Respiratory syncytial virus (RSV) vaccine

Abstract

Provided is a respiratory syncytial virus (RSV) vaccine. The nucleic acid comprises a polynucleotide for encoding a mutant of an RSV F protein. Compared with a wild-type RSV F protein, the mutant at least comprises one or more of the following mutations: a disulfide bond mutation, a cavity-filling mutation and an electrostatic mutation.

Description

Respiratory syncytial virus (RSV) vaccine Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a respiratory syncytial virus (RSV) vaccine. Background Art

[0002] Respiratory syncytial virus (RSV) is a single-stranded RNA virus of the genus Orthopneumovirus in the family Pneumoviridae. The RSV genome consists of 10 genes that encode 11 proteins, including 9 structural proteins and 2 nonstructural proteins. The structural proteins include three transmembrane surface glycoproteins: attachment protein (G protein), fusion protein (F protein), and small hydrophobic protein (SH protein).

[0003] Human respiratory syncytial virus (hRSV) has two subtypes: subtype A and subtype B, which differ primarily in their G protein. HRSV infection can cause upper and lower respiratory tract illness, with high morbidity among infants, immunocompromised individuals, and the elderly. It is the second most common cause of mortality in infants and the leading cause of morbidity and mortality in the elderly (over 60 years).

[0004] Fusion protein (F protein) becomes active only after being cut into two fragments, F1 and F2, by host proteases. It can cause the fusion of the viral envelope and the host cell membrane, play a role in the immunopathology caused by RSV infection, and is currently a hot protein in the research and development of antibodies, vaccines and other therapeutic drugs.

[0005] During RSV entry into the cell, the F protein is rearranged from the pre-fusion state to the post-fusion state through an intermediate extension structure. During this rearrangement, the C-terminal coiled coil of the pre-fusion molecule dissociates into its three component chains, which then coil around the spherical head and engage three other spirals to form a post-fusion six-helix bundle. In order to prevent the virus from entering, before the viral envelope fuses with the cell membrane, F-specific neutralizing antibodies are presumably bound to the pre-fusion configuration of F on the virion, or potentially an extended intermediate. Therefore, the pre-fusion form of the F protein (i.e., "pre-F protein") is considered to be the preferred configuration of the vaccine antigen. However, in the preparation process of the pre-F protein, the pre-F protein is easily converted into the post-fusion form. Therefore, using the pre-F protein as a vaccine antigen remains a challenge.

[0006] Summary of the Invention

[0007] The present disclosure provides a nucleic acid, wherein the protein or polypeptide encoded by the nucleic acid can induce a specific immune response to prevent or at least reduce respiratory syncytial virus (RSV) infection.

[0008] In addition, the present disclosure also provides a delivery vector, a pharmaceutical composition and an RSV vaccine comprising the above-mentioned nucleic acid and their use in preparing drugs.

[0009] A nucleic acid comprising a polynucleotide encoding a mutant of an RSV F protein, the mutant comprising an F1 polypeptide and an F2 polypeptide, wherein the mutant comprises one or more of the following mutations relative to a wild-type RSV F protein: a disulfide bond mutation, a cavity-filling mutation, and an electrostatic mutation.

[0010] In some embodiments, the disulfide bond mutations include one or more of the following: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K191C, I59C and L193C, I59C and L297C, E60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T103C and I148C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C.

[0011] In some embodiments, the disulfide bond mutations include one or more of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C.

[0012] In some embodiments, the cavity-filling mutation includes one or more of the following (1) to (3):

[0013] (1) T at position 54 is substituted by F, W, V, C, A, H, I, Y, L or M; preferably, T at position 54 is substituted by F, W or V;

[0014] (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and

[0015] (3) V at position 296 is replaced by I, L, Y, H, F or M.

[0016] In some embodiments, the electrostatic mutations include one or more of the following:

[0017] (1) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W; and

[0018] (2) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0019] In some embodiments, the mutations of the mutant include at least one of a cavity-filling mutation and an electrostatic mutation and a disulfide bond mutation; preferably, the mutations of the mutant include a cavity-filling mutation and a disulfide bond mutation.

[0020] In some embodiments, the mutations of the mutant are selected from the group consisting of (1) to (20): (1) A102C, I152C, T54V, S190M, and V296L; (2) A102C, I152C, T54F, S190M, and V296L; (3) A102C, I152C, T54C, S190M, and V296L; (4) A102C, I152C, T54A, S190M, and V296L; (5) A102C, I152C, T54V, S190F, and V296L; (6) A102C, I152C, T54F, S190F, and V296L; (7) A102C, I152C, T54C, S190F, and V296L; (8) A102C, I152C, T54A, S190F, and V296L; (9) P101C, I152C, T54V, S190M, and V296L; (10) P101C, I152C, T54F, S190M and V296L; (11) P101C, I152C, T54C, S190M and V296L; (12) P101C, I152C, T54A, S190M and V296L; (13) P101C, I152C, T54V, S190F and V296L; (14) P101C, I152C, T54F, S190F and V296L; (15) P101C, I152C, T54C, S190F and V296L; (16) P101C, I152C, T54A, S190F, and V296L; (17) A102C, I152C, T54V, S190I, and V296L; (18) A102C, I152C, T54F, S190I, and V296L; (19) A102C, I152C, T54C, S190I, and V296L; and (20) A102C, I152C, T54A, S190I, and V296L.

[0021] In some embodiments, the mutant is selected from one of the following (1) to (40): (1) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 61 or 62; (2) a mutant comprising mutations A102C, I152C, T54V, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 61 or 62; (3) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 63 or 64; (4) a mutant comprising mutations A102C, I152C, T54V, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: NO:63 or 64 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:63 or 64; (5) a mutant comprising the amino acid sequence set forth in SEQ ID NO:65 or 66; (6) a mutant comprising the mutations A102C, I152C, T54C, S190M and V296L and comprising the amino acid sequence set forth in SEQ ID NO:66; NO:65 or 66 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:65 or 66; (7) a mutant comprising the amino acid sequence set forth in SEQ ID NO:67 or 68; (8) a mutant comprising the mutations A102C, I152C, T54A, S190M and V296L and comprising the amino acid sequence set forth in SEQ ID NO:67 or 68; NO:67 or 68 has an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical; (9) a mutant comprising the amino acid sequence as set forth in SEQ ID NO:69 or 70; (10) a mutant comprising the mutations A102C, I152C, T54V, S190F and V296L and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:69 or 70.(11) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 71 or 72; (12) a mutant comprising mutations A102C, I152C, T54F, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 71 or 72; (13) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 73 or 74; (14) a mutant comprising mutations A102C, I152C, T54F, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:73 or 74 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:73 or 74; (15) a mutant comprising the amino acid sequence set forth in SEQ ID NO:75 or 76; (16) a mutant comprising the mutations A102C, I152C, T54A, S190F and V296L and comprising the amino acid sequence set forth in SEQ ID NO:73 or 74; NO:75 or 76 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:75 or 76; (17) a mutant comprising the amino acid sequence set forth in SEQ ID NO:77 or 78; (18) a mutant comprising the mutations P101C, I152C, T54V, S190M and V296L and comprising the amino acid sequence set forth in SEQ ID NO:77 or 78; NO:77 or 78 has an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical; (19) a mutant comprising the amino acid sequence as set forth in SEQ ID NO:79 or 80; (20) a mutant comprising the mutations P101C, I152C, T54F, S190M and V296L and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:79 or 80.(21) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 81 or 82; (22) a mutant comprising mutations P101C, I152C, T54C, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 81 or 82; (23) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 83 or 84; (24) a mutant comprising mutations P101C, I152C, T54C, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:83 or 84 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:83 or 84; (25) a mutant comprising the amino acid sequence set forth in SEQ ID NO:85 or 86; (26) a mutant comprising the mutations P101C, I152C, T54V, S190F and V296L and comprising the amino acid sequence set forth in SEQ ID NO:83 or 84; NO:85 or 86 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:85 or 86; (27) a mutant comprising the amino acid sequence set forth in SEQ ID NO:87 or 88; (28) a mutant comprising mutations P101C, I152C, T54F, S190F and V296L and comprising a residue identical to the residue set forth in SEQ ID NO:87 or 88; NO:87 or 88 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:87 or 88; (29) a mutant comprising the amino acid sequence set forth in SEQ ID NO:89 or 90; (30) a mutant comprising the mutations P101C, I152C, T54C, S190F and V296L and comprising at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:89 or 90.(31) a mutant comprising an amino acid sequence as set forth in SEQ ID NO:91 or 92; (32) a mutant comprising mutations P101C, I152C, T54A, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:91 or 92; (33) a mutant comprising an amino acid sequence as set forth in SEQ ID NO:93 or 94; (34) a mutant comprising mutations A102C, I152C, T54V, S190I and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:93 or 94; NO:93 or 94 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:93 or 94; (35) a mutant comprising the amino acid sequence set forth in SEQ ID NO:95 or 96; (36) a mutant comprising the mutations A102C, I152C, T54F, S190I and V296L and comprising the amino acid sequence set forth in SEQ ID NO:93 or 94; NO:95 or 96 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:95 or 96; (37) a mutant comprising the amino acid sequence set forth in SEQ ID NO:97 or 98; (38) a mutant comprising the mutations A102C, I152C, T54C, S190I and V296L and comprising the amino acid sequence set forth in SEQ ID NO:97 or 98; NO:97 or 98 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:97 or 98; (39) a mutant comprising the amino acid sequence set forth in SEQ ID NO:99 or 100; and (40) a mutant comprising the mutations A102C, I152C, T54A, S190I and V296L and comprising the amino acid sequence set forth in SEQ ID NO:99 or 100. A variant of an amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity with the amino acid sequence shown in NO:99 or 100.

[0022] Another nucleic acid, comprising a polynucleotide encoding a mutant of an RSV F protein, the mutant comprising an F1 polypeptide and an F2 polypeptide, wherein relative to the wild-type RSV F protein, the mutant comprises one or more of the following mutations (1) to (8): (1) K or E at position 66 is replaced by I, L, M, F, or V; (2) N at position 67 is replaced by I; (3) S at position 213 is replaced by P; (4) S at position 215 is replaced by P; (5) N at position 216 is replaced by P; (6) I at position 217 is replaced by P, F, Y, or W; (7) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W; and (8) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0023] In some embodiments, the mutations of the mutant include the following (1) to (3): (1) one of the following mutations: S at position 213 is replaced by P, S at position 215 is replaced by P, N at position 216 is replaced by P, and I at position 217 is replaced by P, F, Y, or W; (2) one of the following mutations: K or E at position 66 is replaced by I, L, M, F, or V, and N at position 67 is replaced by I; and (3) one of the following mutations: D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W, and V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0024] In some embodiments, the mutations of the mutant are selected from the group consisting of (1) to (13): (1) I217P, E66I, and D486Q; (2) I217P, N67I, and E487Q; (3) I217P, E66I, and E487T; (4) S215P, N67I, and E487Q; (5) S213P, N67I, and E487Q; (6) S213P, E66I, and E487T; (7) S213P, E66I, and D486Q; (8) N216P, N67I, and E487Q; (9) N216P, E66I, and E487T; (10) N216P, E66I, and D486Q; (11) A102C, I152C, S213P, and E66I; (12) P101C, I152C, S213P, and E66I; and (13) S213P and E66I.

[0025] In some embodiments, the mutant is selected from one of the following (1) to (30): (1) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 31 or 32; (2) a mutant comprising the mutations N216P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 31 or 32; (3) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 33 or 34; (4) a mutant comprising the mutations N216P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: NO:33 or 34 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:33 or 34; (5) a mutant comprising the amino acid sequence set forth in SEQ ID NO:35 or 36; (6) a mutant comprising the mutations N216P, N67I and E487Q and comprising the amino acid sequence set forth in SEQ ID NO:36; NO:35 or 36 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:35 or 36; (7) a mutant comprising the amino acid sequence set forth in SEQ ID NO:37 or 38; (8) a mutant comprising the mutations S213P, E66I and D486Q and comprising the amino acid sequence set forth in SEQ ID NO:37 or 38; NO:37 or 38 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:37 or 38; (9) a mutant comprising the amino acid sequence set forth in SEQ ID NO:39 or 40; (10) a mutant comprising the mutations S213P, E66I and E487T and comprising at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:39 or 40.(11) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 41 or 42; (12) a mutant comprising mutations S213P, N67I and E487Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 41 or 42; (13) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 43 or 44; (14) a mutant comprising mutations S215P, N67I and E487Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:43 or 44 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:43 or 44; (15) a mutant comprising the amino acid sequence set forth in SEQ ID NO:45 or 46; (16) a mutant comprising the mutations I217P, E66I and E487T and comprising the amino acid sequence set forth in SEQ ID NO:43 or 44; NO:45 or 46 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:45 or 46; (17) a mutant comprising the amino acid sequence set forth in SEQ ID NO:47 or 48; (18) a mutant comprising the mutations I217P, N67I and E487Q and comprising the amino acid sequence set forth in SEQ ID NO:47 or 48; NO:47 or 48 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:47 or 48; (19) a mutant comprising the amino acid sequence set forth in SEQ ID NO:49 or 50; (20) a mutant comprising the mutations I217P, E66I and D486Q and comprising at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:49 or 50.(21) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 51 or 52; (22) a mutant comprising mutations S213P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 51 or 52; (23) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 53 or 54; (24) a mutant comprising mutations S213P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:53 or 54 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:53 or 54; (25) a mutant comprising the amino acid sequence set forth in SEQ ID NO:55 or 56; (26) a mutant comprising the mutations A102C, I152C, S213P and E66I and comprising the amino acid sequence set forth in SEQ ID NO:53 or 54; NO:55 or 56 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:55 or 56; (27) a mutant comprising the amino acid sequence set forth in SEQ ID NO:57 or 58; (28) a mutant comprising mutations P101C, I152C, S213P and E66I and comprising a mutant comprising the amino acid sequence set forth in SEQ ID NO:57 or 58; NO:57 or 58 has an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence set forth in SEQ ID NO:57 or 58; (29) a mutant comprising the amino acid sequence set forth in SEQ ID NO:59 or 60; and (30) a mutant comprising the mutations S213P and E66I and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence set forth in SEQ ID NO:59 or 60.

[0026] In some embodiments, the wild-type RSV is subtype A or subtype B.

[0027] In some embodiments, the wild-type RSV is wild-type hRSV.

[0028] In some embodiments, the wild-type hRSV is strain A2, strain Ontario, or strain Buenos Aires.

[0029] In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA; more preferably, the mRNA comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0030] In some embodiments, the nucleic acid is DNA. In some embodiments, the DNA is capable of being transcribed into RNA.

[0031] In some embodiments, the DNA sequence corresponding to the 5'-UTR is shown in SEQ ID NO: 209;

[0032] and / or, the DNA sequence corresponding to the 3'-UTR is shown in SEQ ID NO: 210;

[0033] And / or, the nucleotides constituting the poly(A) tail contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides consecutively; preferably, the nucleotides constituting the poly(A) tail contain one or more nucleotides other than A nucleotides; more preferably, the DNA sequence corresponding to the poly(A) tail is shown in SEQ ID NO: 211.

[0034] In some embodiments, the nucleic acid contains modified nucleotides.

[0035] In some embodiments, the nucleic acid contains modified nucleosides; preferably, the modified nucleosides include at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0036] In some embodiments, the nucleic acid comprises a polynucleotide encoding one or more mutants of the RSV F protein.

[0037] In some embodiments, the nucleic acid further includes polynucleotides encoding other proteins or polypeptides other than mutants of the RSV F protein.

[0038] In some embodiments, the nucleic acid further comprises one or more of the following: a polynucleotide encoding a transmembrane domain of the RSV F protein, a polynucleotide encoding a pep27 polypeptide, a polynucleotide encoding a signal peptide, and a polynucleotide encoding a trimerization domain.

[0039] A genetic engineering vector, comprising the above-mentioned nucleic acid, or comprising a polynucleotide capable of being transcribed into the above-mentioned nucleic acid.

[0040] A host cell comprising the above-mentioned genetic engineering vector.

[0041] a mutant of the RSV F protein encoded by the above nucleic acid;

[0042] In some embodiments, the mutant is a trimer.

[0043] A delivery vector comprising a nucleic acid composition, wherein the nucleic acid composition comprises a first nucleic acid or a first genetically engineered vector, wherein the first nucleic acid is the above-mentioned nucleic acid, and the first genetically engineered vector is the above-mentioned genetically engineered vector;

[0044] Alternatively, the nucleic acid composition includes multiple first nucleic acids or multiple first genetic engineering vectors, the first nucleic acid is the above-mentioned nucleic acid, the first genetic engineering vector is the above-mentioned genetic engineering vector, and the multiple first nucleic acids are independent of each other or the multiple first genetic engineering vectors are independent of each other.

[0045] In some embodiments, the nucleic acid composition further includes a second nucleic acid comprising a polynucleotide encoding a protein or polypeptide other than a mutant of the RSV F protein or a second vector comprising a polynucleotide encoding a protein or polypeptide other than a mutant of the RSV F protein; and / or the first nucleic acid and the second nucleic acid are RNA.

[0046] In some embodiments, the first nucleic acid and the second nucleic acid are mRNA.

[0047] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNPs), a cationic liposome, a cationic protein, or a lipid polymer (LPP).

[0048] In some embodiments, the delivery vehicle is a lipid nanoparticle comprising a cationic lipid comprising the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0049] in:

[0050] L 3 and L 4The same or different, each independently C1-C12 alkylene, C2-C12 alkenylene or C2-C12 alkynylene; preferably L 3 and L 4 are the same or different, and are each independently C3-C10 alkylene, C3-C10 alkenylene or C3-C10 alkynylene; further preferably, L 3 and L 4 The same or different, each independently a C3-C10 alkylene group; most preferably, L 3 and L 4 The same or different, each independently a C5-C8 alkylene group;

[0051] G 4 and G 5 are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S- or -SC(=O)-; preferably, G 4 and G 5 The same or different, each independently being -O-(C=O)-, -(C=O)-O-, -C(=O)- or -O-; most preferably, G 4 and G 5 the same or different, each independently selected from -O-(C=O)- or -(C=O)-O-;

[0052] R 18 and R 19 The same or different, each independently a C5-C27 alkyl group, or a C5-C27 alkenyl group containing one or more double bonds; preferably, R 18 and R 19 The same or different, each independently a C8-C20 alkyl group or a C8-C20 alkenyl group containing one or more double bonds; further preferably, R 18 and R 19 The same or different, each independently a C9-C17 alkyl group or a C9-C18 alkenyl group containing one or two double bonds; most preferably, R 18 and R 19 the same or different, each independently

[0053] R 20 is halogen, hydroxy, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; preferably, R 20is halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; further preferably, R 20 is halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; most preferably, R 20 is fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl or acetamido;

[0054] z is 1, 2 or 3.

[0055] In some embodiments, the cationic lipid is the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0056] Alternatively, the cationic lipid is the following compound, or a pharmaceutically acceptable salt thereof:

[0057] A pharmaceutical composition comprising the above nucleic acid, the above genetically engineered vector, the above host cell, the above RSV F protein mutant or the above delivery vector, and a pharmaceutically acceptable carrier.

[0058] In some embodiments, the pharmaceutical composition includes a plurality of said delivery vehicles;

[0059] Alternatively, the pharmaceutical composition comprises a plurality of said nucleic acids.

[0060] Use of the aforementioned nucleic acid, the aforementioned genetically engineered vector, the aforementioned host cell, the aforementioned RSV F protein mutant, the aforementioned delivery vector or the aforementioned pharmaceutical composition in the preparation of a drug;

[0061] In some embodiments, the medicament is used to prevent or treat RSV infection or a disease caused by RSV infection;

[0062] In some embodiments, the drug is a vaccine.

[0063] A vaccine comprising the above nucleic acid, the above genetic engineering vector, the above mutant of RSV F protein or the above delivery vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figures 1A and 1B show the results of the total IgG anti-titer test (GMT, 95% CI) induced by different mRNA vaccines on day 35 after a single immunization of mice. Figure 1A shows the total IgG anti-titer that specifically binds to the RSV / AF protein, and Figure 1B shows the total IgG anti-titer that specifically binds to the RSV / BF protein. In Figures 1A and 1B, "RSV / AF protein" represents the F protein of RSV subtype A (the same applies to "RSV / AF protein" appearing in other figures), "RSV / BF protein" represents the F protein of RSV subtype B (the same applies to "RSV / BF protein" appearing in other figures), and "598+589" represents a bivalent RSV mRNA vaccine, which means that an LNP formulation encapsulating mRNA numbered 598 and an LNP formulation encapsulating mRNA numbered 589 were mixed and injected into mice. The same applies to the bivalent RSV mRNA vaccines represented by other groups, and the same applies to other figures not otherwise specified.

[0065] Figures 1C to 1D are the test results of total IgG anti-titer (GMT, 95% CI) induced by different mRNA vaccines on the 19th and 28th days after a single immunization of mice, where RSV / A represents RSV subtype A, RSV / B represents RSV subtype B, Figure 1C is the total IgG anti-titer on the 19th day after a single immunization of mice, and Figure 1D is the total IgG anti-titer on the 28th day after a single immunization of mice.

[0066] Figure 1E shows the results of total IgG titer detection (GMT, 95% CI) induced on D14, D28 and D35 after mice were immunized with different doses of mRNA vaccine.

[0067] Figures 2A to 2C are the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum of mice immunized with a single dose of different mRNA vaccines, where the serum dilution is 1:900. Figure 2A shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 7th day after immunization, Figure 2B shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 14th day after immunization, and Figure 2C shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 19th day after immunization.

[0068] Figures 3A to 3C are the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum of mice immunized with a single dose of different mRNA vaccines, where the serum dilution is 1:100. Figure 3A shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 7th day after immunization, Figure 3B shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 14th day after immunization, and Figure 3C shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 21st day after immunization.

[0069] Figures 4A to 4C are the detection results of total IgG antibodies specifically binding to RSV / BF protein in the serum of mice immunized with a single dose of different mRNA vaccines, wherein the serum dilution is 1:100. Figure 4A shows the detection results of total IgG antibodies specifically binding to RSV / BF protein in the serum on the 7th day after immunization, Figure 4B shows the detection results of total IgG antibodies specifically binding to RSV / BF protein in the serum on the 14th day after immunization, and Figure 4C shows the detection results of total IgG antibodies specifically binding to RSV / BF protein in the serum on the 21st day after immunization.

[0070] Figures 5A to 5C are the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum of mice immunized with a single dose of different mRNA vaccines, where the serum dilution is 1:100. Figure 5A shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 7th day after immunization; Figure 5B shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 14th day after immunization; and Figure 5C shows the detection results of total IgG antibodies specifically binding to RSV / AF protein in the serum on the 21st day after immunization.

[0071] Figures 6A and 6B are the detection results of total IgG antibodies specifically binding to RSV / BF protein in the serum of mice immunized with a single dose of different mRNA vaccines, wherein the serum dilution is 1:100. Figure 6A shows the detection results of total IgG antibodies specifically binding to RSV / BF protein in the serum on the 7th day after immunization, and Figure 6B shows the detection results of total IgG antibodies specifically binding to RSV / BF protein in the serum on the 14th day after immunization.

[0072] FIG7 shows the neutralizing antibody test results (GMT, 95% CI) against RSV / A pseudovirus induced on day 21 after single immunization of mice with different mRNA vaccines. The antibody titer is NT50, where RSV / A represents RSV subtype A.

[0073] Figure 8 shows the neutralizing antibody titer test results (GMT, 95% CI) against RSV / A pseudovirus induced on day 19 after single immunization of mice with different mRNA vaccines. The antibody titer is NT50, where RSV / A represents RSV subtype A.

[0074] FIG9 shows the neutralizing antibody test results (GMT, 95% CI) against RSV / A pseudovirus induced on day 31 after single immunization of mice with different mRNA vaccines. The antibody titer is NT50, where RSV / A represents RSV subtype A.

[0075] Figures 10A to 10C are the ELISpot test results of mouse spleen cells on the 35th day after a single immunization of mice with different mRNA vaccines, among which Figure 10A is the ELISpot test result of IFN-γ, Figure 10B is the ELISpot test result of IL-4, and Figure 10C is the result of IFN-γ / IL4.

[0076] Figures 11A to 11C are the ELISpot test results of mouse spleen cells on the 28th day after a single immunization of mice with different mRNA vaccines, wherein Figure 11A is the ELISpot test result of IFN-γ, Figure 11B is the ELISpot test result of IL-4, and Figure 11C is the result of IFN-γ / IL4.

[0077] Figure 12 shows the binding of F proteins expressed in vitro from different mRNAs to different antibodies against RSV F protein, wherein 591 represents the F protein expressed by the engineered plasmid encoding the mRNA shown in coding number 591 (without the Cap1 cap structure).

[0078] FIG13 shows the RSV viral load detection (GMT, 95% CI) in the lungs of mice immunized with RSV mRNA vaccine and RSV inactivated vaccine 5 days after RSV challenge, wherein FI-RSV represents formalin-inactivated RSV.

[0079] Figure 14 shows the results of lung tissue pathology analysis of mice immunized with RSV mRNA vaccine and RSV inactivated vaccine 5 days after RSV challenge, where FI-RSV represents formalin-inactivated RSV; Vs PBS, P<0.05, *; P<0.01, **; P<0.001, ***; P<0.0001, ****; Vs FI-RSV, P<0.05, #; P<0.01, ##; P<0.001, ###; P<0.0001, ####.

[0080] FIG15 shows the neutralizing antibody titer test results (GMT, 95% CI) against RSV / A pseudovirus induced by immunizing rats with different doses of mRNA vaccines.

[0081] Figure 16 is the ELISpot test results of mouse spleen cells on the 7th day (D28) after the second immunization (D21) of mice with different doses of mRNA vaccine.

[0082] Figures 17A to 17B show the binding of F proteins expressed in vitro by different mRNAs with different antibodies against RSV F protein, wherein Figure 17A shows the binding of RSV A subtype F protein expressed in vitro by different mRNAs with different antibodies against RSV F protein, and Figure 17B shows the binding of RSV B subtype F protein expressed in vitro by different mRNAs with different antibodies against RSV F protein. Group 1 is a control group without mRNA transfection, groups 2 to 4 are mRNAs numbered 832 from different batches, group 5 represents mRNA numbered 1119, group 6 represents mRNA numbered 1902, group 7 represents mRNA expressing Post F, groups 8 to 10 are mRNAs numbered 973 from different batches, and group 11 represents mRNA numbered 1903.

[0083] Figures 18A and 18B show RSV viral loads in the lungs and nasal tissues of cotton rats immunized with RSV mRNA vaccines 5 days after RSV challenge. FI-RSV indicates formalin-inactivated RSV. RSV A indicates RSV subtype A, and RSV B indicates RSV subtype B.

[0084] Figures 19A and 19B show the neutralizing antibody test results (GMT, 95% CI) against RSV / A and RSV / B true viruses induced by RSV mRNA vaccine in cotton rats. The antibody titer is PRNT60, where RSV / A represents RSV subtype A and RSV / B represents RSV subtype B.

[0085] FIG20 shows the neutralizing antibody test results (GMT, 95% CI) against RSV / A pseudovirus induced on day 28 after single immunization of mice with different mRNA vaccines. The antibody titer is NT50, where RSV / A represents RSV subtype A.

[0086] Figures 21A to 21D show the binding of F proteins expressed in vitro by different mRNAs to different antibodies against RSV F proteins, wherein Figure 21A shows the binding of RSV A subtype F proteins expressed in vitro by different mRNAs to antibody D25, Figure 21B shows the binding of RSV A subtype F proteins expressed in vitro by different mRNAs to antibody AM22, Figure 21C shows the binding of RSV A subtype F proteins expressed in vitro by different mRNAs to antibody AM14, and Figure 21D shows the binding of RSV A subtype F proteins expressed in vitro by different mRNAs to antibody CR9501; in Figures 21A to 21D, "NC" is a control group that is not transfected with an engineered plasmid, "1988" represents the F protein expressed by the engineered plasmid of the mRNA shown in coding number 1988 (without Cap1 cap structure), "2000" represents the group in which the F protein expressed by the engineered plasmid of the mRNA shown in coding number 2000 (without Cap1 cap structure) belongs, and the same applies to the other numbers representing the groups.

