Modified respiratory syncytial virus f proteins and uses thereof

Abstract

A respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation for increasing the pre-fusion conformational stability of the RSV F protein selected from the group consisting of: (1) introducing one or more disulfide bonds in the RSV F protein; (2) introducing one or more amino acid mutations in the RSV F protein for filling a hydrophobic cavity; (3) introducing one or more amino acid mutations in the RSV F protein for electrostatic remodeling; and (4) removing the p27 peptide segment in the RSV F protein. The RSV F protein has high stability as a recombinant protein antigen, is easy to store and transport, has improved expression, is easy to achieve yield improvement, and the modified RSV F protein has higher immunogenicity and can produce better immune protection.

Description

Modified respiratory syncytial virus F protein and its use Technical Field

[0001] The present disclosure relates to the fields of medicine and immunology. Specifically, one aspect of the present disclosure relates to a modified respiratory syncytial virus F protein and use thereof in preventing and treating respiratory syncytial virus infection. Background Art

[0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus in the family Paramyxoviridae. It has only one serotype, divided into two subtypes, A and B. RSV primarily encodes 10 proteins: three transmembrane proteins: fusion protein (F), adhesion protein (G), and small hydrophobic protein (SH); two matrix proteins, M1 and M2; three proteins that bind to viral RNA to form the nucleocapsid (N, P, and L); and two nonstructural proteins (NS1 and NS2). The G and F surface proteins play important roles in RSV attachment and fusion, respectively, and are the most important antigenic sites for the production of neutralizing antibodies, as well as the primary targets for inducing immunogenicity and antiviral activity.

[0003] The virus is highly contagious, with the source of infection being secretions or contaminants containing the virus. It is primarily transmitted through droplets and direct contact, and is more common in newborns and infants under six months old. Symptoms include high fever, rhinitis, pharyngitis, and laryngitis, followed by bronchiolitis and pneumonia. A small number of children may also develop complications such as otitis media, pleurisy, and myocarditis. Infected adults and older children primarily present with upper respiratory tract infections. Currently, there is no commercial vaccine for RSV. High-income countries such as the United States use the neutralizing monoclonal antibody Palivizumab for immunization prevention, to a certain extent, to prevent RSV disease in extremely premature infants or patients with congenital heart disease. However, the high cost of monthly medication makes it difficult to use in low-income countries. Therefore, the development of an effective RSV vaccine is crucial for the prevention and control of the disease.

[0004] Because the F protein elicits stronger neutralizing antibodies than G protein antibodies and exhibits less variability, current RSV vaccine candidate development focuses on the highly conserved F protein. However, the RSV F protein possesses two conformations. The prefusion conformation (PreF) is metastable. Upon membrane fusion or spontaneous triggering, the prefusion conformation undergoes an irreversible rearrangement to a nonfunctional postfusion conformation. Prior to infecting human cells, it resides in the first conformation. During infection, it transitions to the second conformation. Although approximately 50% of the surface area is shared between the prefusion and postfusion conformations (sites I–IV), the antigenic sites most sensitive to neutralization are located only in the prefusion conformation (sites V and Φ). Therefore, the F protein in the second conformation elicits a weak antibody response. Therefore, engineering the F protein to stabilize it in the prefusion conformation is a key strategy for vaccine development. Pfizer's PF-06928316 RSV vaccine has entered Phase III clinical trials and has demonstrated positive results in pregnant women. Similarly, GlaxoSmithKline's RSV PreF3OA vaccine, based on Phase III clinical data, demonstrates strong efficacy in people aged 60 and older. Both vaccines are recombinant protein vaccines with a modified F protein as their primary antigen.

[0005] At present, the research and development of RSV vaccines in China is still in its infancy. Given the harm caused by RSV, the development of an effective RSV vaccine is of great significance to the prevention and control of RSV in my country.

[0006] Summary of the Invention

[0007] The present disclosure provides a modified respiratory syncytial virus F protein and its use in preventing and treating respiratory syncytial virus infection. Furthermore, the present disclosure provides a nucleic acid sequence encoding the F protein, a vector comprising the nucleic acid sequence, and a corresponding host cell, as well as immunogenic compositions, vaccine compositions, pharmaceutical compositions, and kits related to the F protein, and methods and uses of the F protein in preventing and treating respiratory syncytial virus infection.

[0008] In a first aspect, the present disclosure provides a respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation selected from the following for increasing the stability of the RSV F protein prefusion conformation:

[0009] (1) introducing one or more disulfide bonds into the RSV F protein;

[0010] (2) introducing one or more amino acid mutations into the RSV F protein to fill the hydrophobic cavity;

[0011] (3) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein;

[0012] (4) Remove the p27 peptide from RSV F protein.

[0013] In some embodiments, the introduced disulfide bonds are present at one or more of the following positions:

[0014] (1) between positions 7 and 416 of the RSV F protein;

[0015] (2) between positions 15 and 358 of the RSV F protein;

[0016] (3) between positions 20 and 338 of the RSV F protein;

[0017] (4) between positions 20 and 339 of the RSV F protein;

[0018] (5) between positions 22 and 248 of the RSV F protein;

[0019] (6) between positions 23 and 283 of the RSV F protein;

[0020] (7) between positions 30 and 163 of the RSV F protein;

[0021] (8) between positions 31 and 164 of the RSV F protein;

[0022] (9) between positions 34 and 168 of the RSV F protein;

[0023] (10) between positions 34 and 272 of the RSV F protein;

[0024] (11) between positions 37 and 175 of the RSV F protein;

[0025] (12) between positions 64 and 209 of the RSV F protein;

[0026] (13) between positions 67 and 229 of the RSV F protein;

[0027] (14) between positions 78 and 123 of the RSV F protein;

[0028] (15) between positions 119 and 381 of the RSV F protein;

[0029] (16) between positions 125 and 433 of the RSV F protein;

[0030] (17) between positions 128 and 436 of the RSV F protein;

[0031] (18) between positions 133 and 266 of the RSV F protein;

[0032] (19) between positions 152 and 164 of the RSV F protein;

[0033] (20) between positions 155 and 161 of the RSV F protein;

[0034] (21) between positions 210 and 224 of the RSV F protein;

[0035] (22) between positions 216 and 254 of the RSV F protein;

[0036] (23) between positions 236 and 249 of the RSV F protein;

[0037] (24) between positions 309 and 450 of the RSV F protein;

[0038] (25) between positions 385 and 441 of the RSV F protein;

[0039] (26) between positions 418 and 441 of the RSV F protein;

[0040] (27) between positions 457 and 477 of the RSV F protein;

[0041] (28) Between positions 130 and 265 of the RSV F protein.

[0042] In some embodiments, the amino acid mutation for filling the hydrophobic cavity comprises an amino acid substitution at an amino acid position selected from the group consisting of: asparagine 42 residue, valine 65 residue, leucine 71 residue, leucine 205 residue, methionine 264 residue, isoleucine 267 residue, isoleucine 192 residue and / or serine 165 residue.

[0043] In some embodiments, amino acid mutations for filling the hydrophobic cavity include: 42L, 42F, 42Y, 42W, 42H, 65Y, 65F, 71I, 71Y, 205F, 205Y, 264F, 264Y, 267L, 267F, 267Y, 192L, 165I, 165F, 165Y and / or 165L.

[0044] In some preferred embodiments, the amino acid mutations used to fill the hydrophobic cavity include: N42L, N42F, N42Y, N42W, N42H, V65Y, V65F, L71I, L71Y, L205F, L205Y, M264F, M264Y, I267L, I267F, I267Y, I192L, S165I, S165F, S165Y and / or S165L.

[0045] In some embodiments, the amino acid mutation for electrostatic engineering comprises an amino acid substitution at the amino acid position of aspartic acid residue 461 and / or glutamic acid residue 462.

[0046] In some embodiments, the amino acid mutations used for electrostatic modification include: 461S, 461N, 461Q, 461E, 461G, 461A, 461K, 461R, 461H, 462Q, 462S, 462N, 462D, 462G, 462A, 462K, 462R, 462H.

[0047] In some preferred embodiments, the amino acid mutations used for electrostatic modification include: D461S, D461N, D461Q, D461E, D461G, D461A, D461K, D461R, D461H, E462Q, E462S, E462N, E462D, E462G, E462A, E462K, E462R, and E462H.

[0048] In some embodiments, the RSV F protein comprises a combination of modifications or mutations selected from the group consisting of:

[0049] (1)N42L, S165I;

[0050] (2)N42L, D461S;

[0051] (3) V65Y, D461S;

[0052] (4) V65Y, S165I;

[0053] (5)N42L, V65Y, D461S;

[0054] (6)N42L, D461S, M264F;

[0055] (7) V65Y, D461S, M264F;

[0056] (8)N42L, V65Y, D461S, M264F;

[0057] (9)I192L, N42L, L71Y, I267L, D461S;

[0058] (10)I192L, N42L, L205F, M264F, I267L, D461S;

[0059] (11)I192L, N42L, V65Y, L205F, M264F, D461S;

[0060] (12)I192L, N42L, L71Y, I267L;

[0061] (13)N42L, V65Y, S165I;

[0062] (14)N42L, S165I, M264F;

[0063] (15)V65Y, S165I, M264F;

[0064] (16)N42L, V65Y, S165I, M264F;

[0065] (17)I192L, N42L, V65Y, L205F, M264F;

[0066] (18)I192L, N42L, L71Y, I267L, S165I;

[0067] (19)I192L, N42L, L205F, M264F, I267L, S165I;

[0068] (20)I192L, N42L, V65Y, L205F, M264F, S165I;

[0069] (21)N42L, D461S, S165I;

[0070] (22)V65Y, D461S, S165I;

[0071] (23) N42L, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0072] (24)M264F, D461S, S165I;

[0073] (25)N42L, V65Y, D461S, S165I;

[0074] (26)N42L, D461S, S165I, M264F;

[0075] (27)V65Y, D461S, S165I, M264F;

[0076] (28) N42L, D461S, introduction of a disulfide bond between protein positions 30 and 163;

[0077] (29)I192L, N42L, L71Y, I267L, D461S, S165I;

[0078] (30)I192L, N42L, L205F, M264F, I267L, D461S, S165I;

[0079] (31)I192L, N42L, V65Y, L205F, M264F, D461S, S165I;

[0080] (32) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0081] (33) V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163;

[0082] (34) M264F, D461S, introduction of a disulfide bond between protein positions 30 and 163;

[0083] (35) N42L, V65Y, D461S, and introduction of a disulfide bond between protein positions 30 and 163;

[0084] (36) N42L, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0085] (37) N42L, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0086] (38) V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0087] (39)M264F, S165I;

[0088] (40) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0089] (41) I192L, N42L, L71Y, I267L, D461S, and a disulfide bond was introduced between protein positions 30 and 163;

[0090] (42) I192L, N42L, L205F, M264F, I267L, D461S, and a disulfide bond was introduced between protein positions 30 and 163;

[0091] (43) I192L, N42L, V65Y, L205F, M264F, D461S, and a disulfide bond was introduced between protein positions 30 and 163;

[0092] (44) N42L, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0093] (45) V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0094] (46) M264F, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0095] (47) N42L, V65Y, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0096] (48) N42L, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0097] (49) N42L, S165I, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0098] (50) V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0099] (51) N42L, M264F, and introduction of a disulfide bond between protein positions 457 and 477;

[0100] (52) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0101] (53) I192L, N42L, L71Y, I267L, S165I, and a disulfide bond was introduced between protein positions 30 and 163;

[0102] (54) I192L, N42L, L205F, M264F, I267L, S165I, and a disulfide bond was introduced between protein positions 30 and 163;

[0103] (55) I192L, N42L, V65Y, L205F, M264F, S165I, and a disulfide bond was introduced between protein positions 30 and 163;

[0104] (56) N42L, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0105] (57) V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0106] (58)N42L, M264F;

[0107] (59) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 457 and 477;

[0108] (60) M264F, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0109] (61) N42L, V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0110] (62) N42L, V65Y, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0111] (63) N42L, D461S, and introduction of a disulfide bond between protein positions 78 and 123;

[0112] (64) V65Y, S165I, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0113] (65) V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123;

[0114] (66) M264F, D461S, introduction of a disulfide bond between protein positions 78 and 123;

[0115] (67) N42L, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0116] (68) N42L, D461S, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0117] (69) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 152 and 164;

[0118] (70) V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0119] (71)M264F, D461S;

[0120] (72) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0121] (73)N42L, V65Y, M264F, D461S, S165I;

[0122] (74) I192L, N42L, L71Y, I267L, D461S, and a disulfide bond was introduced between protein positions 78 and 123;

[0123] (75) I192L, N42L, L205F, M264F, I267L, D461S, and a disulfide bond was introduced between protein positions 78 and 123;

[0124] (76) N42L, V65Y, D461S, and introduction of a disulfide bond between protein positions 78 and 123;

[0125] (77) I192L, N42L, V65Y, L205F, M264F, D461S, and a disulfide bond was introduced between protein positions 78 and 123;

[0126] (78) N42L, S165I, introduction of a disulfide bond between protein positions 78 and 123;

[0127] (79) V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123;

[0128] (80) M264F, S165I, introduction of a disulfide bond between protein positions 78 and 123;

[0129] (81) N42L, V65Y, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0130] (82) N42L, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0131] (83) V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0132] (84) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0133] (85) I192L, N42L, L71Y, I267L, S165I, and a disulfide bond was introduced between protein positions 78 and 123;

[0134] (86) I192L, N42L, L205F, M264F, I267L, S165I, and a disulfide bond was introduced between protein positions 78 and 123;

[0135] (87) I192L, N42L, V65Y, L205F, M264F, S165I, and a disulfide bond was introduced between protein positions 78 and 123;

[0136] (88) N42L, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0137] (89) V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0138] (90) M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0139] (91) N42L, V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0140] (92) V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0141] (93) N42L, V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0142] (94) N42L, V65Y, and introduction of a disulfide bond between protein positions 7 and 416;

[0143] (95) N42L, V65Y, and introduction of a disulfide bond between protein positions 15 and 358;

[0144] (96) N42L, V65Y, and introduction of a disulfide bond between protein positions 20 and 338;

[0145] (97) N42L, V65Y, and introduction of a disulfide bond between protein positions 20 and 339;

[0146] (98) N42L, V65Y, and introduction of a disulfide bond between protein positions 22 and 248;

[0147] (99) N42L, V65Y, and introduction of a disulfide bond between protein positions 23 and 283;

[0148] (100) N42L, V65Y, and introduction of a disulfide bond between protein positions 31 and 164;

[0149] (101) N42L, V65Y, and introduction of a disulfide bond between protein positions 34 and 168;

[0150] (102) N42L, V65Y, and introduction of a disulfide bond between protein positions 34 and 272;

[0151] (103) N42L, V65Y, and introduction of a disulfide bond between protein positions 37 and 175;

[0152] (104) N42L, V65Y, and introduction of a disulfide bond between protein positions 64 and 209;

[0153] (105) N42L, V65Y, and introduction of a disulfide bond between protein positions 133 and 266;

[0154] (106) N42L, V65Y, and introduction of a disulfide bond between protein positions 155 and 161;

[0155] (107) N42L, V65Y, and introduction of a disulfide bond between protein positions 152 and 164;

[0156] (108) N42L, V65Y, and introduction of a disulfide bond between protein positions 236 and 249;

[0157] (109) N42L, V65Y, and introduction of a disulfide bond between protein positions 309 and 450;

[0158] (110) N42L, V65Y, and introduction of a disulfide bond between protein positions 418 and 441;

[0159] (111) N42L, V65Y, and introduction of a disulfide bond between protein positions 385 and 441;

[0160] (112) N42L, V65Y, and the introduction of a disulfide bond between protein positions 457 and 477;

[0161] (113) N42L, V65Y, and introduction of a disulfide bond between protein positions 67 and 229;

[0162] (114) N42L, V65Y, and introduction of a disulfide bond between protein positions 119 and 381;

[0163] (115) N42L, V65Y, and introduction of a disulfide bond between protein positions 125 and 433;

[0164] (116) N42L, V65Y, and introduction of a disulfide bond between protein positions 128 and 436;

[0165] (117) N42L, V65Y, and introduction of a disulfide bond between protein positions 210 and 224;

[0166] (118) N42L, V65Y, and introduction of a disulfide bond between protein positions 216 and 254;

[0167] (119) N42L, M264F, introduction of a disulfide bond between protein positions 7 and 416;

[0168] (120) N42L, M264F, introduction of a disulfide bond between protein positions 15 and 358;

[0169] (121) N42L, M264F, introduction of a disulfide bond between protein positions 20 and 338;

[0170] (122) N42L, M264F, introduction of a disulfide bond between protein positions 20 and 339;

[0171] (123) N42L, M264F, introduction of a disulfide bond between protein positions 22 and 248;

[0172] (124) N42L, M264F, and introduction of a disulfide bond between protein positions 23 and 283;

[0173] (125) N42L, M264F, introduction of a disulfide bond between protein positions 31 and 164;

[0174] (126) N42L, M264F, introduction of a disulfide bond between protein positions 34 and 168;

[0175] (127) N42L, M264F, and introduction of a disulfide bond between protein positions 34 and 272;

[0176] (128) N42L, M264F, and introduction of a disulfide bond between protein positions 37 and 175;

[0177] (129) V65Y, M264F, introduction of a disulfide bond between protein positions 7 and 416;

[0178] (130) N42L, M264F, introduction of a disulfide bond between protein positions 133 and 266;

[0179] (131) N42L, M264F, and introduction of a disulfide bond between protein positions 155 and 161;

[0180] (132) N42L, M264F, introduction of a disulfide bond between protein positions 152 and 164;

[0181] (133) N42L, M264F, and introduction of a disulfide bond between protein positions 236 and 249;

[0182] (134) N42L, M264F, and introduction of a disulfide bond between protein positions 309 and 450;

[0183] (135) V65Y, M264F, introduction of a disulfide bond between protein positions 15 and 358;

[0184] (136) N42L, M264F, and introduction of a disulfide bond between protein positions 385 and 441;

[0185] (137) V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 338;

[0186] (138) N42L, M264F, and introduction of a disulfide bond between protein positions 67 and 229;

[0187] (139) N42L, M264F, introduction of a disulfide bond between protein positions 119 and 381;

[0188] (140) N42L, M264F, and introduction of a disulfide bond between protein positions 125 and 433;

[0189] (141) N42L, M264F, introduction of a disulfide bond between protein positions 128 and 436;

[0190] (142) N42L, M264F, introduction of a disulfide bond between protein positions 210 and 224;

[0191] (143) N42L, M264F, introduction of a disulfide bond between protein positions 216 and 254;

[0192] (144) V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 339;

[0193] (145) V65Y, M264F, introduction of a disulfide bond between protein positions 22 and 248;

[0194] (146) V65Y, M264F, introduction of a disulfide bond between protein positions 23 and 283;

[0195] (147) V65Y, M264F, introduction of a disulfide bond between protein positions 31 and 164;

[0196] (148) V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 168;

[0197] (149) V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 272;

[0198] (150) V65Y, M264F, introduction of a disulfide bond between protein positions 37 and 175;

[0199] (151) V65Y, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0200] (152) V65Y, M264F, and introduction of a disulfide bond between protein positions 133 and 266;

[0201] (153) V65Y, M264F, introduction of a disulfide bond between protein positions 155 and 161;

[0202] (154) V65Y, M264F, introduction of a disulfide bond between protein positions 152 and 164;

[0203] (155) V65Y, M264F, introduction of a disulfide bond between protein positions 236 and 249;

[0204] (156) V65Y, M264F, and introduction of a disulfide bond between protein positions 309 and 450;

[0205] (157) V65Y, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0206] (158) V65Y, M264F, and introduction of a disulfide bond between protein positions 385 and 441;

[0207] (159) V65Y, M264F, and introduction of a disulfide bond between protein positions 457 and 477;

[0208] (160) V65Y, M264F, introduction of a disulfide bond between protein positions 67 and 229;

[0209] (161) V65Y, M264F, introduction of a disulfide bond between protein positions 119 and 381;

[0210] (162) V65Y, M264F, introduction of a disulfide bond between protein positions 125 and 433;

[0211] (163) V65Y, M264F, introduction of a disulfide bond between protein positions 128 and 436;

[0212] (164) V65Y, M264F, introduction of a disulfide bond between protein positions 210 and 224;

[0213] (165) V65Y, M264F, introduction of a disulfide bond between protein positions 216 and 254;

[0214] (166) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 7 and 416;

[0215] (167) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 15 and 358;

[0216] (168) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 20 and 338;

[0217] (169) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 20 and 339;

