). 391-2 DS-Cav1 sc9 Q98C-Q361C (SEQ ID NO: 32) elicited the highest titers (geometric mean 11,453). Thus the 1000-fold difference in D25 mAb binding affinities observed between various pre-F immunogens (FIG. 8) did not appear to impact the neutralization titers of elicited sera. To gauge the overall immunogenicity of each immunogen, the sera binding response to pre-F and post-F immunogens was assessed using an ELISA (FIG. 14a). The binding titers of pre-F elicited sera to the six pre-F immunogens were statistically comparable to each other with geometric mean endpoint binding titers ranging from 4,687 to 100,323. Intriguingly, sera from 391-2 sc9 Q98C-Q361C(SEQ ID NO: 32)-immunized mice displayed the lowest titers for pre-F immunogen even though it had the highest titers of neutralizing antibodies. As expected, sera elicited by pre-F immunogens displayed lower binding titers to post-F immunogens.

Example 6: Immunogenic Characterization of bRSV F Immunogens in Calves

[0414] To investigate the effectiveness of pre-F-stabilized RSV F vaccines in bRSV-seronegative calves, the highly stable DS2 immunogen (391-2 DS-Cav1 sc9 Q98C-Q361C; SEQ ID NO: 32), which in mice elicited the highest neutralization titers (geometric mean reciprocal IC.sub.50 11,453), was selected. As controls post-F 391-2 were chose and phosphate buffered saline (PBS) was used to immunize a placebo group. Groups of five 3-6 week old male calves (FIG. 15) were immunized twice at weeks 0 and 4, and sera were collected two weeks after each immunization. Each injection consisted of 50 g protein in 0.6 ml PBS adjuvanted with 1.4 ml of ISA 71G. The reciprocal IC.sub.50 neutralization titers from the DS2-immunized calves were observed to increase exponentially at weeks 2, 4 and 6 relative to week 0, resulting in a final geometric mean titer of 56,055 two weeks after the boost (FIG. 12b and FIG. 16). By week 8, however, four weeks after the boost, titers had dropped to a geometric mean of 21,849. In contrast, by week 8, post-F immunogen elicited minimal titers (geometric mean 172) within the same range as two of the saline control-immunized calves (141 and 100 respectively), which appeared to have maternally-derived serum antibodies at the start of the study. Results from ELISA analysis of sera from DS2-immunized calves mirrored the neutralization titers with titers steadily increasing and then slightly decreasing from weeks 6 to 8 (FIG. 14b). As expected, sera from 391-2 post-F-immunized calves followed a similar trend, reaching geometric mean titers approximately 3.0 and 3.8-fold lower than pre-F by weeks 6 and 8 respectively. It is also clear from the neutralization data (FIG. 12b) that post-F elicited significantly lower levels of neutralizing antibodies. Not surprisingly, sera elicited by 391-2 pre-F immunogen displayed lower binding titers to 391-2 post-F immunogen and 391-2 post-F-elicited sera recognized both pre- and post-F with comparable titers. Results from a competition ELISA showed that the sera from DS2-immunized calves competed with the broadly neutralizing mAbs AM14, D25, RSD5, MPE8 and palivizumab (pali), suggesting that antigenic sites 0, II and V were all targeted (FIG. 17). Although the mAb mota showed considerably less competition than the other antigenic site II mAb, pali, it's high 34.6 M affinity (Wu, H., et al. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. Journal of molecular biology 368, 652-665 (2007)) for RSV F likely limited sera competition. As expected, sera from 391-2 post-F-immunized calves competed to a much lower extent with the pre-F-specific mAbs AM14, RSD5, MPE8, and did not compete at all with D25. Interestingly, two mAbs compatible with post-F RSV F, mota and pali also competed to a much lower extent with post-F-elicited sera. This suggests that a minority of the post-F-elicited sera was directed against site II.

Example 7: bRSV Challenge of Immunized Calves

[0415] Next, all calves were challenged by intranasal and intratracheal routes with the heterologous Snook strain of bRSV, four weeks after the boost. Calves were monitored daily for clinical signs of disease and for viral titers in the nasopharynx for six days after challenge. At day six after challenge, calves were euthanized and bronchoalveolar lavage (BAL) and lung biopsies from three regions of the lung were obtained to determine viral titers, neutrophil infiltration, and the extent of microscopic and macroscopic lesions. Remarkably, calves vaccinated with DS2 (391-2 DS-Cav1 sc9 Q98C-Q361C; SEQ ID NO: 32) had no detectable bRSV viral titers in nasopharyngeal secretions (FIG. 18a and FIG. 19). No detectable bRSV titers were observed from a postmortem lung wash, samples of tracheal epithelium, right apical, right cardiac or left cardiac regions of the lung (FIG. 12d and FIG. 20). In contrast, geometric mean viral titers of up to 1.78 and 1.67 (log.sub.10 pfu/ml) were observed in the daily nasal secretions from the post-F and PBS vaccinated calves respectively. Likewise, virus was isolated post mortem from BAL cells of all of the post-F-immunized and PBS-immunized calves with the greatest extent of lung virus replication in the PBS-immunized control animals (FIG. 18d). Thus, all DS2-immunized calves were protected from bRSV viral replication in both the upper and lower respiratory tracts. Furthermore, four out of five of the DS2-immunized calves were also protected from clinical signs of disease, lung inflammation and macroscopic lung lesions (FIG. 18b-c, FIG. 21 and FIG. 22-24). Clinical scores, based mainly on differences in respiratory rate and body temperature, were minimal for most the pre-F- and post-F-immunized calves. Scores trended higher for the PBS-immunized controls, but were not significantly different from the other two groups (FIG. 22-23). However, both respiratory rate (RR) and body temperature increased in all PBS-immunized calves, 6 days after bRSV challenge, whereas the RR increased in only 2 post-F-immunized and one pre-F-immunized calves at this time (FIG. 21a). Although the one calf in the pre-F-immunized group that had developed a raised RR and body temperature also exhibited signs of lung inflammation, the geometric mean number of cells observed in the BAL, the percentage of polymorph nuclear neutrophils (PMNs) in BAL, and the percentage of macroscopic lung lesions were all statistically lower than in the placebo group (FIG. 18b-c and FIG. 22-24). The post-F-immunized calves displayed intermediate levels of lung inflammation. Although the extent of macroscopic lung lesions in the post-F-immunized calves was less that in the placebo group, the percentage of PMNs in BAL was similar to that seen in BAL from calves in the placebo group. Microscopic lung lesions in the placebo group, six days post challenge, were typical of bRSV bronchiolitis and alveolitis and were characterized by epithelial hypertrophy of small bronchioles, bronchiolar exudate containing desquamated epithelial cells, neutrophils and macrophages, and thickening of alveolar walls due to infiltration by mononuclear cells and granulocytes (FIG. 25c). Bronchiolitis and alveolitis were also seen in in three of the post-F-immunized calves (FIG. 25b). In addition, a peribronchiolar lymphoreticular hyperplasia was seen in all lung sections from these animals. In contrast, bronchiolitis and alveolitis were absent from all but one of the DS2-immunized calves, and the histopathology was essentially restricted to a peribronchiolar lymphoreticular hyperplasia (FIG. 25a).