[0087] Detailed Description of the Invention

[0088] 1. Definition

[0089] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0090] Unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are terms widely used in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). At the same time, in order to better understand this application, definitions and explanations of relevant terms are provided below.

[0091] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0092] As used herein, unless the context indicates otherwise, the singular expressions "a," "an," "the," and similar references used in the context of describing the present application (particularly in the context of the claims) should be interpreted to cover both the singular and the plural. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. The terms "at least one" or "one or more" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0093] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range "1 to 10" should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any individual value (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.) within the range of 1 to 10. This principle also applies to ranges that use only one value as a minimum or maximum value.

[0094] As used herein, the terms "and / or," "any combination thereof," and their grammatical equivalents are used interchangeably. These terms may expressly refer to any combination. For example, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may refer to "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C."

[0095] As used herein, the term "naturally occurring" or "naturally occurring" refers to the fact that a substance can be found in nature. For example, a peptide, amino acid, protein, or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and has not been experimentally modified by man is naturally occurring.

[0096] As used herein, the term "non-naturally occurring" when used to describe a nucleic acid herein is intended to mean that the nucleic acid is not found in nature. For example, a non-naturally occurring nucleic acid encoding a viral peptide or protein has at least one genetic alteration or chemical modification that is not normally found in wild-type strains of the virus in question. Such genetic alterations include, for example, the introduction of an expressible nucleic acid sequence encoding a peptide or polypeptide that is heterologous to the virus in question, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and / or other functional disruptions to the viral genetic material. Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.

[0097] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0098] As used herein, the term "wild type" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.

[0099] As used herein, the term "% identity" or "% identity" refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window." Optimal alignment can be performed manually or with the aid of algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or the similarity search method described by computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the publicly available BLASTN or BLASTP algorithms available on the National Center for Biotechnology Information (NCBI) website.

[0100] "% identity" or "% homology" can be obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100 to obtain % identity. In some embodiments, the degree of identity is given for a region of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the degree of identity is given for the entire length of the reference sequence. Alignment for determining sequence identity can be performed using tools known in the art, preferably utilizing optimal sequence alignment, e.g., utilizing Align, utilizing standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0101] As used herein, "nucleotides" include deoxyribonucleotides, deoxyribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotides" are constituents of ribonucleic acid (RNA), consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotides" are constituents of deoxyribonucleic acid (DNA), also consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen. They are the main chemical components of chromosomes.

[0102] "Nucleotide" is generally referred to by a single letter representing the base, "A" or "A nucleotide" refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine, "C" or "C nucleotide" refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine, "G" or "G nucleotide" refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine, "U" or "U nucleotide" refers to uracil ribonucleotide containing uracil, and "T" or "T nucleotide" refers to thymine deoxyribonucleotide containing thymine.

[0103] As used herein, the term "nucleic acid" generally refers to a polymer comprising deoxyribonucleotides (deoxyribonucleic acid, referred to as DNA) or a polymer comprising ribonucleotides (ribonucleic acid, referred to as RNA) or any compound of a combination thereof. In addition, nucleic acids herein also include derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids on the bases, sugars or phosphates of the nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In addition, herein, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0104] "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. Those skilled in the art will understand that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylate in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes. The RNA corresponding to a DNA is a polynucleotide in which all Ts in the DNA are replaced by Us.

[0105] The polynucleotide may comprise one or more segments (nucleic acid fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments). For example, the polynucleotide may comprise a segment encoding a polypeptide of interest. In a specific embodiment, the polynucleotide may comprise a segment encoding a polypeptide of interest and a regulatory segment (including but not limited to segments for transcriptional regulation and translational regulation). In one embodiment, the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.

[0106] As used herein, the term "promoter" refers to a polynucleotide located upstream of the 5' end of the coding region of a gene, which contains a conserved sequence required for specific binding of RNA polymerase and transcription initiation, can activate RNA polymerase, enable RNA polymerase to accurately bind to template DNA and have the specificity of transcription initiation. Promoters can be derived from viruses, bacteria, fungi, plants, insects and animals. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and CMV IE promoter.

[0107] As used herein, the term "5' untranslated region" or "5'-UTR" can be an RNA sequence in an mRNA that is upstream of the coding sequence and is not translated into protein. The 5'-UTR in a gene generally begins at the transcription start site and ends at the nucleotides upstream of the translation start codon of the coding sequence. The 5'-UTR can contain elements that control gene expression, such as a ribosome binding site, a 5'-terminal oligopyrimidine tract, and a translation initiation signal such as a Kozak sequence. mRNA can be post-transcriptionally modified by the addition of a 5' cap. Therefore, the 5'-UTR in a mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.

[0108] As used herein, the term "3' untranslated region" or "3'-UTR" can be an RNA sequence in an mRNA that is located downstream of a coding sequence and is not translated into protein. The 3'-UTR in an mRNA is located between the stop codon and the poly(A) sequence of the coding sequence, for example, starting from the nucleotides downstream of the stop codon and ending at the nucleotides upstream of the poly(A) sequence.

[0109] As used herein, the terms "poly(A)", "poly(A) sequence", and "poly(A) tail" are used interchangeably. Naturally occurring poly(A) sequences are typically composed of adenine ribonucleotides. In the present application, the term "modified poly(A) sequence" refers to a poly(A) sequence that contains nucleotides or nucleotide segments other than adenine ribonucleotides. The poly(A) sequence is typically located at the 3' end of the mRNA, for example, at the 3' end (downstream) of the 3'-UTR.

[0110] As used herein, the term "5'-cap structure": The 5'-cap structure is typically located at the 5' end of the mature mRNA. In some embodiments, the 5'-cap structure is linked to the 5'-end of the mRNA via a 5'-5'-triphosphate bond. The 5'-cap structure is typically formed by modified (e.g., methylated) ribonucleotides (particularly guanine nucleotide derivatives). For example, m7GpppN (cap 0 or "cap0" is a cap structure formed by the 5' phosphate group of the hnRNA reacting with the 5'-phosphate group of the m7GTP under the action of guanylyltransferase to form a 5',5'-phosphodiester bond), wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure. In some embodiments, the 5'-cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylating the 2'-OH of the first nucleotide sugar group of hnRNA on the basis of cap 0, or referred to as "cap 1"), cap 2 (a cap structure formed by further methylating the 2'-OH of the second nucleotide sugar group of hnRNA on the basis of cap 1, or referred to as "cap 2"), cap 4, cap 0 analogs, cap 1 analogs, cap 2 analogs, or cap 4 analogs.

[0111] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."

[0112] As used herein, the term "host cell" refers to a cell for receiving, maintaining, replicating, expressing a polynucleotide or a vector. The term "host cell" includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells and insect cells). For example, cells from humans, mice, hamsters, pigs, goats, primates. The cell can be derived from a variety of tissue types and includes primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte, or a macrophage. The nucleic acid can be present in the host cell with a single copy or with several copies. In some embodiments, the host cell can be a cell expressing the polypeptide of the present application therein.

[0113] In the context of the present application, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term "plasmid" also encompasses, for example, a circular plasmid digested with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule and the resulting molecule of the circular plasmid linearization. Plasmids can replicate, i.e., amplify the genetic information stored as chromosomal DNA in the cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In an alternative embodiment, the DNA plasmid is a medium copy or high copy plasmid. In another alternative embodiment, the DNA plasmid is a high copy plasmid. Examples of such high copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) comprising a replication origin that supports high copies of the plasmid.

[0114] The term "vaccine" is typically understood as a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0115] The term "treatment" or the like is used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. Therefore, the treatment of the present application may relate to the treatment of a certain disease state, but may also relate to a prophylactic treatment for preventing a disease or its symptoms in whole or in part. In some embodiments, the term "treatment" is understood to be therapeutic in terms of partially or completely curing a disease and / or owing to the adverse effects and / or symptoms of the disease. Treatment may also be prophylactic or preventive treatment, i.e., measures taken to prevent a disease, such as to prevent infection and / or the onset of a disease.

[0116] As used herein, the terms "subject" and "patient" can be used interchangeably. In certain embodiments, the subject is a mammal, such as a human, non-human primate (e.g., ape, chimpanzee, monkey, and orangutan), domesticated animal (including dog and cat and livestock (e.g., horse, cattle, pig, sheep, and goat)), or other mammal. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0117] As used herein, the term "administer" refers to providing or administering a medicament to a subject by any effective route. Exemplary routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous), oral, intracavitary, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation. When used to treat a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom. When used to prevent a disease, disorder, condition, or symptom, administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom.

[0118] "Antibody D25" or "monoclonal antibody D25" is a neutralizing monoclonal antibody that specifically binds to the prefusion conformation of the RSV F protein, but not the postfusion conformation of the RSV F protein. The protein and nucleic acid sequences of antibody D25 are known. For example, the heavy and light chain amino acid sequences of the D25 antibody are described in U.S. Patent Application Publication No. 2010 / 0239593, which is incorporated herein by reference in its entirety; see also Kwakkenbos et al., Nat. Med., 16:123-128, 2009. D25 specifically binds to a quaternary epitope (included in the antigenic site) present in the prefusion conformation of the RSV F protein, but not in the postfusion conformation. This epitope is included within RSV F positions 62-69 and 196-209 and is located at the membrane-distal apex of the RSV F protein in the prefusion conformation. Antibody D25 specifically binds to the preF protein.

[0119] "Antibody AM14" or "monoclonal antibody AM14" is a neutralizing monoclonal antibody that specifically binds to the prefusion conformation of the RSV F protein but not the postfusion conformation of the RSV F protein. The AM14 protein and nucleic acid sequence are known, for example, as described in WO 2008 / 147196 A2.

[0120] "Antibody AM22" or "monoclonal antibody AM22" is a neutralizing monoclonal antibody that specifically binds to the prefusion conformation of the RSV F protein, rather than the postfusion conformation of the RSV F protein. The AM22 protein and nucleic acid sequences are known, for example, the heavy and light chain amino acid sequences of the AM22 antibody are described in U.S. Patent Application Publication No. 2012 / 0070446 (incorporated herein by reference in its entirety). AM22 specifically binds to epitopes (including on antigenic sites), including positions where the RSV F protein exists in its prefusion conformation, rather than in its postfusion conformation. This epitope is included within RSV F positions 62-69 and 196-209 and is located at the distal end of the membrane of the RSV F protein in the prefusion conformation. Antibody AM22 specifically binds to the preF protein disclosed herein.

[0121] "Antibody clone 131-2A" or "monoclonal antibody 131-2A" specifically binds to the site I epitope of the RSV F protein. Antibody clone 131-2A is a monoclonal antibody that preferentially binds to the postfusion conformation of the RSV F protein rather than the prefusion conformation of the RSV F protein.

[0122] The term "mutation" refers to a deletion, addition, or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, an amino acid substitution at a specific position in a protein sequence is referred to using the code "(amino acid residue in the wild-type protein)(amino acid position)(amino acid residue in the engineered protein)". For example, the code P101C means that the proline (P) residue at position 101 of the amino acid sequence of the reference protein is substituted with a cysteine ​​(C) residue (in a mutant of the reference protein).

[0123] Herein, some elements of the present application will be described. These elements are listed together with specific embodiments, but it should be understood that they can be combined in any way and in any number to produce other embodiments. The examples and preferred embodiments described differently should not be interpreted as limiting the present application to only the embodiments clearly described. This specification should be understood to support and include embodiments that combine the clearly described embodiments with any number of disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any permutation and combination of all described elements in this application should be considered to be disclosed by the specification of this application. For example, in one embodiment, the mutation of the mutant includes: P101C and I152C, and in another embodiment, the mutation of the mutant includes: T at position 54 is replaced by F, W or V, S at position 190 is replaced by I, and V at position 296 is replaced by L. Then the following scheme is also an embodiment claimed in this application: the mutation of the mutant includes: P101C and I152C, T at position 54 is replaced by F, W or V, S at position 190 is replaced by I, and V at position 296 is replaced by L.

[0124] 2. Nucleic Acids

[0125] F0 is the precursor of the RSV F protein. F0 is a 574-amino acid polypeptide translated from the RSV F protein mRNA. F0 contains a signal peptide (amino acids 1 to 25) at the N-terminus. After translation, the signal peptide is removed from F0 by a signal peptidase. The remaining F0 (i.e., residues 26 to 574) is further cleaved by cellular proteases (e.g., furin) at two cleavage sites (amino acids 109 / 110 and 136 / 137), removing 27 amino acids (amino acids 110 to 136, i.e., the pep27 polypeptide), forming the F1 polypeptide (C-terminal portion; amino acids 137 to 574) and the F2 polypeptide (N-terminal portion; amino acids 26 to 109). The F1 and F2 polypeptides are linked by a disulfide bond.

[0126] The RSV F protein complex of trimeric form mediates the fusion between virion membrane and the host cell membrane and also promotes the formation of syncytium. Before fusion, RSV F albumen can be identified by monoclonal antibody D25, AM22 and MPE8. Before fusion, F protein trimer can be identified by monoclonal antibody AM14 specificity.

[0127] 1. Nucleic acid 1

[0128] One embodiment of the present disclosure provides a nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein. Relative to the wild-type RSV F protein, the mutant of the RSV F protein (hereinafter referred to as a "mutant") comprises or is one or more of the following mutations: a disulfide bond mutation, a cavity-filling mutation, and an electrostatic mutation. By means of disulfide bond mutations, cavity-filling mutations, and / or electrostatic mutations, the RSV F protein can be stably converted to a pre-F protein. It should be noted that, in this article, "multiple" means at least two, for example, two, three, four, five, or more.

[0129] In some embodiments, the disulfide bond mutations include or are one or more of the following: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K191C, I59C and L193C, I59C and L297C, E60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T103C and I148C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C. In some embodiments, the disulfide bond mutations include or are one or more of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C. In some embodiments, the disulfide bond mutations include or are one or more of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C.

[0130] In view of the substantial conservatism of RSV F sequence, those of ordinary skill in the art can easily compare the amino acid position between different natural RSV F sequences to identify the corresponding RSV F amino acid position between different RSV strains and hypotypes.For example, between the natural RSV F0 precursor proteins of nearly all identifications, the furin cleavage site falls in the same amino acid position.Therefore, the conservatism of natural RSV F protein sequences between strains and hypotypes allows the use of reference RSV F sequences to compare the amino acid at specific positions in RSV F protein. For purposes of this disclosure (unless otherwise indicated by context), RSV F protein amino acid position is with reference to the sequence of the F0 shown in SEQ ID NO:216 (the amino acid sequence of the full-length natural F0 of strain Ontario). However, it should be noted that, and it will be appreciated by those skilled in the art that different RSV F0 sequences can have different numbering systems, for example, compared with SEQ ID NO:216, add or remove additional amino acid residues. Thus, it should be understood that when a particular amino acid residue is referred to by number, the description is not limited to the amino acid at precisely that numbered position when counting from the beginning of a given amino acid sequence, but also refers to the equivalent / corresponding amino acid residue in any and all RSV F sequences, even if that residue is not at the same exact numbered position, for example, if the RSV sequence is shorter or longer than SEQ ID NO: 216, or has insertions or deletions compared to SEQ ID NO: 216.

[0131] In some embodiments, the disulfide bond mutations include or are one of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C. In some embodiments, the disulfide bond mutations include or are one of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C.

[0132] In some embodiments, the disulfide bond mutations include or are: (i) at least one of A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C, and (ii) one of V144C and V406C, and S146C and N460C.

[0133] In some embodiments, the disulfide bond mutation is selected from: (i) one of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C, and (ii) one of V144C and V406C, and S146C and N460C. In some embodiments, the disulfide bond mutation is selected from: (i) one of P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C, and (ii) one of V144C and V406C, and S146C and N460C.

[0134] In some embodiments, the cavity-filling mutation includes or is one or more of the following (1) to (3):

[0135] (1) T at position 54 is substituted by F, W, V, C, A, H, I, Y, L, or M; for example, T at position 54 is substituted by F, W, V, C, or A;

[0136] (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and

[0137] (3) V at position 296 is substituted with I, L, Y, H, F, or M. For example, V at position 296 is substituted with L, F, or M.

[0138] In some embodiments, the mutant of the RSV F protein comprises a cavity-filling mutation comprising or being a substitution of T at position 54 with A.

[0139] In some embodiments, the mutant of the RSV F protein comprises a cavity-filling mutation, the cavity-filling mutation comprising a substitution of T at position 54 with A, and the mutant of the RSV F protein does not comprise a mutation at position 185.

[0140] In some embodiments, the mutant of the RSV F protein comprises a cavity-filling mutation, the cavity-filling mutation comprising a substitution of T at position 54 with A, and the mutant of the RSV F protein does not comprise mutation V185F.

[0141] In some embodiments, the mutant of the RSV F protein comprises a cavity-filling mutation, which includes or is a V to L substitution at position 296.

[0142] In some embodiments, the mutant of the RSV F protein comprises a cavity-filling mutation, the cavity-filling mutation comprising a V-to-L substitution at position 296, and the mutant of the RSV F protein does not comprise a mutation at position 291.

[0143] In some embodiments, the mutant of the RSV F protein comprises a cavity-filling mutation, the cavity-filling mutation comprises a V-to-L substitution at position 296, and the mutant of the RSV F protein does not comprise an I291V mutation.

[0144] In some embodiments, the cavity filling mutation includes or is a substitution of T at position 54 with F, W, V, C, A, H, I, Y, L, or M, and a substitution of S at position 190 with an amino acid other than S. In some embodiments, the cavity filling mutation is a substitution of T at position 54 with F, W, C, A, or V, and a substitution of S at position 190 with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K.

[0145] In some embodiments, the cavity filling mutation comprises or is a substitution of T at position 54 with F, W, V, C, A, H, I, Y, L, or M, and a substitution of V at position 296 with I, L, Y, H, F, or M. In some embodiments, the cavity filling mutation is a substitution of T at position 54 with F, W, C, A, or V, and a substitution of V at position 296 with I, L, Y, H, F, or M. Alternatively, the cavity filling mutation comprises a substitution of T at position 54 with F, W, V, C, A, H, I, Y, L, or M, and a substitution of V at position 296 with L, F, or M. In some embodiments, the cavity filling mutation is a substitution of T at position 54 with F, W, or V, and a substitution of V at position 296 with L, F, or M.

[0146] In some embodiments, the cavity filling mutation comprises or is: S at position 190 is substituted with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is substituted with I, L, Y, H, F, or M. In some embodiments, the cavity filling mutation is S at position 190 is substituted with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is substituted with L, F, or M.

[0147] In some embodiments, the cavity filling mutation comprises or is: T at position 54 is substituted with F, W, V, C, A, H, I, Y, L, or M, S at position 190 is substituted with an amino acid other than S, and V at position 296 is substituted with I, L, Y, H, F, or M. In some embodiments, the cavity filling mutation is T at position 54 is substituted with F, W, C, A, or V, S at position 190 is substituted with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is substituted with I, L, Y, H, F, or M. Alternatively, the cavity-filling mutation comprises a substitution of T at position 54 with F, W, V, C, A, H, I, Y, L, or M, a substitution of S at position 190 with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and a substitution of V at position 296 with L, F, or M. In some embodiments, the cavity-filling mutation is a substitution of T at position 54 with F, W, C, A, or V, a substitution of S at position 190 with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and a substitution of V at position 296 with L, F, or M. In some embodiments, the cavity-filling mutations are substitution of T at position 54 with F, W, or V, substitution of S at position 190 with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and substitution of V at position 296 with L.

[0148] In some embodiments, the cavity-filling mutation comprises or is: T at position 54 is substituted by F, W, V, C, A, or H, S at position 190 is substituted by an amino acid other than S, and V at position 296 is substituted by I or L. In some embodiments, the cavity-filling mutations include or are the group consisting of: (1) T54V, S190M, and V296L; (2) T54F, S190M, and V296L; (3) T54C, S190M, and V296L; (4) T54A, S190M, and V296L; (5) T54V, S190F, and V296L; (6) T54F, S190F, and V296L; (7) T54C, S190F, and V296L; (8) T54A, S190F, and V296L; (9) T54V, S190M, and V296L; (10) T54F, S190 M and V296L; (11) T54C, S190M and V296L; (12) T54A, S190M and V296L; (13) T54V, S190F and V296L; (14) T54F, S190F and V296L; (15) T54C, S190F and V296L; (16) T54A, S190F and V296L; (17) T54V, S190I and V296L; (18) T54F, S190I and V296L; (19) T54C, S190I and V296L; and (20) T54A, S190I and V296L.

[0149] In some embodiments, the mutant has the following (i) and (ii): (i) a group comprising the following mutations: (1) T54V, S190M and V296L; (2) T54F, S190M and V296L; (3) T54C, S190M and V296L; (4) T54A, S190M and V296L; (5) T54V, S190F and V296L; (6) T54F, S190F and V296L; (7) T54C , S190F and V296L; (8) T54A, S190F and V296L; (9) T54V, S190M and V296L; (10) T54F, S190M and V296L; (11) T54C, S190M and V296L; (12) T54A, S190M and V296L; (13) T54V, S190F and V296L; (14) T54F, S190F and V296L; (15) T54C, S190F and V296L; (16) T54A, S190F and V296L; (17) T54V, S190I and V296L; (18) T54F, S190I and V296L; (19) T54C, S190I and V296L; and (20) T54A, S190I and V296L; and (ii) the amino acid sequence of the mutant is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical to the corresponding wild-type RSV F protein.

[0150] In some embodiments, the electrostatic mutation includes or is one or both of the following: (1) D at position 486 is substituted with Q, S, H, N, T, P, C, L, I, F, or W; and (2) V at position 487 is substituted with Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C. In some embodiments, the electrostatic mutation is D at position 486 is substituted with Q, L, or I. In some embodiments, the electrostatic mutation is D at position 486 is substituted with Q.

[0151] In some embodiments, the mutation of the mutant includes or is a disulfide bond mutation.

[0152] In some embodiments, the disulfide bond mutations include or are one or more of the following: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K191C, I59C and L193C, I59C and L297C, E60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T103C and I148C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C. In some embodiments, the disulfide bond mutations include or are one or more of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C. In some embodiments, the disulfide bond mutations include or are one or more of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C.

[0153] In some embodiments, the disulfide bond mutations include or are one of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C. In some embodiments, the disulfide bond mutations include or are one of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C.

[0154] In some embodiments, the disulfide bond mutations include or are: (i) at least one of A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C, and (ii) one of V144C and V406C, and S146C and N460C.

[0155] In some embodiments, the disulfide bond mutation is selected from: (i) one of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C, and (ii) one of V144C and V406C, and S146C and N460C. In some embodiments, the disulfide bond mutation is selected from: (i) one of P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C, and (ii) one of V144C and V406C, and S146C and N460C.

[0156] In some embodiments, the mutant has the following (i) and (ii): (i) comprises one of the following mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C; (ii) the amino acid sequence of the mutant is identical to that of the corresponding wild-type RSV The F protein has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity. In some embodiments, the mutations of the mutant include or are: cavity filling mutations and electrostatic mutations.

[0157] In some embodiments, the cavity-filling mutation includes or is one or more of the following (1) to (3): (1) T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M; preferably, T at position 54 is replaced by F, W, C, A, or V; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and (3) V at position 296 is replaced by I, L, Y, H, F, or M; the electrostatic mutation includes or is one or two of the following (1) to (2): (1) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W; and (2) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0158] In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, C, A, or V, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. Alternatively, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, L, or I. In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M, and an electrostatic mutation in which D at position 486 is replaced by Q. In some embodiments, the mutant includes a cavity-filling mutation in which T at position 54 is replaced by F, W, or V, and an electrostatic mutation in which D at position 486 is replaced by Q.

[0159] In some embodiments, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. In some embodiments, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and an electrostatic mutation in which D at position 486 is replaced by Q, L, or I. In some embodiments, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and an electrostatic mutation in which D at position 486 is replaced by Q.

[0160] In some embodiments, the mutant comprises a cavity-filling mutation in which V at position 296 is replaced by I, L, Y, H, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. In some embodiments, the mutant comprises a cavity-filling mutation in which V at position 296 is replaced by I, L, Y, H, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, L, or I. Alternatively, the mutant comprises a mutation in which V at position 296 is replaced by L, F, or M, and D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. In some embodiments, the mutant comprises a cavity-filling mutation in which V at position 296 is replaced by I, L, Y, H, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q. In some embodiments, the mutant comprises a cavity-filling mutation in which V at position 296 is replaced by L, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q.

[0161] In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M and S at position 190 is replaced by an amino acid other than S, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, C, A, or V and S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. Alternatively, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M and S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and an electrostatic mutation in which D at position 486 is replaced by Q, L, or I. In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, C, A, or V and S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and an electrostatic mutation in which D at position 486 is replaced by Q, L, or I. In some embodiments, the mutant includes a cavity-filling mutation in which T at position 54 is replaced by F, W, or V and S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and an electrostatic mutation in which D at position 486 is replaced by Q.

[0162] In some embodiments, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K and V at position 296 is replaced by I, L, Y, H, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. In some embodiments, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K and V at position 296 is replaced by L, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. Alternatively, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, V at position 296 is replaced by I, L, Y, H, F, or M, and the electrostatic mutation is a substitution of D at position 486 by Q, L, or I. In some embodiments, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, V at position 296 is replaced by I, L, Y, H, F, or M, and the electrostatic mutation is a substitution of D at position 486 by Q. In some embodiments, the mutant comprises a cavity-filling mutation in which S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is replaced by L, F, or M, and D at position 486 is replaced by Q.

[0163] In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M, S at position 190 is replaced by an amino acid other than S, and V at position 296 is replaced by I, L, Y, H, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, C, A, or V, S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is replaced by I, L, Y, H, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. Alternatively, the mutant includes a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M, S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is replaced by L, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. Alternatively, the mutant includes a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M, S at position 190 is replaced by an amino acid other than S, and V at position 296 is replaced by I, L, Y, H, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, L, or I. Alternatively, the mutant includes a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L or M, S at position 190 is replaced by an amino acid other than S, and V at position 296 is replaced by I, L, Y, H, F or M, and an electrostatic mutation in which D at position 486 is replaced by Q.

[0164] In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, C, A, or V, S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is replaced by L, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W. Alternatively, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M, S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K, and V at position 296 is replaced by L, F, or M, and an electrostatic mutation in which D at position 486 is replaced by Q, L, or I. Alternatively, the mutant includes a cavity-filling mutation in which T at position 54 is replaced by F, W, C, A or V, S at position 190 is replaced by an amino acid other than S, and V at position 296 is replaced by I, L, Y, H, F or M, and an electrostatic mutation in which D at position 486 is replaced by Q, L or I.

[0165] In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W or V, S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K, and V at position 296 is replaced by L, F or M, and an electrostatic mutation in which D at position 486 is replaced by Q, L or I. In some embodiments, the mutant comprises a cavity-filling mutation in which T at position 54 is replaced by F, W or V, S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K, V at position 296 is replaced by L, F or M, and D at position 486 is replaced by Q.

[0166] In some embodiments, the mutation of the mutant includes or is one of a cavity filling mutation and an electrostatic mutation and a disulfide bond mutation.

[0167] In some embodiments, the mutations of the mutant include or are: (i) one or more of the following disulfide bond mutations: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K191C, I59C and L193C, I59C and L297C, E60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T104C and I157C; 3C and I148C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C; and (ii) one or more of the following cavity-filling mutations: (1) T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and (3) V at position 296 is replaced by I, L, Y, H, F, or M. In some embodiments, the cavity-filling mutation of (ii) is a substitution of S at position 190 with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K.