[0218] (170) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 22 and 248;

[0219] (171) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 23 and 283;

[0220] (172) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 31 and 164;

[0221] (173) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 34 and 168;

[0222] (174) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 34 and 272;

[0223] (175) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 37 and 175;

[0224] (176) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 64 and 209;

[0225] (177) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 133 and 266;

[0226] (178) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 155 and 161;

[0227] (179) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 216 and 254;

[0228] (180) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 236 and 249;

[0229] (181) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 309 and 450;

[0230] (182) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 210 and 224;

[0231] (183) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 385 and 441;

[0232] (184) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 128 and 436;

[0233] (185) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 67 and 229;

[0234] (186) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 119 and 381;

[0235] (187) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 125 and 433;

[0236] (188) A disulfide bond and S165I were introduced between protein positions 418 and 441;

[0237] (189) A disulfide bond, D461S, was introduced between protein positions 418 and 441;

[0238] (190) A disulfide bond was introduced between protein positions 418 and 441, E462Q;

[0239] (191) A disulfide bond and S165I were introduced between protein positions 457 and 477;

[0240] (192) A disulfide bond, D461S, was introduced between protein positions 457 and 477;

[0241] (193) introduction of a disulfide bond between protein positions 457 and 477, E462Q;

[0242] (194) A disulfide bond and S165I were introduced between protein positions 64 and 209;

[0243] (195) A disulfide bond, D461S, was introduced between protein positions 64 and 209;

[0244] (196) A disulfide bond, E462Q, was introduced between protein positions 64 and 209;

[0245] (197)I192L, N42L, L205F, M264F, I267L;

[0246] (198)N42L, L205F, L71Y, I267L;

[0247] (199)I192L, N42L, V65Y, L205F;

[0248] (200)I192L, N42L, L205F, L71Y;

[0249] (201)I192L, N42L, L205F, I267L;

[0250] (202) I192L, N42L, L205F, I267L, and the introduction of a disulfide bond between protein positions 457 and 477;

[0251] (203) I192L, N42L, V65Y, L205F, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[0252] (204)I192L, N42L, L205F;

[0253] (205) I192L, N42L, L205F, I267L, and a disulfide bond was introduced between protein positions 130 and 265;

[0254] (206) N42L, V65Y, L205F, L71Y, and introduction of a disulfide bond between protein positions 130 and 265;

[0255] (207) I192L, N42L, V65Y, L205F, M264F, and a disulfide bond was introduced between protein positions 130 and 265;

[0256] (208) S165I, M264F, introduction of a disulfide bond between protein positions 418 and 441;

[0257] (209) D461S, M264F, introduction of a disulfide bond between protein positions 418 and 441;

[0258] (210) E462Q, M264F, introduction of a disulfide bond between protein positions 418 and 441;

[0259] (211) S165I, M264F, introduction of a disulfide bond between protein positions 457 and 477;

[0260] (212) D461S, M264F, introduction of a disulfide bond between protein positions 457 and 477;

[0261] (213) E462Q, M264F, introduction of a disulfide bond between protein positions 457 and 477;

[0262] (214) S165I, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0263] (215) D461S, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0264] (216) E462Q, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0265] (217) D461S, S165I, introduction of a disulfide bond between protein positions 64 and 209;

[0266] (218) D461S, S165I, introduction of a disulfide bond between protein positions 418 and 441;

[0267] (219) S165I, N42L, introduction of a disulfide bond between protein positions 418 and 441;

[0268] (220) D461S, N42L, introduction of a disulfide bond between protein positions 418 and 441;

[0269] (221) E462Q, N42L, introduction of a disulfide bond between protein positions 418 and 441;

[0270] (222) S165I, N42L, and introduction of a disulfide bond between protein positions 457 and 477;

[0271] (223) D461S, N42L, and introduction of a disulfide bond between protein positions 457 and 477;

[0272] (224) E462Q, N42L, introduction of a disulfide bond between protein positions 457 and 477;

[0273] (225) S165I, N42L, introduction of a disulfide bond between protein positions 64 and 209;

[0274] (226) D461S, N42L, introduction of a disulfide bond between protein positions 64 and 209;

[0275] (227) E462Q, N42L, introduction of a disulfide bond between protein positions 64 and 209;

[0276] (228) S165I, V65Y, introduction of a disulfide bond between protein positions 418 and 441;

[0277] (229) D461S, V65Y, introduction of a disulfide bond between protein positions 418 and 441;

[0278] (230) E462Q, V65Y, introduction of a disulfide bond between protein positions 418 and 441;

[0279] (231) S165I, V65Y, and introduction of a disulfide bond between protein positions 457 and 477;

[0280] (232) D461S, V65Y, introduction of a disulfide bond between protein positions 457 and 477;

[0281] (233) E462Q, V65Y, introduction of a disulfide bond between protein positions 457 and 477;

[0282] (234) S165I, V65Y, introduction of a disulfide bond between protein positions 64 and 209;

[0283] (235) D461S, V65Y, introduction of a disulfide bond between protein positions 64 and 209;

[0284] (236) E462Q, V65Y, introduction of a disulfide bond between protein positions 64 and 209;

[0285] (237) E462Q, S165I, introduction of a disulfide bond between protein positions 64 and 209;

[0286] (238) E462Q, S165I, introduction of a disulfide bond between protein positions 418 and 441;

[0287] (239) D461N, introduction of a disulfide bond between protein positions 418 and 441;

[0288] (240) D461Q, introduction of a disulfide bond between protein positions 418 and 441;

[0289] (241) D461E, introduction of a disulfide bond between protein positions 418 and 441;

[0290] (242) D461G, introduction of a disulfide bond between protein positions 418 and 441;

[0291] (243) D461A, introduction of a disulfide bond between protein positions 418 and 441;

[0292] (244) D461K, introduction of a disulfide bond between protein positions 418 and 441;

[0293] (245) D461R, introduction of a disulfide bond between protein positions 418 and 441;

[0294] (246) D461H, introduction of a disulfide bond between protein positions 418 and 441;

[0295] (247) D461N, introduction of a disulfide bond between protein positions 457 and 477;

[0296] (248) D461Q, introduction of a disulfide bond between protein positions 457 and 477;

[0297] (249) D461E, introduction of a disulfide bond between protein positions 457 and 477;

[0298] (250) D461G, introduction of a disulfide bond between protein positions 457 and 477;

[0299] (251) D461A, introduction of a disulfide bond between protein positions 457 and 477;

[0300] (252) D461K, introduction of a disulfide bond between protein positions 457 and 477;

[0301] (253) D461R, introduction of a disulfide bond between protein positions 457 and 477;

[0302] (254) D461H, introduction of a disulfide bond between protein positions 457 and 477;

[0303] (255) D461N, introduction of a disulfide bond between protein positions 64 and 209;

[0304] (256) D461Q, introduction of a disulfide bond between protein positions 64 and 209;

[0305] (257) D461E, introduction of a disulfide bond between protein positions 64 and 209;

[0306] (258) D461G, introduction of a disulfide bond between protein positions 64 and 209;

[0307] (259) D461A, introduction of a disulfide bond between protein positions 64 and 209;

[0308] (260) D461K, introduction of a disulfide bond between protein positions 64 and 209;

[0309] (261) D461R, introduction of a disulfide bond between protein positions 64 and 209;

[0310] (262) D461H, introduction of a disulfide bond between protein positions 64 and 209;

[0311] (263) E462S, introduction of a disulfide bond between protein positions 418 and 441;

[0312] (264) E462N, introduction of a disulfide bond between protein positions 418 and 441;

[0313] (265) E462D, introduction of a disulfide bond between protein positions 418 and 441;

[0314] (266) E462G, introduction of a disulfide bond between protein positions 418 and 441;

[0315] (267) E462A, introduction of a disulfide bond between protein positions 418 and 441;

[0316] (268) E462K, introduction of a disulfide bond between protein positions 418 and 441;

[0317] (269) E462R, introduction of a disulfide bond between protein positions 418 and 441;

[0318] (270) E462H, introduction of a disulfide bond between protein positions 418 and 441;

[0319] (271) E462S, introduction of a disulfide bond between protein positions 64 and 209;

[0320] (272) E462N, introduction of a disulfide bond between protein positions 64 and 209;

[0321] (273) E462D, introduction of a disulfide bond between protein positions 64 and 209;

[0322] (274) E462G, introduction of a disulfide bond between protein positions 64 and 209;

[0323] (275) E462A, introduction of a disulfide bond between protein positions 64 and 209;

[0324] (276) E462K, introduction of a disulfide bond between protein positions 64 and 209;

[0325] (277) E462R, introduction of a disulfide bond between protein positions 64 and 209;

[0326] (278) E462H, introduction of a disulfide bond between protein positions 64 and 209;

[0327] (279) E462S, introduction of a disulfide bond between protein positions 457 and 477;

[0328] (280) E462N, introduction of a disulfide bond between protein positions 457 and 477;

[0329] (281) E462D, introduction of a disulfide bond between protein positions 457 and 477;

[0330] (282) E462G, introduction of a disulfide bond between protein positions 457 and 477;

[0331] (283) E462A, introduction of a disulfide bond between protein positions 457 and 477;

[0332] (284) E462K, introduction of a disulfide bond between protein positions 457 and 477;

[0333] (285) E462R, introduction of a disulfide bond between protein positions 457 and 477;

[0334] (286) E462H, introduction of a disulfide bond between protein positions 457 and 477;

[0335] (287) D461N, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0336] (288) D461Q, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0337] (289) D461E, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0338] (290) D461G, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0339] (291) D461A, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0340] (292) D461K, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0341] (293) D461R, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0342] (294) D461H, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0343] (295) D461N, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0344] (296) D461Q, introduction of a disulfide bond between protein positions 457 and 477, N42L;

[0345] (297) D461E, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0346] (298) D461G, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0347] (299) D461A, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0348] (300) D461K, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0349] (301) D461R, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0350] (302) D461H, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0351] (303) D461N, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0352] (304) D461Q, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0353] (305) D461E, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0354] (306) D461G, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0355] (307) D461A, introduction of a disulfide bond between protein positions 64 and 209, and N42L;

[0356] (308) D461K, introduction of a disulfide bond between protein positions 64 and 209, and N42L;

[0357] (309) D461R, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0358] (310) D461H, introduction of a disulfide bond between protein positions 64 and 209, and N42L;

[0359] (311) D461N, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0360] (312) D461Q, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0361] (313) D461E, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0362] (314) D461G, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0363] (315) D461A, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0364] (316) D461K, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0365] (317) D461R, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0366] (318) D461H, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0367] (319) D461N, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0368] (320) D461Q, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0369] (321) D461E, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0370] (322) D461G, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0371] (323) D461A, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0372] (324) D461K, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0373] (325) D461R, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0374] (326) D461H, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0375] (327) D461N, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0376] (328) D461Q, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0377] (329) D461E, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0378] (330) D461G, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0379] (331) D461A, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0380] (332) D461K, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0381] (333) D461R, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0382] (334) D461H, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0383] (335) D461N, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0384] (336) D461Q, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0385] (337) D461E, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0386] (338) D461G, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0387] (339) D461A, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0388] (340) D461K, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0389] (341) D461R, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0390] (342) D461H, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0391] (343) D461N, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0392] (344) D461Q, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0393] (345) D461E, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0394] (346) D461G, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0395] (347) D461A, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0396] (348) D461K, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0397] (349) D461R, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0398] (350) D461H, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0399] (351) D461N, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0400] (352) D461Q, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0401] (353) D461E, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0402] (354) D461G, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0403] (355) D461A, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0404] (356) D461K, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0405] (357) D461R, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0406] (358) D461H, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0407] (359) E462S, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0408] (360) E462N, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0409] (361) E462D, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0410] (362) E462G, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0411] (363) E462A, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0412] (364) E462K, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0413] (365) E462R, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0414] (366) E462H, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0415] (367) E462S, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0416] (368) E462N, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0417] (369) E462D, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0418] (370) E462G, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0419] (371) E462A, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0420] (372) E462K, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0421] (373) E462R, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0422] (374) E462H, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0423] (375) E462S, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0424] (376) E462N, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0425] (377) E462D, introduction of a disulfide bond between protein positions 457 and 477, N42L;

[0426] (378) E462G, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0427] (379) E462A, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0428] (380) E462K, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0429] (381) E462R, introduction of a disulfide bond between protein positions 457 and 477, N42L;

[0430] (382) E462H, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0431] (383) E462S, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0432] (384) E462N, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0433] (385) E462D, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0434] (386) E462G, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0435] (387) E462A, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0436] (388) E462K, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0437] (389) E462R, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0438] (390) E462H, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0439] (391) E462S, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0440] (392) E462N, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0441] (393) E462D, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0442] (394) E462G, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0443] (395) E462A, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0444] (396) E462K, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0445] (397) E462R, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0446] (398) E462H, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[0447] (399) E462S, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0448] (400) E462N, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0449] (401) E462D, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0450] (402) E462G, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0451] (403) E462A, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0452] (404) E462K, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0453] (405) E462R, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0454] (406) E462H, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0455] (407) E462S, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0456] (408) E462N, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0457] (409) E462D, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0458] (410) E462G, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0459] (411) E462A, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0460] (412) E462K, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0461] (413) E462R, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0462] (414) E462H, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[0463] (415) E462S, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0464] (416) E462N, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0465] (417) E462D, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0466] (418) E462G, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0467] (419) E462A, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0468] (420) E462K, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0469] (421) E462R, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0470] (422) E462H, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[0471] (423) E462S, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0472] (424) E462N, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0473] (425) E462D, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0474] (426) E462G, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0475] (427) E462A, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0476] (428) E462K, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0477] (429) E462R, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0478] (430) E462H, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[0479] (431)V65Y, M264F;

[0480] (432)N42L, V65Y, M264F.

[0481] In some preferred embodiments, the RSV F protein comprises a combination of modifications or mutations selected from the group consisting of:

[0482] (1)192L, 42L, 71Y, 267L;

[0483] (2)192L, 42L, 65Y, 205F, 264F;

[0484] (3) 42L, 264F, and the introduction of a disulfide bond between protein positions 64 and 209;

[0485] (4) 42L, 264F, 461S, 165I, and the introduction of a disulfide bond between protein positions 78 and 123;

[0486] (5) introduction of a disulfide bond between 42L, 264F, and protein positions 457 and 477;

[0487] (6) 42L, 65Y, 264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[0488] (7) 42L, 65Y, 264F, 461S, 165I, and the introduction of a disulfide bond between protein positions 30 and 163;

[0489] (8) 42L, 264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0490] (9)42L, 65Y, 264F, 461S, 165I;

[0491] (10) 42L, 65Y, 461S, and introduction of a disulfide bond between protein positions 78 and 123;

[0492] (11)192L, 42L, 205F, 264F, 267L;

[0493] (12)42L, 205F, 71Y, 267L;

[0494] (13)192L, 42L, 65Y, 205F;

[0495] (14)192L, 42L, 205F, 71Y;

[0496] (15)192L, 42L, 205F, 267L;

[0497] (16) 192L, 42L, 205F, 267L, and the introduction of a disulfide bond between protein positions 457 and 477;

[0498] (17) 192L, 42L, 65Y, 205F, 264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[0499] (18)192L, 42L, 205F;

[0500] (19) 192L, 42L, 205F, 267L, and introduction of a disulfide bond between protein positions 130 and 265;

[0501] (20) 42L, 65Y, 205F, 71Y, and a disulfide bond was introduced between protein positions 130 and 265;

[0502] (21) 192L, 42L, 65Y, 205F, 264F, and introduction of a disulfide bond between protein positions 130 and 265;

[0503] (22) 42L, 65Y, 264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0504] (23)264F, 165I;

[0505] (24)42L, 264F;

[0506] (25) 42L, 65Y, 264F, and introduction of a disulfide bond between protein positions 152 and 164;

[0507] (26) 461Q, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[0508] (27) 461G, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[0509] (28) 461K, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[0510] (29) 461H, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[0511] (30) 461A, introduction of a disulfide bond between protein positions 457 and 477, 264F;

[0512] (31) 461H, introduction of a disulfide bond between protein positions 64 and 209, 264F.

[0513] In some more preferred embodiments, the RSV F protein comprises a combination of modifications or mutations selected from the group consisting of:

[0514] (1)I192L, N42L, L71Y, I267L;

[0515] (2)I192L, N42L, V65Y, L205F, M264F;

[0516] (3) N42L, M264F, and the introduction of a disulfide bond between protein positions 64 and 209;

[0517] (4) N42L, M264F, D461S, S165I, and the introduction of a disulfide bond between protein positions 78 and 123;

[0518] (5) N42L, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[0519] (6) N42L, V65Y, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[0520] (7) N42L, V65Y, M264F, D461S, S165I, and the introduction of a disulfide bond between protein positions 30 and 163;

[0521] (8) N42L, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0522] (9)N42L, V65Y, M264F, D461S, S165I;

[0523] (10) N42L, V65Y, D461S, and introduction of a disulfide bond between protein positions 78 and 123;

[0524] (11)I192L, N42L, L205F, M264F, I267L;

[0525] (12)N42L, L205F, L71Y, I267L;

[0526] (13)I192L, N42L, V65Y, L205F;

[0527] (14)I192L, N42L, L205F, L71Y;

[0528] (15)I192L, N42L, L205F, I267L;

[0529] (16) I192L, N42L, L205F, I267L, and the introduction of a disulfide bond between protein positions 457 and 477;

[0530] (17) I192L, N42L, V65Y, L205F, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[0531] (18)I192L, N42L, L205F;

[0532] (19) I192L, N42L, L205F, I267L, and the introduction of a disulfide bond between protein positions 130 and 265;

[0533] (20) N42L, V65Y, L205F, L71Y, and introduction of a disulfide bond between protein positions 130 and 265;

[0534] (21) I192L, N42L, V65Y, L205F, M264F, and the introduction of a disulfide bond between protein positions 130 and 265;

[0535] (22) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0536] (23)M264F, S165I;

[0537] (24)N42L, M264F;

[0538] (25) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 152 and 164;

[0539] (26) D461Q, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0540] (27) D461G, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0541] (28) D461K, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0542] (29) D461H, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0543] (30) D461A, introduction of a disulfide bond between protein positions 457 and 477, and M264F;

[0544] (31) D461H, introduction of a disulfide bond between protein positions 64 and 209, and M264F.

[0545] In some embodiments, the p27 peptide segment of the RSV F protein is replaced with a linker.

[0546] In some preferred embodiments, the linker is a flexible polypeptide.

[0547] In some more preferred embodiments, the above-mentioned flexible polypeptide is selected from (GS)n, (GGSG)n, (GGGGS)n, (GGGSGG)n, (GGGSGGG)n, (EA)n, (EAK)n, (EAKA)n, (AAAAK)n, (EAAAK)n, (LEAAK)n, wherein n is an integer from 1 to 4.

[0548] In some more preferred embodiments, the flexible polypeptide is selected from the amino acid sequences shown in SEQ ID NOs: 500-543.

[0549] In some embodiments, the RSV F protein comprises an amino acid sequence selected from SEQ ID NOs: 2-499, 585-608 having 80% or greater identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity.

[0550] In some embodiments, the N-terminus of the RSV F protein is linked to a signal peptide.

[0551] In some preferred embodiments, the signal peptide is selected from a wild-type RSV F protein signal peptide or a non-RSV F protein signal peptide.

[0552] In some preferred embodiments, the signal peptide comprises an amino acid sequence selected from SEQ ID NO: 544, 546, 548.

[0553] In some preferred embodiments, the C-terminus of the RSV F protein is connected to a trimerization tag.

[0554] In some preferred embodiments, the trimerization tag comprises an amino acid sequence as shown in SEQ ID NO: 550 or any variant thereof.

[0555] In a second aspect, the present disclosure provides a nucleic acid molecule encoding the RSV F protein of any of the above aspects.

[0556] In a third aspect, the present disclosure provides a vector comprising the above-mentioned nucleic acid molecule.

[0557] In some embodiments, the above-mentioned vector is an expression vector.

[0558] In a fourth aspect, the present disclosure provides a host cell comprising the above-mentioned vector.

[0559] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0560] In some preferred embodiments, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0561] In some preferred embodiments, the mammalian cells are selected from CHO cells, HEK293 cells or COS cells.

[0562] In a fifth aspect, the present disclosure provides an immunogenic composition comprising the RSV F protein of any of the above aspects.

[0563] In a sixth aspect, the present disclosure provides a vaccine composition comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[0564] In a seventh aspect, the present disclosure provides a kit for immunizing a subject against viral infection, comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[0565] In some preferred embodiments, the above viral infection is RSV infection.