TABLE-US-00018 TABLEOFSEQUENCESANDSEQIDNUMBERS(SEQUENCELISTING): SEQID NO Sequence Remarks SEQID maatamrmiisiifistymthitlcqniteefyqstcsaysrgylsalr bRSV391-2F0 NO:1 tgwytsvvtielskiqknvckstdskvklikqelerynnavielqsl (GenBankAcc.No: mqnepasfsrakrgipelihytrnstkrfyglmgkkrkrrflgfllgig AAA42808.1) saiasgvavskvlhlegevnkiknallstnkavvslsngvsvltskvl dlknyidkellpkvnnhdcrisnietviefqqknnrlleiarefsvn agittplstymltnsellslindmpitndqkklmssnvqivrqqsys imsvvkeeviayvvqlpiygvidtpcwklhtsplcttdnkegsni cltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlptd vnlcntdifntkydckimtsktdisssvitsigaivscygktkctasn knrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklegkalyikge piinyydplvfpsdefdasiaqvnakinqslafirrsdellhsvdvg ksttnvvittiiivivvvilmliavgllfycktrstpimlgkdqlsginnl sfsk SEQID mattamrmiisiifistyvthitlcqniteefyqstcsavsrgylsalrt bRSVATue51908F0 NO:2 gwytsvvtielskiqknvckstdskvklikqelerynnavvelqsl (NCBIreference mqnepasfsrakrgipelihytrnstkkfyglmgkkrkrrflgfllgi sequence: gsavasgvavskvlhlegeynkiknallstnkavvslsngvsvlts NP_048055.1) kvldlknyidkellpqynnhdcrisnietviefqqknnrlleiaref svnagittplstymltnsellslindmpitndqkklmssnvqivrq qsysimsvvkeeviayvvqlpiygvidtpcwklhtsplcttdnke gsnicltrtdrgwycdnagsvsffpqtetckvqsnrvfcdtmnsltl ptdvnlcntdifntkydckimtsktdisssvitsigaivscygktkct asnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklegkalyi kgepiinyydplvfpsdefdasiaqvnakinqslafirrsdellhsv dvgksttnvvittiiivivvvilmliavgllfycktkstpimlgkdqls ginnlsfsk SEQID matttmrmiisiiiifiyvqhitlcqniteefyqstcsavsrgylsalrt bRSVRB94F0 NO:3 gwytsvvtielskiqknvcnstdsnvklikqelerynnavvelqsl (GenBankAcc.No: mqnepasssrakrgipelihykrnstkkfyglmgkkrkrrflgfllg CAN90052.1) igsaiasgvavskvlhlegevnkiknallstnkavvslsngvsvlts kvldlknyidkellpkvnnhdcqisniatviefqqknnrlleiaref svnagittplstymltnsellslindmpitndqkklmssnvqivrq qsysimsvvkeevmayvvqlpiygvidtpcwklhtsplcttdnk egsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmns ltlptdvnlcntdifnakydckimtsktdisssvitsigaivscygkt kctasnknrgiiktfsngcdyvsnrgvdtvsvgntlyyvnklegka lyikgepiinyydplvfpsdefdasiaqvnakinqslafirrsdell hsvdvgksttnvvittiiivivvvilmliavgllfysktrstpimlgkd qlsginnlsfsk SEQID mattamrmiisiifistyvthitlcqniteefyqstcsavsrgylsalrt bRSVRB94F-11F0 NO:4 gwytsvvtielskiqknvcnstdsnvklikqelerynnavvelqsl (GenBankAcc.No: mqnepasssrakrgipelihykrnstkkfyglmgkkrkrrflgfllg BAA00798.1) igsaiasgvavskvlhlegevnkiknallstnkavvslsngvsvlts kvldlknyidkellpkvnnhdckisniatviefqqknnrlleiaref svnagittplstymltnsellslindmpitndqkklmssnvqivrq qsysimsvvkeevmayvvqlpiygvidtpcwklhtsplcttdnk egsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmns ltlptdvnlcntdifnakydckimtsktdisssvitsigaivscygkt kctasnknrgiiktfsngcdyvsnrgvdtvsvgntlyyvnklegka lyikgepiinyydplvfpsdefdasiaqvnakinqslafirrsdell hsvdvgksttnvvittiiivivvvilmliavgllfysktrstpimlgkd qlsginnlsfsk SEQID matttmrmiisiilistyvphitlcqniteefyqstcsavsrgylsalrt bRSVA51908F0 NO:5 gwytsvvtielskiqknvcngtdskvklikqelerynnavaelqsl (GenBankAcc.No: mqneptsssrakrgipesihytrnstkkfyglmgkkrkrrflgfllgi AAA42804.1) gsaiasgvavskvlhlegevnkiknallstnkavvslsngvsvltsk vldlknyidkellpkvnnhdcrisniatviefqqknnrlleiarefsv nagittplstymltnsellsiindmpitndqkklmsvcqivrqqsys imsvlreviayvvqlplygvidtpcwklhtsplcttdnkegsniclt rtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlptdvnl cntdifnskydckimtsktdisssvitsigaivscygktkctasnknr giiktfsngcdyvsnkgvdtvsvgntlyyvnklegkalyikgepiin yynplvfpsdefdasiaqvnakinqslafirrsdellhsvdvgkstt nvvittiiivivvvilmlitvgllfycktrstpimlgkdqlssinnlsfsk SEQID mrmiisiilistyvphitlcqniteefyqstcsaysrgylsalrtgwyts bRSVA375F0 NO:6 vvtielskiqknvcngtdskvklikqelerynnavvelqslmqne (GenBankAcc.No: ptsssrakrgipesihytrnstkkfyglmgkkrkrrflgfllgigsaias ACL80037.1) gvayskvlhlegevnkiknallstnkavvslsngvsvltskvldlkn yidkkllpkvnnhdcrisnietviefqqknnrlleiarefsvnagitt plstymltnsellslindmpitndqkklmssnvqivrqqsysims vvkeeviayvvqlpiygvidtpcwkvhtsplcttdnkegsnicltr tdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlptdvnl cntdifntkydckimtsktdisssvitsigaivscygktkctasnknr giiktfsngcdyvsnkgvdtvsvgntlyyvnklegkalyikgepiin yynplvfgtyefdasiaqvnak SEQID mgttamrmvisiifistyvthitlcqniteefyqstcsaysrgylsalrt bRSVFS1F0 NO:7 gwytsvvtielskiqknvckstdskvklikqelerynnavielqsl (GenBankAcc.No: mqnepasfsrakrgipelihyprnstkrfyglmgkkrkrrflgfllgi AAB28458.1) gsaiasgvayskvlhlegevnkiknallstnkavvslsngvsvltsk yldknyidkellpkvnnhdcrisnigtviefqqknnrlleiarefsv nagittplstymltnsellslindmpitndqkklmssnvqivrqqs ysimsvvkeeviayevqlpiygvidtpcwkihtsplcttdnkegs nicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpt dvnlcntdifntkydckimtsktdisssvitsigaivscygktkctas nknrgiiktfpigcdyvsnkgvdtvsvgntlyyvnklegkalyikg epiinyydplvfpsdefdasiaqvnakinqslafirrsdellhsvdv gksttnvvittiiivivvvilmliavgllfycktrstpimlgkdqlsgin nlsfsk SEQID mattamtmiisiifistyvthitlcqniteefyqstcsaysrgylsalrt bRSVSnookF0 NO:8 gwytsvvtielskiqknvckstdskvklikqelerynnavvelqsl (GenBankAcc.No: mqnepasfsrakrsipelihytrnstkkfyglmgkkrkrrflgfllgi