[0168] In some embodiments, the mutations of the mutant include or are: (i) one or more of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105 C and S146C, and N105C and A147C; and (ii) one or more of the following cavity-filling mutations: (1) T at position 54 is substituted with F, W, V, C, A, H, I, Y, L, or M; (2) S at position 190 is substituted with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and (3) V at position 296 is substituted with I, L, Y, H, F, or M. In some embodiments, the cavity-filling mutation in (ii) is S at position 190 is substituted with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K.

[0169] In some embodiments, the mutations in the mutant include or are: (i) one or more of the following disulfide bond mutations: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C; and (ii) one or more of the following cavity filling mutations: (1) T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and (3) V at position 296 is replaced by I, L, Y, H, F, or M. In some embodiments, the cavity-filling mutation of (ii) is a substitution of S at position 190 with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K.

[0170] In some embodiments, the mutations of the mutant include or are: (i) one of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N1 05C and S146C, and N105C and A147C; and (ii) one or more of the following cavity-filling mutations: (1) T at position 54 is replaced by F, W, C, A, or V; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and (3) V at position 296 is replaced by I, L, Y, H, F, or M.

[0171] In some embodiments, the mutations of the mutant include or are: (i) one of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G14 5C, N105C and S146C, and N105C and A147C; and (ii) one or more of the following cavity-filling mutations: (1) T at position 54 is replaced by F, W or V; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K; and (3) V at position 296 is replaced by L, F or M.

[0172] In some embodiments, the mutations of the mutant include or are a group selected from the group consisting of (1) to (20): (1) A102C, I152C, T54V, S190M, and V296L; (2) A102C, I152C, T54F, S190M, and V296L; (3) A102C, I152C, T54C, S190M, and V296L; (4) A102C, I152C, T54A, S190M, and V296L; (5) A102C, I152C, T54V, S190F, and V296L; (6) A102C, I152C, T54F, S190F, and V296L; (7) A102C, I152C, T54C, S190F, and V296L; (8) A102C, I152C, T54A, S190F, and V296L; (9) P101C, I152C, T54V, S190M, and V296L; (10) P101C, I152C, T54F, S190M and V296L; (11) P101C, I152C, T54C, S190M and V296L; (12) P101C, I152C, T54A, S190M and V296L; (13) P101C, I152C, T54V, S190F and V296L; (14) P101C, I152C, T54F, S190F and V296L; (15) P101C, I152C, T54C, S190F and V296L; (16) P101C, I152C, T54A, S190F, and V296L; (17) A102C, I152C, T54V, S190I, and V296L; (18) A102C, I152C, T54F, S190I, and V296L; (19) A102C, I152C, T54C, S190I, and V296L; and (20) A102C, I152C, T54A, S190I, and V296L.

[0173] In some embodiments, the mutant is selected from one of the following (1) to (40): (1) a mutant comprising the amino acid sequence as set forth in SEQ ID NO: 61 or 62; (2) a mutant comprising the mutations A102C, I152C, T54V, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 61 or 62; (3) a mutant comprising the amino acid sequence as set forth in SEQ ID NO: 63 or 64; (4) a mutant comprising the mutations A102C, I152C, T54F, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 63 or 64. NO:63 or 64 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:63 or 64; (5) a mutant comprising the amino acid sequence set forth in SEQ ID NO:65 or 66; (6) a mutant comprising the mutations A102C, I152C, T54C, S190M and V296L and comprising the amino acid sequence set forth in SEQ ID NO:66; NO:65 or 66 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:65 or 66; (7) a mutant comprising the amino acid sequence set forth in SEQ ID NO:67 or 68; (8) a mutant comprising the mutations A102C, I152C, T54A, S190M and V296L and comprising the amino acid sequence set forth in SEQ ID NO:67 or 68; NO:67 or 68 has an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical; (9) a mutant comprising the amino acid sequence as set forth in SEQ ID NO:69 or 70; (10) a mutant comprising the mutations A102C, I152C, T54V, S190F and V296L and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:69 or 70.(11) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 71 or 72; (12) a mutant comprising mutations A102C, I152C, T54F, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 71 or 72; (13) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 73 or 74; (14) a mutant comprising mutations A102C, I152C, T54F, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:73 or 74 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:73 or 74; (15) a mutant comprising the amino acid sequence set forth in SEQ ID NO:75 or 76; (16) a mutant comprising the mutations A102C, I152C, T54A, S190F and V296L and comprising the amino acid sequence set forth in SEQ ID NO:73 or 74; NO:75 or 76 has an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical; (17) a mutant comprising the amino acid sequence as shown in SEQ ID NO:77 or 78; (18) a mutant comprising the mutations P101C, I152C, T54V, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO:77 or 78. 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to an amino acid sequence; (19) a mutant comprising the amino acid sequence of SEQ ID NO: 79 or 80; (20) a mutant comprising the mutations P101C, I152C, T54F, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to an amino acid sequence of SEQ ID NO: 79 or 80.(21) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 81 or 82; (22) a mutant comprising mutations P101C, I152C, T54C, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 81 or 82; (23) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 83 or 84; (24) a mutant comprising mutations P101C, I152C, T54C, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:83 or 84 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:83 or 84; (25) a mutant comprising the amino acid sequence set forth in SEQ ID NO:85 or 86; (26) a mutant comprising the mutations P101C, I152C, T54V, S190F and V296L and comprising the amino acid sequence set forth in SEQ ID NO:83 or 84; NO:85 or 86 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:85 or 86; (27) a mutant comprising the amino acid sequence set forth in SEQ ID NO:87 or 88; (28) a mutant comprising mutations P101C, I152C, T54F, S190F and V296L and comprising a residue identical to the residue set forth in SEQ ID NO:87 or 88; NO:87 or 88 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:87 or 88; (29) a mutant comprising the amino acid sequence set forth in SEQ ID NO:89 or 90; (30) a mutant comprising the mutations P101C, I152C, T54C, S190F and V296L and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:89 or 90.(31) a mutant comprising an amino acid sequence as set forth in SEQ ID NO:91 or 92; (32) a mutant comprising mutations P101C, I152C, T54A, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:91 or 92; (33) a mutant comprising an amino acid sequence as set forth in SEQ ID NO:93 or 94; (34) a mutant comprising mutations A102C, I152C, T54V, S190I and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:93 or 94; NO:93 or 94 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:93 or 94; (35) a mutant comprising the amino acid sequence set forth in SEQ ID NO:95 or 96; (36) a mutant comprising the mutations A102C, I152C, T54F, S190I and V296L and comprising the amino acid sequence set forth in SEQ ID NO:93 or 94; NO:95 or 96 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:95 or 96; (37) a mutant comprising the amino acid sequence set forth in SEQ ID NO:97 or 98; (38) a mutant comprising the mutations A102C, I152C, T54C, S190I and V296L and comprising the amino acid sequence set forth in SEQ ID NO:97 or 98; NO:97 or 98 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:97 or 98; (39) a mutant comprising the amino acid sequence set forth in SEQ ID NO:99 or 100; and (40) a mutant comprising the mutations A102C, I152C, T54A, S190I and V296L and comprising the amino acid sequence set forth in SEQ ID NO:99 or 100. A variant of an amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity with the amino acid sequence shown in NO:99 or 100.

[0174] In some embodiments, the mutation of the mutant includes or is a group of the following (1) to (7):

[0175] (1) P101C, I152C, T54A, S190F, and V296L; (2) P101C, I152C, T54C, S190F, and V296L; (3) P101C, I152C, T54A, S190M, and V296L; (4) P101C, I152C, T54C, S190M, and V296L; (5) P101C, I152C, T54V, S190M, and V296L; (6) A102C, I152C, T54A, S190F, and V296; and (7) A102C, I152C, T54C, S190M, and V296L.

[0176] In some embodiments, the mutations of the mutant include or are a group of the following (1) to (3): (1) P101C, I152C, and S190M;

[0177] (2) P101C, I152C, and S190F; and (3) P101C, I152C, and S190I.

[0178] In some embodiments, the mutations of the mutant include or are: (i) one or more of the following disulfide bond mutations: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K191C, I59C and L193C, I59C and L297C, E60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T103C and I148C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C; and (ii) one or two of the following electrostatic mutations: D at position 486 is substituted by Q, S, H, N, T, P, C, L, I, F, or W; and V at position 487 is substituted by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0179] In some embodiments, the mutations in the mutant include or are: (i) one or more of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C; and (ii) one or two of the following electrostatic mutations: D at position 486 is substituted by Q, S, H, N, T, P, C, L, I, F, or W; and V at position 487 is substituted by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0180] In some embodiments, the mutations of the mutant include or are: (i) one of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C; and (ii) the following electrostatic mutation: D at position 486 is substituted with Q, L, or I. In some embodiments, the electrostatic mutation in (ii) is D at position 486 is substituted with Q.

[0181] In some embodiments, the mutations of the mutant include or are disulfide bond mutations, cavity filling mutations, and electrostatic mutations.

[0182] In some embodiments, the mutations of the mutant include or are: (i) one or more of the following disulfide bond mutations: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K 191C, I59C and L193C, I59C and L297C, E60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T103C and I148C, N104C and G145C, N104C and S146C, N105C and G145C , N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C; (ii) one or more of the following cavity-filling mutations: (1) T at position 54 is replaced by F, W, V, C, A, H, I, Y, L, or M; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and (3) V at position 296 is replaced by I, L, Y, H, F, or M; and (iii) one or two of the following electrostatic mutations: D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W, and V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0183] In some embodiments, the disulfide bond mutations in (i) are selected from one or more of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C. In some embodiments, the disulfide bond mutations in (i) are selected from one or more of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C.

[0184] In some embodiments, the cavity-filling mutation in (ii) is at least one of a substitution of T at position 54 with F, W, V, C, A, H, I, Y, L, or M and a substitution of V at position 296 with I, L, Y, H, F, or M, and a substitution of S at position 190 with an amino acid other than S. Alternatively, the cavity-filling mutation in (ii) is one or both of the following: a substitution of T at position 54 with F, W, V, C, A, H, I, Y, L, or M, and a substitution of V at position 296 with I, L, Y, H, F, or M. In some embodiments, the cavity-filling mutation in (ii) is: S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K, and V at position 296 is replaced by at least one of I, L, Y, H, F or M, and T at position 54 is replaced by F, W or V; or the cavity-filling mutation in (ii) is: T at position 54 is replaced by F, W or V and V at position 296 is replaced by L, F or M, and S at position 190 is replaced by an amino acid other than S; or, the cavity-filling mutation in (ii) is: T at position 54 is replaced by F, W or V, and V at position 296 is replaced by L, F or M.

[0185] In some embodiments, the electrostatic mutation in (iii) comprises or is a substitution of D at position 486 by Q, L, or L. In some embodiments, the electrostatic mutation in (iii) is a substitution of D at position 486 by Q.

[0186] In some embodiments, the mutations of the mutant include or are: (i) one or more of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C; (ii) the following cavity filling One or more of the following mutations: (1) T at position 54 is substituted by F, W, C, A or V; (2) S at position 190 is substituted by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K; and (3) V at position 296 is substituted by I, L, Y, H, F or M; and (iii) one or two of the following electrostatic mutations: D at position 486 is substituted by Q, L or I, and V at position 487 is substituted by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C. In some embodiments, the disulfide bond mutation in (i) is one of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C; and / or, the V at position 296 in (ii) is replaced by L, F or M, the T at position 54 in (ii) is replaced by F, W or V; and / or the electrostatic mutation in (iii) is a D at position 486 is replaced by Q.

[0187] In some embodiments, the mutations of the mutant include or are: (i) one of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and (ii) one or more of the following cavity-filling mutations: (1) T at position 54 is substituted with F, W, C, A, or V; (2) S at position 190 is substituted with V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N, or K; and (3) V at position 296 is substituted with L, F, or M; and (iii) an electrostatic mutation: D at position 486 is substituted with Q, L, or I. In some embodiments, the disulfide bond in (i) is selected from one or more of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C. And / or, the electrostatic mutation in (iii) is D at position 486 is substituted with Q.

[0188] In some embodiments, the mutation of the mutant is at least one of substitution of T at position 54 by F, W or V, and substitution of D at position 486 by Q, and one of the following disulfide bond mutations: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C. In some embodiments, the disulfide bond is selected from one or more of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C.

[0189] In some embodiments, the mutations of the mutant include or are: (i) one of the following disulfide bond mutations: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C; (ii) one or more of the following cavity filling mutations: (1) T at position 54 is substituted by F, W, V, C, A, H, I, Y, L, or M; (2) 190 The S at position 486 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K; and (iii) the V at position 296 is replaced by I, L, Y, H, F or M; and (iii) one or two of the following electrostatic mutations: the D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W, and the V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

[0190] In some embodiments, the mutations of the mutant include or are: (i) one of the following disulfide bond mutations: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and one of N105C and A147C; (ii) the following cavity filling mutations: (1) T at position 54 is replaced by F, W, V, C, A, H, I, Y, L or M; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K; and (3) V at position 296 is replaced by I, L, Y, H, F or M; and (iii) the following electrostatic mutation: D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W.

[0191] In some embodiments, the mutations in the mutant include or are: (i) one of the following disulfide bond mutations: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C; (ii) one or more of the following cavity filling mutations: (1) T at position 54 is replaced by H, F, W or V; (2) S at position 190 is replaced by I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K; and (3) V at position 296 is replaced by I, L, F or M; and (iii) the electrostatic mutation is D at position 486 is replaced by S. In some embodiments, the mutations in the mutant are: (i) one of the following disulfide bond mutations: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C; (ii) one or more of the following cavity filling mutations: (1) T at position 54 is substituted by H, F, W or V; (2) S at position 190 is substituted by I; and (3) V at position 296 is substituted by I, L, F or M; and (iii) the electrostatic mutation is D at position 486 is substituted by S.

[0192] In some embodiments, the mutations of the mutant include or are a group of the following (1) to (15): (1) T103C, S146C, T54H, S190I, V296I and D486S; (2) P101C, I152C, T54H, S190I, V296I and D486S; (3) N105C, S146C, T54H, S190I, V296I and D486S; (4) N105C, G145C, T54H, S190I, V296I and D486S; (5) N105C, A147C, T54H, S190I, V296I and D486S; (6) N104C, S146C, T54H, S190I, V296I and D486S; (7) N104C, G145C, T54H, S1 90I, V296I and D486S; (8) A102C, I152C, T54H, S190I, V296I and D486S; (9) A102C, I148C, T54H, S190I, V296I and D486S; (10) A47C, I309C, T54H, V144C, V406C and D486S; (11) S46C, T31 1C, T54H and D486S; (12) S46C, T311C, T54H, V144C, V406C and D486S; (13) L48C, V308C, T54H and D486S; (14) L48C, V308C, T54H, V144C, V406C and D486S; and (15) A47C, I309C, T54H and D486S.

[0193] In some embodiments, the mutant is selected from one of the following (1) to (34): (1) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 1 or 2; (2) a mutant comprising the mutations A47C, I309C, T54H, V144C, V406C and D486S and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 1 or 2; (3) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 3 or 4; (4) a mutant comprising the mutations A47C, I309C, T54H and D486S and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 3 or 4. NO:3 or 4 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:3 or 4; (5) a mutant comprising the amino acid sequence set forth in SEQ ID NO:5 or 6; (6) a mutant comprising the mutations L48C, V308C, T54H, V144C, V406C and D486S and comprising the amino acid sequence set forth in SEQ ID NO:5 or 6; NO:5 or 6 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:5 or 6; (7) a mutant comprising the amino acid sequence set forth in SEQ ID NO:7 or 8; (8) a mutant comprising the mutations L48C, V308C, T54H and D486S and comprising the amino acid sequence set forth in SEQ ID NO: NO:7 or 8 having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:7 or 8; (9) a mutant comprising the amino acid sequence set forth in SEQ ID NO:9 or 10; (10) a mutant comprising the mutations S46C, T311C, T54H, V144C, V406C and D486S and comprising at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:9 or 10.(11) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 11 or 12; (12) a mutant comprising mutations S46C, T311C, T54H and D486S and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 11 or 12; (13) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 13 or 14; (14) a mutant comprising mutations A102C, I148C, T54H, S190I, V296I and D486S and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:13 or 14 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:13 or 14; (15) a mutant comprising the amino acid sequence set forth in SEQ ID NO:15 or 16; (16) comprising mutations A102C, I152C, T54H, S190I, V296I and D486S and comprising a variant identical to the amino acid sequence set forth in SEQ ID NO:13 or 14; NO:15 or 16 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:15 or 16; (17) a mutant comprising the amino acid sequence set forth in SEQ ID NO:17 or 18; (18) comprising mutations N104C, G145C, T54H, S190I, V296I and D486S and comprising a variant identical to the amino acid sequence set forth in SEQ ID NO:17 or 18; NO:17 or 18 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:17 or 18; (19) a mutant comprising the amino acid sequence set forth in SEQ ID NO:19 or 20; (20) comprising mutations N104C, S146C, T54H, S190I, V296I and D486S and comprising a variant identical to the amino acid sequence set forth in SEQ ID NO:19 or 20; The amino acid sequence shown in NO: 19 or 20 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.(21) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 21 or 22; (22) a mutant comprising mutations N105C, A147C, T54H, S190I, V296I and D486S and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 21 or 22; (23) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 23 or 24; (24) a mutant comprising mutations N105C, G145C, T54H, S190I, V296I and D486S and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:23 or 24 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:23 or 24; (25) a mutant comprising the amino acid sequence set forth in SEQ ID NO:25 or 26; (26) comprising mutations N105C, S146C, T54H, S190I, V296I and D486S and comprising a variant identical to the amino acid sequence set forth in SEQ ID NO:23 or 24; NO:25 or 26 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:25 or 26; (27) a mutant comprising the amino acid sequence set forth in SEQ ID NO:27 or 28; (28) comprising mutations P101C, I152C, T54H, S190I, V296I and D486S and comprising a variant identical to the amino acid sequence set forth in SEQ ID NO:27 or 28; NO:27 or 28 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:27 or 28; (29) a mutant comprising the amino acid sequence set forth in SEQ ID NO:29 or 30; (30) comprising mutations T103C, S146C, T54H, S190I, V296I and D486S and comprising a mutant having the same amino acid sequence as set forth in SEQ ID NO:29 or 30; The amino acid sequence shown in NO:29 or 30 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.(31) a mutant comprising the amino acid sequence as set forth in SEQ ID NO: 101 or 102; (32) a mutant comprising mutations T103C, I148C, T54H, S190I, V296I and D486S and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 101 or 102; (33) a mutant comprising the amino acid sequence as set forth in SEQ ID NO: 101 or 102; NO:103 or 104; and (34) a mutant comprising mutations T103C, I148C, T54H, S190I, V296I and D486S and comprising an amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence as shown in SEQ ID NO:101 or 102. . .

[0194] In some embodiments, the mutant of any of the above embodiments further comprises one or more of the following mutations: (1) K or E at position 66 is replaced by I, L, M, F, or V;

[0195] (2) N at position 67 is replaced by I;

[0196] (3) S at position 213 is replaced by P or G;

[0197] (4) S at position 215 is replaced by P or G;

[0198] (5) N at position 216 is replaced by P;

[0199] (6) I at position 217 is replaced by P, C, F, Y or W; and

[0200] (7) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0201] In some embodiments, the wild-type RSV is subtype A or subtype B.

[0202] In some embodiments, wild-type RSV is wild-type hRSV. In some embodiments, wild-type hRSV is strain A2, strain Ontario or strain Buenos Aires. It is understood that in other embodiments, wild-type RSV is not limited to the above enumeration, can also be other.

[0203] In some embodiments, the mutant comprises an F1 polypeptide and an F2 polypeptide. The F1 polypeptide and the F2 polypeptide of the mutant of the RSV protein into which one or more mutations are introduced can be derived from any wild-type RSV F protein known in the art or discovered in the future, including but not limited to RSV subtype A and subtype B strains (e.g., A2, Ontario, Buenos Aires) or any other subtype F protein. In addition, the F1 polypeptide and the F2 polypeptide can be derived from the F protein of the same subtype RSV or from F proteins of different subtypes of RSV. For example, the F1 polypeptide is derived from the F protein of RSV subtype A, and the F2 polypeptide is derived from the F protein of RSV subtype B; or the F1 polypeptide is derived from the F protein of RSV subtype B, and the F2 polypeptide is derived from the F protein of RSV subtype A.

[0204] The amino acid sequence of the F protein of the Ontario strain is shown in SEQ ID NO: 216; the amino acid sequence of the F protein of the Buenos Aires strain is shown in SEQ ID NO: 217.

[0205] In some embodiments, the mutant further comprises one or more of the following: a pep27 polypeptide, a transmembrane domain (TM) of an RSV F protein (e.g., a transmembrane domain of a wild-type RSV F protein), a signal peptide, and a trimerization domain (e.g., a foldon). In some embodiments, the mutant further comprises a pep27 polypeptide. In some embodiments, the mutant further comprises a trimerization domain (e.g., a foldon). In some embodiments, the mutant further comprises a transmembrane domain (TM) of an RSV F protein (e.g., a transmembrane domain of a wild-type hRSV F protein). In some embodiments, the mutant further comprises a signal peptide. In some embodiments, the mutant further comprises one or more of the following: a pep27 polypeptide, a signal peptide, and a trimerization domain (e.g., a foldon). The trimerization domain is used to promote the formation of a trimer of three F1 / F2 heterodimers. The trimerization domain is not particularly limited, and may, for example, be a GCN4 leucine zipper, a trimerization motif from a lung surfactant protein, a bacteriophage T4 fibritin foldon, etc. In some embodiments, the foldon is located at the C-terminus of the F1 polypeptide. In an alternative embodiment, the amino acid sequence of the foldon is: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 212). In some embodiments, the mutant comprises a pep27 polypeptide, a signal peptide, and a trimerization domain. In other embodiments, the mutant comprises a pep27 polypeptide, a signal peptide, and a transmembrane domain.

[0206] In some embodiments, the signal peptide is derived from the RSV F protein.

[0207] In some embodiments, the amino acid sequence of the signal peptide is positions 1 to 26 of SEQ ID NO: 1 or 2.

[0208] In some embodiments, the nucleotide sequence of the signal peptide is positions 1 to 78 of SEQ ID NO: 105 or 106. In an alternative specific example, the mutant of the RSV F protein is derived from the F protein of the Ontario strain, and the amino acid sequence of the signal peptide is positions 1 to 26 of SEQ ID NO: 1. In an alternative specific example, the mutant of the RSV F protein is derived from the F protein of the Buenos Aires strain, and the amino acid sequence of the signal peptide is positions 1 to 26 of SEQ ID NO: 2.

[0209] In some embodiments, the signal peptide and RSV F protein are heterologous. As used herein, the term "heterologous" refers to the two parts contained in the nucleic acid, such as the polynucleotide encoding the RSV F protein and the polynucleotide encoding the signal peptide, which do not exist in this combination naturally (native state). They are usually recombinant. Preferably, the polynucleotide encoding the signal peptide is derived from a gene different from the polynucleotide encoding the RSV F protein, that is, the source gene of the signal peptide is different from the source gene of the polynucleotide encoding the RSV F protein. For example, the source gene of the polynucleotide encoding the RSV F protein and the source gene of the signal peptide are genes that encode different proteins. For another example, the source gene of the polynucleotide encoding the RSV F protein and the source gene encoding the signal peptide are genes that encode the same protein but belong to different subtypes. For example, the source gene of the polynucleotide encoding the signal peptide is a gene encoding A subtype hRSV F protein, and the source gene of the polynucleotide encoding the RSV F protein is a gene encoding B subtype hRSV F protein. In some embodiments, the signal peptide is an IgE signal peptide or an IgGκ signal peptide. In some embodiments, the IgE signal peptide is an IgE HC (Ig heavy chain epsilon-1) signal peptide.

[0210] In some embodiments, the F1 polypeptide of the mutant has the same length as the full-length F1 polypeptide of the corresponding wild-type RSV F protein. The full-length F1 polypeptide of the mutant of the RSV F protein corresponds to amino acid positions 137-574 of the native RSV F0 precursor and includes (from N-terminus to C-terminus) the extracellular region (residues 137-524), the transmembrane domain (residues 525-550), and the cytoplasmic domain (residues 551-574).

[0211] In other embodiments, the mutant F1 polypeptide has a deletion relative to the full-length F1 polypeptide of the corresponding wild-type RSV F protein (or native F1 polypeptide). For example, the optional sequence from amino acid residue 482 onward of the native F1 polypeptide may not be present in the mutant F1 polypeptide. For example, the optional sequence from amino acid residue 514 onward of the native F1 polypeptide may not be present in the mutant F1 polypeptide, that is, the mutant F1 polypeptide is missing 1 to 60 amino acid residues from the C-terminus of the native F1 polypeptide. For example, the mutant F1 polypeptide lacks the entire cytoplasmic domain. For another example, the mutant F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain. For further example, compared to the native F1 polypeptide, the mutant F1 polypeptide lacks amino acid residues at positions 510, 511, 512, 513, 514, 515, 520, 525, or 530 to 574. Typically, for mutants with a trimerization domain (such as a foldon) attached, amino acids 482 to 574 are absent or amino acids 514 to 574 may be absent. Thus, in some embodiments, amino acid residues 482 to 574 or 514 to 574 are absent in the F1 polypeptide of the mutant. In other embodiments, the F1 polypeptide of the mutant comprises or consists of amino acid residues 137 to 513 of the native F0 polypeptide.

[0212] In some embodiments, the mutant F2 polypeptide can have the same length as the full-length F2 polypeptide of the corresponding wild-type RSV F protein, or can have deletions, such as deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from the N-terminus or C-terminus of the F2 polypeptide.

[0213] In addition, the nucleic acid further comprises one or more of the following: a polynucleotide encoding a protease cleavage site (e.g., a thrombin cleavage site (LVPRGS, SEQ ID NO: 213)), a polynucleotide encoding a protein tag (e.g., a 6×His-tag (HHHHHH, SEQ ID NO: 214) and a streptomycin tag II (WSHPGFEK, SEQ ID NO: 215), a polynucleotide encoding a linker sequence (e.g., GG and GS)). The polypeptides encoded by these sequences are not essential for the function of the RSV F protein (e.g., inducing an immune response). Such sequences are well known to those skilled in the art, and it will be appreciated that these sequences are not limited to the above.

[0214] In some embodiments, the nucleic acid described above is an isolated nucleic acid.

[0215] In some embodiments, the nucleic acid is a non-naturally occurring nucleic acid.

[0216] In some embodiments, the nucleic acid is a codon-optimized polynucleotide. Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to: match the codon frequency of the target and host organism to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base stretches that can impair gene structure or expression; customize transcription and translation control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites in the encoded protein (e.g., glycosylation sites); add, remove or reorganize protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; regulate translation rate so that the various domains of the protein can fold properly; or reduce or eliminate problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or patented methods. In some embodiments, an optimization algorithm is used to optimize the open reading frame (ORF) sequence. In some embodiments, the codon-optimized nucleic acid is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the nucleic acid before codon optimization.

[0217] In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is RNA, and the RNA contains an open reading frame (ORF).

[0218] In some embodiments, the nucleic acid is RNA, and the RNA is mRNA.

[0219] In some embodiments, the nucleic acid is a non-self-replicating mRNA.

[0220] In some embodiments, the nucleic acid further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail. In an alternative specific example, the nucleic acid further comprises a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0221] In some embodiments, the 5'-cap structure is selected from m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 and m 7 Gppp(m 2′-O ) At least one of N1; wherein "m 7"G" represents 7-methylguanosine cap nucleoside, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5' most nucleotide, "G" represents guanosine nucleoside, "7" represents the methyl group at the 7-position of guanine, and "m 2′-O " represents a methyl group at the 2'-O position of the nucleotide. In some embodiments, the 5'-cap structure is m 7 Gppp(5')N1 or m 7 Gppp(m 2′-O )N1. It will be appreciated that, in other embodiments, the 5'-cap structure is not limited to the above.