[0566] In some preferred embodiments, the subject is a human subject.

[0567] In an eighth aspect, the present disclosure provides a pharmaceutical composition comprising the RSV F protein according to any of the above aspects and a pharmaceutically acceptable carrier.

[0568] In some preferred embodiments, the above pharmaceutical composition further comprises other therapeutic agents.

[0569] In a ninth aspect, the present disclosure provides a method for generating an immune response against a viral infection, comprising administering the RSV F protein of any of the above aspects in a pharmaceutically acceptable formulation to a subject.

[0570] In some preferred embodiments, the pharmaceutically acceptable formulation comprises an adjuvant.

[0571] In a tenth aspect, the present disclosure provides use of the above-mentioned RSV F protein in the preparation of a medicament for preventing or treating a disease or condition associated with respiratory syncytial virus (RSV) infection.

[0572] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[0573] In an eleventh aspect, the present disclosure provides a method for preventing or treating diseases associated with respiratory syncytial virus (RSV) infection, comprising administering an effective amount of the RSV F protein of any one of the above aspects in a pharmaceutically acceptable formulation to a subject in need thereof.

[0574] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[0575] Compared with the prior art, the RSV F protein involved in the present disclosure has high stability as a recombinant protein antigen, is easy to store and transport, has improved expression level, and is easy to achieve increased production. In addition, the modified RSV F protein has higher immunogenicity and can produce better immune protection effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0576] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the specification and, together with the description, serve to explain the principles of the specification.

[0577] FIG1 shows the gel filtration chromatography results of some preferred mutant purified proteins.

[0578] FIG2 shows the SDS-PAGE results of some preferred mutant purified proteins.

[0579] FIG3 shows the gel filtration chromatography results of some preferred mutants after freezing and thawing once, three times and five times.

[0580] FIG4 shows the SDS-PAGE results of some preferred mutants after freezing and thawing once, three times and five times.

[0581] FIG5 shows the gel filtration chromatography results of some preferred mutants after standing at 4° C. for 3 days and at room temperature for 3 days.

[0582] FIG6 shows the SDS-PAGE results of some preferred mutants after being kept at 4° C. for 3 days and at room temperature for 3 days.

[0583] FIG7 shows the gel filtration chromatography results of some preferred mutants after shaking at 4° C. for 3 days and shaking at room temperature for 3 days.

[0584] FIG8 shows the SDS-PAGE results of some preferred mutants after shaking at 4° C. for 3 days and shaking at room temperature for 3 days.

[0585] FIG9 shows the gel filtration chromatography results of some mutants after heat stress at 40° C. and 50° C. for 1 hour.

[0586] FIG10 shows the SDS-PAGE results of some mutants after heat stress at 40° C. and 50° C. for 1 hour.

[0587] FIG11 shows the thermal stability of some preferred mutants analyzed by differential scanning fluorimetry (DSF).

[0588] FIG12 shows the thermal stability of some preferred mutants analyzed by differential scanning fluorimetry (DSF).

[0589] FIG. 13 shows the results of serum neutralization of RSV A2 by some preferred mutant CpG adjuvant groups.

[0590] FIG. 14 shows the results of serum neutralization of RSV A2 by some preferred mutant MPL adjuvant groups.

[0591] FIG. 15 shows the results of RSV A2 neutralization by sera from mice immunized with Post-F, pXCS847, and CL-073.

[0592] FIG. 16 shows a graph of mouse body weight change showing protection from challenge provided by RSV F protein mutant antigens in mice.

[0593] FIG. 17 shows viral copies in mouse lung tissues that are protected from challenge by RSV F protein mutant antigens in mice.

[0594] FIG. 18 shows live virus titers in mouse lung tissue for protection against challenge provided by RSV F protein mutant antigens in mice.

[0595] FIG19 shows the thermal stability of backbone CL-073 RSV F proteins from different strains analyzed by differential scanning fluorimetry (DSF).

[0596] FIG. 20 shows the thermal stability of backbone CL-073 RSV F proteins from different strains analyzed by differential scanning fluorimetry (DSF).

[0597] FIG21 shows the Elisa detection results of the binding of different strains of CL-073 RSV F protein backbone to specific antibodies Motavizumab, D25 and AM14.

[0598] FIG. 22 shows the results of Elisa assays of the binding of sera from mice immunized with CL-073 RSV F proteins from different strains to CL-073A / B antigens.

[0599] FIG. 23 shows the results of virus neutralization assays of sera from mice immunized with backbone CL-073 RSV F proteins from different strains. DETAILED DESCRIPTION

[0600] I. Definition

[0601] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0602] Provided herein is a kind of respiratory syncytial virus fusion (RSV F) albumen and purposes thereof.In specific aspect, provided herein is the RSV F albumen of modification or mutation (for example, amino acid point mutation, introducing disulfide bond).In specific aspect, provided herein is the RSV F albumen comprising modification or mutation (for example, introducing disulfide bond, filling hydrophobic cavity, electrostatic mutation) for increasing RSV F protein fusion before conformational stability, compared with the wild-type RSV F albumen without modification or mutation, the RSV F albumen of modification can better keep conformation before fusion, increases stability, is conducive to storage and transportation.

[0603] The modified or mutated RSV F proteins provided herein are derived from RSV subtype A or RSV subtype B. In specific aspects, the RSV subtype A includes RSV A2 strain, RSV Long strain, RSV ON1 strain, or RSV Ontario strain; and the RSV subtype B includes RSV B1 strain, RSV BA9 strain, RSV Buenos strain, or 18537 strain.

[0604] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0605] With respect to protein or polypeptide sequences, the phrase "substantially identical" is understood to mean a polypeptide sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the phrase is understood to mean a nucleotide sequence that exhibits at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

[0606] The term "respiratory syncytial virus" or "RSV" is a single-stranded negative-strand RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus. Because the virus can cause cultured cells to produce a unique cell fusion effect, it is named respiratory syncytial virus (RSV). According to the differences in surface antigens, RSV can be divided into two subtypes, RSV-A and RSV-B. The virus is transmitted through airborne droplets and close contact. Respiratory syncytial virus contains 10 genes that encode 11 proteins, of which the surface glycoprotein fusion protein (F protein) and attachment protein (G protein) are the most important viral antigens that stimulate the body to produce protective antibodies. Because the G protein varies greatly between subtypes, the F protein is highly conserved between subtypes, and the F protein directly mediates the fusion of the virus and cells, the penetration of the virus, and the formation of syncytia, it is the main target protein that stimulates the body to produce protective antibodies. Herein, "respiratory syncytial virus" or "RSV" refers to any respiratory syncytial virus or RSV molecule known to those skilled in the art. For example, RSV can include any of the above-mentioned subtypes, for example, RSV can be from mammals, for example, RSV can be from humans.

[0607] The terms "RSV F protein", "F protein" or "RSV fusion protein" refer to a polypeptide or protein having all or part of the amino acid sequence of an RSV fusion protein polypeptide. The F protein is an N-glycosylated type I transmembrane protein with a total length of 574 amino acids, which has a signal peptide cleaved at the N-terminus and a membrane anchor near the C-terminus. The amino acid sequence of the F protein is provided in GenBank under accession number AAX23994. The F protein has a pre-fusion conformation (Pre-F) and a post-fusion conformation (Post-F) structure on the surface of the virus particle. Pre-F exists in the form of a trimer and undergoes a conformational change, which causes the hydrophobic fusion peptide to be inserted into the host cell membrane, and then the F protein is refolded into a stable, elongated post-fusion conformation (Post-F), resulting in the fusion of the virus and host cell membranes.

[0608] The term "p27 polypeptide" or "pep27" refers to a 27-amino acid polypeptide that is cleaved from the RSV F protein precursor during maturation. The p27 polypeptide sequence is flanked by two furin cleavage sites, which are cleaved by cellular proteases during F protein maturation to generate the F1 and F2 polypeptides. Therefore, it should be understood that the multiple peptides formed upon removal of the p27 peptide from the RSV F protein are also encompassed by the term "RSV F protein."

[0609] As used herein, the terms "protein," "polypeptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified (conservatively substituted) variants thereof.

[0610] As used herein, the terms sequence "identity," "identity," or "homology" have their art-recognized meanings, and published techniques can be used to calculate the percentage of sequence identity between two nucleic acids or polypeptides or regions. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. While there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art.

[0611] As used herein, the term "variant" refers to an amino acid sequence having at least one amino acid difference (substitution, insertion, or deletion) compared to a reference sequence. In certain embodiments, a "variant" has a high degree of amino acid sequence homology and / or conservative amino acid substitutions, deletions, and / or insertions compared to a reference sequence. In some embodiments, a variant has no more than 75, 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid differences compared to a reference sequence. In some embodiments, a variant has at least 70%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity compared to a reference sequence.

[0612] As used herein, the term "substitution" variant is a variant in which at least one amino acid residue in the native sequence is removed and a different amino acid is inserted in the same position. The substitution can be single, in which only one amino acid is substituted in the molecule; or it can be multiple, in which two or more amino acids are substituted in the same molecule. Multiple substitutions can be located at consecutive sites. Likewise, an amino acid can be substituted by multiple residues, in which case such a variant includes both substitutions and insertions. An "insertion" variant is a variant in which one or more amino acids are inserted adjacent to an amino acid at a specific position in a native sequence. Adjacent to an amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is a variant in which one or more amino acids in the native amino acid sequence are removed. Typically, a deletion variant has one or two amino acids deleted in a specific region of the molecule. It should be noted that the specific modification or mutation sites described in the present disclosure are generally based on the amino acid sequence of the RSV F protein without a signal peptide (e.g., the amino acid sequence shown in SEQ ID NO: 1). In some embodiments, the amino acid residues at the corresponding sites of the wild-type RSV F protein are limited, such as "N42L" indicating that the 42nd amino acid residue asparagine N of the wild-type RSV F protein without a signal peptide is substituted with leucine L; in some embodiments, the amino acid residues at the corresponding sites of the wild-type RSV F protein are not limited, such as "42L" indicating that the 42nd amino acid residue of the wild-type RSV F protein without a signal peptide, regardless of which amino acid residue it is, is substituted with leucine L.

[0613] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. As used herein, the terms "polynucleotide" and "nucleic acid molecule" are intended to include DNA molecules and RNA molecules, which may be single-stranded or double-stranded, and may be cDNA. The term also includes codon-optimized nucleic acid molecules.

[0614] As used herein, the term "conservative substitutions" or "conservative sequence modifications" of a sequence refers to nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an anti-MASP-2 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abrogate antigen binding are well known in the art.

[0615] Exemplary amino acids that can be conservatively substituted are shown in Table 1 below:

[0616] Table 1. Examples of conservative amino acid substitutions

[0617] As used herein, the term "expression" refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0618] As used herein, the term "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0619] As used herein, the term "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction enzyme digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the purpose of amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are typically kept episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles are well known to those skilled in the art.

[0620] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0621] As used herein, the term "expression vector" includes vectors capable of expressing DNA that is operably linked to regulatory sequences that can affect the expression of such DNA fragments, such as promoter regions. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells and expression vectors that remain episomal or are integrated into the host cell genome.

[0622] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable formulation" refers to one or more non-toxic materials that do not interfere with the biological activity of the active ingredient when administered with a therapeutic agent, including but not limited to buffers, preservatives, compatible carriers, diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, vehicles, and optionally other additives or encapsulating materials. Pharmaceutical carriers suitable for use in the present disclosure can be conventional pharmaceutical formulation excipients; and compositions and formulations suitable for delivering the disclosed neutralizing antibodies.

[0623] The term "vaccine" or "vaccine composition" refers to a composition comprising at least one immunogenic composition that induces an immune response in an animal.

[0624] As used herein, the term "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or the development of the disease. Treatment also includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided, as well as the compositions provided herein.

[0625] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

[0626] As used herein, the term "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A complete prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0627] As used herein, the term "patient" or "subject" refers to a mammal, such as a human.

[0628] As used herein, "DSCav1" or "DS-Cav1" is a genetic modification of the RSV F protein comprising four amino acid substitutions (S155C, S290C, S190F, V207L). See CN112851766A, the contents of which are incorporated herein by reference.

[0629] As used herein, "pXCS847"; "pXCS847 replacement" refers to a genetic modification of the RSV F protein comprising four amino acid replacements (T78C, I23C, S65I, D461S). See WO 2017 / 109629, the contents of which are incorporated herein by reference.

[0630] As used herein, "D25" or "D25 antibody" describes a neutralizing antibody that specifically binds to the prefusion RSV F peptide. This antibody is described in U.S. Patent Application Publication No. US2010 / 0239593, the entire contents of which are incorporated herein by reference.

[0631] As used herein, "Motavizumab" or "Motavizumab antibody" describes a neutralizing antibody that specifically binds to the RSV F peptide. This antibody is described in U.S. Patent Application Publication No. US2010 / 053558, the entire contents of which are incorporated herein by reference.

[0632] As used herein, "AM14" or "AM14 antibody" describes a neutralizing antibody that specifically binds to the RSV F peptide. This antibody is described in Gilman MSA, Moin SM, Mas v, Chen M. Patel NK, Kramer K, et al. (2015) Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein. PLoS Pathog 11(7):e1005035. http: / / doi.org / 10.1371 / joumal.ppat.1005035, the entire contents of which are incorporated herein by reference.

[0633] II. Detailed description of specific implementation plan

[0634] The modified or mutated RSV F proteins provided herein are derived from RSV subtype A or RSV subtype B.

[0635] In some embodiments, the RSV subtype A includes RSV A2 strain (SEQ ID NO: 551), RSV Long strain (SEQ ID NO: 552), RSV ON1 strain (SEQ ID NO: 553) or RSV Ontario strain (SEQ ID NO: 554); the RSV subtype B includes RSV B1 strain (SEQ ID NO: 555), RSV BA9 strain (SEQ ID NO: 556), RSV Buenos strain (SEQ ID NO: 557) or 18537 strain (SEQ ID NO: 558).

[0636] In some embodiments, the RSV subtype A includes RSV A2 strain (SEQ ID NO: 561), RSV RSS-2 strain (SEQ ID NO: 562), RSV HuN13-10 strain (SEQ ID NO: 563), RSV GZ11-18 strain (SEQ ID NO: 564), RSV GS11-13 strain (SEQ ID NO: 565), RSV BJ04-01 strain (SEQ ID NO: 566), RSV MinA strain (SEQ ID NO: 567), RSV S2 strain (SEQ ID NO: 568), RSV QYW strain (SEQ ID NO: 569), RSV GES strain (SEQ ID NO: 570), RSV WRI strain (SEQ ID NO: 571), or RSV ON1 strain (SEQ ID NO: 572); the RSV subtype B includes RSV B1 strain (SEQ ID NO: 573), RSV Buenos strain (SEQ ID NO: 574), RSV 18537 strains (SEQ ID NO:575), RSV Guangzhou10-02 strains (SEQ ID NO:576), RSV B / WI / 629-Q0190 / 10 strains (SEQ ID NO:577), RSV Beijing10-13 strains (SEQ ID NO:578), RSV 13-001273 strains (SEQ ID NO:579), RSV hRSV / B / Australia / VIC-RCH056 / 2019 strain (SEQ ID NO:580), RSV hRSV / B / England / UKHSA_RVU_4120712 / 2023 strain (SEQ ID NO:581), RSV hRSV / B / England / 224640784 / 2022 strain (SEQ ID NO:582), RSV hRSV / B / USA / WA-UW-08111 / 2023 strain (SEQ ID NO:583) or RSV BA9 (SEQ ID NO:584).

[0637] In some preferred embodiments, the RSV subtype A is RSV A2 strain (SEQ ID NO: 551).

[0638] In some more preferred embodiments, the modified or mutated RSV F protein provided herein is derived from the RSV A2 strain, wherein the wild-type RSV F protein carries three natural mutations (P102A, I379V, and M447V) for increasing expression, and its amino acid sequence is shown in SEQ ID NO: 1.

[0639] In one aspect, the present disclosure provides a respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation selected from the following for increasing the stability of the RSV F protein prefusion conformation:

[0640] (1) introducing one or more disulfide bonds into the RSV F protein;

[0641] (2) introducing one or more amino acid mutations in the RSV F protein to fill the hydrophobic cavity;

[0642] (3) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein;

[0643] (4) Remove the p27 peptide from RSV F protein.

[0644] In some embodiments, the introduced disulfide bonds are present at one or more of the following positions:

[0645] (1) between positions 7 and 416 of the RSV F protein;

[0646] (2) between positions 15 and 358 of the RSV F protein;

[0647] (3) between positions 20 and 338 of the RSV F protein;

[0648] (4) between positions 20 and 339 of the RSV F protein;

[0649] (5) between positions 22 and 248 of the RSV F protein;

[0650] (6) between positions 23 and 283 of the RSV F protein;

[0651] (7) between positions 30 and 163 of the RSV F protein;

[0652] (8) between positions 31 and 164 of the RSV F protein;

[0653] (9) between positions 34 and 168 of the RSV F protein;

[0654] (10) between positions 34 and 272 of the RSV F protein;

[0655] (11) between positions 37 and 175 of the RSV F protein;

[0656] (12) between positions 64 and 209 of the RSV F protein;

[0657] (13) between positions 67 and 229 of the RSV F protein;

[0658] (14) between positions 78 and 123 of the RSV F protein;

[0659] (15) between positions 119 and 381 of the RSV F protein;

[0660] (16) between positions 125 and 433 of the RSV F protein;

[0661] (17) between positions 128 and 436 of the RSV F protein;

[0662] (18) between positions 133 and 266 of the RSV F protein;

[0663] (19) between positions 152 and 164 of the RSV F protein;

[0664] (20) between positions 155 and 161 of the RSV F protein;

[0665] (21) between positions 210 and 224 of the RSV F protein;

[0666] (22) between positions 216 and 254 of the RSV F protein;

[0667] (23) between positions 236 and 249 of the RSV F protein;

[0668] (24) between positions 309 and 450 of the RSV F protein;

[0669] (25) between positions 385 and 441 of the RSV F protein;

[0670] (26) between positions 418 and 441 of the RSV F protein;

[0671] (27) between positions 457 and 477 of the RSV F protein;

[0672] (28) Between positions 130 and 265 of the RSV F protein.

[0673] In some embodiments, the amino acid mutation for filling the hydrophobic cavity comprises an amino acid substitution at an amino acid position selected from the group consisting of: asparagine 42 residue, valine 65 residue, leucine 71 residue, leucine 205 residue, methionine 264 residue, isoleucine 267 residue, isoleucine 192 residue and / or serine 165 residue.

[0674] In some embodiments, amino acid mutations for filling the hydrophobic cavity include: 42L, 42F, 42Y, 42W, 42H, 65Y, 65F, 71I, 71Y, 205F, 205Y, 264F, 264Y, 267L, 267F, 267Y, 192L, 165I, 165F, 165Y and / or 165L.

[0675] In some preferred embodiments, the amino acid mutations used to fill the hydrophobic cavity include: N42L, N42F, N42Y, N42W, N42H, V65Y, V65F, L71I, L71Y, L205F, L205Y, M264F, M264Y, I267L, I267F, I267Y, I192L, S165I, S165F, S165Y and / or S165L.

[0676] In some embodiments, the amino acid mutation for electrostatic engineering comprises an amino acid substitution at the amino acid position of aspartic acid residue 461 and / or glutamic acid residue 462.

[0677] In some embodiments, the amino acid mutations used for electrostatic modification include: 461S, 461N, 461Q, 461E, 461G, 461A, 461K, 461R, 461H, 462Q, 462S, 462N, 462D, 462G, 462A, 462K, 462R, 462H.

[0678] In some preferred embodiments, the amino acid mutations used for electrostatic modification include: D461S, D461N, D461Q, D461E, D461G, D461A, D461K, D461R, D461H, E462Q, E462S, E462N, E462D, E462G, E462A, E462K, E462R, and E462H.