CAA76980.1) gsaiasgvayskvlhlegevnkiknallstnkavvslsngvsvltsk vldlknyidkellpkvnnhdcrisniatviefqqknnrlleiarefs vnagittplstymltnsellslindmpitndqkklmssnvqivrqq sysimsvvkeeviayvvqlpiygvidtpcwklhtsplcttdnkeg snicltrtdrgwycdnagsysffpqaetckvqsnrvfcdtmnsltl ptdvnlcntdifntkydckimtsktdisssvitsigaivscygktkct asnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklegkalyi kgepiinyydplvfpsdefdasiaqvnakinqslafirrsdellhsv dvgksttnvvittiiivivvvilmliavgllfycktrstpimlgkdqlsg innlsfsk SEQID mattamrmiisiifistyvthitlcqniteefyqstcsaysrgylsalrt bRSVATCC51908F0 NO:9 gwytsvvtielskiqknycnstdskvklikqelerynnavvelqsl (GenBanAcc.No: mqnepasfsrakrgipelihytrnstkkfyglmgkkrkrrflgfllgi AAL49399.1) gsaiasgvavskvlhlegevnkiknallstnkavvslsngvsvltsk vldlknyidkellpkvnnhdcriskietviefqqknnrlleiarefs vnagittplstymltnsellslindmpitndqkklmssnvqivrqq sysimsvvkeeviayvvqlpiygvidtpcwklhtsplcttdnkeg snicltrtdrgwycdnagsysffpqtetckvqsnrvfcdtmnsltlp tdvnlcntdifntkydckimtsktdisssvitsigaivscygktkcta snknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklegkalyik gepiinyydplvfpsdefdasiaqvnakinqslafirrsdellhsvd vgksttnvvittiiivivvvilmliavgllfycktkstpimlgkdqlsgi nnlsfsk SEQID GSGNVGLGG Linker NO:10 SEQID GSGNWGLGG Linker NO:11 SEQID GSGNIGLGG Linker NO:12 SEQID GSGGNGIGLGG Linker NO:13 SEQID GSGGSGGSGG Linker NO:14 SEQID GSGNVLGG Linker NO:15 SEQID GGSG Linker NO:16 SEQID GGSGGS Linker NO:17 SEQID GGSGGSG Linker NO:18 SEQID GGSGSGG Linker NO:19 SEQID GGSGGGGSGGSG Linker NO:20 SEQID GGSGG Linker NO:21 SEQID GGSGSGSG Linker NO:22 SEQID GSGGGSG Linker NO:23 SEQID GGPGG Linker NO:24 SEQID GGGSGGGSGGGSGGG Linker NO:25 SEQID GGGSGGGSGGG Linker NO:26 SEQID GYIPEAPRDGQAYVRKDGEWVLLSTF Trimerization NO:27 domain SEQID SAIGGYIPEAPRDGQAYVRKDGEWVLLSTF Trimerization NO:28 domain SEQID SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGG Trimerization NO:29 LVPRGSH domain SEQID LVPRGS Thrombinsite NO:30 SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST 391-2sc9-10DS- NO:31 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS Cav1Q98CQ361C TDSKVKLIKQELERYNNAVIELQSLMCNEPASgsG SAIASGVAVCKVLHLEGEVNKIKNALLSTNKAVV SLSNGVSVLTFKVLDLKNYIDKELLPKLNNHDCRI SNIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTY MLTNSELLSLINDMPITNDQKKLMSSNVQIVRQ QSYSIMCVVKEEVIAYVVQLPIYGVIDTPCWKLH TSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVCSNRVFCDTMNSLTLPTDVNLCN TDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVG NTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEF DASIAQVNAKINQSLAFIRRSDELLSaiggyipeaprd gqayvrkdgewvllstflgglvprgshhhhhhsawshpqfek SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST 391-2sc9DS- NO:32 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS Cav1Q98CQ361C TDSKVKLIKQELERYNNAVIELQSLMCNEPASFSgs GSAIASGVAVCKVLHLEGEVNKIKNALLSTNKAV VSLSNGVSVLTFKVLDLKNYIDKELLPKLNNHDC RISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLST YMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQ QSYSIMCVVKEEVIAYVVQLPIYGVIDTPCWKLH TSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVCSNRVFCDTMNSLTLPTDVNLCN TDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVG NTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEF DASIAQVNAKINQSLAFIRRSDELLSaiggyipeaprd gqayvrkdgewvllstflgglvprgshhhhhhsawshpqfek SEQID MDSKGSSQKGSRLLILLVVSNLLLPQGVVGQNI ATue51908sc9- NO:33 TEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKI 10DS-Cav1 QKNVCKSTDSKVKLIKQELERYNNAVVELQSLM A149C-Y458C QNEPASgsGSAVcSGVAVCKVLHLEGEVNKIKNA LLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKELLP QLNNHDCRISNIETVIEFQQKNNRLLEIAREFSVN AGITTPLSTYMLTNSELLSLINDMPITNDQKKLMS SNVQIVRQQSYSIMCVVKEEVIAYVVQLPIYGVI DTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWY CDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTL PTDVNLCNTDIENTKYDCKIMTSKTDISSSVITSIG AIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYcVNKLEGKALYIKGEPIINYYD PLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLSA IGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLV PRGSHHHHHHSAWSHPQFEK SEQID HHHHHH Histag NO:34 SEQID WSHPQFEK Streptag NO:35 SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST bRSV391-2DSCav1 NO:36 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS TDSKVKLIKQELERYNNAVIELQSLMQNEPASFSR AKRGIPELIHYTRNSTKRFYGLMGKKRKRRFLGFL LGIGSAIASGVAVCKVLHLEGEVNKIKNALLSTNK AVVSLSNGVSVLTFKVLDLKNYIDKELLPKLNNH DCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTP LSTYMLINSELLSLINDMPITNDQKKLMSSNVQI VRQQSYSIMCVVKEEVIAYVVQLPIYGVIDTPCW KLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAG SVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVN LCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCY GKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTV SVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPS DEFDASIAQVNAKINQSLAFIRRSDELLSAIGGYIP EAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSH HHHHHSAWSHPQFEK SEQID MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNI bRSVATue51908 NO:37 TEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKI DSCav1 QKNVCKSTDSKVKLIKQELERYNNAVVELQSLM QNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKK RKRRFLGFLLGIGSAVASGVAVCKVLHLEGEVNK IKNALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDK ELLPQLNNHDCRISNIETVIEFQQKNNRLLEIAREF SVNAGITTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMCVVKEEVIAYVVQLPIY GVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRG WYCDNAGSVSFFPQTETCKVQSNRVFCDTMNS LTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVIT SIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVS NKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINY YDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLG GLVPRGSHHHHHHSAWSHPQFEK SEQID MPMGSLQPLATLYLLGMLVASVLAAQNITEEFY bRSVRB94DSCav1 NO:38 QSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNV CNSTDSNVKLIKQELERYNNAVVELQSLMQNEP ASSSRAKRGIPELIHYKRNSTKKFYGLMGKKRKRR FLGFLLGIGSAIASGVAVCKVLHLEGEVNKIKNAL LSTNKAVVSLSNGVSVLTFKVLDLKNYIDKELLPK LNNHDCQISNIATVIEFQQKNNRLLEIAREFSVN AGITTPLSTYMLTNSELLSLINDMPITNDQKKLMS SNVQIVRQQSYSIMCVVKEEVMAYVVQLPIYGVI DTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWY CDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTL PTDVNLCNTDIFNAKYDCKIMTSKTDISSSVITSIG AIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNR GVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYD PLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLSA IGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLV PRGSHHHHHHSAWSHPQFEK SEQID MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNI RB94DS-Cav1sc9 NO:39 TEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKI A149CY458C QKNVCNSTDSNVKLIKQELERYNNAVVELQSLM QNEPASSSgsGSAlcSGVAVCKVLHLEGEVNKIKN ALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKELL PKLNNHDCQISNIATVIEFQQKNNRLLEIAREFSV NAGITTPLSTYMLTNSELLSLINDMPITNDQKKL MSSNVQIVRQQSYSIMCVVKEEVMAYVVQLPIY GVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRG WYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPTDVNLCNTDIFNAKYDCKIMTSKTDISSSVIT SIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVS NRGVDTVSVGNTLYcVNKLEGKALYIKGEPIINY YDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLG GLVPRGSHHHHHHSAWSHPQFEK SEQID MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNI RB94sc9DS-Cav1 NO:40 TEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKI N183GCN428C QKNVCNSTDSNVKLIKQELERYNNAVVELQSLM QNEPASSSgsGSAIASGVAVCKVLHLEGEVNKIKN ALLSTNKAVVSLSgcGVSVLTFKVLDLKNYIDKELL PKLNNHDCQISNIATVIEFQQKNNRLLEIAREFSV NAGITTPLSTYMLTNSELLSLINDMPITNDQKKL MSSNVQ1VRQQSYSIMCVVKEEVMAYVVQLPIY GVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRG WYCDNAGSVSFFPQAETCKVQSNRVFCDTMNS LTLPTDVNLCNTDIFNAKYDCKIMTSKTDISSSVIT SIGAIVSCYGKTKCTASNKcRGIIKTFSNGCDYVS NRGVDTVSVGNTLYYVNKLEGKALYIKGEPIINY YDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDEL LSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLG GLVPRGSHHHHHHSAWSHPQFEK SEQID MELLILKANAITTILTAVTFCFASGQNITEEFYQST sc9-10_bRSV(RB94) NO:41 CSAVSRGYLSALRTGWYTSVITIELSKIQKNVCNS DS- TDSNVKLIKQELERYNNAVVELQSLMQSTPATGS Cav1_fd_hp2_ GSAIASGVAVCKVLHLEGEVNKIKNALLSTNKAV fp2_ig1 VSLSNGVSVLTFKVLDLKNYIDKELLPILNNHDC QISNIATVIEFQQKNNRLLEIAREFSVNAGVTTPV STYMLTNSELLSLINDMPITNDQKKLMSSNVQIV RQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKL HTSPLCTTDNKEGSNICLTRTDRGWYCDNAGS VSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNL CNTDIFNAKYDCKIMTSKTDVSSSVITSLGAIVSC YGKTKCTASNKNRGIIKTFSNGCDYVSNRGVDT VSVGNTLYYVNKQEGKSLYIKGEPIINYYDPLVFP SDEFDASIAQVNAKINQSLAFIRRSDELLSAIGGYI PEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGS HHHHHHSAWSHPQFEK SEQID MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGqnite 391-2-site hRSV NO:42 efyqstcsaysrgylsalrtgwytsvitielsKIQKNVCKSTDSK bovsurfDS-Cav1- VKLIKQELERYNNAVlelqllmqstpatnngsgsaiasgVA BZGJ9Long VCKvlhlegevnkiknallstnkavvslsngvsVLTFkvldlkny idkELLPKLNNHDCRISNIEtviefqqknnrlleitrefsvna gvttpvstymltnsellslindmpitndqkklmssnvqivrqqsyS IMCllkeevlayvvqlpiygvidtpcwklhtsplcttdnkegsnic ltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsrtlptdv nlcntdifntkydckimtsktdvsssvitslgaivscygktkctasnk nrgiiktfsngcdyvsnkgvdtvsvgntlyyvnkqegkslyvkgep iinfydplvfpsdefdasisqvnekinqslafirrsdeLLhnvnagk sttGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGL VPRGSHHHHHHSAWSHPQFEK SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST bRSV391-2sc9 NO:43 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS DS-Cav1 TDSKVKLIKQELERYNNAVIELQSLMQNEPASFS A149CY458C GSGSAlcSGVAVCKVLHLEGEVNKIKNALLSTNK AVVSLSNGVSVLITKVLDLKNYIDKELLPKLNNH DCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTP LSTYMLTNSELLSLINDMPITNDQKKLMSSNVQI VRQQSYSIMCVVKEEVIAYVVQLPIYGVIDTPCW KLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAG SVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVN LCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCY GKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTV SVGNTLYcVNKLEGKALYIKGEPIINYYDPLVFPS DEFDASIAQVNAKINQSLAFIRRSDELLsaiggyipea prdgqayvrkdgewvllstflgglvprgshhhhhhsawshpqfe k SEQID MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNI bRSVATue51908 NO:44 TEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKI sc9-10DS-Cav1 QKNVCKSTDSKVKLIKQELERYNNAVVELQSLM N183GCN428C QNEPASgsGSAVASGVAVCKVLHLEGEVNKIKN ALLSTNKAVVSLSgcGVSVLTFKVLDLKNYIDKELL PQLNNHDCRISNIETVIEFQQKNNRLLEIAREFSV NAGITTPLSTYMLTNSELLSLINDMPITNDQKKL MSSNVQIVRQQSYSIMCVVKEEVIAYVVQLPIYG VIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRG WYCDNAGSVSFFPQTETCKVQSNRVFCDTMNS LTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVIT SIGAIVSCYGKTKCTASNKcRGIIKTFSNGCDYVS NKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINY YDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDEL Lsaiggyipeaprdgqayvrkdgewvllstflgglvprgshhhhh hsawshpqfek SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST bRSV391-2postF NO:45 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS TDSKVKLIKQELERYNNAVIELQSLMQNEPASFSR AKRGIPELIHYTRNSTKRFYGLMGKKRKRRAIASG VAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGV SVLTSKVLDLKNYIDKELLPKVNNHDCRISNIETVI EFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSE LLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMS VVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTD NKEGSNICLTRTDRGWYCDNAGSVSFFPQAETC KVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKY DCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNK NRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVN KLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVN AKINQSLAFIRRSDELLGLEVLFQGPHHHHHHH HSAWSHPQFEK SEQID MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNI bRSVATue51908 NO:46 TEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKI postF QKNVCKSTDSKVKLIKQELERYNNAVVELQSLM QNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKK RKRRAVASGVAVSKVLHLEGEVNKIKNALLSTNK AVVSLSNGVSVLTSKVLDLKNYIDKELLPQVNNH DCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTP LSTYMLTNSELLSLINDMPITNDQKKLMSSNVQI VRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCW KLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAG SVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNL CNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCY GKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTV SVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPS DEFDASIAQVNAKINQSLAFIRRSDELLGLEVLFQ GPHHHHHHHHSAWSHPQFEK SEQID MDSKGSSQKGSRLLLLLVVSNLLLPQGVVGQNI bRSVRB94postF NO:47 TEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKI QKNVCNSTDSNVKLIKQELERYNNAVVELQSLM QNEPASSSRAKRGIPELIHYKRNSTKKFYGLMGK KRKRRAIASGVAVSKVLHLEGEVNKIKNALLSTNK AVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNH DCQISNIATVIEFQQKNNRLLEIAREFSVNAGITTP LSTYMLTNSELLSLINDMPITNDQKKLMSSNVQI VRQQSYSIMSVVKEEVMAYVVQLPIYGVIDTPC WKLHTSPLCTTDNKEGSNICLTRTDRGWYCDN AGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTD VNLCNTDIFNAKYDCKIMTSKTDISSSVITSIGAIV SCYGKTKCTASNKNRGIIKTFSNGCDYVSNRGV DTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLV FPSDEFDASIAQVNAKINQSLAFIRRSDELLGLEVL FQGPHHHHHHHHSAWSHPQFEK SEQID MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYT ferritin NO:48 HSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVP polypeptide VQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINN IVDHAIKGKDHATFNFLQWYVAEQHEEEVLFKD ILDKIELIGNENHGLYLADQYVKGIAKSRKS SEQID MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGR encapsulin NO:49 KFVDVEGPYGWEYAAHPLGEVEVLSDENEVVK polypeptide WGLRKSLPLIELRATFTLDLWELDNLERGKPNVD LSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEER KIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINT DRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIE DALVVSERGGDFKLILGQDLSIGYEDREKDAVRL FITETFTFQVVNPEALILLKF SEQID EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM MPE8HeavyChain NO:50 NWVRQAPGKGLEWVSSISASSSYSDYADSAKGR Variableregion FTISRDNAKTSLFLQMNSLRAEDTAIYFCARARAT GYSSITPYFDIWGQGTLVTVSS SEQID QSVVTQTPSVSGAPGQRVTISCTGSSSNIGAGY MPE8LightChain NO:51 DVHWYQQLPGTAPKLLIYDNNNRPSGVPDRFS Variableregion ASKSGTSASLAITGLQAEDEADYYCQSYDRNLSG VFGTGTKVTVL SEQID DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLN AM14LightChain NO:52 WYHQKPGKVPELLMHDASNLETGVPSRFSGRG Variableregion SGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFG GGTKVEIKR SEQID EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYA AM14HeavyChain NO:53 MHWVRQAPGKGLEWVAVISYDGENTYYADSV Variableregion KGRFSISRDNSKNTVSLQMNSLRPEDTALYYCAR DRIVDDYYYYGMDVWGQGATVTVSS SEQID DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYL D25LightChain NO:54 NWYQQKPGKAPKLLIYVASNLETGVPSRFSGSG Variableregion SGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGG GTKVEIK SEQID QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNY D25HeavyChain NO:55 IINWLRQAPGQGPEWMGGIIPVLGTVHYAPKF Variableregion QGRVTITADESTDTAYIHLISLRSEDTAMYYCATE TALVVSTTYLPHYFDNWGQGTLVTVSS SEQID atggctgctactgctatgcggatgattatctcaattatttttatttcaacct 391-2sc9-10DS- NO:56 acatgactcacattaccctgtgtcagaacattaccgaggaattctac Cav1Q98C- cagagcacttgctccgccgtgtctagaggatacctgtctgctctgag Q361C_nuc gaccggctggtatacaagcgtggtcactattgagctgtccaagatcc agaaaaacgtgtgtaagagtaccgattcaaaggtcaaactgatcaa acaggagctggaaaggtataacaatgccgtgattgagctgcagag cctgatgtgcaatgaacctgctagcgggtctggaagtgccatcgctt ccggagtggccgtctgcaaggtgctgcacctggagggcgaagtca acaagatcaagaatgccctgctgtctacaaacaaagctgtggtctc actgagcaatggcgtgagtgtcctgacttttaaggtgctggacctgaa aaactacatcgataaggagctgctgccaaaactgaacaatcatga ctgtcggatcagcaatattgagacagtgattgaattccagcagaaga acaatcgactgctggagatcgcaagagaattttcagtgaacgccgg cattaccacacccctgagcacctacatgctgacaaattctgagctg ctgagtctgattaacgacatgcctatcaccaatgatcagaagaaact gatgagctccaacgtgcagatcgtcagacagcagtcctattctattat gtgcgtggtcaaggaggaagtgatcgcctacgtggtccagctgcct atctacggcgtgatcgataccccatgctggaagctgcacacaagtc ccctgtgtactaccgacaacaaagagggctcaaatatctgcctgac aaggactgaccgcggctggtactgtgataacgcagggagtgtgtca ttctttccacaggccgaaacttgcaaggtgtgctccaacagggtcttc tgtgataccatgaattctctgaccctgcccacagacgtgaacctgtg caacactgatatctttaataccaagtacgactgtaagattatgactag caagaccgacatctctagttcagtgatcacctccattggagctatcgt ctcttgctacggcaagacaaaatgtactgcatctaacaagaatcgc gggatcatcaagacattctctaacggatgtgattatgtcagtaataag ggggtcgacacagtgagcgtcggaaacactctgtactatgtgaata agctggagggcaaagccctgtacatcaaaggggaacctatcatta actactatgatccactggtgttccccagtgacgagtttgatgcatcaa ttgcccaggtgaacgctaagatcaatcagtccctggccttcatccgg agatcagacgagctgctgagcgcaattggcgggtacatccccgaa gctcctcgcgatggccaggcatatgtgcgaaaagacggggagtgg gtcctgctgagcaccttcctgggaggactggtgcctcgaggatccc accatcaccatcaccatagcgcttggtcccatccacagtttgaaaa g SEQID atggctgctactgctatgcggatgattatctcaattatttttatttcaacct 391-2sc9DS-Cav1 NO:57 acatgactcacattaccctgtgtcagaacattaccgaggaattctac Q98C-Q361C_nuc cagagcacttgctccgccgtgtctagaggatacctgtctgctctgag gaccggctggtatacaagcgtggtcactattgagctgtccaagatcc agaaaaacgtgtgtaagagtaccgattcaaaggtcaaactgatcaa acaggagctggaaaggtataacaatgccgtgattgagctgcagag cctgatgtgcaatgaacctgctagcttctccgggtctggaagtgccat cgcttccggagtggccgtctgcaaggtgctgcacctggagggcga agtcaacaagatcaagaatgccctgctgtctacaaacaaagctgtg gtctcactgagcaatggcgtgagtgtcctgacttttaaggtgctggac ctgaaaaactacatcgataaggagctgctgccaaaactgaacaat catgactgtcggatcagcaatattgagacagtgattgaattccagca gaagaacaatcgactgctggagatcgcaagagaattttcagtgaac gccggcattaccacacccctgagcacctacatgctgacaaattctg agctgctgagtctgattaacgacatgcctatcaccaatgatcagaag aaactgatgagctccaacgtgcagatcgtcagacagcagtcctatt ctattatgtgcgtggtcaaggaggaagtgatcgcctacgtggtccag ctgcctatctacggcgtgatcgataccccatgctggaagctgcaca caagtcccctgtgtactaccgacaacaaagagggctcaaatatctg cctgacaaggactgaccgcggctggtactgtgataacgcagggag tgtgtcattctttccacaggccgaaacttgcaaggtgtgctccaacag ggtcttctgtgataccatgaattctctgaccctgcccacagacgtgaa cctgtgcaacactgatatctttaataccaagtacgactgtaagattat gactagcaagaccgacatctctagttcagtgatcacctccattggag ctatcgtctcttgctacggcaagacaaaatgtactgcatctaacaag aatcgcgggatcatcaagacattctctaacggatgtgattatgtcagt aataagggggtcgacacagtgagcgtcggaaacactctgtactatg tgaataagctggagggcaaagccctgtacatcaaaggggaaccta tcattaactactatgatccactggtgttccccagtgacgagtttgatgc atcaattgcccaggtgaacgctaagatcaatcagtccctggccttca tccggagatcagacgagctgctgagcgcaattggcgggtacatcc ccgaagctcctcgcgatggccaggcatatgtgcgaaaagacggg gagtgggtcctgctgagcaccttcctgggaggactggtgcctcgag gatcccaccatcaccatcaccatagcgcttggtcccatccacagttt gaaaagtga SEQID atggattccaaggggagctcccagaaaggatctaggctgctgctgc ATue51908sc9- NO:58 tgctggtggtctccaacctgctgctgccacagggagtggtcggaca 10DS-Cav1 gaatatcacagaggaattctaccagagcacttgctccgcagtgtctc A149C- ggggatacctgtctgccctgagaactggctggtatacctctgtggtca Y458C_nuc caattgagctgagtaagatccagaagaacgtgtgcaaaagtaccg actcaaaggtcaaactgatcaagcaggagctggaacggtataaca atgccgtggtcgagctgcagagcctgatgcagaacgaacctgcttc tggcagcggatctgccgtgtgtagtggagtggccgtctgcaaagtgc tgcatctggagggcgaagtcaacaagatcaagaatgcactgctgtc tactaacaaggccgtggtctcactgagcaatggcgtgagtgtcctga cctttaaggtgctggacctgaaaaactacatcgataaggagctgctg cctcagctgaacaatcacgattgtaggatctccaatattgagacagt gattgaattccagcagaagaacaatcgcctgctggagatcgctcga gagttcagcgtgaacgcaggcattaccacaccactgtcaacatac atgctgactaattcagagctgctgagcctgattaacgacatgcccat caccaatgatcagaagaaactgatgtctagtaacgtgcagatcgtc cgccagcagtcctattctattatgtgcgtggtcaaggaggaagtgatc gcatacgtggtccagctgcctatctacggcgtgatcgataccccatg ctggaaactgcatacatctcccctgtgcactaccgacaacaagga aggaagtaatatttgcctgacaagaactgacaggggctggtactgtg ataacgctggcagcgtgagcttcttccctcagaccgaaacatgcaa