[0222] In some embodiments, the DNA sequence corresponding to the 5'-UTR is shown in SEQ ID NO: 209; and / or, the DNA sequence corresponding to the 3'-UTR is shown in SEQ ID NO: 210. It should be noted that the "DNA sequence corresponding to the 5'-UTR" refers to the nucleotide sequence of the 5'-UTR in DNA form, and the same applies to the "DNA sequence corresponding to the 3'-UTR" and the "DNA sequence corresponding to the poly(A) tail" below.

[0223] It is understood that, in other embodiments, the 5'-UTR and 3'-UTR are not limited to the above, and may be others, such as the 5'-UTR and 3'-UTR described in patents such as CN108291230A, CN104321432A, and CN107849574A.

[0224] In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides. In some embodiments, the nucleotides comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively.

[0225] In some embodiments, the nucleotides constituting the poly(A) tail include one or more nucleotides other than A nucleotides. In some embodiments, the poly(A) tail includes two or more consecutive nucleotides other than A nucleotides. In an optional specific example, the nucleotide DNA sequence corresponding to the poly(A) tail is shown in SEQ ID NO: 211. It is understood that the poly(A) tail contained in the nucleic acid of the present disclosure is not limited to the above, and may also be other poly(A) tails, such as the poly(A) tails described in patents such as US20170166905A1 and WO2020074642A1.

[0226] In some embodiments, the nucleic acids described above do not contain modified nucleotides.

[0227] In some embodiments, the nucleic acids described above contain modified nucleotides.

[0228] In some embodiments, the nucleic acid comprises modified nucleosides. In some embodiments, the modified nucleosides comprised by the nucleic acid comprise at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0229] In some embodiments, the modified nucleoside in the nucleic acid is a modified uridine. In some embodiments, 0.1% to 100% of the uridine in the nucleic acid is modified. In some embodiments, 80% to 100% of the uridine is modified. In some embodiments, 100% of the uridine is modified. Exemplary modified uridines include pseudouridine (ψ), N1-methyl pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine-5-oxyethyl acid (cmo5U), uridine-5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio -uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine Uridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3 -carboxypropyl) pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl -uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-methoxyformylvinyl)uridine (5-(2-carbomethoxyvinyl)uridine) and 5-[3-(1-E-propenylamino)uridine.

[0230] In some embodiments, the modified nucleosides in the nucleic acids are modified cytidines. In some embodiments, 0.1% to 100% of the cytidines in the nucleic acids are modified. In some embodiments, 80% to 100% of the cytidines are modified. In some embodiments, 100% of the cytidines are modified. Exemplary modified cytidines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1- Deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine and 2'-OH-ara-cytidine.

[0231] In some embodiments, the modified nucleoside in the above nucleic acid is a modified adenosine. In some embodiments, 0.1% to 100% of the adenosine in the above nucleic acid is modified. In some embodiments, 80% to 100% of the adenosine is modified. In some embodiments, 100% of the adenosine is modified. Exemplary modified adenosines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl -adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio -adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabino-adenosine, 2'-F-adenosine, 2'-OH-arabino-adenosine and N6-(19-amino-pentaoxahonadecyl)-adenosine.

[0232] In some embodiments, the modified nucleoside in the above nucleic acid is a modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the above nucleic acid is modified. In some embodiments, 80% to 100% of the guanosine is modified. In some embodiments, 100% of the guanosine is modified. Exemplary modified guanosines include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), iso-wyosine (imG2), yW, peroxyyW (o2yW), hydroxyyW (OHyW), undermodified hydroxyyW (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (o Q), galactosyl-braided guanosine (galQ), mannosyl-braided guanosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (M2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thioguanosine, O6-methyl-guanosine, 2'-F-arabinoguanosine and 2'-F-guanosine.

[0233] In some embodiments, the modified nucleotides in the nucleic acids described above comprise nucleotides containing isotopes.

[0234] In some embodiments, the nucleic acid comprises nucleotides containing isotopes of hydrogen. Hydrogen isotopes are not limited to deuterium and tritium. In addition, in some embodiments, the nucleic acid further comprises or contains nucleotides containing isotopes of elements other than hydrogen, including but not limited to carbon, oxygen, nitrogen, and phosphorus.

[0235] In some embodiments, the nucleic acid comprises RNA corresponding to a polynucleotide having a nucleotide sequence as shown in any one of SEQ ID NOs: 105-134 and 165-208, and all uridine in the nucleic acid is replaced by N1-methylpseudouridine.

[0236] In other embodiments, the nucleic acid is DNA.

[0237] In some embodiments, the DNA is capable of being transcribed into RNA in vitro.

[0238] In some embodiments, the nucleic acid comprises a polynucleotide having a nucleotide sequence as shown in any one of SEQ ID NOs: 105-134 and 165-208.

[0239] In some embodiments, the nucleic acid comprises a polynucleotide encoding a mutant of RSV F protein. It should be noted that "a mutant of RSV F protein" herein and below refers to any one of the multiple mutants of RSV F protein mentioned above.

[0240] In other embodiments, the nucleic acid comprises a polynucleotide for encoding the multiple mutants of RSV F protein. At this point, the coding strand of the multiple mutants of RSV F protein is located on the same nucleic acid chain, and each polynucleotide encoding the multiple mutants of RSV F protein is directly connected or indirectly connected by a connecting element (such as a polynucleotide encoding a 2A peptide or a polynucleotide encoding a flexible connecting peptide). The flexible connecting peptide includes a "GS" connecting peptide, and the "GS" connecting peptide is composed of Gly and Ser residues, such as (Gly-Gly-Gly-Gly-Ser) n, and by adjusting the numerical value (repeat number) of n, the length of the "GS" connecting peptide can be changed, so that the two connected proteins can be optimally separated, or it can interact. It should be noted that, herein and below, "the multiple mutants of RSV F protein" refers to any multiple (such as two, three, four or more) in the mutant of the multiple RSV F protein mentioned above. For example, the multiple mutants of RSV F protein are different RSV F proteins from the same subtype strain but with different mutations. For example, there are two multiple mutants of RSV F protein, the first mutant being derived from strain Ontario and having P101C, I152C, and S190M mutations, and the second mutant being derived from strain Ontario and having P101C, I152C, and S190F mutations. Correspondingly, the nucleic acid comprises a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations and a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190F mutations.

[0241] For another example, the multiple mutants of RSV F protein are different RSV F proteins from different subtypes but with the same mutations. For a further example, there are two multiple mutants of RSV F protein, the first mutant being an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations, and the second mutant being an RSV F protein derived from strain Buenos Aires and having P101C, I152C, and S190M mutations. Correspondingly, the nucleic acid comprises a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations and a polynucleotide encoding an RSV F protein derived from strain Buenos Aires and having P101C, I152C, and S190M mutations.

[0242] For another example, the multiple mutants of RSV F protein are different RSV F proteins from different subtypes and with different mutations. For a further example, there are two multiple mutants of RSV F protein, the first mutant being derived from strain Ontario and having P101C, I152C, and S190M mutations, and the second mutant being derived from strain Buenos Aires and having P101C, I152C, and S190F mutations. Correspondingly, the nucleic acid comprises a nucleic acid encoding a polynucleotide of an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations and a polynucleotide of an RSV F protein derived from strain Buenos Aires and having P101C, I152C, and S190F mutations.

[0243] For another example, there are three multiple mutants of RSV F protein. The first mutant is a polynucleotide of RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations. The second mutant is a polynucleotide of RSV F protein derived from strain Buenos Aires and having P101C, I152C, and S190M mutations. The third mutant is a polynucleotide of RSV F protein derived from strain Ontario and having P101C, I152C, and S190F mutations. Correspondingly, the above-mentioned nucleic acid comprises a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C and S190M mutations, a polynucleotide encoding an RSV F protein derived from strain Buenos Aires and having P101C, I152C and S190M mutations, and a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C and S190F mutations.

[0244] In some embodiments, the nucleic acid further comprises polynucleotides encoding other proteins or polypeptides other than mutants of RSV F protein. It is understood that other proteins or polypeptides other than mutants of RSV F protein refer to proteins or polypeptides with biological significance (e.g., immunogenicity), such as other proteins or polypeptides or mutants thereof of RSV (e.g., G protein of RSV or mutants thereof), or proteins or polypeptides or mutants thereof of other viruses (e.g., HA protein of influenza virus or mutants thereof, or S protein of SARS-CoV-2 or mutants thereof, etc.).

[0245] In addition, it is understood that in the above nucleic acids, whether it is a polynucleotide encoding a single mutant of RSV F protein or a polynucleotide encoding multiple mutants of RSV F protein, multiple (e.g., two or three) repeat units can be present on the same chain. For example, for a polynucleotide encoding a single mutant of RSV F protein, a polynucleotide encoding one of the mutants of RSV F protein (e.g., a mutant having P101C, I152C, T54A, S190M, and V296L mutations) is referred to as "A segment," and multiple A segments can be present in the same nucleic acid. For another example, a polynucleotide encoding multiple mutants of RSV F protein, a polynucleotide encoding one mutant of RSV F protein (e.g., a mutant having P101C, I152C, T54A, S190M, and V296L mutations) is referred to as “A segment”, and a polynucleotide encoding another mutant of RSV F protein (e.g., a mutant having P101C, I152C, T54C, S190M, and V296L mutations) is referred to as “B segment”. The same nucleic acid may contain multiple A segments and multiple B segments, one A segment and multiple B segments, or multiple A segments and one B segment.

[0246] In some embodiments, the RSV F protein produced by the nucleic acid is a pre-F protein.

[0247] In some embodiments, the RSV F protein produced by the nucleic acid described above can bind to at least one of the following antibodies: AM14, D25, AM22, and CR9501. In some embodiments, the RSV F protein produced by the nucleic acid described above can bind to the following antibodies: AM14, D25, AM22, and CR9501.

[0248] In some embodiments, the RSV F protein produced by the nucleic acids described above has a higher affinity for antibodies AM14, D25, AM22, or CR9501 than for the Post-F protein. In some embodiments, the affinity of the RSV F protein produced by the nucleic acids for antibody AM14, D25, AM22, or CR9501 is at least 1.1-fold, 1.3-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater than the affinity of the Post-F protein for antibody AM14, D25, AM22, or CR9501.

[0249] In some embodiments, the RSV F protein produced by the nucleic acid described above has a lower affinity for the antibody clone 131-2A than the Post-F protein. In some embodiments, the affinity of the Post-F protein for antibody clone 131-2A is at least 1.1-fold, 1.3-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater than the affinity of the RSV F protein produced by the nucleic acid for antibody clone 131-2A.

[0250] In some embodiments, the nucleic acid can induce the production of neutralizing antibodies against subtype A hRSV.

[0251] In some embodiments, the nucleic acid can induce the production of neutralizing antibodies against subtype B hRSV.

[0252] In some embodiments, the nucleic acid can induce the production of neutralizing antibodies against subtype A hRSV and subtype B hRSV.

[0253] The nucleic acid comprises a polynucleotide encoding a mutant of an RSV F protein having one or more of the following mutations: a disulfide bond mutation, a cavity-filling mutation, and an electrostatic mutation. The disulfide bond mutation, the cavity-filling mutation, and / or the electrostatic mutation of the RSV F protein can stably form the RSV F protein as a pre-F protein in vitro. It has been demonstrated that the protein or polypeptide encoded by the nucleic acid disclosed herein can induce a specific immune response that prevents or at least reduces respiratory syncytial virus infection, and that the nucleic acid disclosed herein induces at least the same immune response as an inactivated RSV vaccine composed of the whole virus.

[0254] 2. Nucleic acid 2

[0255] Another embodiment of the present disclosure provides another nucleic acid, which differs from nucleic acid 1 in that the mutant of RSV F protein corresponding to the nucleic acid has at least partially different mutations from the mutant of RSV F protein corresponding to nucleic acid 1. Specifically, relative to the wild-type RSV F protein, the mutant of RSV F protein encoded by the nucleic acid comprises or is one or more of the following mutations (1) to (8):

[0256] (1) K or E at position 66 is replaced by I, L, M, F or V;

[0257] (2) N at position 67 is replaced by I;

[0258] (3) S at position 213 is replaced by P or G;

[0259] (4) S at position 215 is replaced by P or G;

[0260] (5) N at position 216 is replaced by P;

[0261] (6) I at position 217 is replaced by P, C, F, Y, or W;

[0262] (7) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W; and

[0263] (8) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0264] In some embodiments, the mutant of the RSV F protein encoded by the above nucleic acid comprises or is one or more of the following mutations (1) to (8): (1) K or E at position 66 is replaced by I, L, M, F, or V; (2) N at position 67 is replaced by I; (3) S at position 213 is replaced by P; (4) S at position 215 is replaced by P; (5) N at position 216 is replaced by P; (6) I at position 217 is replaced by P, F, Y, or W; (7) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F, or W; and (8) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H, or C.

[0265] In some embodiments, the mutations in the mutant encoded by the above-mentioned nucleic acid include or are: (i) K or E at position 66 is replaced by I, L, M, F or V; (ii) one of the following mutations: (1) S at position 213 is replaced by P or G, (2) S at position 215 is replaced by P or G, (3) N at position 216 is replaced by P, and (4) I at position 217 is replaced by P, C, F, Y or W; and (iii) one or two of the following mutations: (1) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W, and (2) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

[0266] Alternatively, the mutations in the mutant encoded by the nucleic acid include or are: (i) N at position 67 is replaced by I; (ii) one of the following mutations: (1) S at position 213 is replaced by P or G, (2) S at position 215 is replaced by P or G, (3) N at position 216 is replaced by P, and (4) I at position 217 is replaced by P, C, F, Y or W; and (iii) one or two of the following mutations: (1) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W, and (2) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

[0267] In some embodiments, the mutations in the mutant encoded by the above-mentioned nucleic acid include or are: (i) K or E at position 66 is replaced by I, L, M, F or V; (ii) one of the following mutations: (1) S at position 213 is replaced by P, (2) S at position 215 is replaced by P, G, (3) N at position 216 is replaced by P, and (4) I at position 217 is replaced by P, C, F, Y or W; and (iii) one or two of the following mutations: (1) D at position 486 is replaced by Q, L or I, and (2) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

[0268] Alternatively, the mutations in the mutant encoded by the above nucleic acid include or are: (i) N at position 67 is replaced by I; (ii) one of the following mutations: (1) S at position 213 is replaced by P, (2) S at position 215 is replaced by P or G, (3) N at position 216 is replaced by P, and (4) I at position 217 is replaced by P, C, F, Y or W; and (iii) one or two of the following mutations: (1) D at position 486 is replaced by Q, L or I, and (2) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

[0269] In some embodiments, the mutation in (ii) above is one of the following: (1) S at position 213 is substituted by P or G, (2) N at position 216 is substituted by P, and (3) I at position 217 is substituted by P, C, F, Y, or W.

[0270] In some embodiments, the mutations of the mutant encoded by the above-mentioned nucleic acid include or are: K or E at position 66 is replaced by I, L, M, F or V, S at position 213 is replaced by P, and one of the following mutations: (1) D at position 486 is replaced by Q, L or I, and (2) V at position 487 is replaced by T.

[0271] In some embodiments, the mutations of the mutant encoded by the above-mentioned nucleic acid include or are a group of the following (1) to (11): (1) I217P, E66I and D486Q; (2) I217P, N67I and E487Q; (3) I217P, E66I and E487T; (4) S215P, N67I and E487Q; (5) S213P, N67I and E487Q; (6) S213P, E66I and E487T; (7) S213P, E66I and D486Q; (8) N216P, N67I and E487Q; (9) N216P, E66I and E487T; (10) N216P, E66I and D486Q; and (11) S213P and E66I.

[0272] In some embodiments, the mutant is selected from one of the following (1) to (26): (1) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 31 or 32; (2) a mutant comprising the mutations N216P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 31 or 32; (3) a mutant comprising the amino acid sequence as shown in SEQ ID NO: 33 or 34; (4) a mutant comprising the mutations N216P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: NO:33 or 34 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:33 or 34; (5) a mutant comprising the amino acid sequence set forth in SEQ ID NO:35 or 36; (6) a mutant comprising the mutations N216P, N67I and E487Q and comprising the amino acid sequence set forth in SEQ ID NO:36; NO:35 or 36 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:35 or 36; (7) a mutant comprising the amino acid sequence set forth in SEQ ID NO:37 or 38; (8) a mutant comprising the mutations S213P, E66I and D486Q and comprising the amino acid sequence set forth in SEQ ID NO:37 or 38; NO:37 or 38 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:37 or 38; (9) a mutant comprising the amino acid sequence set forth in SEQ ID NO:39 or 40; (10) a mutant comprising the mutations S213P, E66I and E487T and comprising at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:39 or 40.(11) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 41 or 42; (12) a mutant comprising mutations S213P, N67I and E487Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 41 or 42; (13) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 43 or 44; (14) a mutant comprising mutations S215P, N67I and E487Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:43 or 44 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:43 or 44; (15) a mutant comprising the amino acid sequence set forth in SEQ ID NO:45 or 46; (16) a mutant comprising the mutations I217P, E66I and E487T and comprising the amino acid sequence set forth in SEQ ID NO:43 or 44; NO:45 or 46 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:45 or 46; (17) a mutant comprising the amino acid sequence set forth in SEQ ID NO:47 or 48; (18) a mutant comprising the mutations I217P, N67I and E487Q and comprising the amino acid sequence set forth in SEQ ID NO:47 or 48; NO:47 or 48 has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:47 or 48; (19) a mutant comprising the amino acid sequence set forth in SEQ ID NO:49 or 50; (20) a mutant comprising the mutations I217P, E66I and D486Q and comprising at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO:49 or 50.(21) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 51 or 52; (22) a mutant comprising mutations S213P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 51 or 52; (23) a mutant comprising an amino acid sequence as set forth in SEQ ID NO: 53 or 54; (24) a mutant comprising mutations S213P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: NO:53 or 54 has an amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity; (25) a mutant comprising the amino acid sequence as shown in SEQ ID NO:59 or 60; (26) a mutant comprising the mutations S213P and E66I and comprising an amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the amino acid sequence as shown in SEQ ID NO:59 or 60.

[0273] In some embodiments, the mutant encoded by the nucleic acid of any of the above embodiments further comprises a disulfide bond mutation. In some embodiments, the disulfide bond mutation is selected from one or more of the following: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K191C, I59C and L193C, I59C and L297C, E60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T103C and I148C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C. In some embodiments, the disulfide bond mutations are selected from one or more of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C. In some embodiments, the disulfide bond mutations are selected from one or more of the following: P101C and I152C, A102C and I148C, A102C and I152C, N104C and S146C, and N105C and A147C.

[0274] In some embodiments, the mutations of the mutant encoded by the nucleic acid include or are a group of the following (1) to (2): (1) A102C, I152C, S213P and E66I; and (2) P101C, I152C, S213P and E66I.

[0275] In some embodiments, the mutant is selected from one of the following (1) to (4): (1) a mutant comprising the amino acid sequence as set forth in SEQ ID NO: 55 or 56; (2) a mutant comprising mutations A102C, I152C, S213P, and E66I and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: 55 or 56; (3) a mutant comprising the amino acid sequence as set forth in SEQ ID NO: 57 or 58; (4) a mutant comprising mutations P101C, I152C, S213P, and E66I and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO: A variant of an amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity with the amino acid sequence shown in NO:57 or 58.

[0276] In some embodiments, the amino acid sequence of the mutant encoded by the nucleic acid is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence of the F protein of the corresponding wild-type RSV. For specific species of wild-type RSV, please refer to the description of nucleic acid 1.

[0277] In some embodiments, the mutant encoded by the above nucleic acid comprises an F1 polypeptide and an F2 polypeptide. For detailed descriptions of the F1 polypeptide and the F2 polypeptide, please refer to the corresponding description in Nucleic Acid 1.

[0278] In some embodiments, the mutant encoded by the nucleic acid also comprises one or more of the following: a pep27 polypeptide, a transmembrane domain (TM) of the RSV F protein (e.g., the transmembrane domain of the wild-type RSV F protein), a signal peptide, and a trimerization domain (e.g., a foldon). The trimerization domain is used to promote the formation of a trimer of three F1 / F2 heterodimers. The trimerization domain is not particularly limited and may, for example, be a GCN4 leucine zipper, a trimerization motif from lung surfactant protein, a bacteriophage T4 fibritin foldon, etc. In some embodiments, the foldon is at the C-terminus of the F1 polypeptide. In an alternative specific example, the amino acid sequence of the foldon is: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 212). In some embodiments, the mutant encoded by the nucleic acid comprises a pep27 polypeptide, a signal peptide, and a trimerization domain. In other embodiments, the mutant encoded by the nucleic acid comprises a pep27 polypeptide, a signal peptide, and a transmembrane domain.

[0279] In addition, the nucleic acid further comprises one or more of the following: a polynucleotide encoding a protease cleavage site (e.g., a thrombin cleavage site (LVPRGS, SEQ ID NO: 213)), a polynucleotide encoding a protein tag (e.g., a 6×His-tag (HHHHHH, SEQ ID NO: 214) and a streptomycin tag II (WSHPGFEK, SEQ ID NO: 215), a polynucleotide encoding a linker sequence (e.g., GG and GS)). The polypeptides encoded by the above sequences are not essential for the function of the RSV F protein (e.g., inducing an immune response). Those skilled in the art are familiar with such sequences and will appreciate that these sequences are not limited to the above.

[0280] In some embodiments, the nucleic acid is isolated.

[0281] In some embodiments, the nucleic acid is a non-naturally occurring nucleic acid.

[0282] In some embodiments, the nucleic acid is a codon-optimized polynucleotide. In some embodiments, the codon-optimized nucleic acid is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the nucleic acid before codon optimization.

[0283] In some embodiments, the nucleic acid is RNA.

[0284] In some embodiments, the nucleic acid is RNA, and the RNA contains an open reading frame (ORF).

[0285] In some embodiments, the nucleic acid is RNA, and the RNA is mRNA.

[0286] In some embodiments, the nucleic acid is a non-self-replicating mRNA.

[0287] In some embodiments, the nucleic acid further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail. In some embodiments, the 5'-cap structure, 5'-UTR, 3'-UTR, and poly(A) tail in the nucleic acid are described in detail in Nucleic Acid 1 and are not further described here.

[0288] In some embodiments, the nucleic acids described above do not contain modified nucleotides.

[0289] In some embodiments, the nucleic acid contains modified nucleotides. In some embodiments, the nucleic acid contains modified nucleosides. In some embodiments, the modified nucleosides contained in the nucleic acid include at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine. The modified nucleotides, modified uridine, modified cytidine, modified adenosine, and modified guanosine of the nucleic acid are described in detail in the modified nucleotides, modified uridine, modified cytidine, modified adenosine, and modified guanosine of nucleic acid 1, and are not further described herein.

[0290] In some embodiments, the nucleic acid comprises RNA corresponding to a polynucleotide having a nucleotide sequence as shown in any one of SEQ ID NOs: 135 to 164, and all uridine in the nucleic acid is replaced by N1-methylpseudouridine.

[0291] In other embodiments, the nucleic acid is DNA.

[0292] In some embodiments, the DNA is capable of being transcribed into RNA in vitro.

[0293] In some embodiments, the nucleic acid comprises a polynucleotide having a nucleotide sequence as shown in any one of SEQ ID NOs: 135-164.

[0294] In some embodiments, the nucleic acid comprises a polynucleotide encoding a mutant of the RSV F protein.

[0295] In other embodiments, the nucleic acid comprises polynucleotides for encoding the various mutants of RSV F protein. At this point, the coding strands of the various mutants of RSV F protein are located on the same nucleic acid chain, and the various polynucleotides encoding the various mutants of RSV F protein are directly connected or indirectly connected by a connecting element (e.g., a polynucleotide encoding a 2A peptide or a polynucleotide encoding a flexible connecting peptide). The flexible connecting peptide includes a "GS" connecting peptide, which is composed of Gly and Ser residues, such as (Gly-Gly-Gly-Gly-Ser)n. By adjusting the numerical value (number of repeats) of n, the length of the "GS" connecting peptide can be changed, thereby optimally separating the two connected proteins or allowing them to interact.

[0296] In some embodiments, the nucleic acid further comprises polynucleotides encoding other proteins or polypeptides other than mutants of RSV F protein. It will be appreciated that other proteins or polypeptides other than mutants of RSV F protein refer to proteins or polypeptides with biological significance (e.g., immunogenicity), such as other proteins or polypeptides or mutants thereof of RSV (e.g., G protein of RSV or mutants thereof), or proteins or polypeptides or mutants thereof of other viruses (e.g., HA protein of influenza virus or mutants thereof, or S protein of SARS-CoV-2 or mutants thereof).

[0297] In addition, similarly, in the above nucleic acids, whether it is a polynucleotide encoding a single mutant of RSV F protein or a polynucleotide encoding multiple mutants of RSV F protein, there can be multiple (eg, two or three) repeating units on the same chain.

[0298] In some embodiments, the RSV F protein produced by the nucleic acid is a pre-F protein.

[0299] In some embodiments, the RSV F protein produced by the above nucleic acids can bind to at least one of the following antibodies: AM14, D25, AM22, and CR9501. In some embodiments, the RSV F protein produced by the above nucleic acids can bind to at least one of the following antibodies: AM14, D25, AM22, and CR9501.

[0300] In some embodiments, the RSV F protein produced by the nucleic acids described above has a higher affinity for antibodies AM14, D25, AM22, or CR9501 than for the Post-F protein. In some embodiments, the affinity of the RSV F protein produced by the nucleic acids for antibody AM14, D25, AM22, or CR9501 is at least 1.1-fold, 1.3-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater than the affinity of the Post-F protein for antibody AM14, D25, AM22, or CR9501.

[0301] In some embodiments, the RSV F protein produced by the nucleic acid described above has a lower affinity for the antibody clone 131-2A than the Post-F protein. In some embodiments, the affinity of the Post-F protein for antibody clone 131-2A is at least 1.1-fold, 1.3-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater than the affinity of the RSV F protein produced by the nucleic acid for antibody clone 131-2A.

[0302] In some embodiments, the nucleic acid can induce the production of neutralizing antibodies against subtype A hRSV.

[0303] In some embodiments, the nucleic acid can induce the production of neutralizing antibodies against subtype B hRSV.

[0304] In some embodiments, the nucleic acid can induce the production of neutralizing antibodies against subtype A hRSV and subtype B hRSV.

[0305] In some embodiments, the nucleic acid is any one of the mRNAs listed in Table 1. The mRNA in Table 1 is an mRNA encoding a mutant of the RSV F protein, comprising a Cap1-type cap structure, a 5'-UTR corresponding to the sequence set forth in SEQ ID NO: 209, a 3'-UTR corresponding to the sequence set forth in SEQ ID NO: 210, a poly(A) tail corresponding to the sequence set forth in SEQ ID NO: 211, and an F protein coding region corresponding to one of SEQ ID NOs: 105 to 208, wherein the F protein coding region is an RNA, and all uridine residues in all mRNAs listed in Table 1 are replaced with N1-methyl pseudouridine. For example, the mRNA indicated by number 832 comprises a Cap1-type cap structure, a 5'-UTR corresponding to the sequence set forth in SEQ ID NO: 209, a 3'-UTR corresponding to the sequence set forth in SEQ ID NO: 210, a poly(A) tail corresponding to the sequence set forth in SEQ ID NO: 211, and an F protein coding region corresponding to the sequence set forth in SEQ ID NO: 187, and all uridine residues in the mRNA indicated by number 832 are replaced with N1-methyl pseudouridine. In addition, in Table 1, all mRNAs in groups G1 to G25 and G51 contain a polynucleotide encoding a foldon derived from T4 phage fiber protein as shown in SEQ ID NO: 212 at their C-termini. For example, positions 1552-1632 of SEQ ID NO: 105 in group G1 encode the foldon derived from T4 phage fiber protein as shown in SEQ ID NO: 212 (i.e., positions 518-544 of SEQ ID NO: 1).