[0679] In some embodiments, the RSV F protein comprises a combination of modifications or mutations selected from the group consisting of:

[0680] (1)N42L, S165I;

[0681] (2)N42L, D461S;

[0682] (3) V65Y, D461S;

[0683] (4) V65Y, S165I;

[0684] (5)N42L, V65Y, D461S;

[0685] (6)N42L, D461S, M264F;

[0686] (7) V65Y, D461S, M264F;

[0687] (8)N42L, V65Y, D461S, M264F;

[0688] (9)I192L, N42L, L71Y, I267L, D461S;

[0689] (10)I192L, N42L, L205F, M264F, I267L, D461S;

[0690] (11)I192L, N42L, V65Y, L205F, M264F, D461S;

[0691] (12)I192L, N42L, L71Y, I267L;

[0692] (13)N42L, V65Y, S165I;

[0693] (14)N42L, S165I, M264F;

[0694] (15)V65Y, S165I, M264F;

[0695] (16)N42L, V65Y, S165I, M264F;

[0696] (17)I192L, N42L, V65Y, L205F, M264F;

[0697] (18)I192L, N42L, L71Y, I267L, S165I;

[0698] (19)I192L, N42L, L205F, M264F, I267L, S165I;

[0699] (20)I192L, N42L, V65Y, L205F, M264F, S165I;

[0700] (21)N42L, D461S, S165I;

[0701] (22)V65Y, D461S, S165I;

[0702] (23) N42L, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0703] (24)M264F, D461S, S165I;

[0704] (25)N42L, V65Y, D461S, S165I;

[0705] (26)N42L, D461S, S165I, M264F;

[0706] (27)V65Y, D461S, S165I, M264F;

[0707] (28) N42L, D461S, introduction of a disulfide bond between protein positions 30 and 163;

[0708] (29)I192L, N42L, L71Y, I267L, D461S, S165I;

[0709] (30)I192L, N42L, L205F, M264F, I267L, D461S, S165I;

[0710] (31)I192L, N42L, V65Y, L205F, M264F, D461S, S165I;

[0711] (32) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0712] (33) V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163;

[0713] (34) M264F, D461S, introduction of a disulfide bond between protein positions 30 and 163;

[0714] (35) N42L, V65Y, D461S, and introduction of a disulfide bond between protein positions 30 and 163;

[0715] (36) N42L, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0716] (37) N42L, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0717] (38) V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0718] (39)M264F, S165I;

[0719] (40) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0720] (41) I192L, N42L, L71Y, I267L, D461S, and a disulfide bond was introduced between protein positions 30 and 163;

[0721] (42) I192L, N42L, L205F, M264F, I267L, D461S, and a disulfide bond was introduced between protein positions 30 and 163;

[0722] (43) I192L, N42L, V65Y, L205F, M264F, D461S, and a disulfide bond was introduced between protein positions 30 and 163;

[0723] (44) N42L, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0724] (45) V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0725] (46) M264F, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0726] (47) N42L, V65Y, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0727] (48) N42L, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0728] (49) N42L, S165I, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0729] (50) V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0730] (51) N42L, M264F, and introduction of a disulfide bond between protein positions 457 and 477;

[0731] (52) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0732] (53) I192L, N42L, L71Y, I267L, S165I, and a disulfide bond was introduced between protein positions 30 and 163;

[0733] (54) I192L, N42L, L205F, M264F, I267L, S165I, and a disulfide bond was introduced between protein positions 30 and 163;

[0734] (55) I192L, N42L, V65Y, L205F, M264F, S165I, and a disulfide bond was introduced between protein positions 30 and 163;

[0735] (56) N42L, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0736] (57) V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0737] (58)N42L, M264F;

[0738] (59) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 457 and 477;

[0739] (60) M264F, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163;

[0740] (61) N42L, V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0741] (62) N42L, V65Y, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163;

[0742] (63) N42L, D461S, and introduction of a disulfide bond between protein positions 78 and 123;

[0743] (64) V65Y, S165I, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F;

[0744] (65) V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123;

[0745] (66) M264F, D461S, introduction of a disulfide bond between protein positions 78 and 123;

[0746] (67) N42L, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0747] (68) N42L, D461S, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0748] (69) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 152 and 164;

[0749] (70) V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0750] (71)M264F, D461S;

[0751] (72) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0752] (73)N42L, V65Y, M264F, D461S, S165I;

[0753] (74) I192L, N42L, L71Y, I267L, D461S, and a disulfide bond was introduced between protein positions 78 and 123;

[0754] (75) I192L, N42L, L205F, M264F, I267L, D461S, and a disulfide bond was introduced between protein positions 78 and 123;

[0755] (76) N42L, V65Y, D461S, and introduction of a disulfide bond between protein positions 78 and 123;

[0756] (77) I192L, N42L, V65Y, L205F, M264F, D461S, and a disulfide bond was introduced between protein positions 78 and 123;

[0757] (78) N42L, S165I, introduction of a disulfide bond between protein positions 78 and 123;

[0758] (79) V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123;

[0759] (80) M264F, S165I, introduction of a disulfide bond between protein positions 78 and 123;

[0760] (81) N42L, V65Y, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0761] (82) N42L, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0762] (83) V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0763] (84) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0764] (85) I192L, N42L, L71Y, I267L, S165I, and a disulfide bond was introduced between protein positions 78 and 123;

[0765] (86) I192L, N42L, L205F, M264F, I267L, S165I, and a disulfide bond was introduced between protein positions 78 and 123;

[0766] (87) I192L, N42L, V65Y, L205F, M264F, S165I, and a disulfide bond was introduced between protein positions 78 and 123;

[0767] (88) N42L, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0768] (89) V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0769] (90) M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0770] (91) N42L, V65Y, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123;

[0771] (92) V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0772] (93) N42L, V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F;

[0773] (94) N42L, V65Y, and introduction of a disulfide bond between protein positions 7 and 416;

[0774] (95) N42L, V65Y, and introduction of a disulfide bond between protein positions 15 and 358;

[0775] (96) N42L, V65Y, and introduction of a disulfide bond between protein positions 20 and 338;

[0776] (97) N42L, V65Y, and introduction of a disulfide bond between protein positions 20 and 339;

[0777] (98) N42L, V65Y, and introduction of a disulfide bond between protein positions 22 and 248;

[0778] (99) N42L, V65Y, and introduction of a disulfide bond between protein positions 23 and 283;

[0779] (100) N42L, V65Y, and introduction of a disulfide bond between protein positions 31 and 164;

[0780] (101) N42L, V65Y, and introduction of a disulfide bond between protein positions 34 and 168;

[0781] (102) N42L, V65Y, and introduction of a disulfide bond between protein positions 34 and 272;

[0782] (103) N42L, V65Y, and introduction of a disulfide bond between protein positions 37 and 175;

[0783] (104) N42L, V65Y, and introduction of a disulfide bond between protein positions 64 and 209;

[0784] (105) N42L, V65Y, and introduction of a disulfide bond between protein positions 133 and 266;

[0785] (106) N42L, V65Y, and introduction of a disulfide bond between protein positions 155 and 161;

[0786] (107) N42L, V65Y, and introduction of a disulfide bond between protein positions 152 and 164;

[0787] (108) N42L, V65Y, and introduction of a disulfide bond between protein positions 236 and 249;

[0788] (109) N42L, V65Y, and introduction of a disulfide bond between protein positions 309 and 450;

[0789] (110) N42L, V65Y, and introduction of a disulfide bond between protein positions 418 and 441;

[0790] (111) N42L, V65Y, and introduction of a disulfide bond between protein positions 385 and 441;

[0791] (112) N42L, V65Y, and the introduction of a disulfide bond between protein positions 457 and 477;

[0792] (113) N42L, V65Y, and introduction of a disulfide bond between protein positions 67 and 229;

[0793] (114) N42L, V65Y, and introduction of a disulfide bond between protein positions 119 and 381;

[0794] (115) N42L, V65Y, and introduction of a disulfide bond between protein positions 125 and 433;

[0795] (116) N42L, V65Y, and introduction of a disulfide bond between protein positions 128 and 436;

[0796] (117) N42L, V65Y, and introduction of a disulfide bond between protein positions 210 and 224;

[0797] (118) N42L, V65Y, and introduction of a disulfide bond between protein positions 216 and 254;

[0798] (119) N42L, M264F, introduction of a disulfide bond between protein positions 7 and 416;

[0799] (120) N42L, M264F, introduction of a disulfide bond between protein positions 15 and 358;

[0800] (121) N42L, M264F, introduction of a disulfide bond between protein positions 20 and 338;

[0801] (122) N42L, M264F, introduction of a disulfide bond between protein positions 20 and 339;

[0802] (123) N42L, M264F, introduction of a disulfide bond between protein positions 22 and 248;

[0803] (124) N42L, M264F, and introduction of a disulfide bond between protein positions 23 and 283;

[0804] (125) N42L, M264F, introduction of a disulfide bond between protein positions 31 and 164;

[0805] (126) N42L, M264F, introduction of a disulfide bond between protein positions 34 and 168;

[0806] (127) N42L, M264F, and introduction of a disulfide bond between protein positions 34 and 272;

[0807] (128) N42L, M264F, and introduction of a disulfide bond between protein positions 37 and 175;

[0808] (129) V65Y, M264F, introduction of a disulfide bond between protein positions 7 and 416;

[0809] (130) N42L, M264F, introduction of a disulfide bond between protein positions 133 and 266;

[0810] (131) N42L, M264F, and introduction of a disulfide bond between protein positions 155 and 161;

[0811] (132) N42L, M264F, introduction of a disulfide bond between protein positions 152 and 164;

[0812] (133) N42L, M264F, and introduction of a disulfide bond between protein positions 236 and 249;

[0813] (134) N42L, M264F, and introduction of a disulfide bond between protein positions 309 and 450;

[0814] (135) V65Y, M264F, introduction of a disulfide bond between protein positions 15 and 358;

[0815] (136) N42L, M264F, and introduction of a disulfide bond between protein positions 385 and 441;

[0816] (137) V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 338;

[0817] (138) N42L, M264F, and introduction of a disulfide bond between protein positions 67 and 229;

[0818] (139) N42L, M264F, introduction of a disulfide bond between protein positions 119 and 381;

[0819] (140) N42L, M264F, and introduction of a disulfide bond between protein positions 125 and 433;

[0820] (141) N42L, M264F, introduction of a disulfide bond between protein positions 128 and 436;

[0821] (142) N42L, M264F, introduction of a disulfide bond between protein positions 210 and 224;

[0822] (143) N42L, M264F, introduction of a disulfide bond between protein positions 216 and 254;

[0823] (144) V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 339;

[0824] (145) V65Y, M264F, introduction of a disulfide bond between protein positions 22 and 248;

[0825] (146) V65Y, M264F, introduction of a disulfide bond between protein positions 23 and 283;

[0826] (147) V65Y, M264F, introduction of a disulfide bond between protein positions 31 and 164;

[0827] (148) V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 168;

[0828] (149) V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 272;

[0829] (150) V65Y, M264F, introduction of a disulfide bond between protein positions 37 and 175;

[0830] (151) V65Y, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0831] (152) V65Y, M264F, and introduction of a disulfide bond between protein positions 133 and 266;

[0832] (153) V65Y, M264F, introduction of a disulfide bond between protein positions 155 and 161;

[0833] (154) V65Y, M264F, introduction of a disulfide bond between protein positions 152 and 164;

[0834] (155) V65Y, M264F, introduction of a disulfide bond between protein positions 236 and 249;

[0835] (156) V65Y, M264F, and introduction of a disulfide bond between protein positions 309 and 450;

[0836] (157) V65Y, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[0837] (158) V65Y, M264F, and introduction of a disulfide bond between protein positions 385 and 441;

[0838] (159) V65Y, M264F, and introduction of a disulfide bond between protein positions 457 and 477;

[0839] (160) V65Y, M264F, introduction of a disulfide bond between protein positions 67 and 229;

[0840] (161) V65Y, M264F, introduction of a disulfide bond between protein positions 119 and 381;

[0841] (162) V65Y, M264F, introduction of a disulfide bond between protein positions 125 and 433;

[0842] (163) V65Y, M264F, introduction of a disulfide bond between protein positions 128 and 436;

[0843] (164) V65Y, M264F, introduction of a disulfide bond between protein positions 210 and 224;

[0844] (165) V65Y, M264F, introduction of a disulfide bond between protein positions 216 and 254;

[0845] (166) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 7 and 416;

[0846] (167) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 15 and 358;

[0847] (168) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 20 and 338;

[0848] (169) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 20 and 339;

[0849] (170) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 22 and 248;

[0850] (171) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 23 and 283;

[0851] (172) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 31 and 164;

[0852] (173) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 34 and 168;

[0853] (174) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 34 and 272;

[0854] (175) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 37 and 175;

[0855] (176) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 64 and 209;

[0856] (177) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 133 and 266;

[0857] (178) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 155 and 161;

[0858] (179) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 216 and 254;

[0859] (180) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 236 and 249;

[0860] (181) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 309 and 450;

[0861] (182) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 210 and 224;

[0862] (183) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 385 and 441;

[0863] (184) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 128 and 436;

[0864] (185) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 67 and 229;

[0865] (186) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 119 and 381;

[0866] (187) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 125 and 433;

[0867] (188) A disulfide bond and S165I were introduced between protein positions 418 and 441;

[0868] (189) A disulfide bond, D461S, was introduced between protein positions 418 and 441;

[0869] (190) A disulfide bond was introduced between protein positions 418 and 441, E462Q;

[0870] (191) A disulfide bond and S165I were introduced between protein positions 457 and 477;

[0871] (192) A disulfide bond, D461S, was introduced between protein positions 457 and 477;

[0872] (193) introduction of a disulfide bond between protein positions 457 and 477, E462Q;

[0873] (194) A disulfide bond and S165I were introduced between protein positions 64 and 209;

[0874] (195) A disulfide bond, D461S, was introduced between protein positions 64 and 209;

[0875] (196) A disulfide bond, E462Q, was introduced between protein positions 64 and 209;

[0876] (197)I192L, N42L, L205F, M264F, I267L;

[0877] (198)N42L, L205F, L71Y, I267L;

[0878] (199)I192L, N42L, V65Y, L205F;

[0879] (200)I192L, N42L, L205F, L71Y;

[0880] (201)I192L, N42L, L205F, I267L;

[0881] (202) I192L, N42L, L205F, I267L, and the introduction of a disulfide bond between protein positions 457 and 477;

[0882] (203) I192L, N42L, V65Y, L205F, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[0883] (204)I192L, N42L, L205F;

[0884] (205) I192L, N42L, L205F, I267L, and a disulfide bond was introduced between protein positions 130 and 265;

[0885] (206) N42L, V65Y, L205F, L71Y, and introduction of a disulfide bond between protein positions 130 and 265;

[0886] (207) I192L, N42L, V65Y, L205F, M264F, and a disulfide bond was introduced between protein positions 130 and 265;

[0887] (208) S165I, M264F, introduction of a disulfide bond between protein positions 418 and 441;

[0888] (209) D461S, M264F, introduction of a disulfide bond between protein positions 418 and 441;

[0889] (210) E462Q, M264F, introduction of a disulfide bond between protein positions 418 and 441;

[0890] (211) S165I, M264F, introduction of a disulfide bond between protein positions 457 and 477;

[0891] (212) D461S, M264F, introduction of a disulfide bond between protein positions 457 and 477;

[0892] (213) E462Q, M264F, introduction of a disulfide bond between protein positions 457 and 477;

[0893] (214) S165I, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0894] (215) D461S, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0895] (216) E462Q, M264F, introduction of a disulfide bond between protein positions 64 and 209;

[0896] (217) D461S, S165I, introduction of a disulfide bond between protein positions 64 and 209;

[0897] (218) D461S, S165I, introduction of a disulfide bond between protein positions 418 and 441;

[0898] (219) S165I, N42L, introduction of a disulfide bond between protein positions 418 and 441;

[0899] (220) D461S, N42L, introduction of a disulfide bond between protein positions 418 and 441;

[0900] (221) E462Q, N42L, introduction of a disulfide bond between protein positions 418 and 441;

[0901] (222) S165I, N42L, and introduction of a disulfide bond between protein positions 457 and 477;

[0902] (223) D461S, N42L, and introduction of a disulfide bond between protein positions 457 and 477;

[0903] (224) E462Q, N42L, introduction of a disulfide bond between protein positions 457 and 477;

[0904] (225) S165I, N42L, introduction of a disulfide bond between protein positions 64 and 209;

[0905] (226) D461S, N42L, introduction of a disulfide bond between protein positions 64 and 209;

[0906] (227) E462Q, N42L, introduction of a disulfide bond between protein positions 64 and 209;

[0907] (228) S165I, V65Y, introduction of a disulfide bond between protein positions 418 and 441;

[0908] (229) D461S, V65Y, introduction of a disulfide bond between protein positions 418 and 441;

[0909] (230) E462Q, V65Y, introduction of a disulfide bond between protein positions 418 and 441;

[0910] (231) S165I, V65Y, and introduction of a disulfide bond between protein positions 457 and 477;

[0911] (232) D461S, V65Y, introduction of a disulfide bond between protein positions 457 and 477;

[0912] (233) E462Q, V65Y, introduction of a disulfide bond between protein positions 457 and 477;

[0913] (234) S165I, V65Y, introduction of a disulfide bond between protein positions 64 and 209;

[0914] (235) D461S, V65Y, introduction of a disulfide bond between protein positions 64 and 209;

[0915] (236) E462Q, V65Y, introduction of a disulfide bond between protein positions 64 and 209;

[0916] (237) E462Q, S165I, introduction of a disulfide bond between protein positions 64 and 209;

[0917] (238) E462Q, S165I, introduction of a disulfide bond between protein positions 418 and 441;

[0918] (239) D461N, introduction of a disulfide bond between protein positions 418 and 441;

[0919] (240) D461Q, introduction of a disulfide bond between protein positions 418 and 441;

[0920] (241) D461E, introduction of a disulfide bond between protein positions 418 and 441;

[0921] (242) D461G, introduction of a disulfide bond between protein positions 418 and 441;

[0922] (243) D461A, introduction of a disulfide bond between protein positions 418 and 441;

[0923] (244) D461K, introduction of a disulfide bond between protein positions 418 and 441;

[0924] (245) D461R, introduction of a disulfide bond between protein positions 418 and 441;

[0925] (246) D461H, introduction of a disulfide bond between protein positions 418 and 441;

[0926] (247) D461N, introduction of a disulfide bond between protein positions 457 and 477;

[0927] (248) D461Q, introduction of a disulfide bond between protein positions 457 and 477;

[0928] (249) D461E, introduction of a disulfide bond between protein positions 457 and 477;

[0929] (250) D461G, introduction of a disulfide bond between protein positions 457 and 477;

[0930] (251) D461A, introduction of a disulfide bond between protein positions 457 and 477;

[0931] (252) D461K, introduction of a disulfide bond between protein positions 457 and 477;

[0932] (253) D461R, introduction of a disulfide bond between protein positions 457 and 477;

[0933] (254) D461H, introduction of a disulfide bond between protein positions 457 and 477;

[0934] (255) D461N, introduction of a disulfide bond between protein positions 64 and 209;

[0935] (256) D461Q, introduction of a disulfide bond between protein positions 64 and 209;

[0936] (257) D461E, introduction of a disulfide bond between protein positions 64 and 209;

[0937] (258) D461G, introduction of a disulfide bond between protein positions 64 and 209;

[0938] (259) D461A, introduction of a disulfide bond between protein positions 64 and 209;

[0939] (260) D461K, introduction of a disulfide bond between protein positions 64 and 209;

[0940] (261) D461R, introduction of a disulfide bond between protein positions 64 and 209;

[0941] (262) D461H, introduction of a disulfide bond between protein positions 64 and 209;

[0942] (263) E462S, introduction of a disulfide bond between protein positions 418 and 441;

[0943] (264) E462N, introduction of a disulfide bond between protein positions 418 and 441;

[0944] (265) E462D, introduction of a disulfide bond between protein positions 418 and 441;

[0945] (266) E462G, introduction of a disulfide bond between protein positions 418 and 441;

[0946] (267) E462A, introduction of a disulfide bond between protein positions 418 and 441;

[0947] (268) E462K, introduction of a disulfide bond between protein positions 418 and 441;

[0948] (269) E462R, introduction of a disulfide bond between protein positions 418 and 441;

[0949] (270) E462H, introduction of a disulfide bond between protein positions 418 and 441;

[0950] (271) E462S, introduction of a disulfide bond between protein positions 64 and 209;

[0951] (272) E462N, introduction of a disulfide bond between protein positions 64 and 209;

[0952] (273) E462D, introduction of a disulfide bond between protein positions 64 and 209;

[0953] (274) E462G, introduction of a disulfide bond between protein positions 64 and 209;

[0954] (275) E462A, introduction of a disulfide bond between protein positions 64 and 209;

[0955] (276) E462K, introduction of a disulfide bond between protein positions 64 and 209;

[0956] (277) E462R, introduction of a disulfide bond between protein positions 64 and 209;

[0957] (278) E462H, introduction of a disulfide bond between protein positions 64 and 209;

[0958] (279) E462S, introduction of a disulfide bond between protein positions 457 and 477;

[0959] (280) E462N, introduction of a disulfide bond between protein positions 457 and 477;