ggtgcagagcaaccgggtcttctgtgatacaatgaattccctgactct gccaaccgacgtgaacctgtgcaacaccgatatctttaatacaaag tacgactgtaagatcatgacaagcaagactgacatctcaagctccg tgatcacaagtattggagctatcgtgtcatgctacggcaagaccaaa tgtacagcatctaacaaaaacagagggatcattaagactttctcaaa cggatgtgattatgtgagcaacaagggggtcgacactgtgagcgtc ggaaacaccctgtactgtgtgaataagctggagggcaaagccctgt acatcaagggggaacccatcattaactactatgatccactggtgttc cccagcgacgagtttgatgcatccattgcccaggtgaacgccaaa atcaatcagtccctggcttttattaggcgctccgacgagctgctgtct gccattggcgggtacatccccgaagcccctagggatggccaggct tatgtgcgcaaggacggggagtgggtcctgctgtcaaccttcctggg aggactggtgccaagaggctcccaccatcaccatcaccatagcg cctggtcccaccctcagtttgaaaag SEQID atggattctaagggttccagccagaaaggttccaggctgctgctgct RB94DS-Cav1sc9 NO:59 gctggtggtgagcaatctgctgctgcctcagggagtggtgggacag A149C-Y458C_nuc aacatcaccgaggagttctaccagtcaacctgcagcgccgtgagc cggggctacctgagcgcactgagaaccggatggtatacatccgtg gtcactattgagctgtctaagatccagaaaaacgtgtgtaattctaca gatagtaacgtcaagctgatcaaacaggagctggaaaggtataac aatgctgtggtcgagctgcagtccctgatgcagaacgaacctgcca gcagcagcggcagcggcagcgccatctgttctggggtggcagtct gcaaggtgctgcatctggagggagaagtcaacaagatcaaaaatg cactgctgagtactaacaaagccgtggtcagtctgtcaaatggggtg agcgtcctgacctttaaggtgctggacctgaaaaactacatcgataa ggagctgctgcccaaactgaacaatcacgactgtcagatcagcaa tattgccactgtgattgagttccagcagaagaacaatcgcctgctgg agatcgcccgggagttcagcgtgaacgcaggcattaccacacca ctgtccacctacatgctgacaaatagtgagctgctgtcactgattaac gacatgcccatcaccaatgatcagaagaaactgatgagttcaaac gtgcagatcgtcaggcagcagagctattccattatgtgcgtggtcaa ggaggaagtgatggcctacgtggtccagctgcctatctacggcgtg atcgatacaccatgctggaagctgcatacttcacccctgtgtactac cgacaacaaagaggggagcaatatctgcctgacaagaactgaca ggggatggtactgtgataacgctggctctgtgagtttctttcctcaggc agaaacctgcaaggtgcagtctaaccgcgtcttctgtgatacaatga atagtctgaccctgccaacagacgtgaacctgtgcaatacagatat ctttaatgccaagtacgactgtaagattatgacttccaagaccgacat cagctcctctgtgatcacttctattggggccatcgtcagttgctacgg aaagacaaaatgtactgctagcaacaagaatcggggcatcatcaa gacattcagtaacgggtgtgattatgtgtcaaatagaggcgtggaca ctgtgagcgtcgggaacaccctgtactgtgtgaataagctggaggg aaaagctctgtacatcaagggcgaacctatcattaactactatgatc cactggtgttcccctcagacgagtttgatgcaagcattgcccaggtg aacgccaaaatcaatcagtctctggcttttattaggcgcagcgacga gctgctgtccgcaattggcgggtacatccccgaagcccctagggat ggacaggcttatgtgcgcaaggacggcgagtgggtcctgctgtcca ccttcctgggaggcctggtgcccagaggctctcaccatcaccatca ccattcagcctggagccaccctcagtttgaaaaa SEQID atggattctaagggttccagccagaaaggttccaggctgctgctgct RB94sc9DS-Cav1 NO:60 gctggtggtgagcaatctgctgctgcctcagggagtggtgggacag N183GC-N428C_ aacatcaccgaggagttctaccagtcaacctgcagcgccgtgagc nuc cggggctacctgagcgcactgagaaccggatggtatacatccgtg gtcactattgagctgtctaagatccagaaaaacgtgtgtaattctaca gatagtaacgtcaagctgatcaaacaggagctggaaaggtataac aatgctgtggtcgagctgcagtccctgatgcagaacgaacctgcca gcagcagcggcagcggcagcgccatcgcttctggggtggcagtct gcaaggtgctgcatctggagggagaagtcaacaagatcaaaaatg cactgctgagtactaacaaagccgtggtcagtctgtcaggttgtggg gtgagcgtcctgacctttaaggtgctggacctgaaaaactacatcga taaggagctgctgcccaaactgaacaatcacgactgtcagatcag caatattgccactgtgattgagttccagcagaagaacaatcgcctgc tggagatcgcccgggagttcagcgtgaacgcaggcattaccacac cactgtccacctacatgctgacaaatagtgagctgctgtcactgatta acgacatgcccatcaccaatgatcagaagaaactgatgagttcaa acgtgcagatcgtcaggcagcagagctattccattatgtgcgtggtc aaggaggaagtgatggcctacgtggtccagctgcctatctacggcg tgatcgatacaccatgctggaagctgcatacttcacccctgtgtacta ccgacaacaaagaggggagcaatatctgcctgacaagaactgac aggggatggtactgtgataacgctggctctgtgagtttctttcctcagg cagaaacctgcaaggtgcagtctaaccgcgtcttctgtgatacaatg aatagtctgaccctgccaacagacgtgaacctgtgcaatacagata tctttaatgccaagtacgactgtaagattatgacttccaagaccgaca tcagctcctctgtgatcacttctattggggccatcgtcagttgctacgg aaagacaaaatgtactgctagcaacaagtgtcggggcatcatcaa gacattcagtaacgggtgtgattatgtgtcaaatagaggcgtggaca ctgtgagcgtcgggaacaccctgtactatgtgaataagctggaggg aaaagctctgtacatcaagggcgaacctatcattaactactatgatc cactggtgttcccctcagacgagtttgatgcaagcattgcccaggtg aacgccaaaatcaatcagtctctggcttttattaggcgcagcgacga gctgctgtccgcaattggcgggtacatccccgaagcccctagggat ggacaggcttatgtgcgcaaggacggcgagtgggtcctgctgtcca ccttcctgggaggcctggtgcccagaggctctcaccatcaccatca ccattcagcctggagccaccctcagtttgaaaaa SEQID atggctgctactgctatgcggatgattatctcaattatttttatttcaacct bRSV391-2sc9 NO:61 acatgactcacattaccctgtgtcagaacattaccgaggaattctac DS-Cav1 cagagcacttgctccgccgtgtctagaggatacctgtctgctctgag A149C- gaccggctggtatacaagcgtggtcactattgagctgtccaagatcc Y458C_nuc agaaaaacgtgtgtaagagtaccgattcaaaggtcaaactgatcaa acaggagctggaaaggtataacaatgccgtgattgagctgcagag cctgatgcagaatgaacctgctagcttctccgggtctggaagtgcca tctgttccggagtggccgtctgcaaggtgctgcacctggagggcga agtcaacaagatcaagaatgccctgctgtctacaaacaaagctgtg gtctcactgagcaatggcgtgagtgtcctgacttttaaggtgctggac ctgaaaaactacatcgataaggagctgctgccaaaactgaacaat catgactgtcggatcagcaatattgagacagtgattgaattccagca gaagaacaatcgactgctggagatcgcaagagaattttcagtgaac gccggcattaccacacccctgagcacctacatgctgacaaattctg agctgctgagtctgattaacgacatgcctatcaccaatgatcagaag