[0306] Table 1

[0307] III. Genetic Engineering Vectors, Nucleic Acid Preparation Methods, Host Cells, and Protein Mutants

[0308] The disclosure also provides a kind of genetic engineering vector, this genetic engineering vector comprises the nucleic acid of any one of the above-mentioned embodiments, or this genetic engineering vector comprises the polynucleotide that can be transcribed into the nucleic acid of any one of the above-mentioned embodiments.In some embodiments, genetic engineering vector is plasmid, cosmid, virus, phage or another carrier conventionally used in genetic engineering.In an alternative specific example, genetic engineering vector is plasmid.In some embodiments, genetic engineering vector also at least comprises following one or more replication origins (ORI), marker gene or its fragment, reporter gene or its fragment and restriction site that allows to insert DNA element.Preferably the restriction site of multiple cloning site (MCS) form.

[0309] In some embodiments, the genetically engineered vector is an expression vector. In some embodiments, the genetically engineered vector comprises a promoter, a 5'-UTR, a coding region of a mutant of the RSV F protein of any of the above embodiments (referred to as "mutant coding region"), a 3'-UTR, and a poly(A) tail, wherein the poly(A) tail, the promoter, the 5'-UTR, the mutant coding region, and the 3'-UTR are operably linked to each other.

[0310] In other embodiments, the genetic engineering vector is a cloning vector. It is understood that the nucleic acid contained in the genetic engineering vector does not contain a 5'-cap structure.

[0311] The present disclosure also provides a method for preparing the nucleic acid of any of the above embodiments, which comprises the steps of introducing (for example, in the form of a plasmid) the genetic engineering vector of any of the above embodiments into a host cell (for example, Escherichia coli) and then culturing the host cell containing the nucleic acid.

[0312] In addition, the present disclosure also provides another method for preparing the above-mentioned nucleic acid, which comprises the step of preparing the nucleic acid by chemical synthesis according to the nucleotide sequence of the nucleic acid of any of the above-mentioned embodiments. It is understood that the specific method of the chemical synthesis method can be a method known in the art, such as the solid phase phosphoramidite method.

[0313] It is understood that the method for preparing the nucleic acid in any of the above embodiments is not limited to the above, and other methods may also be used.

[0314] In addition, the present disclosure also provides a host cell, which comprises the nucleic acid of any of the above embodiments or the genetic engineering vector of any of the above embodiments.

[0315] In some embodiments, the host cell is an isolated cell.

[0316] In some embodiments, the host cells are used to store and / or amplify the nucleic acids described above.

[0317] In some embodiments, the host cell is a bacterial cell. Bacterial host cells include Escherichia coli (E. coli) cells well known to those skilled in the art.

[0318] Host cells of the present disclosure can be prepared by transforming competent host cells using the genetically engineered vectors of any of the above-mentioned embodiments. Competent host cells are cells with the ability of free extracellular genetic material (such as DNA plasmids) that does not rely on sequence uptake. Various bacterial cells well known to those skilled in the art are naturally able to take in exogenous DNA from the environment, and therefore can serve as bacterial host cells according to the present disclosure. In addition, it is known to those skilled in the art that competent bacterial host cells can be obtained from natural non-competent bacterial cells using, for example, electroporation or chemicals (such as, for example, calcium ion treatment and accompanying high temperature exposure). After uptake, exogenous DNA is preferably neither degraded nor integrated in the genome of the bacterial host cell.

[0319] In addition, the present disclosure also provides a mutant of RSV F protein, which is encoded by the nucleic acid of any of the above embodiments.

[0320] In some embodiments, the mutant of the RSV F protein is a unimolecular or multimolecular complex (eg, a trimer).

[0321] In addition, the present disclosure also provides an RSV immunogen, which comprises a mutant of the RSV F protein encoded by the nucleic acid of any of the above embodiments.

[0322] In some embodiments, the RSV immunogen is a trimer. In some embodiments, the mutations of the trimer's monomers are identical. In other embodiments, the mutations of the three trimer's monomers are partially identical or completely different.

[0323] In addition, the present disclosure also provides a method for preparing a mutant of RSV F protein, the preparation method comprising: transcribing a nucleic acid comprising a polynucleotide encoding the mutant of RSV F protein into mRNA; and translating the transcribed mRNA into a polypeptide or protein.

[0324] In some embodiments, the method for preparing the RSV F protein mutant is performed entirely in vitro or partially in vitro.

[0325] 4. RNA and RNA Preparation Methods

[0326] The present disclosure also provides a method for preparing RNA, which comprises the step of performing transcription using the genetic engineering vector according to any of the above embodiments of the present disclosure.

[0327] In some embodiments, the method for preparing RNA is an in vitro method. In some embodiments, the method for preparing RNA comprises contacting the genetic engineering vector (e.g., plasmid) of any of the above embodiments with an RNA polymerase. In some embodiments, the method for preparing RNA further comprises the step of linearizing the genetic engineering vector (e.g., plasmid). In some embodiments, before linearization, the supercoil rate of the genetic engineering vector (e.g., plasmid) is at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, etc.). In some embodiments, the method for preparing RNA further comprises the step of purifying the linearized genetic engineering vector. In some embodiments, the method for preparing RNA further comprises the step of purifying RNA.

[0328] The present disclosure also provides another method for preparing RNA, comprising the step of preparing the RNA by chemical synthesis based on the nucleotide sequence of the RNA or DNA corresponding to any of the above embodiments. It is understood that the specific method of the chemical synthesis method can be a method known in the art, such as the solid-phase phosphoramidite method.

[0329] In some embodiments, the RNA is mRNA.

[0330] In some embodiments, any of the above methods for preparing RNA further comprises the steps of capping and optionally purifying the capped product. In some embodiments, the cap is a Cap1 cap. The Cap1 cap structure is as follows:

[0331] cap G 1 G 2 =m 7 G-5'-ppp-5'-Gm2'-3'-p-[m7=7-CH3; m2'=2'-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-=-PO2H-].

[0332] The capping reaction is as follows:

[0333] pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi

[0334] ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi

[0335] G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc

[0336] m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3')+AdoHyc.

[0337] In other embodiments, the method for preparing RNA is a partially in vitro method. In this case, the RNA preparation method includes the following steps: preparing a genetically engineered vector according to any of the above embodiments in vitro; and introducing the genetically engineered vector into the body (e.g., in the form of a plasmid). In some embodiments, the genetically engineered vector is encapsulated in a delivery vehicle. In this case, the genetically engineered vector is delivered into the body via the delivery vehicle.

[0338] In some embodiments, the RNA prepared in the method for preparing RNA according to any of the above embodiments comprises modified nucleosides or modified nucleotides. Correspondingly, the raw materials for preparing the RNA include one or more modified nucleosides or nucleotides. It is understood that the amount and type of the modified nucleosides or nucleotides correspond to the RNA to be prepared.

[0339] In addition, the present disclosure also provides an RNA, which is prepared by the method for preparing RNA according to any of the above embodiments. In some embodiments, the RNA is mRNA.

[0340] 5. Nucleic Acid Composition

[0341] The present disclosure also provides a nucleic acid composition, which comprises a first nucleic acid or a first genetic engineering vector, wherein the first nucleic acid is the nucleic acid of any of the above embodiments, and the first genetic engineering vector is the genetic engineering vector of any of the above embodiments.

[0342] In some embodiments, the first nucleic acid is RNA. In some embodiments, the first nucleic acid is mRNA.

[0343] In some embodiments, the nucleic acid composition comprises a first nucleic acid or a first genetic engineering vector, wherein a first nucleic acid is a nucleic acid comprising a polynucleotide encoding any one or more of the multiple mutants of the RSV F protein, and a first genetic engineering vector is a genetic engineering vector comprising a polynucleotide encoding any one or more of the multiple mutants of the RSV F protein.

[0344] In some embodiments, the nucleic acid composition includes a first nucleic acid or a first genetic engineering vector, wherein a first nucleic acid is a nucleic acid comprising a polynucleotide encoding one of the multiple mutants of the RSV F protein, and a first genetic engineering vector is a genetic engineering vector comprising a polynucleotide encoding one of the multiple mutants of the RSV F protein.

[0345] In other embodiments, the nucleic acid composition includes a first nucleic acid or a first genetic engineering vector, wherein the first nucleic acid is a nucleic acid comprising a polynucleotide encoding multiple mutants of the multiple mutants of the RSV F protein, and the first genetic engineering vector is a genetic engineering vector comprising a polynucleotide encoding multiple mutants of the multiple mutants of the RSV F protein, wherein the polynucleotides encoding the multiple mutants of the multiple mutants of the RSV F protein are located on the same nucleic acid chain.

[0346] In some embodiments, the nucleic acid composition includes multiple first nucleic acids or multiple first genetic engineering vectors, multiple first nucleic acids are independent of each other or multiple first genetic engineering vectors are independent of each other, multiple first nucleic acids are used to encode multiple mutants of RSV F protein, and multiple first genetic engineering vectors are used to clone or express multiple polynucleotides encoding RSV F protein. It should be noted that "independent of each other" herein refers to the absence of a connecting element between the multiple first nucleic acids to connect them, or the absence of a connecting element between the multiple first genetic engineering vectors to connect them. At this point, each polynucleotide encoding the multiple mutants of RSV F protein is not on the same nucleic acid chain.

[0347] In some embodiments, the multiple mutants of RSV F proteins corresponding to the multiple first nucleic acids are RSV F proteins derived from different subtype strains and having the same mutation, RSV F proteins derived from different subtype strains and having different mutations, and / or RSV F proteins derived from the same subtype strain but having different mutations.

[0348] For example, the plurality of first nucleic acids are nucleic acids comprising polynucleotides encoding RSV F proteins derived from strain Ontario and having P101C, I152C, and S190M mutations (or first first nucleic acids), and nucleic acids comprising polynucleotides encoding RSV F proteins derived from strain Buenos Aires and having P101C, I152C, and S190M mutations (or second first nucleic acids). In other words, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid being a nucleic acid comprising polynucleotides encoding RSV F proteins derived from strain Ontario and having P101C, I152C, and S190M mutations, and the second first nucleic acid being a nucleic acid comprising polynucleotides encoding RSV F proteins derived from strain Buenos Aires and having P101C, I152C, and S190M mutations. In some embodiments, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Ontario having P101C, I152C, T54A, S190M, and V296L mutations, and the second first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Buenos Aires having P101C, I152C, T54A, S190M, and V296L mutations.

[0349] In some embodiments, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 83; and the second first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in NO:84 has a mutant of the amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity.

[0350] In some embodiments, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, wherein the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 83; and the second first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, wherein the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 84.

[0351] In some embodiments, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid comprising a DNA encoding the RSV F protein as set forth in SEQ ID NO:83 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identity to the DNA as set forth in SEQ ID NO:187, or its corresponding RNA; and the second first nucleic acid comprising a DNA encoding the RSV F protein as set forth in SEQ ID NO:84 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identity to the DNA as set forth in SEQ ID NO:187, or its corresponding RNA; The DNA shown in NO:188 has a DNA or its corresponding RNA that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical.

[0352] In some embodiments, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid comprising a DNA as set forth in SEQ ID NO: 187 or its corresponding RNA; and the second first nucleic acid comprising a DNA as set forth in SEQ ID NO: 188 or its corresponding RNA. For example, the plurality of first nucleic acids comprises a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Ontario having P101C, I152C, and S190M mutations (the first first nucleic acid), and a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Buenos Aires having P101C, I152C, and S190F mutations (the second first nucleic acid). In other words, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Ontario having P101C, I152C, and S190M mutations, and the second first nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Buenos Aires having P101C, I152C, and S190F mutations.

[0353] For example, the plurality of first nucleic acids are nucleic acids comprising polynucleotides encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations (first first nucleic acid), and nucleic acids comprising polynucleotides encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190F mutations (second first nucleic acid). In other words, the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid being a nucleic acid comprising polynucleotides encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations, and the second first nucleic acid being a nucleic acid comprising polynucleotides encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190F mutations.

[0354] For another example, the plurality of first nucleic acids are nucleic acids comprising polynucleotides encoding RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations, nucleic acids comprising polynucleotides encoding RSV F protein derived from strain Buenos Aires and having P101C, I152C, and S190M mutations, and nucleic acids comprising polynucleotides encoding RSV F protein derived from strain Ontario and having P101C, I152C, and S190F mutations. In other words, the nucleic acid composition comprises three first nucleic acids, the first first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations, the second first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Buenos Aires and having P101C, I152C, and S190M mutations, and the third first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190F mutations.

[0355] It is understandable that the above-mentioned nucleic acid composition is not limited to including one or more of the above-mentioned first nucleic acids or first genetic engineering vectors, but also includes other nucleic acids (such as nucleic acids encoding other immunogens) and / or other substances (such as buffers or lyophilization protectants, etc.).

[0356] In some embodiments, the nucleic acid composition further comprises a second nucleic acid or a second vector, wherein the second nucleic acid comprises a polynucleotide encoding a protein or polypeptide other than a mutant RSV F protein, and the second vector comprises a polynucleotide encoding a protein or polypeptide other than a mutant RSV F protein. Proteins or polypeptides other than mutant RSV F protein are described above. In some embodiments, the second nucleic acid is RNA. In some embodiments, the second nucleic acid is mRNA. It is understood that the nucleic acid composition may not be limited to one second nucleic acid but may comprise multiple second nucleic acids. For example, the nucleic acid composition may further comprise one second nucleic acid comprising a polynucleotide encoding a protein or polypeptide other than a mutant RSV F protein. The nucleic acid composition may also further comprise multiple second nucleic acids, each of which is independent of the other, comprising multiple polynucleotides encoding proteins or polypeptides other than mutant RSV F protein (e.g., a nucleic acid comprising a polynucleotide encoding influenza virus HA or a variant thereof, and a nucleic acid comprising a polynucleotide encoding SARS-CoV-2 S protein or a variant thereof). The second vector is also not limited to one but may comprise multiple second vectors.

[0357] VI. Delivery vectors, pharmaceutical compositions, and their applications

[0358] The present disclosure also provides a delivery vector comprising the nucleic acid according to any of the above embodiments, the RNA according to any of the above embodiments, the genetic engineering vector according to any of the above embodiments, or the nucleic acid composition according to any of the above embodiments.

[0359] In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.

[0360] In some embodiments, the delivery vehicle is selected from a plurality of composites or one of the following: lipid nanoparticles (LNPs), liposomes, cationic proteins, vesicles, microparticles, polymers and micelles. In some embodiments, the delivery vehicle is selected from one of the following: lipid nanoparticles, liposomes, cationic proteins, vesicles, microparticles, polymers and micelles.

[0361] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP) comprising the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetically engineered vector of any of the above embodiments, or the nucleic acid composition of any of the above embodiments.

[0362] In some embodiments, lipid nanoparticles refer to particles having a nanometer scale (eg, 1 nm to 1000 nm) that include one or more lipids.

[0363] In some embodiments, the average diameter of the lipid nanoparticles is 20 nm to 800 nm, 20 nm to 500 nm, 20 nm to 400 nm, 20 nm to 300 nm, 20 nm to 200 nm, 20 nm to 100 nm, 30 nm to 700 nm, 30 nm to 500 nm, 30 nm to 300 nm, 30 nm to 200 nm, 30 nm to 100 nm, 40 nm to 800 nm, 40 nm to 600 nm, 40 nm to 500 nm, 40 nm to 300 nm, 40 nm to 200 nm, 40nm~100nm, 50nm~800nm, 50nm~600nm, 50nm~500nm, 50nm~500nm, 50nm~400nm, 50nm~500nm, 50nm~400nm, 50nm~300nm, 50nm~200nm, 50nm~100nm, 60nm~800nm, 60nm~600nm, 60nm~500nm, 60nm~400nm, 60nm~300nm, 60nm~200nm or 60nm~100nm. In some optional specific examples, the average diameter of the lipid nanoparticles is 26 nm, 31 nm, 36 nm, 41 nm, 46 nm, 51 nm, 56 nm, 61 nm, 66 nm, 71 nm, 76 nm, 81 nm, 86 nm, 91 nm, 96 nm, 101 nm, 106 nm, 111 nm, 116 nm, 121 nm, 126 nm, 131 nm, 136 nm, 141 nm, 146 nm, 151 nm, 156 nm, 161 nm, 166 nm, 171 nm, 176 nm, 181 nm, 186 nm, 191 nm, 196 nm, 201 nm, 206 nm, 211 nm, 216 nm, 221 nm, 226 nm, 231 nm, 236 nm, 241 nm, 246 nm, or 249 nm. Herein, the average diameter of lipid nanoparticles can be expressed as the z-average value determined by dynamic light scattering.

[0364] In some embodiments, the lipid nanoparticles include one of the following: cationic lipid nanoparticles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), nonlamellar lipid nanoparticles. In an optional specific example, the lipid nanoparticles are cationic lipid nanoparticles.

[0365] In some embodiments, the lipid nanoparticles contain one or more of the following: cationic lipids, helper lipids, structural lipids, and polymer-lipids.

[0366] The term "cationic lipid" refers to a lipid that becomes positively charged when the pH drops below the pKa of the ionizable group of the lipid, but gradually becomes neutral at higher pH values. At pH values ​​below the pKa, the positively charged lipid is able to bind to negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid.

[0367] In some embodiments, the cationic lipid comprises the following compound (I), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0368] in:

[0369] R1 is selected from the group consisting of: C5-C30 alkyl, C5-C20 alkenyl, -R*YR", -YR", and -R"M'R';

[0370] R2 and R3 are independently selected from the group consisting of H, C1-C14 alkyl, C2-C14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring;

[0371] R4 is selected from the group consisting of hydrogen, C3-C6 carbocycle, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2 and unsubstituted C1-C6 alkyl, wherein Q is selected from carbocyclic, heterocyclic, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O) R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, N(R)R8, -N(R)S(O)2R8, -O(CH2) nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N( R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9) R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, each o is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5;

[0372] each R5 is independently selected from the group consisting of OH, C1-C3 alkyl, C2-C3 alkenyl, and H;

[0373] each R6 is independently selected from the group consisting of OH, C1-C3 alkyl, C2-C3 alkenyl, and H;

[0374] M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, wherein M" is a bond, a C1-C13 alkyl group, or a C2-C13 alkenyl group;

[0375] R7 is selected from the group consisting of C1-C3 alkyl, C2-C3 alkenyl and H;

[0376] R8 is selected from the group consisting of C3-C6 carbocycle and heterocycle;

[0377] R9 is selected from the group consisting of H, CN, NO2, C1-C6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-C6 alkenyl, C3-C6 carbocycle and heterocycle;

[0378] R 10 selected from the group consisting of H, C1-C3 alkyl and C2-C3 alkenyl;

[0379] Each R is independently selected from the group consisting of: C1-C3 alkyl, C2-C3 alkenyl, (CH2) q OR* and H,

[0380] and each q is independently selected from 1, 2, and 3;

[0381] Each R' is independently selected from the group consisting of C1-C18 alkyl, C2-C18 alkenyl, -R*YR", -YR", H and And R 11 、R 12 and R 13 are independently selected from the group consisting of C1-C12 alkyl and C2-C12 alkenyl;

[0382] Each R" is independently selected from the group consisting of C3-C15 alkyl and C3-C15 alkenyl;

[0383] each R* is independently selected from the group consisting of absence, C1-C12 alkyl, and C2-C12 alkenyl;

[0384] Each Y is independently a C3-C6 carbocycle;

[0385] each X is independently selected from the group consisting of: F, Cl, Br, and I; and

[0386] m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; and wherein when R4 is -(CH2) n Q, -(CH2) n When CHQR, -CHQR or -CQ(R)2, then (i) when n is 1, 2, 3, 4 or 5, Q is not -N(R)2; or (ii) when n is 1 or 2, Q is not a 5-, 6- or 7-membered heterocycloalkyl.

[0387] In an optional specific example, the cationic lipid is the following compound (I), its N-oxide, its salt or its isomer:

[0388] wherein R1-R7, M and m are as defined above.

[0389] In some embodiments, the cationic lipid comprises the following compound (II), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0390] in:

[0391] R1, R2, R3, R5, R6, M and R7 are as described above,

[0392] R N is H or C1-C3 alkyl;

[0393] X a and X b each independently O or S;

[0394] R 14 Selected from H, halogen, -OH, R b 、-N(Rb )2, -CN, -N3, -C(O)OH, -C(O)OR b 、-OC(O)R b 、-OR b 、-SR b 、-S(O)R b 、-S(O)OR b 、-S(O)2OR b 、-NO2、-S(O)2N(R b )2、-N(R b )S(O)2R b 、-NH(CH2) t1 N(R b )2、-NH(CH2) p1 O(CH2) q1 N(R b )2、-NH(CH2) s1 OR b 、-N((CH2) S OR b )2、-N(R b )-carbocyclic ring, -N(R b )-heterocyclic, N(R b )-aryl, -N(R b )-heteroaryl, -N(R b )(CH2) t1 -Carbocyclic ring, -N(R b )(CH2) t1 -heterocycle, -N(R b )(CH2) t1- Aryl, -N(R b )(CH2) t1 - the group consisting of heteroaryl, carbocycle, heterocycle, aryl and heteroaryl;

[0395] Each R b independently selected from the group consisting of C1-C3 alkyl, C2-C3 alkenyl, and H;

[0396] u is 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0397] w is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0398] r is 0 or 1;

[0399] t 1 is 1, 2, 3, 4, or 5;

[0400] p 1 is 1, 2, 5, 4, or 5;

[0401] q 1 is 1, 2, 5, 4, or 5; and

[0402] s 1 1, 2, 3, 4, or 5.

[0403] In an optional specific example, the cationic lipid is the following compound (II), its N-oxide, its salt or its isomer:

[0404] Among them, R1-R3, R5-R7, R 14 、X a 、X b 、R N , M, u, w and r are as defined above.

[0405] In some embodiments, the cationic lipid comprises the following compound (III), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0406] in:

[0407] L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other one is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O- or bond;

[0408] G 1 and G 2 Each is independently an unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0409] G 3 C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0410] R a is H or a C1-C12 hydrocarbon group;

[0411] R 15 and R 16 Each is independently a C6-C24 alkyl or a C6-C24 alkenyl;

[0412] R 17 H, OR 18 、CN、-C(=O)OR 19 、-OC(=O)R 19 or –NR 18 C(=O)R 19 ;

[0413] R 19 is a C1-C12 hydrocarbon group;

[0414] R 18 is H or a C1-C6 hydrocarbon group; and

[0415] x is 0, 1, or 2.

[0416] In an optional specific example, the cationic lipid is the following compound (III), its N-oxide, its salt or its isomer:

[0417] Where: R 15 -R 17 , G 1 -G 3 、L 1 -L 2 As defined above.

[0418] In some embodiments, the cationic lipid comprises the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0419] in:

[0420] L 3 and L 4 are the same or different, and are each independently C1-C12 alkylene, C2-C12 alkenylene, or C2-C12 alkynylene; in some embodiments, L 3 and L 4 are the same or different, and are each independently C3-C10 alkylene, C3-C10 alkenylene, or C3-C10 alkynylene; in some embodiments, L 3 and L4 are the same or different, and are each independently C3-C10 alkylene; in some embodiments, L 3 and L 4 The same or different, each independently a C5-C8 alkylene group;

[0421] G 4 and G 5 are the same or different and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S- or -SC(=O)-; in some embodiments, G 4 and G 5 are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, or -O-; in some embodiments, G 4 and G 5 the same or different, each independently selected from -O-(C=O)- or -(C=O)-O-;

[0422] R 18 and R 19 are the same or different, and are each independently a C5-C27 alkyl group, or a C5-C27 alkenyl group containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently a C8-C20 alkyl group or a C8-C20 alkenyl group containing one or more double bonds; In some embodiments, R 18 and R 19 are the same or different and are each independently C9-C17 alkyl or C9-C18 alkenyl containing one or two double bonds; in some embodiments, R 18 and R 19 the same or different, each independently

[0423] R 20 is halogen, hydroxy, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; in some embodiments, R 20is fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl or acetamido;

[0424] z is 1, 2 or 3.

[0425] In some embodiments, the cationic lipid is the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0426] Where: R 18 -R 20 , G 4 -G 5 、L 3 -L 4 and z are as defined above.

[0427] In some embodiments, the cationic lipid comprises the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0428] In some embodiments, the cationic lipid comprises the following compound, or a pharmaceutically acceptable salt thereof:

[0429] In some embodiments, the cationic lipids include one or more of the following: ALC-0315 (CAS No. 2036272-55-4), SM-102 (CAS No. 2089251-47-6), and

[0430] In some embodiments, the helper lipids of the lipid nanoparticles include phospholipids. Phospholipids are generally semi-synthetic, but can also be of natural origin or chemically modified. In an alternative specific example, the helper lipids of the lipid nanoparticles are phospholipids. In some embodiments, the phospholipids of the lipid nanoparticles include one or more of the following: DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), DOPS (dioleoylphosphatidylserine), DSPG (1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)homoserine), and lysophospholipids. In some embodiments, the helper lipid of the lipid nanoparticle is one or more selected from the group consisting of: DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid of the lipid nanoparticle is DSPC and / or DOPE.

[0431] In some embodiments, the structural lipids of the lipid nanoparticles include sterols. In an alternative specific example, the structural lipids of the lipid nanoparticles are sterols. In some embodiments, the sterols of the lipid nanoparticles include one or more of the following: 20α-hydroxycholesterol, cholesterol, cholesterol esters, steroid hormones, steroid vitamins, bile acids, cholesterol, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipids of the lipid nanoparticles include at least one of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and bile acids. In some embodiments, the structural lipid of the lipid nanoparticles is cholesterol. In an alternative specific example, the structural lipid of the lipid nanoparticles is high-purity cholesterol, particularly injection-grade high-purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0432] Polymer-lipid refers to a conjugate comprising a polymer and a lipid coupled to the polymer. Polymer-lipid (e.g., polyethylene glycol-lipid) in lipid nanoparticles can improve the stability of lipid nanoparticles in vivo.

[0433] In some embodiments, the lipids of the polymer-lipid used to form lipid nanoparticles include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG) and 1,2-dipalmitoyl-rac-glycero (DPG).

[0434] In some embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles includes one or both of the following: a hydrophilic polymer and an amphiphilic polymer.

[0435] In some embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles is a hydrophilic polymer. In other embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles is an amphoteric polymer.

[0436] In some embodiments, the hydrophilic polymer comprises one or more of the following: polyethylene glycol (PEG), polyoxazolines (POX), polyglycerols (PGs), polyhydroxypropyl methacrylate (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyaminoacids, glycosaminoglycans (GAGs), heparin, hyaluronic acid (HA), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond acid (HA), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin and CD47.

[0437] Correspondingly, polymer-lipid includes one or more of the following: polyethylene glycol-lipid (PEG-lipid), polyoxazoline-lipid, polyglycerol-lipid, polyhydroxypropyl methacrylate-lipid, polymethacrylate-2-hydroxyethyl ester-lipid, poly N-(2-hydroxypropyl) methacrylamide-lipid, polyvinyl pyrrolidone-lipid, poly N, N-dimethylacrylamide-lipid, poly N acryloylmorpholine-lipid, glycosaminoglycan-lipid, heparin-lipid, hyaluronic acid-lipid, polysialic acid-lipid, elastin-lipid, serum albumin-lipid and CD47-lipid. It should be noted that "PEG-lipid" is a conjugate of polyethylene glycol and lipid, "polyoxazoline-lipid" refers to a conjugate formed by coupling polyoxazoline with lipid, "polyglycerol-lipid" refers to a conjugate formed by coupling polyglycerol with lipid, and the same applies to other polymer-lipids. In an optional specific example, the hydrophilic polymer includes polyethylene glycol.