[0960] (281) E462D, introduction of a disulfide bond between protein positions 457 and 477;

[0961] (282) E462G, introduction of a disulfide bond between protein positions 457 and 477;

[0962] (283) E462A, introduction of a disulfide bond between protein positions 457 and 477;

[0963] (284) E462K, introduction of a disulfide bond between protein positions 457 and 477;

[0964] (285) E462R, introduction of a disulfide bond between protein positions 457 and 477;

[0965] (286) E462H, introduction of a disulfide bond between protein positions 457 and 477;

[0966] (287) D461N, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0967] (288) D461Q, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0968] (289) D461E, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0969] (290) D461G, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0970] (291) D461A, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0971] (292) D461K, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0972] (293) D461R, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[0973] (294) D461H, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[0974] (295) D461N, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0975] (296) D461Q, introduction of a disulfide bond between protein positions 457 and 477, N42L;

[0976] (297) D461E, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0977] (298) D461G, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0978] (299) D461A, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0979] (300) D461K, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0980] (301) D461R, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0981] (302) D461H, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[0982] (303) D461N, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0983] (304) D461Q, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0984] (305) D461E, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0985] (306) D461G, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0986] (307) D461A, introduction of a disulfide bond between protein positions 64 and 209, and N42L;

[0987] (308) D461K, introduction of a disulfide bond between protein positions 64 and 209, and N42L;

[0988] (309) D461R, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[0989] (310) D461H, introduction of a disulfide bond between protein positions 64 and 209, and N42L;

[0990] (311) D461N, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0991] (312) D461Q, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0992] (313) D461E, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0993] (314) D461G, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0994] (315) D461A, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0995] (316) D461K, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0996] (317) D461R, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0997] (318) D461H, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[0998] (319) D461N, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[0999] (320) D461Q, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1000] (321) D461E, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1001] (322) D461G, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1002] (323) D461A, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1003] (324) D461K, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1004] (325) D461R, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1005] (326) D461H, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1006] (327) D461N, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1007] (328) D461Q, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1008] (329) D461E, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1009] (330) D461G, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1010] (331) D461A, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1011] (332) D461K, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1012] (333) D461R, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1013] (334) D461H, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1014] (335) D461N, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1015] (336) D461Q, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1016] (337) D461E, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1017] (338) D461G, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1018] (339) D461A, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1019] (340) D461K, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1020] (341) D461R, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1021] (342) D461H, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1022] (343) D461N, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1023] (344) D461Q, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1024] (345) D461E, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1025] (346) D461G, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1026] (347) D461A, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1027] (348) D461K, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1028] (349) D461R, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1029] (350) D461H, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1030] (351) D461N, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1031] (352) D461Q, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1032] (353) D461E, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1033] (354) D461G, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1034] (355) D461A, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1035] (356) D461K, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1036] (357) D461R, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1037] (358) D461H, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1038] (359) E462S, introduction of a disulfide bond between protein positions 418 and 441, and N42L;

[1039] (360) E462N, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[1040] (361) E462D, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[1041] (362) E462G, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[1042] (363) E462A, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[1043] (364) E462K, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[1044] (365) E462R, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[1045] (366) E462H, introduction of a disulfide bond between protein positions 418 and 441, N42L;

[1046] (367) E462S, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1047] (368) E462N, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1048] (369) E462D, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1049] (370) E462G, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1050] (371) E462A, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1051] (372) E462K, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1052] (373) E462R, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1053] (374) E462H, introduction of a disulfide bond between protein positions 64 and 209, N42L;

[1054] (375) E462S, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1055] (376) E462N, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1056] (377) E462D, introduction of a disulfide bond between protein positions 457 and 477, N42L;

[1057] (378) E462G, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1058] (379) E462A, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1059] (380) E462K, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1060] (381) E462R, introduction of a disulfide bond between protein positions 457 and 477, N42L;

[1061] (382) E462H, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1062] (383) E462S, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1063] (384) E462N, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1064] (385) E462D, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1065] (386) E462G, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1066] (387) E462A, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1067] (388) E462K, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1068] (389) E462R, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1069] (390) E462H, introduction of a disulfide bond between protein positions 418 and 441, V65Y;

[1070] (391) E462S, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1071] (392) E462N, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1072] (393) E462D, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1073] (394) E462G, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1074] (395) E462A, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1075] (396) E462K, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1076] (397) E462R, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1077] (398) E462H, introduction of a disulfide bond between protein positions 64 and 209, V65Y;

[1078] (399) E462S, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1079] (400) E462N, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1080] (401) E462D, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1081] (402) E462G, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1082] (403) E462A, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1083] (404) E462K, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1084] (405) E462R, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1085] (406) E462H, introduction of a disulfide bond between protein positions 457 and 477, V65Y;

[1086] (407) E462S, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1087] (408) E462N, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1088] (409) E462D, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1089] (410) E462G, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1090] (411) E462A, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1091] (412) E462K, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1092] (413) E462R, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1093] (414) E462H, introduction of a disulfide bond between protein positions 418 and 441, M264F;

[1094] (415) E462S, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1095] (416) E462N, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1096] (417) E462D, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1097] (418) E462G, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1098] (419) E462A, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1099] (420) E462K, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1100] (421) E462R, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1101] (422) E462H, introduction of a disulfide bond between protein positions 64 and 209, M264F;

[1102] (423) E462S, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1103] (424) E462N, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1104] (425) E462D, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1105] (426) E462G, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1106] (427) E462A, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1107] (428) E462K, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1108] (429) E462R, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1109] (430) E462H, introduction of a disulfide bond between protein positions 457 and 477, M264F;

[1110] (431)V65Y, M264F;

[1111] (432)N42L, V65Y, M264F.

[1112] In some preferred embodiments, the RSV F protein comprises a combination of modifications or mutations selected from the group consisting of:

[1113] (1)192L, 42L, 71Y, 267L;

[1114] (2)192L, 42L, 65Y, 205F, 264F;

[1115] (3) 42L, 264F, and the introduction of a disulfide bond between protein positions 64 and 209;

[1116] (4) 42L, 264F, 461S, 165I, and the introduction of a disulfide bond between protein positions 78 and 123;

[1117] (5) introduction of a disulfide bond between 42L, 264F, and protein positions 457 and 477;

[1118] (6) 42L, 65Y, 264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[1119] (7) 42L, 65Y, 264F, 461S, 165I, and the introduction of a disulfide bond between protein positions 30 and 163;

[1120] (8) 42L, 264F, and introduction of a disulfide bond between protein positions 418 and 441;

[1121] (9)42L, 65Y, 264F, 461S, 165I;

[1122] (10) 42L, 65Y, 461S, and introduction of a disulfide bond between protein positions 78 and 123;

[1123] (11)192L, 42L, 205F, 264F, 267L;

[1124] (12)42L, 205F, 71Y, 267L;

[1125] (13)192L, 42L, 65Y, 205F;

[1126] (14)192L, 42L, 205F, 71Y;

[1127] (15)192L, 42L, 205F, 267L;

[1128] (16) 192L, 42L, 205F, 267L, and the introduction of a disulfide bond between protein positions 457 and 477;

[1129] (17) 192L, 42L, 65Y, 205F, 264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[1130] (18)192L, 42L, 205F;

[1131] (19) 192L, 42L, 205F, 267L, and introduction of a disulfide bond between protein positions 130 and 265;

[1132] (20) 42L, 65Y, 205F, 71Y, and a disulfide bond was introduced between protein positions 130 and 265;

[1133] (21) 192L, 42L, 65Y, 205F, 264F, and introduction of a disulfide bond between protein positions 130 and 265;

[1134] (22) 42L, 65Y, 264F, and introduction of a disulfide bond between protein positions 418 and 441;

[1135] (23)264F, 165I;

[1136] (24)42L, 264F;

[1137] (25) 42L, 65Y, 264F, and introduction of a disulfide bond between protein positions 152 and 164;

[1138] (26) 461Q, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[1139] (27) 461G, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[1140] (28) 461K, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[1141] (29) 461H, introduction of a disulfide bond between protein positions 457 and 477, 42L;

[1142] (30) 461A, introduction of a disulfide bond between protein positions 457 and 477, 264F;

[1143] (31) 461H, introduction of a disulfide bond between protein positions 64 and 209, 264F.

[1144] In some more preferred embodiments, the RSV F protein comprises a combination of modifications or mutations selected from the group consisting of:

[1145] (1)I192L, N42L, L71Y, I267L;

[1146] (2)I192L, N42L, V65Y, L205F, M264F;

[1147] (3) N42L, M264F, and the introduction of a disulfide bond between protein positions 64 and 209;

[1148] (4) N42L, M264F, D461S, S165I, and the introduction of a disulfide bond between protein positions 78 and 123;

[1149] (5) N42L, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[1150] (6) N42L, V65Y, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[1151] (7) N42L, V65Y, M264F, D461S, S165I, and the introduction of a disulfide bond between protein positions 30 and 163;

[1152] (8) N42L, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[1153] (9)N42L, V65Y, M264F, D461S, S165I;

[1154] (10) N42L, V65Y, D461S, and introduction of a disulfide bond between protein positions 78 and 123;

[1155] (11)I192L, N42L, L205F, M264F, I267L;

[1156] (12)N42L, L205F, L71Y, I267L;

[1157] (13)I192L, N42L, V65Y, L205F;

[1158] (14)I192L, N42L, L205F, L71Y;

[1159] (15)I192L, N42L, L205F, I267L;

[1160] (16) I192L, N42L, L205F, I267L, and the introduction of a disulfide bond between protein positions 457 and 477;

[1161] (17) I192L, N42L, V65Y, L205F, M264F, and the introduction of a disulfide bond between protein positions 457 and 477;

[1162] (18)I192L, N42L, L205F;

[1163] (19) I192L, N42L, L205F, I267L, and the introduction of a disulfide bond between protein positions 130 and 265;

[1164] (20) N42L, V65Y, L205F, L71Y, and introduction of a disulfide bond between protein positions 130 and 265;

[1165] (21) I192L, N42L, V65Y, L205F, M264F, and the introduction of a disulfide bond between protein positions 130 and 265;

[1166] (22) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 418 and 441;

[1167] (23)M264F, S165I;

[1168] (24)N42L, M264F;

[1169] (25) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 152 and 164;

[1170] (26) D461Q, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1171] (27) D461G, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1172] (28) D461K, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1173] (29) D461H, introduction of a disulfide bond between protein positions 457 and 477, and N42L;

[1174] (30) D461A, introduction of a disulfide bond between protein positions 457 and 477, and M264F;

[1175] (31) D461H, introduction of a disulfide bond between protein positions 64 and 209, and M264F.

[1176] In some embodiments, the p27 peptide segment (SEQ ID NO: 560) of the RSV F protein is replaced with a linker.

[1177] In some embodiments, when the p27 peptide segment is replaced with a linker, some of the residues upstream and downstream of the p27 peptide segment may be replaced together.

[1178] In some embodiments, when the p27 peptide segment is replaced by a linker, the p27 peptide segment may be partially or completely replaced by the linker.

[1179] In some preferred embodiments, when the p27 peptide is replaced by a linker, the starting point of the linker connection is any site between amino acid residue positions 75-84 of the RSV F protein, and the end point is any site between amino acid residue positions 112-125 of the RSV F protein.

[1180] In some preferred embodiments, the linker is a flexible polypeptide.

[1181] In some preferred embodiments, the above-mentioned flexible polypeptide is selected from (GS)n, (GGSG)n, (GGGGS)n, (GGGSGG)n, (GGGSGGG)n, (EA)n, (EAK)n, (EAKA)n, (AAAAK)n, (EAAAK)n, (LEAAK)n, wherein n is an integer from 1 to 4.

[1182] In some preferred embodiments, the flexible polypeptide is selected from the amino acid sequences shown in Table 2 below:

[1183] Table 2. Optional flexible polypeptide sequences

[1184] In some embodiments, the RSV F protein comprises an amino acid sequence selected from SEQ ID NOs: 2-499, 585-608 having 80% or greater identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity.

[1185] In some embodiments, the N-terminus of the RSV F protein is linked to a signal peptide.

[1186] In some preferred embodiments, the signal peptide is selected from a wild-type RSV F protein signal peptide or a non-RSV F protein signal peptide.

[1187] In some preferred embodiments, the signal peptide comprises an amino acid sequence selected from SEQ ID NO: 544, 546, 548.

[1188] In some preferred embodiments, the RSV F protein sequence does not contain a signal peptide, and its N-terminus may be linked to an RSV A2 subtype F protein signal peptide as shown in SEQ ID NO: 544 or any variant thereof, or may not be linked to a signal peptide.

[1189] In some preferred embodiments, the RSV F protein sequence does not contain a signal peptide, and its N-terminus may be connected to another signal peptide, which optionally has one or more amino acid mutations, substitutions, insertions or deletions compared to the RSV F protein signal peptide shown in SEQ ID NO: 544.

[1190] In some preferred embodiments, the RSV F protein sequence does not contain a signal peptide, and its N-terminus may be linked to a non-RSV F protein signal peptide, for example, an amino acid sequence as shown in SEQ ID NO: 546, 548 or any variant thereof, or other common signal peptide sequences.

[1191] In some embodiments, the C-terminus of the RSV F protein is linked to a trimerization tag.

[1192] In some preferred embodiments, the trimerization tag comprises an amino acid sequence as shown in SEQ ID NO: 550 or any variant thereof.

[1193] In some preferred embodiments, the trimerization tag can be other short peptides that are connected to the C-terminus of the RSV F protein and form a trimer in vitro after expression.

[1194] In a second aspect, the present disclosure provides a nucleic acid molecule encoding the RSV F protein of any of the above aspects.

[1195] In a third aspect, the present disclosure provides a vector comprising the above-mentioned nucleic acid molecule.

[1196] In some embodiments, the above-mentioned vector is an expression vector.

[1197] In a fourth aspect, the present disclosure provides a host cell comprising the above-mentioned vector.

[1198] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[1199] In some preferred embodiments, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[1200] In some preferred embodiments, the mammalian cells are selected from CHO cells, HEK293 cells or COS cells.

[1201] In a fifth aspect, the present disclosure provides an immunogenic composition comprising the RSV F protein of any of the above aspects.

[1202] In a sixth aspect, the present disclosure provides a vaccine composition comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[1203] In a seventh aspect, the present disclosure provides a kit for immunizing a subject against viral infection, comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[1204] In some preferred embodiments, the above viral infection is RSV infection.

[1205] In some preferred embodiments, the subject is a human subject.

[1206] In an eighth aspect, the present disclosure provides a pharmaceutical composition comprising the RSV F protein according to any of the above aspects and a pharmaceutically acceptable carrier.

[1207] In some preferred embodiments, the above pharmaceutical composition further comprises other therapeutic agents.

[1208] In a ninth aspect, the present disclosure provides a method for generating an immune response against a viral infection, comprising administering the RSV F protein of any of the above aspects in a pharmaceutically acceptable formulation to a subject.

[1209] In some preferred embodiments, the pharmaceutically acceptable formulation comprises an adjuvant.

[1210] In a tenth aspect, the present disclosure provides use of the above-mentioned RSV F protein in the preparation of a medicament for preventing or treating a disease or condition associated with respiratory syncytial virus (RSV) infection.

[1211] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[1212] In an eleventh aspect, the present disclosure provides a method for preventing or treating diseases associated with respiratory syncytial virus (RSV) infection, comprising administering an effective amount of the RSV F protein of any one of the above aspects in a pharmaceutically acceptable formulation to a subject in need thereof.

[1213] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[1214] In a twelfth aspect, the present disclosure provides a RSV F protein for preventing or treating diseases associated with respiratory syncytial virus (RSV) infection, wherein the RSV F protein is as described in any one of the above aspects.

[1215] For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[1216] Example

[1217] Example 1: Design and construction of RSV F protein mutants

[1218] 1. Design ideas for RSV F protein mutants

[1219] Using the wild-type RSV F protein from strain A2 as the parent, mutants were initially designed using single-point or single-pair mutations, including disulfide bond introduction, hydrophobic cavity filling, and electrostatic mutagenesis. Subsequently, based on ELISA data, these three modification methods were combined internally, and the optimal combination within each method was selected. Finally, the optimal combination of the three modification methods was selected and combined. The single-point or single-pair mutation designs are shown in Table 3 below, and the mutants generated by these mutation combinations are shown in Table 4.

[1220] Table 3. Transformation methods and single-point / single-pair mutations involved in the mutants

[1221] Table 4. Mutant combinations

[1222] 2. Construction of RSV F protein mutants

[1223] The A2 strain wild-type RSV F protein expression vector was synthesized by GenScript Biotech Co., Ltd., using the pcDNA3.1(+) backbone. Point mutation primers were designed using an online tool (https: / / crm.vazyme.com / cetool / singlepoint.html). Using wild-type VF-1 (SEQ ID NO: 1) as a template, PCR was performed using standard molecular biology techniques to introduce the desired amino acid mutations. The PCR product was transformed into competent DH5α medium and cultured overnight at 37°C. A single colony was picked and sequenced the next day. After sequencing confirmed the results, the culture was expanded to 15 mL, and the endotoxin-free plasmid was extracted according to the instructions of the EndoFree Mini Plasmid Kit II (Cat: #DP118-02). This yielded the expression vector for the RSV F protein mutant.

[1224] Example 2: Preliminary ELISA detection of antigenic properties of RSV F protein mutants

[1225] (1) Transfection of mutant expression plasmid

[1226] According to Nulen PlusTrans TM Transfection of mutant expression vectors into 96-well plates containing 293T cells was performed according to the transfection kit (Cat: CT801) instructions. The steps are as follows: 293T cells were plated one day in advance. The next day, cells were observed to grow to a confluence of 80-100% and then transfected. 0.5 μg of expression vector and 0.9 μL of transfection reagent were diluted in 25 μL of Opti-Men medium, respectively. The mixture was then mixed to form a transfection complex. The mixture was incubated at room temperature for 20 minutes and then added dropwise to the 96-well plate. After incubation at 37°C in a CO2 incubator for 72 hours, the supernatant was aspirated for subsequent analysis.

[1227] (2) ELISA detection of mutant antigen characteristics

[1228] The coating antibodies used were D25 and Motavizumab, the former recognizes the Φ site, and the latter recognizes the Ⅱ site on the RSV F protein.

[1229] Coating: Antibody D25 and Motavizumab were diluted to 3 μg / mL and 1 μg / mL, respectively, using ELISA coating solution. 100 μL was added to each well of a 96-well plate, sealed, and coated at 4°C overnight.

[1230] Washing: Discard the liquid in the wells and pat dry with absorbent paper. Add 300 μL of PBST to each well, let it stand for 5 minutes, spin dry, pat dry the ELISA plate, and repeat three times;

[1231] Blocking: Prepare 2% BSA solution with PBST, add 200 μL per well to block the ELISA plate, and block at 37°C for 2 h;

[1232] Washing: Same as above;

[1233] Antigen incubation: After transfection, the supernatant was diluted with PBST 1:5 and 100 μL was added to each ELISA plate. PBS negative control wells were set up at the same time and incubated at 37°C for 2 h.

[1234] Washing: Same as above;

[1235] Primary antibody incubation: Add 100 μL HRP conjugated mouse anti-His tag antibody (1:5000 dilution) and incubate at 37°C for 1 h.

[1236] Washing: Same as above;

[1237] Color development: Add 100 μL TMB substrate to each well and incubate at room temperature in the dark for 15 min;

[1238] Termination: Add 50 μL ELISA reaction stop solution to each well to terminate the reaction

[1239] Detection: Enable the microplate reader and read the OD value at a wavelength of 450 nm.

[1240] Analysis: A higher ratio (S / NP) of the sample well's OD value to the positive reference DS-Cav1's OD value indicates better binding between the sample and the corresponding antibody. Based on the test and analysis results, preferred mutants were selected. The S / NP ratios of some of these preferred mutants are shown in Table 5.

[1241] Table 5. S / NP values ​​of crude culture supernatants of some preferred mutants

[1242] Example 3: Stability of RSV F protein mutant culture supernatant

[1243] The stability (as shown in Table 6 and Table 7) of the RSV F protein mutant designed by stress test and storage stability experimental assessment. During the heat stress test, the crude culture supernatant of the mutant designed was hatched at 50 ℃, 55 ℃ or 60 ℃ for 1 hour or 2 hours and detected with anti-RSV F protein monoclonal antibody Motavizumab and pre-fusion specific monoclonal antibody D25 in ELISA assay. The ratio of the antibody reactivity of the sample of stress compared to the sample of no stress is defined as stress resistance parameter. It is expected that the more stable mutant has higher stress resistance.