aaactgatgagctccaacgtgcagatcgtcagacagcagtcctatt ctattatgtgcgtggtcaaggaggaagtgatcgcctacgtggtccag ctgcctatctacggcgtgatcgataccccatgctggaagctgcaca caagtcccctgtgtactaccgacaacaaagagggctcaaatatctg cctgacaaggactgaccgcggctggtactgtgataacgcagggag tgtgtcattctttccacaggccgaaacttgcaaggtgcagtccaaca gggtcttctgtgataccatgaattctctgaccctgcccacagacgtga acctgtgcaacactgatatctttaataccaagtacgactgtaagatta tgactagcaagaccgacatctctagttcagtgatcacctccattgga gctatcgtctcttgctacggcaagacaaaatgtactgcatctaacaa gaatcgcgggatcatcaagacattctctaacggatgtgattatgtcag taataagggggtcgacacagtgagcgtcggaaacactctgtactgt gtgaataagctggagggcaaagccctgtacatcaaaggggaacct atcattaactactatgatccactggtgttccccagtgacgagtttgatg catcaattgcccaggtgaacgctaagatcaatcagtccctggccttc atccggagatcagacgagctgctgagcgcaattggcgggtacatc cccgaagctcctcgcgatggccaggcatatgtgcgaaaagacgg ggagtgggtcctgctgagcaccttcctgggaggactggtgcctcga ggatcccaccatcaccatcaccatagcgcttggtcccatccacagt ttgaaaag SEQID atggattccaaggggagctcccagaaaggatctaggctgctgctgc bRSVATue51908 NO:62 tgctggtggtctccaacctgctgctgccacagggagtggtcggaca sc9-10DS-Cav1 gaatatcacagaggaattctaccagagcacttgctccgcagtgtctc N183GC-N428C_ ggggatacctgtctgccctgagaactggctggtatacctctgtggtca nuc caattgagctgagtaagatccagaagaacgtgtgcaaaagtaccg actcaaaggtcaaactgatcaagcaggagctggaacggtataaca atgccgtggtcgagctgcagagcctgatgcagaacgaacctgcttc tggcagcggatctgccgtggctagtggagtggccgtctgcaaagtg ctgcatctggagggcgaagtcaacaagatcaagaatgcactgctgt ctactaacaaggccgtggtctcactgagcggctgcggcgtgagtgt cctgacctttaaggtgctggacctgaaaaactacatcgataaggag ctgctgcctcagctgaacaatcacgattgtaggatctccaatattgag acagtgattgaattccagcagaagaacaatcgcctgctggagatcg ctcgagagttcagcgtgaacgcaggcattaccacaccactgtcaa catacatgctgactaattcagagctgctgagcctgattaacgacatg cccatcaccaatgatcagaagaaactgatgtctagtaacgtgcaga tcgtccgccagcagtcctattctattatgtgcgtggtcaaggaggaag tgatcgcatacgtggtccagctgcctatctacggcgtgatcgatacc ccatgctggaaactgcatacatctcccctgtgcactaccgacaaca aggaaggaagtaatatttgcctgacaagaactgacaggggctggta ctgtgataacgctggcagcgtgagcttcttccctcagaccgaaacat gcaaggtgcagagcaaccgggtcttctgtgatacaatgaattccctg actctgccaaccgacgtgaacctgtgcaacaccgatatctttaatac aaagtacgactgtaagatcatgacaagcaagactgacatctcaag ctccgtgatcacaagtattggagctatcgtgtcatgctacggcaaga ccaaatgtacagcatctaacaaatgcagagggatcattaagactttc tcaaacggatgtgattatgtgagcaacaagggggtcgacactgtga gcgtcggaaacaccctgtactatgtgaataagctggagggcaaag ccctgtacatcaagggggaacccatcattaactactatgatccactg gtgttccccagcgacgagtttgatgcatccattgcccaggtgaacgc caaaatcaatcagtccctggcttttattaggcgctccgacgagctgct gtctgccattggcgggtacatccccgaagcccctagggatggcca ggcttatgtgcgcaaggacggggagtgggtcctgctgtcaaccttcc tgggaggactggtgccaagaggctcccaccatcaccatcaccata gcgcctggtcccaccctcagtttgaaaag SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST 391-2sc9DS- NO:63 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS Cav1N88C TDSKVKLIKQELERYNcAVIELQSLMQNEPASFSgs N254C GSAIASGVAVCKVLHLEGEVNKIKNALLSTNKAV VSLSNGVSVLTFKVLDLKNYIDKELLPKLNNHDC RISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLST YMLTcSELLSLINDMPITNDQKKLMSSNVQIVRQ QSYSIMCVVKEEVIAYVVQLPIYGVIDTPCWKLH TSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVS FFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCN TDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVG NTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEF DASIAQVNAKINQSLAFIRRSDELLSAIGGYIPEAP RDGQAYVRKDGEWVLLSTFLGGLVPRGSHHH HHHSAWSHPQFEK SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST 391-2sc9DS- NO:64 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS Cav1sc9E92C TDSKVKLIKQELERYNNAVlcLQSLMQNEPASFSg N254C sGSAIASGVAVCKVLHLEGEVNKIKNALLSTNKA VVSLSNGVSVLITKVLDLKNYIDKELLPKLNNHD CRISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLS TYMLTcSELLSLINDMPITNDQKKLMSSNVQIVR QQSYSIMCVVKEEVIAYVVQLPIYGVIDTPCWKL HTSPLCTTDNKEGSNICLTRTDRGWYCDNAGS VSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNL CNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCY GKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTV SVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPS DEFDASIAQVNAKINQSLAFIRRSDELLSAIGGYIP EAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSH HHHHHSAWSHPQFEK SEQID MAATAMRMIISIIFISTYMTHITLCQNITEEFYQST 391-2sc9DS- NO:65 CSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKS Cav1sc9S238C TDSKVKLIKQELERYNNAVIELQSLMQNEPASFSg Q279C sGSAIASGVAVCKVLHLEGEVNKIKNALLSTNKA VVSLSNGVSVLTFKVLDLKNYIDKELLPKLNNHD CRISNIETVIEFQQKNNRLLEIAREFcVNAGITTPLS TYMLTNSELLSLINDMPITNDQKKLMSSNVcIVR QQSYSIMCVVKEEVIAYVVQLPIYGVIDTPCWKL HTSPLCTTDNKEGSNICLTRTDRGWYCDNAGS VSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNL CNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCY GKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTV SVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPS DEFDASIAQVNAKINQSLAFIRRSDELLSAIGGYIP EAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSH HHHHHSAWSHPQFEK