[0438] In some embodiments, the polymer-lipid includes a PEG-lipid. In an alternative specific example, the polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid includes one or more of the following: PEG-myristoyl diglycerol (PEG-DMG), PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-diacylglycerol (PEG-DAG), PEG-dialkyloxypropyl (PEG-DAA), PEG-phospholipids, PEG-ceramide (PEG-ceramide, PEG-Cer), PEG-1,2-distearoyl-rac-glycerol (PEG-DSG) and PEG-1,2-dipalmitoyl-rac-glycerol (PEG-DPG). The PEG-lipid is preferably PEG-DMG, PEG-DSG, or PEG-DPG. PEG-DMG is a polyethylene glycol derivative of 1,2-dimyristoylglycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000 to 5000. In an alternative specific example, the average molecular weight of PEG in the PEG-lipid is about 2000. In some embodiments, the amphoteric polymer comprises one or more of the following: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers.In some embodiments, the amphoteric polymer includes one or more of the following: poly(carboxybetaine acrylamide, pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylpyridine.

[0439] Correspondingly, the polymer-lipid comprises one or more of the following: polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphobetaine-based polymer-lipid and phosphorylcholine polymer-lipid. In some embodiments, the polymer-lipid comprises one or more of the following: poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethylphosphorylcholine)-lipid, poly(vinylpyridylpropanesulfonate)-lipid, polyvinylimidazolylbetaine-lipid, polyvinylimidazolylsulfobetaine-lipid, polyvinylpyridylsulfobetaine-lipid.

[0440] In addition, in some embodiments, the polymers used in nanoparticles in "The Importance of Poly(ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and Bioconjugation." Polymers vol. 12, 2 298 by Hoang Thi, Thai Thanh et al. are also incorporated herein.

[0441] In some embodiments, the lipid nanoparticles contain cationic lipids, helper lipids, structural lipids and polymer-lipids. In some embodiments, the lipid nanoparticles include the following amount (molar percentage) of cationic lipids based on the total amount of cationic lipids, helper lipids, structural lipids and polymer-lipids: about 25.0% to 75.0%, such as about 25.0% to 28.0%, 28.0% to 32.0%, 32.0% to 35.0%, 35.0% to 40.0%, 40.0% to 42.0%, 42.0% to 45.0%, 45.0% to 46.3%, 46.3% to 48.0%, 48.0% to 49.5%, 49.5% to 50.0%, 50.0% to 55.0%, 55.0% to 60.0%, 60.0% to 65.0% or 65.0% to 75.0%.

[0442] In some embodiments, the lipid nanoparticles comprise cationic lipids, helper lipids, structural lipids and polymer-lipids, wherein the cationic lipids account for 25 mol% to 75 mol% of the total lipids present in the lipid nanoparticles, the helper lipids account for 0 mol% to 45 mol% of the total lipids present in the lipid nanoparticles, the structural lipids account for 0 mol% to 60 mol% of the total lipids present in the lipid nanoparticles, and the polymer-lipids account for 0.5 mol% to 5 mol% of the total lipids present in the lipid nanoparticles.

[0443] In some embodiments, the cationic lipid in the lipid nanoparticles comprises 25 mol% to 75 mol% of the total lipid present in the lipid nanoparticles, for example, 25 mol%, 28 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, 49.5 mol%, 50 mol%, 50.5 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 mol%, 79 mol%, 80 mol%, 81 mol%, 82 .5mol%, 53mol%, 53.5mol%, 54mol%, 54.5mol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59. 5 mol%, 60 mol%, 60.5 mol%, 61 mol%, 61.5 mol%, 62 mol%, 62.5 mol%, 63 mol%, 63.5 mol%, 64 mol%, 64.5 mol%, 65 mol%, 68 mol%, 70 mol% or 75 mol%. In some embodiments, the cationic lipid in the above-mentioned lipid nanoparticles accounts for 30 mol% to 65 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 45 mol% to 55 mol% or 50 mol% to 55 mol% of the total lipids present in the lipid nanoparticles.

[0444] In some embodiments, the auxiliary lipid (e.g., DSPC) in the above-mentioned lipid nanoparticles accounts for 0 mol% to 45 mol% of the total lipid present in the lipid nanoparticles. For example, 0.5 mol%, 1 mol%, 3 mol%, 5 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, 15 mol%, 15.5 mol%, 16 mol%, 16.5 mol%, 17 mol%, 17.5 mol%, 18 mol%, 18.5 mol%, 19 mol%, 19.5 mol%. %, 20 mol%, 20.5 mol%, 21 mol%, 21.5 mol%, 22 mol%, 22.5 mol%, 23 mol%, 23.5 mol%, 24 mol%, 24.5 mol%, 25 mol%, 25.5 mol%, 26 mol%, 26.5 mol%, 27 mol%, 27.5 mol%, 28 mol%, 28.5 mol%, 29 mol%, 29.5 mol%, 30 mol%, 34 mol%, 35 mol%, 36 mol%, 38 mol%, 40 mol%, 42 mol%, 44 mol% or 45 mol%. In some embodiments, the helper lipid (e.g., DSPC) in the above-mentioned lipid nanoparticles accounts for 1 mol% to 40 mol%, 5 mol% to 40 mol%, 5 mol% to 35 mol%, 5 mol% to 30 mol% or 5 mol% to 25 mol% of the total lipid present in the lipid nanoparticles.

[0445] In some embodiments, the structural lipid (e.g., cholesterol) in the lipid nanoparticles described above accounts for 0 mol% to 60 mol% of the total lipid present in the lipid nanoparticles. For example, 0 mol%, 1 mol%, 5 mol%, 8 mol%, 10 mol%, 12 mol%, 14 mol%, 15 mol%, 16 mol%, 18 mol%, 20 mol%, 22 mol%, 24 mol%, 25 mol%, 27 mol%, 27.5 mol%, 28 mol%, 28.5 mol%, 29 mol%, 29.5 mol%, 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35mol%, 35.5mol%, 36mol%, 36.5mol%, 37mol%, 37.5mol%, 38mol%, 38.5mol%, 39mol%, 39.5mol%, 40mol%, 40.5mol%, 41mol%, 41.5mol%, 42mol%, 42.5mol% , 43mol%, 44mol%, 45mol%, 46mol%, 47mol%, 48mol%, 49mol%, 50mol%, 51mol%, 52mol%, 53mol%, 54mol%, 55mol%, 56mol%, 57mol%, 58mol%, 59mol% or 60mol%. In some embodiments, the structural lipid (e.g., cholesterol) in the above-mentioned lipid nanoparticles accounts for 1 mol% to 60 mol%, 1 mol% to 55 mol%, 5 mol% to 55 mol%, 10 mol% to 50 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 20 mol% to 45 mol%, or 25 mol% to 40 mol% of the total lipids present in the lipid nanoparticles.

[0446] In some embodiments, the polymer-lipid (e.g., PEG lipid) in the lipid nanoparticles accounts for 0.5 mol% to 5 mol% of the total lipid present in the lipid nanoparticles. For example, 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol% or 5 mol%. In some embodiments, the polymer-lipid (e.g., PEG lipid) in the lipid nanoparticles accounts for 0.5 mol% to 4.5 mol%, 1 mol% to 4.5 mol%, 1 mol% to 4 mol%, 1.5 mol% to 4 mol%, 1.5 mol% to 3.5 mol% or 1.5 mol% to 3 mol% of the total lipid present in the lipid nanoparticles.

[0447] In some embodiments, the non-lamellar lipid nanoparticles are selected from one of the following: ethosomes and echogenic liposomes.

[0448] In some embodiments, the delivery vector is a liposome containing a nucleic acid according to any of the above embodiments, an RNA according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, or a nucleic acid composition according to any of the above embodiments. The liposome encapsulates the nucleic acid, RNA, vector, or nucleic acid composition according to any of the above embodiments using a vesicle formed by a phospholipid bilayer membrane. In some embodiments, the liposome comprises phospholipids and cholesterol.

[0449] In some embodiments, the delivery vector is a cationic protein loaded with the nucleic acid of any of the above embodiments, the RNA of any of the embodiments, the genetic engineering vector of any of the embodiments, or the nucleic acid composition of any of the embodiments. In some embodiments, the cationic protein includes but is not limited to protamine.

[0450] In some embodiments, the above-mentioned delivery vector is a polymer comprising the nucleic acid of any of the above-mentioned embodiments, the RNA of any of the above-mentioned embodiments, the genetic engineering vector of any of the above-mentioned embodiments, or the nucleic acid composition of any of the above-mentioned embodiments. In some embodiments, the polymer is a lipid polymer (lipopolyplex, LPP) and / or a hyaluronic acid polymer (such as hyaluronic acid gel) comprising the nucleic acid, RNA, genetic engineering vector or nucleic acid composition of any of the above-mentioned embodiments. In an alternative specific example, the polymer is a lipid polymer or a hyaluronic acid gel. Lipid polymer is a double-layer structure with a polymer-encapsulated nucleic acid (such as mRNA) as a core and a lipid (such as phospholipid) wrapped as an outer shell.

[0451] It is understood that the delivery vectors applicable to the present disclosure are not limited to the above, and may also be other substances capable of delivering the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetically engineered vector of any of the above embodiments, or the nucleic acid composition of any of the above embodiments into the body, such as vesicles (e.g., exosomes).

[0452] In addition, the present disclosure also provides a method for preparing the above-mentioned compound (IV), and the reaction of the preparation method is as follows:

[0453] Among them, R 18 ~R 20 , G 4 -G 5 、L 3 -L 4and z are as defined above, and X is halogen, preferably bromine.

[0454] The above preparation method includes step S11 and step S12.

[0455] Step S11: Intermediate compound (V) and intermediate compound (VI) are reacted at room temperature (16°C to 30°C, the same below) in an organic solvent in the presence of an acid-binding agent to obtain intermediate compound (VII). The organic solvent in step S1 is selected from one or more of nitrile organic solvents, alcohol organic solvents, halogenated hydrocarbon organic solvents, amide organic solvents, and aromatic hydrocarbon organic solvents. For example, the organic solvent in step S1 is selected from one or more of acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE). The acid-binding agent in step S1 is selected from one or more of organic bases and inorganic bases. For example, the acid-binding agent in step S1 is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA.

[0456] Step S12: by making intermediate compound (VII) and intermediate compound (VIII) carry out substitution reaction in an organic solvent at room temperature in the presence or absence of an acid binding agent and an iodide to obtain a compound of formula (IV). The organic solvent of step S2 is selected from one or more of nitrile organic solvents, alcohol organic solvents, halogenated hydrocarbon organic solvents, amide organic solvents and aromatic hydrocarbon organic solvents. For example, the organic solvent of step S2 is selected from one or more of acetonitrile, methanol, ethanol, dichloromethane and dichloroethane. The acid binding agent is selected from one or more of organic bases and inorganic bases. For example, the acid binding agent is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine and DIPEA. The iodide is a common iodide, such as potassium iodide.

[0457] In addition, the present disclosure also provides a method for preparing an optical isomer (IV-1-i) of the above compound (IV-1), and the reaction of the preparation method is as follows:

[0458] Wherein, X is a halogen, preferably bromine.

[0459] Specifically, the preparation step of the optical isomer (IV-1-i) of the compound (IV-1) of the present disclosure includes step S21 and step S22.

[0460] Step S21: Compound (1-X) and compound (1-1) are subjected to an N-alkylation reaction at 30°C to 50°C in a solvent (e.g., a nitrile, alcohol, halogenated hydrocarbon, amide, or aromatic hydrocarbon solvent, specifically acetonitrile, methanol, ethanol, dichloromethane, or dichloroethane (DCE)) to obtain compound (1-2);

[0461] Step S22: In a solvent (e.g., nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, or ether solvents, specifically acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), cyclopentane methyl ether, or methyl tert-butyl ether), in the presence or absence of an acid-binding agent and a catalyst, compound (1-2) is subjected to an N-alkylation reaction with 8-halogenated octanoic acid nonyl ester at 60° C. to 110° C. to prepare compound (IV-1-i), wherein the acid-binding agent is selected from one or more of the following: an organic base and an inorganic base. For example, one or more of the following: sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA; the catalyst is an iodide, preferably KI.

[0462] In some embodiments, according to the above method, when the compound (1-X) used is (1S, 3R)-3-aminocyclohexanol and the compound (1-2) is represented by the structural formula (1-2-1):

[0463] Obtain the optical isomer compound (IV-1-1) of compound (IV-1):

[0464] When the compound (1-X) used is (1S,3S)-3-aminocyclohexanol and the compound (1-2) is as shown in the structural formula (1-2-2):

[0465] Obtain the optical isomer compound (IV-1-2) of compound (IV-1):

[0466] When the compound (1-X) used is (1R, 3R)-3-aminocyclohexanol and the compound (1-2) is represented by the structural formula (1-2-3):

[0467] Obtain the optical isomer compound (IV-1-3) of compound (IV-1):

[0468] When the compound (1-X) used is (1R, 3S)-3-aminocyclohexanol and the compound (1-2) is as shown in the structural formula (1-2-4):

[0469] Obtain the optical isomer compound (IV-1-4) of compound (IV-1):

[0470] In addition, the present disclosure also provides a pharmaceutical composition, which comprises the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the host cell of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the immunogen of any of the above embodiments, the mutant of the RSV F protein of any of the above embodiments, or the delivery vector of any of the above embodiments, and a pharmaceutically acceptable carrier.

[0471] In some embodiments, the pharmaceutical compositions described above comprise a plurality of nucleic acids.

[0472] In some embodiments, the pharmaceutical composition comprises multiple nucleic acids, and the multiple nucleic acids are contained in the same delivery vehicle (eg, LNPs) or in different delivery vehicles (eg, LNPs).

[0473] In some embodiments, the pharmaceutical compositions described above comprise one or more delivery vehicles.

[0474] In some embodiments, the pharmaceutical composition described above comprises a delivery vehicle comprising the nucleic acid or nucleic acid composition of any of the above embodiments.

[0475] In some embodiments, the pharmaceutical composition comprises a delivery vector comprising a first nucleic acid and a second nucleic acid, the first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 83; and the second nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 83. The amino acid sequence shown in NO:84 has a mutant of the amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity.

[0476] In some embodiments, the pharmaceutical composition comprises a delivery vector comprising two first nucleic acids, the first first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, wherein the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 83; and the second first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, wherein the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 84.

[0477] In some embodiments, the pharmaceutical composition comprises a delivery vector comprising two first nucleic acids, the first first nucleic acid comprising a DNA encoding the RSV F protein as set forth in SEQ ID NO:83 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA as set forth in SEQ ID NO:187, or its corresponding RNA; and the second first nucleic acid comprising a DNA encoding the RSV F protein as set forth in SEQ ID NO:84 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA as set forth in SEQ ID NO:187, or its corresponding RNA; The DNA shown in NO:188 has a DNA or its corresponding RNA that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical.

[0478] In some embodiments, the above-mentioned pharmaceutical composition comprises a delivery vector comprising two first nucleic acids, the first first nucleic acid comprising the DNA shown in SEQ ID NO: 187 or its corresponding RNA; the second first nucleic acid comprising the DNA shown in SEQ ID NO: 188 or its corresponding RNA.

[0479] In some embodiments, the pharmaceutical composition comprises a delivery vehicle, which is LNPs comprising the nucleic acid of any of the above embodiments or the nucleic acid composition of any of the above embodiments.

[0480] In some embodiments, the pharmaceutical composition described above comprises multiple delivery vehicles, each of which independently comprises the nucleic acid or nucleic acid composition of any of the above embodiments.

[0481] In some embodiments, the pharmaceutical composition comprises a plurality of delivery vectors, wherein the RSV F protein mutants encoded by the nucleic acids contained in the plurality of delivery vectors are from the same subtype strain but with different mutations, different subtype strains but with the same mutations, or different subtype strains with different mutations.

[0482] For example, the pharmaceutical composition comprises a first delivery vector and a second delivery vector, wherein the first delivery vector comprises a first nucleic acid, and the second delivery vector comprises a second nucleic acid, wherein the first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations, and the second nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Buenos Aires and having P101C, I152C, and S190M mutations. In some embodiments, the pharmaceutical composition comprises a first delivery vector comprising a first nucleic acid and a second delivery vector comprising a second nucleic acid, wherein the first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, T54A, S190M, and V296L mutations, and the second nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from strain Buenos Aires and having P101C, I152C, T54A, S190M, and V296L mutations.

[0483] In some embodiments, the pharmaceutical composition comprises a first delivery vector and a second delivery vector, the first delivery vector comprising a first nucleic acid, the second delivery vector comprising a second nucleic acid, the first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 83; the second nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in NO:84 has a mutant of the amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity.

[0484] In some embodiments, the pharmaceutical composition comprises a first delivery vector and a second delivery vector, the first delivery vector comprising a first nucleic acid, the second delivery vector comprising a second nucleic acid, the first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein being a mutant comprising the amino acid sequence shown in SEQ ID NO: 83; the second nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein being a mutant comprising the amino acid sequence shown in SEQ ID NO: 84.

[0485] In some embodiments, the pharmaceutical composition comprises a first delivery vector and a second delivery vector, the first delivery vector comprising a first nucleic acid, the second delivery vector comprising a second nucleic acid, the first nucleic acid comprising a DNA encoding an RSV F protein with an amino acid sequence as set forth in SEQ ID NO: 83 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA set forth in SEQ ID NO: 187, or its corresponding RNA; the second nucleic acid comprising a DNA encoding an RSV F protein with an amino acid sequence as set forth in SEQ ID NO: 84 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA set forth in SEQ ID NO: 187, or its corresponding RNA; The DNA shown in NO:188 has a DNA or its corresponding RNA that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical.

[0486] In some embodiments, the pharmaceutical composition comprises a first delivery vector and a second delivery vector, wherein the first delivery vector comprises a first nucleic acid and the second delivery vector comprises a second nucleic acid, wherein the first nucleic acid comprises a DNA as set forth in SEQ ID NO: 187 or its corresponding RNA; and the second nucleic acid comprises a DNA as set forth in SEQ ID NO: 188 or its corresponding RNA. For another example, the pharmaceutical composition comprises a first delivery vector and a second delivery vector, wherein the first delivery vector comprises a first nucleic acid and the second delivery vector comprises a second nucleic acid, wherein the first nucleic acid comprises a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190M mutations, and the second nucleic acid comprises a polynucleotide encoding an RSV F protein derived from strain Ontario and having P101C, I152C, and S190F mutations. In some embodiments, the first delivery vector and the second delivery vector are LNPs comprising the nucleic acid of any of the above embodiments or the nucleic acid combination of any of the above embodiments.

[0487] For another example, the above-mentioned pharmaceutical composition comprises a delivery vector, the delivery vector comprises a first nucleic acid and a second nucleic acid, the first nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from the Ontario strain and having P101C, I152C, and S190M mutations, and the second nucleic acid is a nucleic acid comprising a polynucleotide encoding an RSV F protein derived from the Buenos Aires strain and having P101C, I152C, and S190M mutations.

[0488] In some embodiments, the pharmaceutical composition comprises multiple nucleic acids according to any of the above embodiments, multiple RNAs according to any of the above embodiments, multiple genetically engineered vectors according to any of the above embodiments, or multiple host cells according to any of the above embodiments.

[0489] The term "pharmaceutically acceptable" as used herein means approved for use in animals and / or humans by a regulatory agency (e.g., the State Food and Drug Administration (CFDA), the U.S. Food and Drug Administration (FDA)) or a recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopeia). The term "pharmaceutically acceptable carrier" refers to a substance that can be administered together with the nucleic acid, genetic engineering vector, nucleic acid composition, protein mutant or delivery vector of the present disclosure, including but not limited to diluents, sweeteners, flavoring agents, wetting agents, adjuvants, glidants, preservatives, dyes / colorants, surfactants, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.

[0490] In some embodiments, the above pharmaceutical compositions do not include an adjuvant.

[0491] In some embodiments, the above pharmaceutical composition further comprises an adjuvant.

[0492] In some embodiments, the adjuvant comprises a flagellin adjuvant. In some embodiments, the flagellin adjuvant is an mRNA encoding flagellin.

[0493] In addition, the present disclosure also provides a use of a nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a host cell according to any of the above embodiments, a nucleic acid composition according to any of the above embodiments, a mutant of the RSV F protein according to any of the above embodiments, an RNA according to any of the above embodiments, an immunogen according to any of the above embodiments, a delivery vector according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments in the preparation of a drug.

[0494] In some embodiments, the medicament is for preventing or treating RSV infection or a disease caused by RSV infection. In some embodiments, the medicament is a vaccine.

[0495] 7. Vaccines

[0496] The present disclosure also provides a vaccine, comprising the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the mutant of the RSV F protein of any of the above embodiments, the RNA of any of the above embodiments, the immunogen of any of the above embodiments, or the delivery vector of any of the above embodiments.

[0497] In some embodiments, the above-mentioned vaccine is an RSV vaccine.

[0498] In some embodiments, the above-mentioned vaccine is a multivalent vaccine.

[0499] In some embodiments, the above-mentioned vaccines comprise any of the above-mentioned nucleic acids or any of the above-mentioned mutants of RSV F protein.

[0500] In some embodiments, the above vaccine is a combination vaccine.

[0501] In some embodiments, the vaccine is a concatenated nucleic acid vaccine. In some embodiments, in addition to comprising any one or more of the aforementioned nucleic acids comprising polynucleotides encoding mutants of the RSV F protein, the vaccine further comprises nucleic acids encoding proteins or polypeptides other than the RSV F protein or from other viruses that can serve as immunogens. Of course, the other viruses can be a single subtype, multiple viruses, or multiple subtypes. For example, the other viruses can be influenza A, influenza A and SARS-CoV-2, or influenza A and influenza B.

[0502] In some embodiments, the above vaccine is a polyprotein vaccine.

[0503] In some embodiments, the vaccine comprises, in addition to any one or more of the RSV F protein mutants, proteins or polypeptides from other viruses that can serve as immunogens. Of course, the other viruses can be one or more.

[0504] In some embodiments, the above vaccines do not include an adjuvant.

[0505] In some embodiments, the above-mentioned vaccine further comprises an adjuvant.

[0506] In some embodiments, the adjuvant includes a flagellin adjuvant. In some embodiments, the flagellin adjuvant is an mRNA encoding flagellin. When combined with a flagellin adjuvant, particularly when one or more mRNAs encoding mutants of the RSV F protein are combined with an mRNA encoding flagellin, the efficacy of the RNA (e.g., mRNA) in the vaccine can be significantly enhanced. In some embodiments, the above-mentioned vaccine comprises one or more delivery vectors.

[0507] In some embodiments, the vaccine comprises a delivery vehicle, which is LNPs comprising the nucleic acid or nucleic acid composition of any of the above embodiments.

[0508] In some embodiments, the vaccine comprises a delivery vector comprising a first nucleic acid and a second nucleic acid, the first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 83; and the second nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 83. The amino acid sequence shown in NO:84 has a mutant of the amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity.

[0509] In some embodiments, the above-mentioned vaccine comprises a delivery vector comprising two first nucleic acids, the first first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, wherein the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 83; and the second first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, wherein the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 84.

[0510] In some embodiments, the vaccine comprises a delivery vector comprising two first nucleic acids, the first first nucleic acid comprising a DNA encoding the RSV F protein as set forth in SEQ ID NO:83 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA as set forth in SEQ ID NO:187, or its corresponding RNA; and the second first nucleic acid comprising a DNA encoding the RSV F protein as set forth in SEQ ID NO:84 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA as set forth in SEQ ID NO:187, or its corresponding RNA; The DNA shown in NO:188 has a DNA or its corresponding RNA that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical.

[0511] In some embodiments, the above-mentioned vaccine comprises a delivery vector comprising two first nucleic acids, the first first nucleic acid comprising the DNA shown in SEQ ID NO: 187 or its corresponding RNA; the second first nucleic acid comprising the DNA shown in SEQ ID NO: 188 or its corresponding RNA.

[0512] In some embodiments, the vaccine comprises a plurality of delivery vehicles, each of which is independently LNPs comprising the nucleic acid or nucleic acid composition of any of the above embodiments.

[0513] In some embodiments, the vaccine comprises a first delivery vector and a second delivery vector, the first delivery vector comprising a first nucleic acid, the second delivery vector comprising a second nucleic acid, the first nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 83; the second nucleic acid comprising a polynucleotide encoding a mutant of the RSV F protein, the mutant of the RSV F protein comprising mutations P101C, I152C, T54A, S190M, and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in NO:84 has a mutant of the amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity.

[0514] In some embodiments, the above-mentioned vaccine comprises a first delivery vector and a second delivery vector, the first delivery vector comprises a first nucleic acid, and the second delivery vector comprises a second nucleic acid, the first nucleic acid comprises a polynucleotide encoding a mutant of the RSV F protein, and the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 83; the second nucleic acid comprises a polynucleotide encoding a mutant of the RSV F protein, and the mutant of the RSV F protein is a mutant comprising the amino acid sequence shown in SEQ ID NO: 84.

[0515] In some embodiments, the vaccine comprises a first delivery vector and a second delivery vector, the first delivery vector comprising a first nucleic acid, the second delivery vector comprising a second nucleic acid, the first nucleic acid comprising a DNA encoding the RSV F protein with an amino acid sequence as set forth in SEQ ID NO: 83 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA set forth in SEQ ID NO: 187, or its corresponding RNA; the second nucleic acid comprising a DNA encoding the RSV F protein with an amino acid sequence as set forth in SEQ ID NO: 84 and having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity to the DNA set forth in SEQ ID NO: 187, or its corresponding RNA; The DNA shown in NO:188 has a DNA or its corresponding RNA that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical.

[0516] In some embodiments, the above-mentioned vaccine comprises a first delivery vector and a second delivery vector, the first delivery vector comprises a first nucleic acid, the second delivery vector comprises a second nucleic acid, the first nucleic acid comprises a DNA as shown in SEQ ID NO: 187 or its corresponding RNA; the second nucleic acid comprises a DNA as shown in SEQ ID NO: 188 or its corresponding RNA.

[0517] In some embodiments, the above-mentioned vaccine is an mRNA vaccine.

[0518] In some embodiments, the vaccine is an mRNA vaccine, and the vaccine is administered nasally, intratracheally, or injectably (e.g., intravenously, intraocularly, intravitreally, intramuscularly, intradermally, intracardially, intraperitoneally, and subcutaneously).

[0519] It is understood that the dosage form of the above-mentioned vaccine is not particularly limited.

[0520] 8. Prevention or Treatment Methods

[0521] The present disclosure also provides a method for preventing or treating RSV virus infection, comprising administering to a subject a nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a nucleic acid composition according to any of the above embodiments, a mutant of the RSV F protein according to any of the above embodiments, an RNA according to any of the above embodiments, an immunogen according to any of the above embodiments, a delivery vector according to any of the above embodiments, a pharmaceutical composition according to any of the above embodiments, or a vaccine according to any of the above embodiments.

[0522] In some embodiments, the subject is a mammal (e.g., a human, a non-human primate (e.g., apes, chimpanzees, monkeys, and orangutans)), a domestic animal (e.g., a dog, a cat, and livestock (e.g., a horse, a cow, a pig, a sheep, and a goat)), or other mammal. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In an alternative specific example, the subject is a human.