[1244] During the storage stability assay, the reactivity of prefusion antibodies in crude culture supernatants after storage for one or two weeks at 4°C was compared with that in fresh culture supernatants. The activity ratio was defined as the storage stability of the mutant.

[1245] The results are presented in Tables 6 and 7. Some preferred mutants remained stable after incubation at 50°C, 55°C or 60°C for 1 or 2 hours, and their reactivity with Motavizumab and D25 remained at a high level.

[1246] Table 6. Storage stability of some preferred mutants at 4°C

[1247] Table 7. Heat shock stability of crude culture supernatant of some preferred mutants

[1248] Example 4: Expression and purification of RSV F protein

[1249] HEK293F cells were cultured in 800 ml shake flasks at 37°C and 5% CO2. 6 When the cells / mL was 1:1, transfection was performed using the transfection reagent PEI at a ratio of plasmid amount to transfection reagent amount = 1:3. Five days after transfection, the cell supernatant was recovered by centrifugation at 5000g for 20 minutes. The recovered cell supernatant was added to a Ni-NTA affinity chromatography column equilibrated with PBS pH 7.4 for enrichment, followed by washing with PBS pH 7.4, 20mM imidazole, and elution with PBS pH 7.4, 200mM imidazole. The eluted fusion protein was concentrated using an ultrafiltration tube and further purified by gel filtration chromatography after concentration in a PBS solution buffer (Figure 1). The protein obtained in the above process was identified for purity by SDS-PAGE (Figure 2). It should be noted that the use of nickel column affinity chromatography for protein purification of the fusion protein depends on the tag carried by the fusion protein. In other embodiments, other purification methods can also be selected by replacing the tag. The crude culture supernatant and purified protein were used for in vitro and in vivo assays described herein.

[1250] Example 5: Study on the stability of purified RSV F protein mutants

[1251] Purified RSV F protein mutants were subjected to various treatments to assess their stability, including five freeze-thaw cycles, storage at 4°C and room temperature (25°C) for 3 days, shaking at 4°C and 25°C for 3 days, and heat stress at 40°C and 50°C for 1 hour. The treated proteins were then assessed for stability by gel filtration chromatography, SDS-PAGE electrophoresis, and ELISA. The gel filtration chromatography and SDS-PAGE results are shown in Figures 3-10, and the ELISA results are shown in Tables 8 and 9. Mutants CL-012, CL-032, CL-051, CL-058, CL-059, CL-067, CL-073, CL-199, CL-017, CL-209, CL-204, and CL-205 showed comparable effects to the positive reference materials pXCS847 and DS-Cav1, with no significant changes observed after these treatments.

[1252] Table 8. Freeze-thaw stability ELISA of some preferred mutants

[1253] Table 9. Heat shock stability ELISA of some preferred mutants

[1254] pXCS847, DS-Cav1, and purified RSV F protein mutants were analyzed by differential scanning fluorimetry (DSF) to evaluate their thermal stability. Protein samples were diluted to a final concentration of 1 mg / ml in PBS (pH 7.4), and Sypro@orange dye was diluted to a 50× concentration in ultrapure water. The reaction system consisted of 3 μl of diluted protein sample, 3 μl of diluted dye, and 24 μl of PBS (pH 7.4). The reaction system was prepared as directed and assayed using a quantitative fluorescence PCR instrument (Bio-Rad CFX96 Touch). The PCR program used the melting curve module and HEX channel detection. The temperature was raised from 20°C and increased by 1°C per minute until the temperature reached 80°C. During this time, the fluorescence intensity was recorded as a function of temperature using the instrument's default parameters. The instrument has its own data processing function. The temperature corresponding to the minimum negative value of the first derivative of fluorescence intensity with respect to temperature is the Tm value. The results are shown in Figures 11 and 12. The measured Tm values ​​of all mutants are higher than 50°C, indicating that all mutants have good stability. The Tm values ​​of some preferred mutants are shown in Table 10.

[1255] Table 10. Tm values ​​of some preferred mutants

[1256] Example 6: Study on the immunogenicity of RSV F protein mutants

[1257] 1. Immunization of mice with different RSV F protein mutants

[1258] Female SPF-grade BALB / c mice, 6-8 weeks old, were purchased from Jicui Pharmaceutical Co., Ltd. (5 mice per group). On days 0 and 21, mice were immunized intramuscularly with 50 μl of 10 μg of antigen mixed with adjuvants such as MPL, CpG, or aluminum. Blood was collected by enucleation two weeks after the second immunization (5 weeks after the first immunization) to isolate serum for virus neutralization assays or ELISA tests.

[1259] Immunogen preparation method for the CpG adjuvant group: 150 μg CpG (ODN 1826) was dissolved in 75 μl of saline to prepare CpG working solution (enough for 5 mice). 50 μg of protein was diluted into 200 μl of saline and mixed with 75 μl of CpG solution. 50 μl of the mixture of immunogen and CpG was injected into the leg muscle of each mouse.

[1260] The preparation method of the MPL adjuvant immunogen is as follows:

[1261] Materials: (1) MPL, monophosphoryl lipid A (Sigma, L6895); (2) squalene (Sigma, S3626); (3) Tween 80; (4) soybean lecithin.

[1262] Procedure - Preliminary Preparation

[1263] (1) Prepare a 12% lecithin / squalene (w / v) stock solution:

[1264] a. Use a pipette to transfer 5 ml of squalene to a 15 ml polypropylene tube. b. Weigh out 600 mg of lecithin.

[1265] c. Add the lecithin to the squalene; cap the tube and place in a 65°C water bath until the lecithin dissolves. This will take 30–60 minutes, mixing occasionally by gentle inversion.

[1266] (2) Prepare a 0.5% Tween 80 / water (v / v) stock solution:

[1267] a. Use a pipette to transfer 250 μl of Tween 80 into a 50 ml polypropylene tube.

[1268] b. Add 49.75 ml of dH2O to the Tween 80; cap the tube and place in a 65°C water bath until the Tween 80 dissolves in the water. This will take 15–30 minutes, mixing occasionally by gentle inversion. Sterilize by 0.22 mm filter.

[1269] (3) Dissolve MPL in 12% lecithin / squalene stock solution:

[1270] a. Heat 12% lecithin / squalene stock solution in a 65°C water bath.

[1271] b. Add 100 μl of pre-warmed squalene / phosphatidylcholine mixture to the 1 mg MPL vial and recap the vial.

[1272] c. Sonicate in a 65°C incubator for 30–60 minutes, or until the MPL is completely dissolved.

[1273] (4) Preparation of emulsion

[1274] a. Pipette 0.1 ml of preheated MPL / lecithin / squalene into a 2.5 ml glass syringe. Pour 0.9 ml of preheated Tween 80 / water solution into a second syringe of equal size. Expel excess air from both syringes and securely connect them using a sterile 18-gauge microemulsion needle. Immediately push the Tween 80 / water solution through the microemulsion needle into the MPL / squalene / lecithin mixture. Continue to depress the syringe plunger to force the mixture through the needle to ensure thorough and uniform emulsification.

[1275] (5) Application of MPL oil-in-water emulsion and vaccine antigen

[1276] a. Before use, warm the stock MPL oil-in-water emulsion to room temperature and vortex vigorously. Mix 1 part of this emulsion with 8 parts of the antigen-containing solution to a 1% oil concentration. Mix 30 μl of this emulsion containing 30 μg of MPL (for 5 mice) with 240 μl of PBS containing 50 μg of protein. Inject 50 μl of this mixture into the leg muscle of each mouse.

[1277] 2. Detection of neutralizing antibodies using real virus method

[1278] 1. Sample information

[1279] Serum samples from BALA / c mice after immunization with RSV Fusion protein.

[1280] 2. Cell Plating

[1281] 1) One day in advance, HEp-2 cells (1.3*10 4 / each well) and cultured for 20 hours.

[1282] 3. Sample neutralization

[1283] 1) The neutralization titer of the sample (serum) against RSV A2 virus was detected using the CPE method on HEp-2 cells.

[1284] 2) Each sample was diluted at an initial dilution ratio of 1:10, and the second to eighth gradients were diluted three-fold (i.e., 1:10, 1:30, 1:90, 1:270, 1:810, 1:2430, 1:7290, 1:21870). Eight dilutions were set for each sample, and two replicates were set for each dilution.

[1285] 3) Add fresh culture medium to the deep-well plate. Add 216 μL of DMEM culture medium containing 2% FBS to each well of the initial dilution gradient well. Add 180 μL of DMEM culture medium containing 2% FBS to each well of the second to eighth gradient wells.

[1286] 4) Sample addition: initially add 54 μL of serum sample to each well.

[1287] 5) Gradient Dilution: Adjust the dispenser to a 90 μL volume and perform dilutions sequentially, mixing each well 12 times. Pipette 90 μL from the previous gradient into the next gradient and mix thoroughly. Note: Discard 90 μL after mixing the last gradient (maintaining consistent volume across all wells).

[1288] 6) Virus Neutralization: 180 μL / deep well * 96 wells * 5.5 blocks (prepare two more samples) = 95 mL of 2% FBSDMEM medium. The virus volume used is RSV A2 (3.3 virus titer is 9.14 * 105 PFU / mL) (2.4 times * 2.84 * 5.5 * 96 = 3605 μL). After incubation at 37°C for 1 hour, 150 μL of neutralization sample was added to each well and incubated at 37°C, 5% CO2 for 3 days.

[1289] 4. Plate Reading

[1290] Celigo plate reader: reads CPE and calculates NT50.

[1291] 5. Calculation of Neutralizing Antibody Titer

[1292] 1) When cytopathic effect is observed in one of the two wells of serum at a certain dilution, but not in the other well, the dilution is considered the neutralizing titer of the serum specimen;

[1293] 2) When two wells with a high dilution show complete pathology and two adjacent wells with a low dilution show no pathology, the average dilution of the two wells is the neutralizing titer of the serum specimen;

[1294] 3) When two adjacent dilutions of serum show cytopathic effect in one well and no cytopathic effect in the other well, the average dilution of the two is the neutralizing titer of the serum specimen.

[1295] The results of RSV A2 neutralization by sera from the CpG and MPL adjuvanted groups are shown in Figures 13 and 14, respectively. The results show that after two immunizations in mice, wild-type F, DS-Cav1, and all mutants tested (CL-012, CL-017, CL-199, CL-200, CL-201, CL-202, CL-203, CL-204, CL-205, CL-206, CL-207, CL-208, and CL-209) elicited neutralizing antibody responses. For the combined mutants CL-012, CL-017, CL-199, CL-205, and CL-208, neutralization efficacy was consistently higher with both adjuvants, indicating that these mutants are more immunogenic forms of the stabilized RSV prefusion F glycoprotein.

[1296] Example 7: CL-073 immunogenicity study

[1297] Female SPF BALB / c mice aged 6-8 weeks were purchased from Jicui Pharmaceutical Co., Ltd., with 5 mice per group. On day 0 and day 21, 50 μl of 50 μg aluminum hydroxide adjuvant ( Cat: #AJV3012) Post-F antigen (3 μg, Cat: #RSF-V52H6), CL-073 antigen (10 μg, 3 μg and 0.3 μg) and pXCS847 antigen (10 μg, 3 μg and 0.3 μg). The immunization scheme is shown in Table 11. Blood was collected by removing the eyeball 14 days after the second immunization (5 weeks after the first immunization) to separate serum for virus neutralization experiment or ELISA experiment.

[1298] Table 11. Example 7 Mouse Immunization Scheme

[1299] The neutralizing titres (NT) of the immune serum samples were determined by cytopathic effect assay.

[1300] (1) The day before, HEp-2 cells (Cat: ATCC#CCL-23) were cultured at 1.5×10 4 / well were inoculated into 96-well plates.

[1301] (2) On the second day, the test sample and the control sample were diluted three times at the highest dilution ratio of 1:20, with two replicate wells for each dilution, for a total of six dilutions.

[1302] (3) In a BSL-2 laboratory, RSV A2 virus suspension with a multiplicity of infection (MOI) of 0.2 was added to each well and incubated at 37°C for 1 hour.

[1303] (4) Aspirate and discard the cell culture supernatant, add 150 μL of serum-virus mixture to each well, and then place in a 37°C, 5% CO2 constant temperature and humidity incubator for culture.

[1304] (5) Observe and record the cell pathological changes under a microscope every 24 hours.

[1305] (6) After 72 hours of culture, the cytopathic rate was read using a Celigo Image Cytometer (Celigo) and the NT95 value was calculated.

[1306] The results showed that after two immunizations in mice, all tested mutants elicited neutralizing antibody responses. CL-073 demonstrated strong inhibitory activity against RSV A2 strains at the cellular level. Neutralization efficacy and NT95 values ​​are shown in Figure 15. As shown in Figure 15, CL-073 exhibited superior RSV A2 neutralization activity compared to the control, pXCS847, at the same immunization dose. Overall, antibody titers for mutant CL0-73 were consistently high across all three antigen doses, indicating that this mutant represents a more immunogenic form of the stabilized RSV prefusion F glycoprotein.

[1307] Example 8: CL-073 provides protection against RSV challenge

[1308] Female SPF BALB / c mice aged 6-8 weeks were purchased from Jicui Pharmaceutical Co., Ltd., with 5 mice per group. On day 0 and day 21, 50 μl of 50 μg aluminum hydroxide adjuvant ( Cat: #AJV3012) Post-F antigen (3μg, Cat: #RSF-V52H6), CL-073 antigen (10μg, 3μg and 0.3μg) and pXCS847 antigen (10μg, 3μg and 0.3μg), the immunization scheme is shown in Table 12.

[1309] Table 12. Example 8 mouse immunization scheme

[1310] BALB / c mice were infected with intranasal drops 14 days after the booster immunization. General clinical observations were performed once a day after the challenge, including the mice's mobility and survival status; eyes and hair; death and any observable clinical manifestations.

[1311] (1) 12-week-old female BALB / c mice that had been immunized for 35 days were lightly anesthetized with isoflurane.

[1312] (2) Each mouse was infected by intranasal drip with 50 μL containing 1×10 6 PFU of RSV A2 (stored in the BSL-2 laboratory of Guangzhou National Laboratory).

[1313] (3) The animals were weighed on the day of challenge (i.e., D0) and daily thereafter (D1, D2, D3, and D4).

[1314] (4) Detect the viral load and titer in lung tissue.

[1315] On the morning of day 4 after infection, mice were euthanized, and half of the right lung was removed, weighed, and thoroughly homogenized by adding 1 mL of sterile PBS; the viral load (Fusion gene) was detected by qRT / PCR; and the viral titer was determined by FFA method.

[1316] Intranasal infection was adopted, and the mice were weighed once a day after the virus attack. The results showed that after two immunizations in mice, the mutants tested all had a protective effect against RSV-attacked mice, among which the weight of mice immunized in the CL-073 group recovered faster than that in the control group (Figure 16). As can be seen from Figure 17, the number of viral copies in the lung tissue of mice immunized in the CL-073 group was significantly lower than that in the control group. As can be seen from Figure 18, the live virus titer did not detect the presence of live virus in the lung tissue of mice immunized in the CL-073 group. In short, for the mutant CL-073, the effect of protecting mice was always good under the three antigen doses, indicating that this mutant is a more immunogenic form of stabilizing the RSV prefusion F glycoprotein.

[1317] Example 9: Design and construction of RSV F protein mutants with different strain backbones CL-073

[1318] Wild-type F protein sequences from various RSV strains were searched using databases such as NCBI, GenBank, and Uniprot (Table 13). CL-073 mutants were then designed using these wild-type F protein sequences as the backbone sequences. Expression vectors for the wild-type RSV F proteins from various strains (SEQ ID NOs: 561-584) were synthesized by GenScript Biotech Co., Ltd., using the pcDNA3.1(+) backbone. Point mutation primers were designed using an online tool (https: / / crm.vazyme.com / cetool / singlepoint.html). Using the wild-type F protein sequences from various RSV strains as templates, PCR was performed using standard molecular biology techniques to introduce amino acid mutations corresponding to the mutation combination N42L, V65Y, M264F, D461S, and S165I found in the CL-073 mutant of the RSV A2 strain F protein. The PCR products were transformed into competent DH5α cells and cultured overnight at 37°C. Single colonies were picked and sequenced the next day. After sequencing was confirmed, the bacterial culture was expanded to 15 mL and the endotoxin-free plasmid was extracted according to the instructions of the EndoFree Mini Plasmid Kit II (Cat: #DP118-02) to obtain the expression vector of the RSV F protein mutant. The obtained CL-073 mutants of the F protein of different strains are shown in Table 13. The CL-073 mutants of the F protein of different strains (SEQ ID NO: 585-608) were expressed and purified using the method of Example 4, and the purified proteins were used for further in vitro and in vivo assays.

[1319] Table 13. Wild-type F protein of different CL-073 RSV A / B strains and their CL-073 mutants

[1320] Example 10: Study on the thermal stability of RSV F protein mutants with different strain backbones CL-073

[1321] Purified RSV F protein mutants from different strains of the backbone CL-073 were analyzed by differential scanning fluorimetry (DSF) to evaluate their thermal stability. Protein samples were diluted to a final concentration of 1 mg / ml in PBS (pH 7.4), and Sypro@orange dye was diluted to a 50× concentration in ultrapure water. The reaction system consisted of 3 μl of diluted protein sample, 3 μl of diluted dye, and 24 μl of PBS (pH 7.4). The reaction system was prepared as directed and assayed using a quantitative fluorescence PCR instrument (Bio-Rad CFX96 Touch). The PCR program used a melting curve module and HEX channel detection. The temperature was raised from 20°C and increased by 1°C per minute until the temperature reached 80°C. During this time, the fluorescence intensity was recorded as a function of temperature using the instrument's default parameters. The instrument has its own data processing function. The temperature corresponding to the minimum negative value of the first derivative of fluorescence intensity with respect to temperature is the Tm value. The results are shown in Figures 19-20 and Table 14. The measured Tm values ​​of all mutants are higher than 50°C, indicating that all mutants have good stability.

[1322] Table 14 Tm values ​​of RSV F protein mutants with different strains of backbone CL-073

[1323] Example 11: Binding study of different strain backbone CL-073 RSV F protein mutants with specific antibodies

[1324] ELISA was used to detect differences in binding between RSV F protein mutants with different strains and specific antibodies. The specific antibodies included the anti-RSV F protein monoclonal antibody Motavizumab, the prefusion-specific monoclonal antibody D25, and the prefusion trimer-specific monoclonal antibody AM14. The results are shown in Figure 21. The binding of RSV F protein mutants with different strains and specific antibodies showed high response values, indicating that the RSV F protein mutants with different strains were expressed in the prefusion trimer conformation.

[1325] Example 12: Study on the immunogenicity of RSV F protein mutants with different strain backbones CL-073

[1326] Female SPF BALB / c mice aged 6-8 weeks were purchased from Jicui Pharmaceutical Co., Ltd., with 5 mice per group. On day 0 and day 21, 50 μl of 50 μg aluminum hydroxide adjuvant ( Cat: #AJV3012) with different strains of backbone CL-073 RSV F protein (immunization scheme see Table 15), blood was collected by enucleation 14 days after the second immunization (5 weeks after the first immunization) to separate serum for virus neutralization test or ELISA test.

[1327] Table 15. Example 12 Mouse Immunization Scheme

[1328] Elisa was used to detect specific antibodies in mouse serum.

[1329] (1) Elisa plates were coated with CL-073A / B antigen at a concentration of 2 μg / ml and incubated overnight.

[1330] (2) Wash three times with 1×PBST, each time for five minutes, and add 300 μl 1×PBST to each well.

[1331] (3) Blocking: Add 200 μl of 2% BSA to each well and block at 37°C for two hours. Wash as in (2).

[1332] (4) Sample loading: dilute mouse serum (derived from immune cells immunized with proteins from different RSV strains) by 100, 1000, or 10,000 times, add 100 μl to each well, and incubate at 37°C for two hours. Wash as in (2).

[1333] (5) Enzyme labeling-antibody binding: dilute anti-mouse IgG (H+L)-HRP 5000 times, add 100 μl to each well, and incubate at 37°C for one hour. Wash as in (2) - four times.

[1334] (6) Color development: Add 100 μl of Elisa color development solution and react for 10 min in the dark.

[1335] (7) Termination: Add ELISA stop solution, 50ul per well.

[1336] (8) Elisa test OD450.

[1337] The neutralization concentration of immune serum samples was determined by the neutralization experiment method of fixed virus diluted serum.

[1338] (1) The day before, HEp-2 cells (Cell Bank of Chinese Academy of Sciences, catalog number: TCHu 21) or Vero E6 cells (Cell Bank of Chinese Academy of Sciences, catalog number: GNO17) were cultured at a rate of 2×10 4 / well were inoculated into 96-well plates.

[1339] (2) On the second day, the test sample and the control sample were diluted three times at the highest dilution ratio of 1:20, with two replicate wells for each dilution, for a total of six dilutions.

[1340] (3) In the BSL-2 laboratory, 100 TCID 50 RSV A or B virus suspension was incubated at 33°C for 2 hours.

[1341] (4) Aspirate and discard the cell culture supernatant, add 150 μL of serum-virus mixture to each well, and then place in a 33°C, 5% CO2 constant temperature and humidity incubator for culture.

[1342] (5) Observe and record the cell pathological changes under a microscope every 24 hours.

[1343] (6) After 72 hours of culture, observe the cytopathic effect (syncytia formation) or fix the cells for IFA experiment, read the cytopathic effect rate, and calculate the NT50 value.

[1344] The results are shown in Figures 22-23. CL-073 RSV F proteins with different strain backbones all produced strong specific antibodies and good neutralizing effects after immunizing mice.

[1345] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation selected from the following that increases the stability of the RSV F protein prefusion conformation: (1) introducing one or more disulfide bonds into the RSV F protein; (2) introducing one or more amino acid mutations in the RSV F protein to fill the hydrophobic cavity; (3) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein; (4) Removal of the p27 peptide from RSV F protein.

2. RSV F protein according to claim 1, wherein, The introduced disulfide bonds are present at one or more of the following positions: (1) between positions 7 and 416 of the RSV F protein; (2) between positions 15 and 358 of the RSV F protein; (3) between positions 20 and 338 of the RSV F protein; (4) between positions 20 and 339 of the RSV F protein; (5) between positions 22 and 248 of the RSV F protein; (6) between positions 23 and 283 of the RSV F protein; (7) between positions 30 and 163 of the RSV F protein; (8) between positions 31 and 164 of the RSV F protein; (9) between positions 34 and 168 of the RSV F protein; (10) between positions 34 and 272 of the RSV F protein; (11) between positions 37 and 175 of the RSV F protein; (12) between positions 64 and 209 of the RSV F protein; (13) between positions 67 and 229 of the RSV F protein; (14) between positions 78 and 123 of the RSV F protein; (15) between positions 119 and 381 of the RSV F protein; (16) between positions 125 and 433 of the RSV F protein; (17) between positions 128 and 436 of the RSV F protein; (18) between positions 133 and 266 of the RSV F protein; (19) between positions 152 and 164 of the RSV F protein; (20) between positions 155 and 161 of the RSV F protein; (21) between positions 210 and 224 of the RSV F protein; (22) between positions 216 and 254 of the RSV F protein; (23) between positions 236 and 249 of the RSV F protein; (24) between positions 309 and 450 of the RSV F protein; (25) between positions 385 and 441 of the RSV F protein; (26) between positions 418 and 441 of the RSV F protein; (27) between positions 457 and 477 of the RSV F protein; (28) Between positions 130 and 265 of the RSV F protein.

3. The RSV F protein according to claim 1 or 2, wherein The amino acid mutation for filling the hydrophobic cavity comprises an amino acid substitution at an amino acid position selected from the group consisting of: asparagine 42 residue, valine 65 residue, leucine 71 residue, leucine 205 residue, methionine 264 residue, isoleucine 267 residue, isoleucine 192 residue and / or serine 165 residue.

4. The RSV F protein according to any one of claims 1 to 3, wherein The amino acid mutations for filling the hydrophobic cavity include: 42L, 42F, 42Y, 42W, 42H, 65Y, 65F, 71I, 71Y, 205F, 205Y, 264F, 264Y, 267L, 267F, 267Y, 192L, 165I, 165F, 165Y and / or 165L; Preferably, the amino acid mutations used to fill the hydrophobic cavity include: N42L, N42F, N42Y, N42W, N42H, V65Y, V65F, L71I, L71Y, L205F, L205Y, M264F, M264Y, I267L, I267F, I267Y, I192L, S165I, S165F, S165Y and / or S165L.

5. The RSV F protein according to any one of claims 1 to 4, wherein The amino acid mutation for electrostatic modification comprises an amino acid substitution at the amino acid position of aspartic acid 461 residue and / or glutamic acid 462 residue.

6. The RSV F protein according to any one of claims 1 to 5, wherein The amino acid mutations used for electrostatic modification include: 461S, 461N, 461Q, 461E, 461G, 461A, 461K, 461R, 461H, 462Q, 462S, 462N, 462D, 462G, 462A, 462K, 462R, 462H; Preferably, the amino acid mutations used for electrostatic modification include: D461S, D461N, D461Q, D461E, D461G, D461A, D461K, D461R, D461H, E462Q, E462S, E462N, E462D, E462G, E462A, E462K, E462R, and E462H.

7. The RSV F protein according to any one of claims 1 to 6, wherein The RSV F protein comprises a modification or mutation combination selected from: (1) N42L, S165I; (2) N42L, D461S; (3) V65Y, D461S; (4) V65Y, S165I; (5) N42L, V65Y, D461S; (6) N42L, D461S, M264F; (7) V65Y, D461S, M264F; (8)N42L, V65Y, D461S, M264F; (9)I192L, N42L, L71Y, I267L, D461S; (10)I192L, N42L, L205F, M264F, I267L, D461S; (11)I192L, N42L, V65Y, L205F, M264F, D461S; (12)I192L, N42L, L71Y, I267L; (13) N42L, V65Y, S165I; (14) N42L, S165I, M264F; (15) V65Y, S165I, M264F; (16)N42L, V65Y, S165I, M264F; (17)I192L, N42L, V65Y, L205F, M264F; (18)I192L, N42L, L71Y, I267L, S165I; (19)I192L, N42L, L205F, M264F, I267L, S165I; (20)I192L, N42L, V65Y, L205F, M264F, S165I; (21) N42L, D461S, S165I; (22) V65Y, D461S, S165I; (23) N42L, M264F, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163; (24)M264F, D461S, S165I; (25)N42L, V65Y, D461S, S165I; (26)N42L, D461S, S165I, M264F; (27)V65Y, D461S, S165I, M264F; (28) N42L, D461S, introduction of a disulfide bond between protein positions 30 and 163; (29)I192L, N42L, L71Y, I267L, D461S, S165I; (30)I192L, N42L, L205F, M264F, I267L, D461S, S165I; (31)I192L, N42L, V65Y, L205F, M264F, D461S, S165I; (32) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 418 and 441; (33) V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163; (34) M264F, D461S, introduction of a disulfide bond between protein positions 30 and 163; (35) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163; (36) N42L, M264F, introduction of a disulfide bond between protein positions 64 and 209; (37) N42L, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F; (38) V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163, M264F; (39)M264F, S165I; (40) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 30 and 163, and M264F; (41) I192L, N42L, L71Y, I267L, D461S, introduction of a disulfide bond between protein positions 30 and 163; (42) I192L, N42L, L205F, M264F, I267L, D461S, introduction of a disulfide bond between protein positions 30 and 163; (43) I192L, N42L, V65Y, L205F, M264F, D461S, introduction of a disulfide bond between protein positions 30 and 163; (44) N42L, S165I, introduction of a disulfide bond between protein positions 30 and 163; (45) V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163; (46) M264F, S165I, introduction of a disulfide bond between protein positions 30 and 163; (47) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163; (48) N42L, M264F, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123; (49) N42L, S165I, introduction of a disulfide bond between protein positions 30 and 163, M264F; (50) V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163, M264F; (51) N42L, M264F, introduction of a disulfide bond between protein positions 457 and 477; (52) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 30 and 163, and M264F; (53) I192L, N42L, L71Y, I267L, S165I, introduction of a disulfide bond between protein positions 30 and 163; (54) I192L, N42L, L205F, M264F, I267L, S165I, introduction of a disulfide bond between protein positions 30 and 163; (55) I192L, N42L, V65Y, L205F, M264F, S165I, introduction of a disulfide bond between protein positions 30 and 163; (56) N42L, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163; (57) V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163; (58)N42L, M264F; (59) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 457 and 477; (60) M264F, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163; (61) N42L, V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163; (62) N42L, V65Y, M264F, D461S, S165I, introduction of a disulfide bond between protein positions 30 and 163; (63) N42L, D461S, introduction of a disulfide bond between protein positions 78 and 123; (64) V65Y, S165I, D461S, introduction of a disulfide bond between protein positions 30 and 163, M264F; (65) V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123; (66) M264F, D461S, introduction of a disulfide bond between protein positions 78 and 123; (67) N42L, M264F, introduction of a disulfide bond between protein positions 418 and 441; (68) N42L, D461S, introduction of a disulfide bond between protein positions 78 and 123, M264F; (69) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 152 and 164; (70) V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123, M264F; (71)M264F, D461S; (72) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123, and M264F; (73)N42L, V65Y, M264F, D461S, S165I; (74) I192L, N42L, L71Y, I267L, D461S, introduction of a disulfide bond between protein positions 78 and 123; (75) I192L, N42L, L205F, M264F, I267L, D461S, introduction of a disulfide bond between protein positions 78 and 123; (76) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123; (77) I192L, N42L, V65Y, L205F, M264F, D461S, introduction of a disulfide bond between protein positions 78 and 123; (78) N42L, S165I, introduction of a disulfide bond between protein positions 78 and 123; (79) V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123; (80) M264F, S165I, introduction of a disulfide bond between protein positions 78 and 123; (81) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123; (82) N42L, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F; (83) V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123, M264F; (84) N42L, V65Y, S165I, introduction of a disulfide bond between protein positions 78 and 123, and M264F; (85) I192L, N42L, L71Y, I267L, S165I, introduction of a disulfide bond between protein positions 78 and 123; (86) I192L, N42L, L205F, M264F, I267L, S165I, introduction of a disulfide bond between protein positions 78 and 123; (87) I192L, N42L, V65Y, L205F, M264F, S165I, introduction of a disulfide bond between protein positions 78 and 123; (88) N42L, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123; (89) V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123; (90) M264F, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123; (91) N42L, V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123; (92) V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123, M264F; (93) N42L, V65Y, D461S, S165I, introduction of a disulfide bond between protein positions 78 and 123, M264F; (94) N42L, V65Y, introduction of a disulfide bond between protein positions 7 and 416; (95) N42L, V65Y, introduction of a disulfide bond between protein positions 15 and 358; (96) N42L, V65Y, introduction of a disulfide bond between protein positions 20 and 338; (97) N42L, V65Y, introduction of a disulfide bond between protein positions 20 and 339; (98) N42L, V65Y, introduction of a disulfide bond between protein positions 22 and 248; (99) N42L, V65Y, introduction of a disulfide bond between protein positions 23 and 283; (100) N42L, V65Y, introduction of a disulfide bond between protein positions 31 and 164; (101) N42L, V65Y, introduction of a disulfide bond between protein positions 34 and 168; (102) N42L, V65Y, introduction of a disulfide bond between protein positions 34 and 272; (103) N42L, V65Y, introduction of a disulfide bond between protein positions 37 and 175; (104) N42L, V65Y, introduction of a disulfide bond between protein positions 64 and 209; (105) N42L, V65Y, introduction of a disulfide bond between protein positions 133 and 266; (106) N42L, V65Y, introduction of a disulfide bond between protein positions 155 and 161; (107) N42L, V65Y, introduction of a disulfide bond between protein positions 152 and 164; (108) N42L, V65Y, introduction of a disulfide bond between protein positions 236 and 249; (109) N42L, V65Y, introduction of a disulfide bond between protein positions 309 and 450; (110) N42L, V65Y, introduction of a disulfide bond between protein positions 418 and 441; (111) N42L, V65Y, introduction of a disulfide bond between protein positions 385 and 441; (112) N42L, V65Y, and introduction of a disulfide bond between protein positions 457 and 477; (113) N42L, V65Y, introduction of a disulfide bond between protein positions 67 and 229; (114) N42L, V65Y, introduction of a disulfide bond between protein positions 119 and 381; (115) N42L, V65Y, introduction of a disulfide bond between protein positions 125 and 433; (116) N42L, V65Y, introduction of a disulfide bond between protein positions 128 and 436; (117) N42L, V65Y, introduction of a disulfide bond between protein positions 210 and 224; (118) N42L, V65Y, introduction of a disulfide bond between protein positions 216 and 254; (119) N42L, M264F, introduction of a disulfide bond between protein positions 7 and 416; (120) N42L, M264F, introduction of a disulfide bond between protein positions 15 and 358; (121) N42L, M264F, introduction of a disulfide bond between protein positions 20 and 338; (122) N42L, M264F, introduction of a disulfide bond between protein positions 20 and 339; (123) N42L, M264F, introduction of a disulfide bond between protein positions 22 and 248; (124) N42L, M264F, introduction of a disulfide bond between protein positions 23 and 283; (125) N42L, M264F, introduction of a disulfide bond between protein positions 31 and 164; (126) N42L, M264F, introduction of a disulfide bond between protein positions 34 and 168; (127) N42L, M264F, introduction of a disulfide bond between protein positions 34 and 272; (128) N42L, M264F, introduction of a disulfide bond between protein positions 37 and 175; (129) V65Y, M264F, introduction of a disulfide bond between protein positions 7 and 416; (130) N42L, M264F, introduction of a disulfide bond between protein positions 133 and 266; (131) N42L, M264F, introduction of a disulfide bond between protein positions 155 and 161; (132) N42L, M264F, introduction of a disulfide bond between protein positions 152 and 164; (133) N42L, M264F, introduction of a disulfide bond between protein positions 236 and 249; (134) N42L, M264F, introduction of a disulfide bond between protein positions 309 and 450; (135) V65Y, M264F, introduction of a disulfide bond between protein positions 15 and 358; (136) N42L, M264F, introduction of a disulfide bond between protein positions 385 and 441; (137) V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 338; (138) N42L, M264F, introduction of a disulfide bond between protein positions 67 and 229; (139) N42L, M264F, introduction of a disulfide bond between protein positions 119 and 381; (140) N42L, M264F, introduction of a disulfide bond between protein positions 125 and 433; (141) N42L, M264F, introduction of a disulfide bond between protein positions 128 and 436; (142) N42L, M264F, introduction of a disulfide bond between protein positions 210 and 224; (143) N42L, M264F, introduction of a disulfide bond between protein positions 216 and 254; (144) V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 339; (145) V65Y, M264F, introduction of a disulfide bond between protein positions 22 and 248; (146) V65Y, M264F, introduction of a disulfide bond between protein positions 23 and 283; (147) V65Y, M264F, introduction of a disulfide bond between protein positions 31 and 164; (148) V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 168; (149) V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 272; (150) V65Y, M264F, introduction of a disulfide bond between protein positions 37 and 175; (151) V65Y, M264F, introduction of a disulfide bond between protein positions 64 and 209; (152) V65Y, M264F, introduction of a disulfide bond between protein positions 133 and 266; (153) V65Y, M264F, introduction of a disulfide bond between protein positions 155 and 161; (154) V65Y, M264F, introduction of a disulfide bond between protein positions 152 and 164; (155) V65Y, M264F, introduction of a disulfide bond between protein positions 236 and 249; (156) V65Y, M264F, introduction of a disulfide bond between protein positions 309 and 450; (157) V65Y, M264F, introduction of a disulfide bond between protein positions 418 and 441; (158) V65Y, M264F, introduction of a disulfide bond between protein positions 385 and 441; (159) V65Y, M264F, introduction of a disulfide bond between protein positions 457 and 477; (160) V65Y, M264F, introduction of a disulfide bond between protein positions 67 and 229; (161) V65Y, M264F, introduction of a disulfide bond between protein positions 119 and 381; (162) V65Y, M264F, introduction of a disulfide bond between protein positions 125 and 433; (163) V65Y, M264F, introduction of a disulfide bond between protein positions 128 and 436; (164) V65Y, M264F, introduction of a disulfide bond between protein positions 210 and 224; (165) V65Y, M264F, introduction of a disulfide bond between protein positions 216 and 254; (166) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 7 and 416; (167) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 15 and 358; (168) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 338; (169) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 20 and 339; (170) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 22 and 248; (171) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 23 and 283; (172) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 31 and 164; (173) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 168; (174) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 34 and 272; (175) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 37 and 175; (176) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 64 and 209; (177) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 133 and 266; (178) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 155 and 161; (179) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 216 and 254; (180) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 236 and 249; (181) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 309 and 450; (182) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 210 and 224; (183) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 385 and 441; (184) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 128 and 436; (185) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 67 and 229; (186) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 119 and 381; (187) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 125 and 433; (188) introduced a disulfide bond and S165I between protein positions 418 and 441; (189) introduced a disulfide bond, D461S, between protein positions 418 and 441; (190) introduction of a disulfide bond between protein positions 418 and 441, E462Q; (191) introduced a disulfide bond and S165I between protein positions 457 and 477; (192) introduced a disulfide bond, D461S, between protein positions 457 and 477; (193) introduction of a disulfide bond between protein positions 457 and 477, E462Q; (194) A disulfide bond, S165I, was introduced between protein positions 64 and 209; (195) A disulfide bond, D461S, was introduced between protein positions 64 and 209; (196) introduction of a disulfide bond between protein positions 64 and 209, E462Q; (197)I192L, N42L, L205F, M264F, I267L; (198)N42L, L205F, L71Y, I267L; (199)I192L, N42L, V65Y, L205F; (200)I192L, N42L, L205F, L71Y; (201)I192L, N42L, L205F, I267L; (202) I192L, N42L, L205F, I267L, and introduction of a disulfide bond between protein positions 457 and 477; (203) I192L, N42L, V65Y, L205F, M264F, introduction of a disulfide bond between protein positions 457 and 477; (204)I192L, N42L, L205F; (205) I192L, N42L, L205F, I267L, introduction of a disulfide bond between protein positions 130 and 265; (206) N42L, V65Y, L205F, L71Y, introduction of a disulfide bond between protein positions 130 and 265; (207) I192L, N42L, V65Y, L205F, M264F, introduction of a disulfide bond between protein positions 130 and 265; (208) S165I, M264F, introduction of a disulfide bond between protein positions 418 and 441; (209) D461S, M264F, introduction of a disulfide bond between protein positions 418 and 441; (210) E462Q, M264F, introduction of a disulfide bond between protein positions 418 and 441; (211) S165I, M264F, introduction of a disulfide bond between protein positions 457 and 477; (212) D461S, M264F, introduction of a disulfide bond between protein positions 457 and 477; (213) E462Q, M264F, introduction of a disulfide bond between protein positions 457 and 477; (214) S165I, M264F, introduction of a disulfide bond between protein positions 64 and 209; (215) D461S, M264F, introduction of a disulfide bond between protein positions 64 and 209; (216) E462Q, M264F, introduction of a disulfide bond between protein positions 64 and 209; (217) D461S, S165I, introduction of a disulfide bond between protein positions 64 and 209; (218) D461S, S165I, introduction of a disulfide bond between protein positions 418 and 441; (219) S165I, N42L, introduction of a disulfide bond between protein positions 418 and 441; (220) D461S, N42L, introduction of a disulfide bond between protein positions 418 and 441; (221) E462Q, N42L, introduction of a disulfide bond between protein positions 418 and 441; (222) S165I, N42L, introduction of a disulfide bond between protein positions 457 and 477; (223) D461S, N42L, introduction of a disulfide bond between protein positions 457 and 477; (224) E462Q, N42L, introduction of a disulfide bond between protein positions 457 and 477; (225) S165I, N42L, introduction of a disulfide bond between protein positions 64 and 209; (226) D461S, N42L, introduction of a disulfide bond between protein positions 64 and 209; (227) E462Q, N42L, introduction of a disulfide bond between protein positions 64 and 209; (228) S165I, V65Y, introduction of a disulfide bond between protein positions 418 and 441; (229) D461S, V65Y, introduction of a disulfide bond between protein positions 418 and 441; (230) E462Q, V65Y, introduction of a disulfide bond between protein positions 418 and 441; (231) S165I, V65Y, introduction of a disulfide bond between protein positions 457 and 477; (232) D461S, V65Y, introduction of a disulfide bond between protein positions 457 and 477; (233) E462Q, V65Y, introduction of a disulfide bond between protein positions 457 and 477; (234) S165I, V65Y, introduction of a disulfide bond between protein positions 64 and 209; (235) D461S, V65Y, introduction of a disulfide bond between protein positions 64 and 209; (236) E462Q, V65Y, introduction of a disulfide bond between protein positions 64 and 209; (237) E462Q, S165I, introduction of a disulfide bond between protein positions 64 and 209; (238) E462Q, S165I, introduction of a disulfide bond between protein positions 418 and 441; (239) D461N, introduction of a disulfide bond between protein positions 418 and 441; (240) D461Q, introduction of a disulfide bond between protein positions 418 and 441; (241) D461E, introduction of a disulfide bond between protein positions 418 and 441; (242) D461G, introduction of a disulfide bond between protein positions 418 and 441; (243) D461A, introduction of a disulfide bond between protein positions 418 and 441; (244) D461K, introduction of a disulfide bond between protein positions 418 and 441; (245) D461R, introduction of a disulfide bond between protein positions 418 and 441; (246) D461H, introduction of a disulfide bond between protein positions 418 and 441; (247) D461N, introduction of a disulfide bond between protein positions 457 and 477; (248) D461Q, introduction of a disulfide bond between protein positions 457 and 477; (249) D461E, introduction of a disulfide bond between protein positions 457 and 477; (250) D461G, introduction of a disulfide bond between protein positions 457 and 477; (251) D461A, introduction of a disulfide bond between protein positions 457 and 477; (252) D461K, introduction of a disulfide bond between protein positions 457 and 477; (253) D461R, introduction of a disulfide bond between protein positions 457 and 477; (254) D461H, introduction of a disulfide bond between protein positions 457 and 477; (255) D461N, introduction of a disulfide bond between protein positions 64 and 209; (256) D461Q, introduction of a disulfide bond between protein positions 64 and 209; (257) D461E, introduction of a disulfide bond between protein positions 64 and 209; (258) D461G, introduction of a disulfide bond between protein positions 64 and 209; (259) D461A, introduction of a disulfide bond between protein positions 64 and 209; (260) D461K, introduction of a disulfide bond between protein positions 64 and 209; (261) D461R, introduction of a disulfide bond between protein positions 64 and 209; (262) D461H, introduction of a disulfide bond between protein positions 64 and 209; (263) E462S, introduction of a disulfide bond between protein positions 418 and 441; (264) E462N, introduction of a disulfide bond between protein positions 418 and 441; (265) E462D, introduction of a disulfide bond between protein positions 418 and 441; (266) E462G, introduction of a disulfide bond between protein positions 418 and 441; (267) E462A, introduction of a disulfide bond between protein positions 418 and 441; (268) E462K, introduction of a disulfide bond between protein positions 418 and 441; (269) E462R, introduction of a disulfide bond between protein positions 418 and 441; (270) E462H, introduction of a disulfide bond between protein positions 418 and 441; (271) E462S, introduction of a disulfide bond between protein positions 64 and 209; (272) E462N, introduction of a disulfide bond between protein positions 64 and 209; (273) E462D, introduction of a disulfide bond between protein positions 64 and 209; (274) E462G, introduction of a disulfide bond between protein positions 64 and 209; (275) E462A, introduction of a disulfide bond between protein positions 64 and 209; (276) E462K, introduction of a disulfide bond between protein positions 64 and 209; (277) E462R, introduction of a disulfide bond between protein positions 64 and 209; (278) E462H, introduction of a disulfide bond between protein positions 64 and 209; (279) E462S, introduction of a disulfide bond between protein positions 457 and 477; (280) E462N, introduction of a disulfide bond between protein positions 457 and 477; (281) E462D, introduction of a disulfide bond between protein positions 457 and 477; (282) E462G, introduction of a disulfide bond between protein positions 457 and 477; (283) E462A, introduction of a disulfide bond between protein positions 457 and 477; (284) E462K, introduction of a disulfide bond between protein positions 457 and 477; (285) E462R, introduction of a disulfide bond between protein positions 457 and 477; (286) E462H, introduction of a disulfide bond between protein positions 457 and 477; (287) D461N, introduction of a disulfide bond between protein positions 418 and 441, N42L; (288) D461Q, introduction of a disulfide bond between protein positions 418 and 441, N42L; (289) D461E, introduction of a disulfide bond between protein positions 418 and 441, N42L; (290) D461G, introduction of a disulfide bond between protein positions 418 and 441, N42L; (291) D461A, introduction of a disulfide bond between protein positions 418 and 441, N42L; (292) D461K, introduction of a disulfide bond between protein positions 418 and 441, N42L; (293) D461R, introduction of a disulfide bond between protein positions 418 and 441, N42L; (294) D461H, introduction of a disulfide bond between protein positions 418 and 441, N42L; (295) D461N, introduction of a disulfide bond between protein positions 457 and 477, N42L; (296) D461Q, introduction of a disulfide bond between protein positions 457 and 477, N42L; (297) D461E, introduction of a disulfide bond between protein positions 457 and 477, N42L; (298) D461G, introduction of a disulfide bond between protein positions 457 and 477, N42L; (299) D461A, introduction of a disulfide bond between protein positions 457 and 477, N42L; (300) D461K, introduction of a disulfide bond between protein positions 457 and 477, N42L; (301) D461R, introduction of a disulfide bond between protein positions 457 and 477, N42L; (302) D461H, introduction of a disulfide bond between protein positions 457 and 477, N42L; (303) D461N, introduction of a disulfide bond between protein positions 64 and 209, N42L; (304) D461Q, introduction of a disulfide bond between protein positions 64 and 209, N42L; (305) D461E, introduction of a disulfide bond between protein positions 64 and 209, N42L; (306) D461G, introduction of a disulfide bond between protein positions 64 and 209, N42L; (307) D461A, introduction of a disulfide bond between protein positions 64 and 209, N42L; (308) D461K, introduction of a disulfide bond between protein positions 64 and 209, N42L; (309) D461R, introduction of a disulfide bond between protein positions 64 and 209, N42L; (310) D461H, introduction of a disulfide bond between protein positions 64 and 209, N42L; (311) D461N, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (312) D461Q, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (313) D461E, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (314) D461G, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (315) D461A, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (316) D461K, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (317) D461R, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (318) D461H, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (319) D461N, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (320) D461Q, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (321) D461E, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (322) D461G, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (323) D461A, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (324) D461K, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (325) D461R, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (326) D461H, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (327) D461N, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (328) D461Q, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (329) D461E, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (330) D461G, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (331) D461A, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (332) D461K, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (333) D461R, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (334) D461H, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (335) D461N, introduction of a disulfide bond between protein positions 418 and 441, M264F; (336) D461Q, introduction of a disulfide bond between protein positions 418 and 441, M264F; (337) D461E, introduction of a disulfide bond between protein positions 418 and 441, M264F; (338) D461G, introduction of a disulfide bond between protein positions 418 and 441, M264F; (339) D461A, introduction of a disulfide bond between protein positions 418 and 441, M264F; (340) D461K, introduction of a disulfide bond between protein positions 418 and 441, M264F; (341) D461R, introduction of a disulfide bond between protein positions 418 and 441, M264F; (342) D461H, introduction of a disulfide bond between protein positions 418 and 441, M264F; (343) D461N, introduction of a disulfide bond between protein positions 457 and 477, M264F; (344) D461Q, introduction of a disulfide bond between protein positions 457 and 477, M264F; (345) D461E, introduction of a disulfide bond between protein positions 457 and 477, M264F; (346) D461G, introduction of a disulfide bond between protein positions 457 and 477, M264F; (347) D461A, introduction of a disulfide bond between protein positions 457 and 477, M264F; (348) D461K, introduction of a disulfide bond between protein positions 457 and 477, M264F; (349) D461R, introduction of a disulfide bond between protein positions 457 and 477, M264F; (350) D461H, introduction of a disulfide bond between protein positions 457 and 477, M264F; (351) D461N, introduction of a disulfide bond between protein positions 64 and 209, M264F; (352) D461Q, introduction of a disulfide bond between protein positions 64 and 209, M264F; (353) D461E, introduction of a disulfide bond between protein positions 64 and 209, M264F; (354) D461G, introduction of a disulfide bond between protein positions 64 and 209, M264F; (355) D461A, introduction of a disulfide bond between protein positions 64 and 209, M264F; (356) D461K, introduction of a disulfide bond between protein positions 64 and 209, M264F; (357) D461R, introduction of a disulfide bond between protein positions 64 and 209, M264F; (358) D461H, introduction of a disulfide bond between protein positions 64 and 209, M264F; (359) E462S, introduction of a disulfide bond between protein positions 418 and 441, N42L; (360) E462N, introduction of a disulfide bond between protein positions 418 and 441, N42L; (361) E462D, introduction of a disulfide bond between protein positions 418 and 441, N42L; (362) E462G, introduction of a disulfide bond between protein positions 418 and 441, N42L; (363) E462A, introduction of a disulfide bond between protein positions 418 and 441, N42L; (364) E462K, introduction of a disulfide bond between protein positions 418 and 441, N42L; (365) E462R, introduction of a disulfide bond between protein positions 418 and 441, N42L; (366) E462H, introduction of a disulfide bond between protein positions 418 and 441, N42L; (367) E462S, introduction of a disulfide bond between protein positions 64 and 209, N42L; (368) E462N, introduction of a disulfide bond between protein positions 64 and 209, N42L; (369) E462D, introduction of a disulfide bond between protein positions 64 and 209, N42L; (370) E462G, introduction of a disulfide bond between protein positions 64 and 209, N42L; (371) E462A, introduction of a disulfide bond between protein positions 64 and 209, N42L; (372) E462K, introduction of a disulfide bond between protein positions 64 and 209, N42L; (373) E462R, introduction of a disulfide bond between protein positions 64 and 209, N42L; (374) E462H, introduction of a disulfide bond between protein positions 64 and 209, N42L; (375) E462S, introduction of a disulfide bond between protein positions 457 and 477, N42L; (376) E462N, introduction of a disulfide bond between protein positions 457 and 477, N42L; (377) E462D, introduction of a disulfide bond between protein positions 457 and 477, N42L; (378) E462G, introduction of a disulfide bond between protein positions 457 and 477, N42L; (379) E462A, introduction of a disulfide bond between protein positions 457 and 477, N42L; (380) E462K, introduction of a disulfide bond between protein positions 457 and 477, N42L; (381) E462R, introduction of a disulfide bond between protein positions 457 and 477, N42L; (382) E462H, introduction of a disulfide bond between protein positions 457 and 477, N42L; (383) E462S, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (384) E462N, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (385) E462D, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (386) E462G, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (387) E462A, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (388) E462K, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (389) E462R, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (390) E462H, introduction of a disulfide bond between protein positions 418 and 441, V65Y; (391) E462S, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (392) E462N, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (393) E462D, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (394) E462G, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (395) E462A, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (396) E462K, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (397) E462R, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (398) E462H, introduction of a disulfide bond between protein positions 64 and 209, V65Y; (399) E462S, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (400) E462N, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (401) E462D, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (402) E462G, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (403) E462A, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (404) E462K, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (405) E462R, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (406) E462H, introduction of a disulfide bond between protein positions 457 and 477, V65Y; (407) E462S, introduction of a disulfide bond between protein positions 418 and 441, M264F; (408) E462N, introduction of a disulfide bond between protein positions 418 and 441, M264F; (409) E462D, introduction of a disulfide bond between protein positions 418 and 441, M264F; (410) E462G, introduction of a disulfide bond between protein positions 418 and 441, M264F; (411) E462A, introduction of a disulfide bond between protein positions 418 and 441, M264F; (412) E462K, introduction of a disulfide bond between protein positions 418 and 441, M264F; (413) E462R, introduction of a disulfide bond between protein positions 418 and 441, M264F; (414) E462H, introduction of a disulfide bond between protein positions 418 and 441, M264F; (415) E462S, introduction of a disulfide bond between protein positions 64 and 209, M264F; (416) E462N, introduction of a disulfide bond between protein positions 64 and 209, M264F; (417) E462D, introduction of a disulfide bond between protein positions 64 and 209, M264F; (418) E462G, introduction of a disulfide bond between protein positions 64 and 209, M264F; (419) E462A, introduction of a disulfide bond between protein positions 64 and 209, M264F; (420) E462K, introduction of a disulfide bond between protein positions 64 and 209, M264F; (421) E462R, introduction of a disulfide bond between protein positions 64 and 209, M264F; (422) E462H, introduction of a disulfide bond between protein positions 64 and 209, M264F; (423) E462S, introduction of a disulfide bond between protein positions 457 and 477, M264F; (424) E462N, introduction of a disulfide bond between protein positions 457 and 477, M264F; (425) E462D, introduction of a disulfide bond between protein positions 457 and 477, M264F; (426) E462G, introduction of a disulfide bond between protein positions 457 and 477, M264F; (427) E462A, introduction of a disulfide bond between protein positions 457 and 477, M264F; (428) E462K, introduction of a disulfide bond between protein positions 457 and 477, M264F; (429) E462R, introduction of a disulfide bond between protein positions 457 and 477, M264F; (430) E462H, introduction of a disulfide bond between protein positions 457 and 477, M264F; (431) V65Y, M264F; (432)N42L, V65Y, M264F.