[0523] In some embodiments, the number of times used is once, twice, three times, four times or more times. In addition, the disclosure also provides a method for inducing an immune response for RSV in a subject. In some embodiments, the method includes administering to the subject a nucleic acid of any of the above-mentioned embodiments, a genetically engineered vector of any of the above-mentioned embodiments, a nucleic acid composition of any of the above-mentioned embodiments, a mutant of the RSV F protein of any of the above-mentioned embodiments, an RNA of any of the above-mentioned embodiments, an immunogen of any of the above-mentioned embodiments, a delivery vector of any of the above-mentioned embodiments, a pharmaceutical composition of any of the above-mentioned embodiments, or a vaccine of any of the above-mentioned embodiments. Relative to the anti-antigen antibody titer in a subject inoculated with a conventional vaccination for RSV of a preventive effective dose, the anti-antigen antibody titer in the subject after vaccination of the present disclosure increases." anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0524] In some embodiments, the above-mentioned method for preventing or treating RSV viral infection or the method for eliciting an immune response against RSV in a subject comprises administering to the subject one or two (or more) doses of a nucleic acid, genetically engineered vector, nucleic acid composition, RSV F protein mutant, RNA, immunogen, delivery vehicle, pharmaceutical composition or vaccine (e.g., mRNA vaccine) according to any of the above embodiments at a dose level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg or 0.025 mg / kg to 1.0 mg / kg, based on the subject's body weight.

[0525] In some embodiments, the above-mentioned method for preventing or treating RSV viral infection or the method for eliciting an immune response against RSV in a subject comprises administering to the subject once or twice (or more than twice) a total dose of 0.0100 mg to 1.0 mg or at a dose level sufficient to deliver the total dose, the nucleic acid, genetically engineered vector, nucleic acid composition, RSV F protein mutant, RNA, immunogen, delivery vector, pharmaceutical composition or vaccine (e.g., mRNA vaccine) of any of the above embodiments.

[0526] In some embodiments, the above method for preventing or treating RSV virus infection or the method for eliciting an immune response against RSV in a subject comprises administering the nucleic acid, genetic engineering vector, nucleic acid composition, RSV according to any of the above embodiments. The mutants, RNA, immunogens, delivery vehicles, pharmaceutical compositions or vaccines (e.g., mRNA vaccines) of the F protein are administered at 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg. In some embodiments, the present invention provides an agent that is administered to a subject twice (e.g., day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later) at a dose level sufficient to deliver the total dose. The present disclosure encompasses higher and lower dosages and frequencies of administration. For example, the above-mentioned vaccine (e.g., mRNA vaccine) can be administered three or four times.

[0527] In addition, the present disclosure also provides a use of a nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a nucleic acid composition according to any of the above embodiments, a mutant of the RSV F protein according to any of the above embodiments, an RNA according to any of the above embodiments, an immunogen according to any of the above embodiments, a delivery vector according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments in preparing a detection reagent or kit for RSV F protein-binding antibodies.

[0528] In addition, the present disclosure also provides a detection reagent or kit for RSV F protein-binding antibodies, which comprises the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the mutant of RSV F protein of any of the above embodiments, the RNA of any of the above embodiments, the immunogen of any of the above embodiments, the delivery vector of any of the above embodiments, or the composition of the drug of any of the above embodiments.

[0529] In addition, the present disclosure also provides a method for detecting or isolating RSV F protein-binding antibodies in a subject, the method comprising: providing an effective amount of the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the mutant of the RSV F protein of any of the above embodiments, the immunogen of any of the above embodiments, or a pharmaceutical composition or vaccine containing the mutant of the RSV F protein; contacting a biological sample from the subject with the mutant of the RSV F protein under conditions sufficient to form an immune complex between the mutant and the RSV F-binding antibody; and detecting the immune complex, thereby detecting or isolating the RSV F-binding antibody in the subject. Example

[0530] In order to make the purpose and technical solutions of the present disclosure clearer, the following detailed description is given in conjunction with specific examples. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other implementation methods obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure. Unless otherwise specified, the reagents and instruments used in the examples are all conventionally selected in the art. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the manufacturer.

[0531] Example 1

[0532] 1. Synthesis of Compound (IV-1)

[0533] Step 1): Synthesis of 8-((3-hydroxycyclohexyl)amino)nonyl octanoate (Compound (IV-1-p1))

[0534] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester.

[0535] 1 H NMR (600MHz, CDCl3) δ4.20-4.13(m,0.5H),4.05(t,2H),3.83(m,0.5H),3.09(m,0.5H),2.87(m,0.5H),2.76-2.59(m,2H), 2.29(t,2H),2.00(m,0.5H),1.93-1.78(m,1.5H),1.78-1.65(m,2H),1.65-1.46(m,8H),1.40-1.18(m,20H),0.88(t,3H).

[0536] LCMS: 384.3 [M+H] + .

[0537] Step 2): Synthesis of compound (IV-1)

[0538] In a 100 mL reaction flask, 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times. It was then dried over anhydrous sodium sulfate and concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound (IV-1).

[0539] 1H NMR (600MHz, CDCl3) δ4.95-4.81(m,1H),4.30-4.12(m,0.5H),4.07(t,2H),3.72-3.61(m,0.5H),2.97(m,0.5H),2.64-2.52(m ,0.5H),2.48-2.37(m,4H),2.30(q,4H),1.93-1.81(m,2H),1.72-1.57(m,8H),1.51(t,4H),1.43-1.18(m,56H),0.89(m,9H).

[0540] LCMS: 765.3 [M+H] + .

[0541] 2. Preparation of Compound (IV-1-1)

[0542] To a 250 mL single-necked flask, 8 g (17.3 mmol) of 9-heptadecyl-8-bromooctanoate, 10 g (76.5 mmol) of (1S,3R)-3-aminocyclohexanol, and 100 mL of ethanol were added and reacted at 50°C for 15 h. The 9-heptadecyl-8-bromooctanoate reacted completely. After removing the solvent by rotary evaporation, the crude product was added to 200 mL of ethyl acetate (EA) and washed twice with 100 mL of water to thoroughly remove the (1S,3R)-3-aminocyclohexanol. The EA phase was then dried to afford crude compound (IV-1-1-p) (i.e., 9-heptadecyl-8-(((1R,3S)-3-hydroxycyclohexyl)amino)octanoate). The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to afford compound (IV-1-1-p).

[0543] NMR data:

[0544] 1 H NMR (600MHz, CDCl3): δ4.91-4.81(m,1H),3.86-3.82(m,1H),2.84-2.81(m,1H),2.69-2.54(qt,J=11.2,7.3Hz,2H),2.29-2.26(t,J=7.5Hz,2H),1. 93-1.86(m,1H),1.77-1.74(d,J=12.0Hz,1H),1.72-1.57(m,6H),1.54-1 .45(m,6H),1.37-1.30(m,8H),1.30-1.22(m,24H),0.88(t,J=7.0Hz,6H).

[0545] LCMS: 496.7 [M+H] + .

[0546] Take 6 g (12.1 mmol) of the above compound (IV-1-1-p) in a 250 mL single-necked bottle, add 90 mL of acetonitrile and 60 mL of cyclopentane methyl ether, then add 6.7 g of potassium carbonate powder (48.4 mmol), 2 g of potassium iodide (12.1 mmol) and 5 g of 8-bromooctanoic acid nonyl ester (17.5 mmol), and place at 90 ° C to react for 24 hours. The raw material compound ((IV-1-1-p) is completely reacted. Remove the oil bath and wait for the reaction to cool to room temperature. After the solid is removed by filtration, the filtrate is spin-dried to obtain a crude product, and finally purified by column chromatography (dichloromethane: methanol = 20: 1 to 5: 1) to obtain compound (IV-1-1).

[0547] NMR data:

[0548] 1 H NMR (600MHz, CDCl3): δ4.93-4.84(m,1H),4.09(t,J=6.8Hz,2H),3.68-3.61(m,1H),2.62-2.54(m,1H),2.53-2.41(m,4H),2.31-2.26(q,J= 7.4Hz,4H),1.97(m,1H),1.91-1.78(m,2H),1.71-1.61(m,7H),1.54(d,J=6.0Hz,4H),1.43-1.37(m,4H),1.39-1.23(m,52H),0.91(m,9H).

[0549] Specific rotation: +4.3°

[0550] LCMS: 765.2 [M+H] + .

[0551] 3. Preparation of Compound (IV-1-2)

[0552] To a 100 mL single-necked flask, 2 g of 9-heptadecyl-8-bromooctanoate (4.3 mmol), 2.5 g of (1S,3S)-3-aminocyclohexanol (20.2 mmol), and 20 mL of ethanol were added and reacted at 50°C for 15 h. The 9-heptadecyl-8-bromooctanoate reacted completely. After removing the solvent by rotary evaporation, the crude product was added with 50 mL of EA and washed twice with 25 mL of water to thoroughly remove the (1S,3S)-3-aminocyclohexanol. The EA phase was then dried to afford crude compound (IV-1-2-p) (i.e., 9-heptadecyl-8-(((1S,3S)-3-hydroxycyclohexyl)amino)octanoate). The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to afford compound (IV-1-2-p).

[0553] NMR data:

[0554] 1 H NMR (600MHz, CDCl3): δ4.91-4.82(m,1H),4.16-4.10(m,1H),2.96-2.87(m,1H),2.65-2.55(qt,J=11.2,7.3Hz,2H),2.28-2.26(t,J =7.5Hz,2H),1.93-1.77(m,4H),1.67-1.55(m,6H),1.54-1.45(m,6H),1.33-1.31(m,6H),1.30-1.22(m,24H),0.88(t,J=7.0Hz,6H).

[0555] LCMS: 496.7 [M+H] + .

[0556] Take 1.5 g (3.0 mmol) of the above compound (IV-1-2-p) in a 100 mL single-necked bottle, add 25 mL of acetonitrile and 15 mL of cyclopentane methyl ether, then add 1.67 g of potassium carbonate powder (12 mmol), 0.5 g of potassium iodide (3 mmol) and 1.26 g of 8-bromooctanoic acid nonyl ester (3.6 mmol), and place at 90 ° C to react for 24 hours. The raw material compound (IV-1-2-p) is completely reacted. Remove the oil bath and wait for the reaction to cool to room temperature. After the solid is removed by filtration, the filtrate is spin-dried to obtain a crude product, and finally purified by column chromatography (dichloromethane: methanol = 20:1 to 5:1) to obtain compound (IV-1-2).

[0557] NMR data:

[0558] 1 H NMR (600MHz, CDCl3): δ4.91-4.80(m,1H),4.28-4.22(m,1H),4.06-4.04(t,J=6.8Hz,2H ),3.03-2.97(m,1H),2.47-2.41(m,4H),2.33-2.25(q,J=7.5Hz,4H),1.89-1.85(d,J=12 .1Hz,1H),1.80-1.77(t,J=12.2Hz,1H),1.73-1.65(m,2H),1.64-1.59(m,6H),1.54-1.4 8(m,4H),1.48-1.39(m,4H),1.34-1.29(m,14H),1.29-1.23(m,38H),0.89-0.87(m,9H).

[0559] Specific rotation: -12.9°

[0560] LCMS: 765.2[M+H] + .

[0561] 4. Preparation of Compound (IV-1-3)

[0562] To a 100 mL single-necked flask, 2 g of 9-heptadecyl-8-bromooctanoate (4.3 mmol), 2.5 g of (1R,3R)-3-aminocyclohexanol (20.2 mmol), and 20 mL of ethanol were added and reacted at 50°C for 15 h. The 9-heptadecyl-8-bromooctanoate reacted completely. After removing the solvent by rotary evaporation, the crude product was added with 50 mL of EA and washed twice with 25 mL of water to thoroughly remove the (1R,3R)-3-aminocyclohexanol. The EA phase was then dried to afford crude compound (IV-1-3-p) (i.e., 9-heptadecyl-8-(((1R,3R)-3-hydroxycyclohexyl)amino)octanoate). The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to afford compound (IV-1-3-p).

[0563] NMR data:

[0564] 1 H NMR (600MHz, CDCl3): δ4.91-4.81(m,1H),4.17-4.08(m,1H),2.94-2.84(m,1H),2.64-2.5(qt,J=11.2,7.3Hz,2H),2.28-2.26(t,J =7.5Hz,2H),1.88-1.76(m,2H),1.75-1.54(m,8H),1.54-1.40(m,6H),1.33-1.3(m,6H),1.30-1.23(m,24H),0.88(t,J=7.0Hz,6H).

[0565] LCMS: 496.7 [M+H] + .

[0566] Take 1.5 g (3.0 mmol) of the above compound (IV-1-3-p) in a 100 mL single-necked bottle, add 25 mL of acetonitrile and 15 mL of cyclopentane methyl ether, then add 1.67 g of potassium carbonate powder (12 mmol), 0.5 g of potassium iodide (3 mmol) and 1.26 g of 8-bromooctanoic acid nonyl ester (3.6 mmol), and place at 90 ° C to react for 24 hours. The raw material compound (IV-1-3-p) is completely reacted. Remove the oil bath and wait for the reaction to cool to room temperature. After the solid is removed by filtration, the filtrate is spin-dried to obtain a crude product, and finally purified by column chromatography (dichloromethane: methanol = 20: 1 to 5: 1) to obtain compound (IV-1-3).

[0567] NMR data:

[0568] 1 H NMR (600MHz, CDCl3): δ4.89-4.82(m,1H),4.24-4.22(m,1H),4.06-4.04(t,J=6. 8Hz,2H),3.00-2.95(m,1H),2.47-2.36(m,4H),2.30-2.26(q,J=7.5Hz,4H),1.86 -1.84(d,J=12.1Hz,1H),1.79-1.77(t,J=12.2Hz,1H),1.70-1.66(m,2H),1.65-1 .57(m,6H),1.53-1.48(m,4H),1.45-1.38(m,4H),1.35-1.30(m,14H),1.29-1.22 (m,38H),0.89-0.87(m,9H).

[0569] Specific rotation: +12.6°

[0570] LCMS: 765.2[M+H] + .

[0571] V. Preparation of Compound (IV-1-4)

[0572] To a 250 mL single-necked flask, 8 g of 9-heptadecyl-8-bromooctanoate (17.3 mmol), 10 g of (1R,3S)-3-aminocyclohexanol (76.5 mmol), and 100 mL of ethanol were added and reacted at 50°C for 15 h. The 9-heptadecyl-8-bromooctanoate reacted completely. The solvent was removed by rotary evaporation, and the crude product was added to 200 mL of EA and washed twice with 100 mL of water to thoroughly remove the (1R,3S)-3-aminocyclohexanol. The EA phase was then dried to afford crude compound (IV-1-4-p) (i.e., 9-heptadecyl-8-(((1S,3R)-3-hydroxycyclohexyl)amino)octanoate). The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to afford compound (IV-1-4-p).

[0573] NMR data:

[0574] 1H NMR (600MHz, CDCl3): δ4.90-4.83(m,1H),3.86-3.82(m,1H),2.86-2.82(m,1H) ),2.69-2.58(qt,J=11.2,7.3Hz,2H),2.29-2.26(t,J=7.5Hz,2H),1.94-1.86( m,1H),1.79-1.77(d,J=12.0Hz,1H),1.74-1.64(m,3H),1.64-1.59(m,3H),1. 51-1.46(m,6H),1.36-1.30(m,8H),1.30-1.22(m,24H),0.88(t,J=7.0Hz,6H).

[0575] LCMS: 496.7 [M+H] + .

[0576] Take 6 g (12.1 mmol) of the above compound (IV-1-4-p) in a 250 mL single-necked bottle, add 90 mL of acetonitrile and 60 mL of cyclopentane methyl ether, then add 6.7 g of potassium carbonate powder (48.4 mmol), 2 g of potassium iodide (12.1 mmol) and 5 g of 8-bromooctanoic acid nonyl ester (17.4), and place at 90 ° C to react for 24 hours. The raw material compound (IV-1-4-p) is completely reacted. Remove the oil bath and wait for the reaction to cool to room temperature. After the solid is removed by filtration, the filtrate is spin-dried to obtain a crude product, and finally purified by column chromatography (dichloromethane: methanol = 20:1 to 5:1) to obtain compound (IV-1-4).

[0577] NMR data:

[0578] 1 H NMR (600MHz, CDCl3): δ4.90-4.82(m,1H),4.05(t,J=6.8Hz,2H),3.70-3.60(m,1H),2.64-2.54(m,1H),2.51-2.41(m,4H),2.30-2.26(q,J= 7.5Hz,4H),1.95(m,1H),1.91-1.77(m,2H),1.69-1.56(m,7H),1.50(d,J=6.0Hz,4H),1.41-1.37(m,4H),1.34-1.18(m,52H),0.88(m,9H).

[0579] Specific rotation: -4.5°

[0580] LCMS: 765.2[M+H] + .

[0581] Example 2 Design of RSV F protein mutants and their coding sequences

[0582] RSV F protein mutants as shown in Table 1 were designed. Based on the amino acid sequences of the mutants, codon-optimized DNA sequences (DNA sequences corresponding to the RSV F protein coding regions in Table 1) were further designed and commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. Test results showed that all DNA sequences were correct.

[0583] Example 3 Construction of engineered plasmid

[0584] The construction method is as follows:

[0585] 1. Construction of plasmid B: Suzhou GENEWIZ Biotechnology Co., Ltd. (GENEWIZ) was commissioned to replace the Amp resistance gene in plasmid pcDNA3.1 with the Kana resistance gene. New restriction sites HindIII, BamHI, KpnI, and ApaI were added after the T7 promoter, and the NeoR / KanR sequence from 2136 bp to 2930 bp was deleted to obtain plasmid B.

[0586] 2. Construction of plasmid C: Plasmid B was linearized with ApaI and homologously recombined with a nucleic acid fragment synthesized by IDT (Integrated DNA Technologies) with the nucleotide sequence shown in SEQ ID NO: 211 to obtain plasmid C.

[0587] 3. Construction of plasmid D: Plasmid C was double-digested with KpnI and ApaI, and a large fragment of approximately 4.7 kb was recovered by agarose gel electrophoresis. The fragment was then ligated with a nucleic acid fragment synthesized by IDT (SEQ ID NO: 210) using T4 enzyme to obtain plasmid D.

[0588] 4. Construction of plasmid F: Plasmid D was double-digested with BamHI and HindIII, and a large fragment of approximately 4.8 kb was recovered by agarose gel electrophoresis. The fragment was then ligated with the nucleic acid fragment synthesized by IDT (SEQ ID NO: 209) using T4 enzyme to obtain plasmid F.

[0589] 5. Construction of engineered plasmids: Homologous arm sequences were introduced at both ends of the DNA sequences corresponding to the protein coding regions of different RSV F protein mutants (see Table 1 for details). The PCR products were then digested with plasmid F by BamHI and KpnI, and large molecular fragments of approximately 4.8 kb were recovered and homologous recombination was performed. Finally, multiple engineered plasmids with completely correct sequencing were obtained.

[0590] Example 4 Preparation of mRNA encoding mutants of RSV F protein

[0591] 1. Extract the engineering plasmid and ensure that the supercoil rate of the plasmid is above 85%.

[0592] 2. Plasmid linearization

[0593] (1) Enzyme linearization: Take 20 μg of different engineered plasmids containing the coding sequence of RSV F protein mutants prepared in Example 3, prepare the reaction system according to Table 2, mix well, and place in a 37°C incubator for 16 hours for enzyme digestion reaction.

[0594] Table 2

[0595] (2) Purification of linearized plasmid: After ensuring that the enzyme digestion is complete, use a DNA fragment purification kit (Takara, catalog number: 9761) to recover the linearized plasmid.

[0596] (3) Identification: 100 ng of the purified linear plasmid and the original circular plasmid were subjected to 1% agarose gel electrophoresis to confirm whether the plasmid linearization was complete.

[0597] 3. In vitro transcription of mRNA

[0598] (1) Prepare the IVT reaction system according to Table 3.

[0599] Table 3

[0600] (2) The reaction was carried out at 37°C for 2 h. After the reaction was completed, 5 μL of DNase was added to the reaction system and incubated at 37°C for 15 min (DNase was used to remove the linearized plasmid template).

[0601] (3) Refer to the purification magnetic beads ( The IVT product was purified by magnetic beads according to the instructions of RNA Cleaner (YEASEN, catalog number: 12602ES56).

[0602] (4) Capping:

[0603] mRNACap1 capping reaction:

[0604] The Cap1 type cap structure and reaction principle are as follows:

[0605] pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi

[0606] ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi

[0607] G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc

[0608] m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3')+AdoHyc

[0609] 5'-Cap1 type cap structure:

[0610] cap G 1 G 2 =m 7 G-5'-ppp-5'-Gm 2 '-3'-p-[m 7 =7-CH3;m 2 '=2'-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-= -PO2H-].

[0611] A 67 μL mRNA solution (25 pmol uncapped mRNA was made up to 67 μL with water) was preheated at 65°C for 5 min and then mixed with a mixture containing 10 μL 10× ScriptCap Capping Buffer, 10 μL 10 mM GTP, 2.5 μL 20 mM SAM, 2.5 μL ScriptGuard RNase Inhibitor, 4 μL ScriptCap 2'-O-Methyltransferase (100 U / μL), and 4 μL ScriptCap Capping Enzyme (10 U / μL) and incubated at 37°C for 1 h. The above reagents used for the capping reaction were purchased from Cellscript.

[0612] (5) Purification of capped product:

[0613] Reference purification magnetic beads (Hieff The capped product was purified by magnetic beads according to the instructions of RNA Cleaner (YEASEN, Cat. No. 12602ES56). Characterization data: Concentration was determined using onedrop or Qubit.

[0614] Experimental results: mRNAs encoding different mutants of RSV F protein containing a Cap1-type cap structure, 5'-UTR, 3'-UTR, a poly(A) tail, and all uridines replaced by N1-methylpseudouridine were obtained. The amino acid sequences of the RSV F protein mutants after translation of the protein coding regions of each mRNA are shown in Table 1.

[0615] Example 5 Mouse Immunization Experiment with Encapsulated RSV mRNA Vaccine

[0616] (1) Encapsulation: Using a microsyringe and a microfluidic chip (SN.000038), mRNA was encapsulated at a rate of 9 mL / min in the aqueous phase and 3 mL / min in the alcohol phase to prepare crude LNP preparations containing mRNA encoding RSV F protein mutants. The difference between the LNP preparations was only the encapsulated mRNA. The mRNA used in this example was prepared according to the method described in Example 4. The compositions of the aqueous and alcohol phases of the various LNP preparations are shown in Table 4 below.

[0617] Table 4

[0618] (2) Dialysis fluid exchange:

[0619] a. Dialysate preparation: 1×PBS + 8% (m / v) sucrose solution: Take 2 packets of 1×PBS pre-made powder into a beaker, dissolve and mix with 2L of DEPC water, then add 160g of sucrose and mix to obtain 1×PBS + 8% sucrose solution.

[0620] b. Dialysis: The encapsulated crude product was placed into a 100KD dialysis bag and immersed in a beaker filled with 1L of dialysis solution. The beaker was wrapped with aluminum foil and dialyzed at 100 rpm for 1 hour at room temperature. The dialysate was then replaced and dialysis continued for 1 hour. The dialyzed formulation samples were sterile filtered using a 0.22μm disposable filter membrane to prepare various LNP formulations encapsulating mRNA encoding RSV F protein mutants. The mRNA concentration in the LNP formulations was 0.2μg / μL, the mass ratio of mRNA to Lipid was 1:10, the particle size was 80nm to 130nm, and the encapsulation efficiency was greater than 85%.

[0621] (3) In vivo immunization: 7- to 9-week-old Balb / c female mice (17 g to 22 g, raised in an SPF environment) were divided into a blank LNP group and a test group. The test sample was injected intramuscularly at 10 μg / mouse, wherein each test sample included an equal amount of LNP preparation encapsulating mRNA encoding a subtype A RSV F protein mutant and an LNP preparation encapsulating mRNA encoding a subtype B RSV F protein mutant having the same mutation. The blank LNP group was injected with an LNP preparation without mRNA encapsulation. For example, an LNP preparation encapsulating 5 μg of mRNA numbered 832 and an LNP preparation encapsulating 5 μg of mRNA numbered 973 were mixed and injected into mice. The injection time was recorded as Day 0. Orbital blood was collected on the 7th, 14th, 21st (or 19th), 28th (or 31st) and 35th day after immunization (after injection). The blood was collected in a non-anticoagulant tube, placed on ice for 30 minutes, and centrifuged at 4°C 3500 rpm for 10 minutes. After stratification, the upper layer of light yellow liquid was carefully aspirated with a pipette to prepare immune serum.

[0622] For some experimental batches of mice, spleens were collected on day 28 and day 35, and spleen cells were isolated for subsequent ELISpot detection (Example 8).

[0623] Example 6 IgG antibody titer experiment

[0624] The immune serum used in this example was provided by the experiment in Example 5.

[0625] Reagents: Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein / RSV-F Protein (ECD, His Tag), Human respiratory syncytial virus (RSV) (B, strain 18537) Fusion glycoprotein F0 / RSV-F Protein (His Tag), Goat anti-Mouse IgG (H+L) HRP Conjugate, PBS, FBS, one-component TMB colorimetric solution, stop solution.

[0626] The steps include:

[0627] (1) Dilute RSV A subtype or RSV B subtype F protein to 1 μg / mL using coating solution, mix well, and set aside. Add 100 μL per well to a 96-well ELISA plate, seal with sealing film, and incubate at 2-8°C overnight (16-20 hours).

[0628] (2) After incubation, wash the wells three times with 300 μL of washing solution and pat dry on clean paper.

[0629] (3) Add 250 μL / well of blocking solution to the ELISA plate, seal the plate with a sealing film, and incubate at 37°C for 60 min.

[0630] (4) After blocking, wash the wells three times with 300 μL / well washing solution and pat dry on clean paper.

[0631] (5) Take out the serum that has been separated in advance, vortex mix it, and use it for IgG titer detection.

[0632] (6) Take the separated serum and determine its first dilution factor according to different immunization times. Use this dilution factor as the first dilution factor and make a gradient dilution. Add the diluted serum to the ELISA plate at 100 μL / well and incubate at 37°C for 2 hours.

[0633] (7) After incubation, wash the wells three times with 300 μL / well washing solution and pat dry on clean paper.

[0634] (8) Dilute the enzyme-labeled secondary antibody 10,000-fold with sample diluent, add 100 μL / well, and incubate at 37°C for 60 min.

[0635] (9) After incubation, wash the wells with washing solution 3 times at 300 μL / well and pat dry on clean paper.

[0636] (10) Equilibrate the TMB single-component colorimetric solution to room temperature in advance, add 100 μL to each well, and incubate at room temperature in the dark for 15 min.

[0637] (11) After color development, add 50 μL / well of stop solution.

[0638] (12) Select a detection wavelength of 450 nm and a reference wavelength of 630 nm, and read the results in an enzyme-labeled instrument.

[0639] The IgG antibody titer test results of RSV mRNA vaccines containing different mRNAs are shown in Figures 1A to 6B. Except for the RSV mRNA vaccine represented by 573+567, other RSV mRNA vaccines induced significantly higher IgG antibodies against RSV subtype A F protein and / or RSV subtype B F protein than the blank LNP group (LNP group without mRNA encapsulation).

[0640] In addition, referring to the above steps, the LNP preparation encapsulating 0.1 μg of mRNA numbered 832 and the LNP preparation encapsulating 0.1 μg of mRNA numbered 973 were mixed to obtain a bivalent mRNA vaccine (832+973 0.2 μg). The bivalent mRNA vaccine was injected into BALB / c mice on D0 and D21, and the immune sera were collected on the 14th day (D14) after the first immunization (D0), the 7th day (D28) and the 14th day (D35) after the second immunization (D21), and the IgG antibody titer was detected. At the same time, the IgG antibody titers of other doses of bivalent mRNA vaccines were tested. The other doses of mRNA vaccines were: a bivalent mRNA vaccine (832+973 2μg) obtained by mixing an LNP preparation encapsulating 1μg of mRNA numbered 832 and an LNP preparation encapsulating 1μg of mRNA numbered 973, and a bivalent mRNA vaccine (832+973 10μg) obtained by mixing an LNP preparation encapsulating 5μg of mRNA numbered 832 and an LNP preparation encapsulating 5μg of mRNA numbered 973. The results are shown in Figure 1E.