8. The RSV F protein according to any one of claims 1 to 7, wherein The RSV F protein comprises a modification or mutation combination selected from: (1)192L, 42L, 71Y, 267L; (2)192L, 42L, 65Y, 205F, 264F; (3) 42L, 264F, and introduction of a disulfide bond between protein positions 64 and 209; (4) 42L, 264F, 461S, 165I, and introduction of a disulfide bond between protein positions 78 and 123; (5) 42L, 264F, and introduction of a disulfide bond between protein positions 457 and 477; (6) 42L, 65Y, 264F, and introduction of a disulfide bond between protein positions 457 and 477; (7) 42L, 65Y, 264F, 461S, 165I, and introduction of a disulfide bond between protein positions 30 and 163; (8) 42L, 264F, introduction of a disulfide bond between protein positions 418 and 441; (9)42L, 65Y, 264F, 461S, 165I; (10) 42L, 65Y, 461S, introduction of a disulfide bond between protein positions 78 and 123; (11)192L, 42L, 205F, 264F, 267L; (12)42L, 205F, 71Y, 267L; (13)192L, 42L, 65Y, 205F; (14)192L, 42L, 205F, 71Y; (15)192L, 42L, 205F, 267L; (16) 192L, 42L, 205F, 267L, and introduction of a disulfide bond between protein positions 457 and 477; (17) 192L, 42L, 65Y, 205F, 264F, and introduction of a disulfide bond between protein positions 457 and 477; (18)192L, 42L, 205F; (19) 192L, 42L, 205F, 267L, introduction of a disulfide bond between protein positions 130 and 265; (20) 42L, 65Y, 205F, 71Y, introduction of a disulfide bond between protein positions 130 and 265; (21) 192L, 42L, 65Y, 205F, 264F, introduction of a disulfide bond between protein positions 130 and 265; (22) 42L, 65Y, 264F, introduction of a disulfide bond between protein positions 418 and 441; (23) 264F, 165I; (24) 42L, 264F; (25) 42L, 65Y, 264F, introduction of a disulfide bond between protein positions 152 and 164; (26) 461Q, introduction of a disulfide bond between protein positions 457 and 477, 42L; (27) 461G, introduction of a disulfide bond between protein positions 457 and 477, 42L; (28) 461K, introduction of a disulfide bond between protein positions 457 and 477, 42L; (29) 461H, introduction of a disulfide bond between protein positions 457 and 477, 42L; (30) 461A, introduction of a disulfide bond between protein positions 457 and 477, 264F; (31) 461H, introduction of a disulfide bond between protein positions 64 and 209, 264F; Preferably, the RSV F protein comprises a combination of modifications or mutations selected from: (1)I192L, N42L, L71Y, I267L; (2)I192L, N42L, V65Y, L205F, M264F; (3) N42L, M264F, and introduction of a disulfide bond between protein positions 64 and 209; (4) N42L, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 78 and 123; (5) N42L, M264F, and introduction of a disulfide bond between protein positions 457 and 477; (6) N42L, V65Y, M264F, and introduction of a disulfide bond between protein positions 457 and 477; (7) N42L, V65Y, M264F, D461S, S165I, and introduction of a disulfide bond between protein positions 30 and 163; (8) N42L, M264F, introduction of a disulfide bond between protein positions 418 and 441; (9)N42L, V65Y, M264F, D461S, S165I; (10) N42L, V65Y, D461S, introduction of a disulfide bond between protein positions 78 and 123; (11)I192L, N42L, L205F, M264F, I267L; (12)N42L, L205F, L71Y, I267L; (13)I192L, N42L, V65Y, L205F; (14)I192L, N42L, L205F, L71Y; (15)I192L, N42L, L205F, I267L; (16) I192L, N42L, L205F, I267L, and introduction of a disulfide bond between protein positions 457 and 477; (17) I192L, N42L, V65Y, L205F, M264F, and introduction of a disulfide bond between protein positions 457 and 477; (18) I192L, N42L, L205F; (19) I192L, N42L, L205F, I267L, and introduction of a disulfide bond between protein positions 130 and 265; (20) N42L, V65Y, L205F, L71Y, introduction of a disulfide bond between protein positions 130 and 265; (21) I192L, N42L, V65Y, L205F, M264F, and introduction of a disulfide bond between protein positions 130 and 265; (22) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 418 and 441; (23)M264F, S165I; (24) N42L, M264F; (25) N42L, V65Y, M264F, introduction of a disulfide bond between protein positions 152 and 164; (26) D461Q, introduction of a disulfide bond between protein positions 457 and 477, and N42L; (27) D461G, introduction of a disulfide bond between protein positions 457 and 477, and N42L; (28) D461K, introduction of a disulfide bond between protein positions 457 and 477, and N42L; (29) D461H, introduction of a disulfide bond between protein positions 457 and 477, and N42L; (30) D461A, introduction of a disulfide bond between protein positions 457 and 477, M264F; (31) D461H, introduction of a disulfide bond between protein positions 64 and 209, and M264F.

9. The RSV F protein according to any one of claims 1 to 8, wherein The p27 peptide segment of the RSV F protein is replaced by a linker; Preferably, the linker is a flexible polypeptide; Preferably, the flexible polypeptide is selected from (GS)n, (GGSG)n, (GGGGS)n, (GGGSGG)n, (GGGSGGG)n, (EA)n, (EAK)n, (EAKA)n, (AAAAK)n, (EAAAK)n, (LEAAK)n, wherein n is an integer from 1 to 4; More preferably, the flexible polypeptide is selected from the amino acid sequences shown in SEQ ID NOs: 500-543.

10. The RSV F protein according to any one of claims 1 to 9, wherein The RSV F protein comprises an amino acid sequence selected from SEQ ID NOs: 2-499, 585-608 having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity.

11. The RSV F protein according to any one of claims 1 to 10, wherein The N-terminus of the RSV F protein is connected to a signal peptide; Preferably, the signal peptide is selected from a wild-type RSV F protein signal peptide or a non-RSV F protein signal peptide; More preferably, the signal peptide comprises an amino acid sequence selected from SEQ ID NO: 544, 546, 548 or any variant thereof.

12. The RSV F protein according to any one of claims 1 to 11, wherein The C-terminus of the RSV F protein is connected to a trimerization tag; Preferably, the trimerization tag comprises an amino acid sequence as shown in SEQ ID NO: 550 or any variant thereof.

13. A nucleic acid molecule encoding the RSV F protein of any one of claims 1-12.

14. A vector comprising the nucleic acid molecule of claim 13; Preferably, the vector is an expression vector.

15. A host cell comprising the vector of claim 14; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof; Preferably, the mammalian cell is selected from CHO cells, HEK293 cells or COS cells.

16. An immunogenic composition comprising the RSV F protein of any one of claims 1-12.

17. A vaccine composition comprising the RSV F protein of any one of claims 1-12, the nucleic acid molecule of claim 13, the vector of claim 14 and / or the host cell of claim 15.

18. A kit for immunizing a subject against viral infection, comprising the RSV F protein of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the vector of claim 14 and / or the host cell of claim 15; Preferably, the viral infection is RSV infection; Preferably, the subject is a human subject.

19. A pharmaceutical composition comprising the RSV F protein of any one of claims 1-12 and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises other therapeutic agents.

20. A method for generating an immune response to a viral infection, comprising administering the RSV F protein of any one of claims 1-12 in a pharmaceutically acceptable formulation to a subject; Preferably, the pharmaceutically acceptable formulation comprises an adjuvant.

21. Use of the RSV F protein according to any one of claims 1 to 11 in the preparation of a medicament for preventing or treating a disease or condition associated with respiratory syncytial virus (RSV) infection; Preferably, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

22. A method for preventing or treating a disease associated with respiratory syncytial virus (RSV) infection, comprising administering an effective amount of the RSV F protein of any one of claims 1-11 in a pharmaceutically acceptable formulation to a subject in need thereof; Preferably, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

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