[0641] Example 7 RSV-Luc5 pseudovirus neutralization experiment

[0642] The immune serum required for the pseudovirus neutralization test was provided by the experiment in Example 5.

[0643] Reagents and consumables: RSV-Luc5 pseudovirus (6.2log 10 TCID50 / mL), mouse serum inactivated by incubation at 56°C for 30 min, Hep2 cells, DMEM, FBS, and Luciferase reporter gene detection reagent.

[0644] The steps include:

[0645] (1) Hep2 cells (3×10 4 The cells were cultured in DMEM medium containing 5% FBS for 24 hours.

[0646] (2) Serum samples were diluted threefold with FBS-free DMEM, with an initial dilution of 1:30. 310 μL of serum-free DMEM medium was added to H7 to H12 of a 96-well deep-well plate as a cell control (CC), and 155 μL of DMEM medium was added to H1 to H6 as a virus control (VC).

[0647] (3) Add 233 μL of diluted serum sample to each of A1 to A12 as the initial dilution. Add 155 μL of serum-free DMEM medium to each of B, C, D, E, F, and G. Take 78 μL of each of A1 to A12 and transfer it to the next row, mix thoroughly, and perform a 3-fold serial dilution for a total of 7 serial dilutions. Discard the excess 78 μL of liquid from the last row.

[0648] (4) Except for H7 to H12, 155 μL of diluted virus mixture was added to each well. The titer of the mixed virus was diluted to 1800 TCID50 / mL and incubated at 37°C for 1 h.

[0649] (5) Add 93 μL of the mixture to each well of the 96-well white plate on which Hep2 cells were plated one day in advance (the cells were washed once with PBS in advance). Make three replicate wells for each dilution and culture for 24 h.

[0650] (6) Remove the 96-well white plate, aspirate the culture medium from the plate, add 100 μL PBS to wash once, and then add 14 μL 1x lysis buffer to incubate at room temperature for 5 minutes. After incubation on ice for 5 minutes, add 70 μL luciferase substrate and immediately detect the chemiluminescence value (RLU) using a microplate reader.

[0651] The neutralizing antibody titer test results of RSV mRNA vaccines containing different mRNA combinations are shown in Figures 7 to 9 and Figure 20.

[0652] In addition, referring to the above steps, an LNP preparation encapsulating 25 μg of mRNA numbered 832 and an LNP preparation encapsulating 25 μg of mRNA numbered 973 were mixed to obtain a bivalent mRNA vaccine (832+973 50 μg). The mRNA vaccine was injected into rats on D0, and the immune serum was collected on the 14th day (D14) and 21st day (D21) after the first immunization (D0). After sampling on the 21st day, the mRNA vaccine was injected again, and the immune serum was collected on the 7th day (D28) and 21st day (D42) after the second immunization (D21), and the pseudovirus neutralizing antibody titer was tested. At the same time, the IgG antibody titers of other mRNA vaccines were tested. The other mRNA vaccines were: a bivalent mRNA vaccine (832+973 75 μg) obtained by mixing an LNP preparation encapsulating 37.5 μg of mRNA numbered 832 and an LNP preparation encapsulating 37.5 μg of mRNA numbered 973, an LNP preparation encapsulating 50 μg of mRNA numbered 1119 (1119 50 μg), and an LNP preparation encapsulating 100 μg of mRNA numbered 1119 (1119 100 μg). The results are shown in Figure 15.

[0653] Example 8 Mouse splenocyte ELISpot experiment

[0654] The mouse spleen cells required for the ELISpot experiment were provided by the experiment in Example 5.

[0655] Reagents: RPMI Medium 1640 basic (1×), mouse lymphocyte separation medium, positive reference PMA + Ionomycin, ELISpot Plus Mouse IFN-gamma (HRP), RSV F peptide library, ELISpot Plus Mouse IL-4 (HRP), red blood cell lysis buffer.

[0656] The steps include:

[0657] (1) The mice were killed by cervical dislocation and immersed in 75% ethanol. The spleens of the mice were removed in a clean bench.

[0658] (2) In a 6-well cell culture plate, add 7 mL of mouse lymphocyte separation solution to each well (return to room temperature and shake well before use). Grind with a syringe piston, using the upward rebound force of the cell screen to control the grinding force and minimize possible mechanical damage to the cells.

[0659] (3) Immediately transfer the separation solution containing suspended spleen cells into a 15 mL centrifuge tube and slowly add 10 mL of RPMI 1640 culture medium, keeping a clear liquid boundary.

[0660] (4) Centrifuge at 800 g in a swing-out rotor for 30 min at room temperature.

[0661] (5) Aspirate the lymphocyte layer, add 10 mL of RPMI 1640 medium, and wash by inversion. Centrifuge at 250 g for 10 min at room temperature to collect the cells.

[0662] (6) Perform erythrocyte lysis according to the instructions of the erythrocyte lysis buffer. Add 2 mL of erythrocyte lysis buffer to each tube and resuspend. Incubate at 4°C for 2 minutes, then add 10 mL of DPBS to terminate the erythrocyte lysis reaction. Centrifuge at 250 g for 10 minutes at 4°C to collect the cells.

[0663] (7) Add 10 mL of RPMI 1640 medium to the cells collected in the previous step and wash by inversion. Centrifuge at 250 g for 10 min at room temperature to collect the cells.

[0664] (8) Finally, resuspend the cells in culture medium, count, and dilute.

[0665] (9) Add the cell suspension with adjusted concentration to each experimental well, 1×10 5 cells / 100 μL / well.

[0666] (10) Add stimulators (PMA+Ionomycin working solution and F protein peptide library (1 μg / mL)), 10 μL / well.

[0667] (11) After all samples and stimulants have been added, cover the plate and place in a 37°C, 5% CO2 incubator for 21 hours.

[0668] (12) Discard the liquid in the wells and add PBS buffer at 200 μL / well. Repeat five times, blot dry on absorbent paper each time.

[0669] (13) Dilute the detection antibody to 1 μg / mL with sample diluent, 100 μL / well. Incubate at room temperature for 2 hours.

[0670] (14) Repeat the above plate washing operation.

[0671] (15) Dilute the enzyme-labeled avidin (Streptavidin-HRP) with sample diluent 1000-fold, 100 μL / well. Incubate at room temperature for 1 hour.

[0672] (16) Repeat the above plate washing operation.

[0673] (17) Equilibrate the TMB substrate colorimetric solution to room temperature in advance, add 100 μL to each well, and incubate at room temperature in the dark for 10 min.

[0674] (18) Pour out the liquid in the wells, remove the base of the plate, and wash the front and back surfaces and the base with deionized water 3 to 5 times to stop the color development. Place the plate in a cool, dark place at room temperature and allow it to dry naturally before closing the base.

[0675] (19) The enzyme-linked immunosorbent assay (ELISA) analyzer reads the plate and records various parameters of the spots for statistical analysis.

[0676] The Elispot test results of RSV mRNA vaccines containing different mRNAs are shown in Figures 10A to 11C. RSV mRNA vaccines containing different mRNAs can all induce the body to produce obvious T cell immune responses, and the frequency of F protein-specific cells secreting IFN-γ is significantly higher than that of F protein-specific cells secreting IL-4, revealing that the immune response is Th1-biased rather than Th2-biased.

[0677] In addition, referring to the above steps, the LNP preparation encapsulating 0.5 μg of mRNA No. 832 and the LNP preparation encapsulating 0.5 μg of mRNA No. 973 were mixed to obtain a bivalent mRNA vaccine (832+973 1 μg), the mRNA vaccine was injected into BALB / c mice on D0 and D21, the spleen cells were taken on D28, and ELISpot detection was performed. The IgG antibody titers of blank LNP (i.e., LNP preparation without mRNA encapsulation) and other doses of bivalent mRNA vaccines were also detected. The other doses of bivalent mRNA vaccines were: a bivalent mRNA vaccine (832+9735μg) obtained by mixing an LNP preparation encapsulating 2.5μg of mRNA No. 832 and an LNP preparation encapsulating 2.5μg of mRNA No. 973, and a bivalent mRNA vaccine (832+973 10μg) obtained by mixing an LNP preparation encapsulating 5μg of mRNA No. 832 and an LNP preparation encapsulating 5μg of mRNA No. 973. The results are shown in Figure 16.

[0678] Example 9 Detection of protein conformation in vitro

[0679] Reagent: Lipofectamine TM MessengerMAX TM Transfection reagent, Opti-MEM TM I reduced serum medium, DMEM basic (1×), FBS, TrypLE Express, DPBS, AM14 (Cambridge Bio, Cat No.: 01-07-0119), D25 (Cambridge Bio, Cat No.: 01-07-0120), AM22 (Cambridge Bio, Cat No.: 01-07-0144), clone 131-2A (Sigma-Aldrich, Cat No.: MAB8599), PE anti-human IgG Fc Recombinant Antibody (PE-IgG), CR9501 (Cambridge Bio, Cat No.: 01-07-0145).

[0680] The steps include:

[0681] (1) Use Lipofectamine TM MessengerMAX TMThe transfection reagent was used to transfect HKE293 cells with the engineered plasmids expressing different RSV F protein mutants prepared in Example 3 or the mRNAs expressing different RSV F protein mutants prepared in Example 4. The transfected cells were incubated in a CO2 incubator at 37°C for 24 h.

[0682] (2) Remove the cell supernatant and add 1 mL of cell digestion solution to each dish. Digest at room temperature for 1 min. Add 1 mL of DPBS to terminate the digestion. Pipette the cell suspension into a 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 3 min. Discard the supernatant.

[0683] (3) After discarding the supernatant, add 1 mL of DPBS to resuspend the solution, centrifuge at 1000 rpm for 3 min, and discard the supernatant.

[0684] (4) Add 100 μL of DPBS containing primary antibodies (AM14, D25, AM22, clone 131-2A) to each well of cells and stain at 4°C in the dark for 60 min; centrifuge at 1000 rpm for 3 min and discard the supernatant.

[0685] (5) Wash the cells once with 200 μL of DPBS per well, centrifuge at 1300 rpm for 3 min at room temperature, and discard the supernatant.

[0686] (6) Add 100 μL of DPBS containing secondary antibody (PE-IgG), stain at 4°C in the dark for 30 min, centrifuge at 1300 rpm for 3 min at room temperature, and discard the supernatant.

[0687] (7) Wash the cells once with 200 μL of DPBS per well, centrifuge at 1300 rpm for 3 min at room temperature, discard the supernatant, resuspend the cells in 200 μL of DPBS, and analyze on the microscope.

[0688] The conformational detection results of F proteins expressed by different mRNAs are shown in Figures 12, 17A to 17B, and 21A to 21D, where Group 7 is the test group expressing Post F (Jason S. McLellan, et al., J Virol., 2011 Aug; 85(15): 7788–7796).

[0689] As can be seen from Figure 12, the proportion of cells binding to antibodies AM14, D25 and AM22 is much higher than the proportion of cells binding to antibody Clone 131-2A. It can be seen that the F proteins expressed by the mRNA are mainly in the pre-fusion conformation, that is, the F proteins expressed by the mRNA construction are mainly pre-F proteins that can serve as ideal vaccine antigens.

[0690] As can be seen from Figures 17A and 17B, the RSV / A subtype F protein expressed after cells were transfected with the mRNA numbered 832, and the RSV / B subtype F protein expressed after cells were transfected with the mRNA numbered 973, were both recognized by the four pre-F-specific antibodies D25, AM22, CR9501, and AM14, indicating that the F proteins encoded by the mRNA numbers 832 and 973 were in the pre-F conformation. At the same time, the F protein produced after cells were transfected with the mRNA encoding the RSV / A subtype post-F protein was not recognized by any of the antibodies used, which was in line with expectations and simultaneously verified the pre-F specificity of the above four antibodies.

[0691] As can be seen from Figures 21A to 21D, the F proteins expressed by most mRNAs are mainly in the pre-fusion conformation, such as the mRNAs numbered 1998, 2000, 2001, 2002, 2003, 2006, 2007, 2008, 2009 and 2010.

[0692] Example 10: Mouse challenge experiment

[0693] Balb / c female mice aged 5 to 6 weeks (housed in a SPF environment) were divided into three groups, each consisting of 6 mice: a PBS group, a FI-RSV group (formalin-inactivated RSV), and an mRNA vaccine group. On days 0 and 21 of the experiment, mice were inoculated intramuscularly with PBS buffer, FI-RSV (100 ng / mouse), and an mRNA vaccine (10 μg / mouse). On day 35, RSV / A2 virus was inoculated intranasally at a dose of 5.0E+05 PFU / mouse. Lung tissue was collected on day 40, and viral titers in the lung tissue were determined using a plaque assay. Lung tissue sections were then sectioned and stained with H&E to detect inflammatory markers.

[0694] 1. Plaque assay to detect RSV titer in lung tissue samples

[0695] (1) Cell plating: Add 1 mL of cells to each well of a 12-well cell plate at a density of 3.0 × 10 5 cells / mL and placed in a CO2 cell incubator overnight to obtain a monolayer of cells.

[0696] (2) Sample incubation: The lung tissue samples collected on the 40th day were thawed and ground with a tissue homogenizer. The supernatant was centrifuged and diluted 2-fold using the experimental culture medium. The first group of samples was diluted 6 times in total, and the last three dilutions were used for detection. 0.05 mL of the tissue homogenate supernatant or its dilution was added to a 12-well plate with cells and incubated in a 37°C cell culture incubator for 4 hours to allow the virus to fully adsorb. The liquid in the 12-well plate cells was replaced with agarose-containing culture medium. After the agar solidified, the cells were placed in a 37°C cell culture incubator and cultured for 7 days.

[0697] (3) Fixation and staining: On the 8th day, cells were fixed with paraformaldehyde at room temperature for 4 h, then removed and rinsed with water. 0.2 mL / well of 0.5% crystal violet solution was added and shaken on a shaker for 15 min. The crystal violet was rinsed with water and then dried.

[0698] (4) Plaque counting: Scan the image and read the number of plaques in it to calculate the virus titer in the sample. The virus titer is expressed as Log 10 (number of plaques per gram of tissue homogenate).

[0699] 2. H&E staining to detect lung tissue inflammation indicators

[0700] Lung tissue sections were sectioned and stained with hematoxylin and eosin (H&E) for pathological analysis. H&E staining was used to assess lung lesions, including perivasculitis (PV), peribronchiolar inflammation (PB), alveolitis (A), and interstitial pneumonia (IP). The specific procedures were as follows: on day 40, lung tissue was fully fixed, harvested, dehydrated, paraffin-embedded, and sectioned. The sections were then baked at 60–65°C, dewaxed, stained with hematoxylin and eosin, and mounted. Finally, manual semiquantitative scoring was performed. The scoring criteria are shown in Table 5.

[0701] Table 5 Histopathology semi-quantitative scoring criteria

[0702] The results of the challenge experiment are shown in Figures 13 and 14. From the results of the RSV virus titer in the lungs of mice immunized with PBS, FI-RSV and mRNA vaccines, it can be seen that compared with the PBS group, the mRNA vaccine containing mRNA832+973 can significantly reduce the RSV virus load in the lungs of mice after challenge. From the lung tissue pathology of mice immunized with PBS, FI-RSV and mRNA vaccines, it can be seen that the mRNA vaccination group did not show enhanced RSV disease (ERD) as shown by the FI-RSV vaccination group. Compared with the FI-RSV vaccination group, the pathological scores of the mRNA vaccination group for interstitial pneumonia, alveolitis, peribronchiolar inflammation and perivasculitis were significantly reduced.

[0703] Example 11: Poison challenge experiment on cotton rats

[0704] Animals: Female cotton rats, 6-8 weeks old.

[0705] On day 0, the anim...

Claims

1. A nucleic acid comprising a polynucleotide for encoding a mutant of an RSV F protein, wherein the mutant comprises an F1 polypeptide and an F2 polypeptide, and relative to a wild-type RSV F protein, the mutant comprises one or more of the following mutations: a disulfide bond mutation, a cavity filling mutation, and an electrostatic mutation.

2. The nucleic acid of claim 1, wherein the disulfide bond mutations comprise one or more of the following: S46C and T311C, A47C and I309C, L48C and V308C, S55C and L188C, V56C and T189C, V56C and V300C, I57C and S190C, I57C and Y299C, T58C and K191C, I59C and L193C, I59C and L297C, E 60C and D194C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, T103C and I148C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, N105C and A147C, V144C and V406C, and S146C and N460C; Preferably, the disulfide bond mutations include one or more of the following: A47C and I309C, I57C and Y299C, P101C and I152C, A102C and I148C, A102C and I152C, T103C and I146C, N104C and G145C, N104C and S146C, N105C and G145C, N105C and S146C, and N105C and A147C.

3. The nucleic acid according to any one of claims 1 to 2, wherein the cavity filling mutation comprises one or more of the following (1) to (3): (1) T at position 54 is substituted by F, W, V, C, A, H, I, Y, L or M; preferably, T at position 54 is substituted by F, W or V; (2) S at position 190 is replaced by V, I, M, F, L, W, Y, H, R, E, A, Q, G, P, T, C, D, N or K; and (3) V at position 296 is replaced by I, L, Y, H, F or M.

4. The nucleic acid according to any one of claims 1 to 3, wherein the electrostatic mutation comprises one or more of the following: (1) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W; and (2) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

5. The nucleic acid according to any one of claims 1 to 4, wherein the mutation of the mutant comprises at least one of a cavity filling mutation and an electrostatic mutation and a disulfide bond mutation; preferably, the mutation of the mutant comprises a cavity filling mutation and a disulfide bond mutation.

6. The nucleic acid according to any one of claims 1 to 5, wherein the mutation of the mutant is selected from the group consisting of (1) to (20): (1) A102C, I152C, T54V, S190M and V296L; (2) A102C, I152C, T54F, S190M, and V296L; (3) A102C, I152C, T54C, S190M and V296L; (4) A102C, I152C, T54A, S190M and V296L; (5) A102C, I152C, T54V, S190F and V296L; (6) A102C, I152C, T54F, S190F and V296L; (7) A102C, I152C, T54C, S190F and V296L; (8) A102C, I152C, T54A, S190F and V296L; (9) P101C, I152C, T54V, S190M and V296L; (10) P101C, I152C, T54F, S190M and V296L; (11) P101C, I152C, T54C, S190M and V296L; (12) P101C, I152C, T54A, S190M and V296L; (13) P101C, I152C, T54V, S190F, and V296L; (14) P101C, I152C, T54F, S190F, and V296L; (15) P101C, I152C, T54C, S190F, and V296L; (16) P101C, I152C, T54A, S190F, and V296L; (17) A102C, I152C, T54V, S190I and V296L; (18) A102C, I152C, T54F, S190I, and V296L; (19) A102C, I152C, T54C, S190I, and V296L; and (20)A102C, I152C, T54A, S190I and V296L.

7. The nucleic acid according to claim 6, wherein the mutant is selected from one of the following (1) to (40): (1) a mutant comprising the amino acid sequence shown in SEQ ID NO: 61 or 62; (2) a mutant comprising mutations A102C, I152C, T54V, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 61 or 62; (3) a mutant comprising the amino acid sequence shown in SEQ ID NO: 63 or 64; (4) a mutant comprising mutations A102C, I152C, T54F, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 63 or 64; (5) a mutant comprising the amino acid sequence shown in SEQ ID NO: 65 or 66; (6) a mutant comprising mutations A102C, I152C, T54C, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 65 or 66; (7) a mutant comprising the amino acid sequence shown in SEQ ID NO: 67 or 68; (8) a mutant comprising mutations A102C, I152C, T54A, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 67 or 68; (9) a mutant comprising the amino acid sequence shown in SEQ ID NO: 69 or 70; (10) a mutant comprising mutations A102C, I152C, T54V, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 69 or 70; (11) a mutant comprising the amino acid sequence shown in SEQ ID NO: 71 or 72; (12) a mutant comprising mutations A102C, I152C, T54F, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 71 or 72; (13) a mutant comprising the amino acid sequence shown in SEQ ID NO: 73 or 74; (14) a mutant comprising mutations A102C, I152C, T54C, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 73 or 74; (15) a mutant comprising the amino acid sequence shown in SEQ ID NO: 75 or 76; (16) a mutant comprising mutations A102C, I152C, T54A, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 75 or 76; (17) a mutant comprising the amino acid sequence shown in SEQ ID NO: 77 or 78; (18) a mutant comprising mutations P101C, I152C, T54V, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 77 or 78; (19) a mutant comprising the amino acid sequence shown in SEQ ID NO: 79 or 80; (20) a mutant comprising mutations P101C, I152C, T54F, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 79 or 80; (21) a mutant comprising the amino acid sequence shown in SEQ ID NO: 81 or 82; (22) a mutant comprising mutations P101C, I152C, T54C, S190M and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 81 or 82; (23) a mutant comprising the amino acid sequence shown in SEQ ID NO: 83 or 84; (24) comprising mutations P101C, I152C, T54A, S190M and V296L and comprising the amino acid sequence as shown in SEQ ID NO: 83 or 84 mutants of amino acid sequences having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identity; (25) a mutant comprising the amino acid sequence shown in SEQ ID NO: 85 or 86; (26) a mutant comprising mutations P101C, I152C, T54V, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 85 or 86; (27) a mutant comprising the amino acid sequence shown in SEQ ID NO: 87 or 88; (28) a mutant comprising mutations P101C, I152C, T54F, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 87 or 88; (29) a mutant comprising the amino acid sequence shown in SEQ ID NO: 89 or 90; (30) a mutant comprising mutations P101C, I152C, T54C, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 89 or 90; (31) a mutant comprising the amino acid sequence shown in SEQ ID NO: 91 or 92; (32) a mutant comprising mutations P101C, I152C, T54A, S190F and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 91 or 92; (33) a mutant comprising the amino acid sequence shown in SEQ ID NO: 93 or 94; (34) a mutant comprising mutations A102C, I152C, T54V, S190I and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 93 or 94; (35) a mutant comprising the amino acid sequence shown in SEQ ID NO: 95 or 96; (36) a mutant comprising mutations A102C, I152C, T54F, S190I and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 95 or 96; (37) a mutant comprising the amino acid sequence shown in SEQ ID NO: 97 or 98; (38) a mutant comprising mutations A102C, I152C, T54C, S190I and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 97 or 98; (39) a mutant comprising the amino acid sequence shown in SEQ ID NO: 99 or 100; and (40) A mutant comprising mutations A102C, I152C, T54A, S190I and V296L and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO:99 or 100.

8. A nucleic acid comprising a polynucleotide encoding a mutant of an RSV F protein, wherein the mutant comprises an F1 polypeptide and an F2 polypeptide, and relative to a wild-type RSV F protein, the mutant comprises one or more mutations of (1) to (8): (1) K or E at position 66 is replaced by I, L, M, F or V; (2) N at position 67 is replaced by I; (3) S at position 213 is replaced by P; (4) S at position 215 is replaced by P; (5) N at position 216 is replaced by P; (6) I at position 217 is replaced by P, F, Y or W; (7) D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W; and (8) V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

9. The nucleic acid according to claim 8, wherein the mutations of the mutant include the following (1) to (3): (1) One of the following mutations: S at position 213 is replaced by P, S at position 215 is replaced by P, N at position 216 is replaced by P, and N at position 217 is replaced by P. The I at position is replaced by P, F, Y or W; (2) one of the following mutations: K or E at position 66 is replaced by I, L, M, F or V and N at position 67 is replaced by I; and (3) One of the following mutations: D at position 486 is replaced by Q, S, H, N, T, P, C, L, I, F or W and V at position 487 is replaced by Q, T, N, I, K, P, R, S, Y, D, F, L, H or C.

10. The nucleic acid according to any one of claims 1 to 9, wherein the mutation of the mutant is selected from the group consisting of (1) to (13): (1) I217P, E66I and D486Q; (2) I217P, N67I and E487Q; (3) I217P, E66I and E487T; (4) S215P, N67I and E487Q; (5) S213P, N67I and E487Q; (6) S213P, E66I and E487T; (7) S213P, E66I and D486Q; (8) N216P, N67I and E487Q; (9) N216P, E66I and E487T; (10) N216P, E66I and D486Q; (11) A102C, I152C, S213P and E66I; (12) P101C, I152C, S213P and E66I; and (13)S213P and E66I.

11. The nucleic acid according to claim 10, wherein the mutant is selected from one of the following (1) to (30): (1) a mutant comprising the amino acid sequence shown in SEQ ID NO: 31 or 32; (2) a mutant comprising mutations N216P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 31 or 32; (3) a mutant comprising the amino acid sequence shown in SEQ ID NO: 33 or 34; (4) a mutant comprising the mutations N216P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 33 or 34; (5) a mutant comprising the amino acid sequence shown in SEQ ID NO: 35 or 36; (6) a mutant comprising mutations N216P, N67I and E487Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 35 or 36; (7) a mutant comprising the amino acid sequence shown in SEQ ID NO: 37 or 38; (8) a mutant comprising mutations S213P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 37 or 38; (9) a mutant comprising the amino acid sequence shown in SEQ ID NO: 39 or 40; (10) a mutant comprising mutations S213P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 39 or 40; (11) a mutant comprising the amino acid sequence shown in SEQ ID NO: 41 or 42; (12) a mutant comprising mutations S213P, N67I and E487Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 41 or 42; (13) a mutant comprising the amino acid sequence shown in SEQ ID NO: 43 or 44; (14) comprising mutations S215P, N67I and E487Q and comprising an amino acid sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% similarity to the amino acid sequence of SEQ ID NO: 43 or 44. or a mutant with an amino acid sequence that is 99.5% identical; (15) a mutant comprising the amino acid sequence shown in SEQ ID NO: 45 or 46; (16) a mutant comprising mutations I217P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 45 or 46; (17) a mutant comprising the amino acid sequence shown in SEQ ID NO: 47 or 48; (18) a mutant comprising mutations I217P, N67I and E487Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 47 or 48; (19) a mutant comprising the amino acid sequence shown in SEQ ID NO: 49 or 50; (20) a mutant comprising mutations I217P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 49 or 50; (21) a mutant comprising the amino acid sequence shown in SEQ ID NO: 51 or 52; (22) a mutant comprising mutations S213P, E66I and E487T and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 51 or 52; (23) a mutant comprising the amino acid sequence shown in SEQ ID NO: 53 or 54; (24) a mutant comprising mutations S213P, E66I and D486Q and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 53 or 54; (25) a mutant comprising the amino acid sequence shown in SEQ ID NO: 55 or 56; (26) a mutant comprising mutations A102C, I152C, S213P and E66I and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 55 or 56; (27) a mutant comprising the amino acid sequence shown in SEQ ID NO: 57 or 58; (28) a mutant comprising mutations P101C, I152C, S213P and E66I and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 57 or 58; (29) a mutant comprising the amino acid sequence shown in SEQ ID NO: 59 or 60; and (30) A mutant comprising mutations S213P and E66I and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO:59 or 60.

12. The nucleic acid according to any one of claims 1 to 11, wherein the wild-type RSV is subtype A or subtype B; Preferably, the wild-type RSV is wild-type hRSV; Preferably, the wild-type hRSV is strain A2, strain Ontario or strain Buenos Aires.

13. The nucleic acid according to any one of claims 1 to 12, wherein the nucleic acid is RNA; preferably, the RNA is mRNA; more preferably, the mRNA comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR and a poly(A) tail; Alternatively, the nucleic acid is DNA; preferably, the DNA is capable of being transcribed into RNA.

14. The nucleic acid according to claim 13, wherein the DNA sequence corresponding to the 5'-UTR is shown in SEQ ID NO: 209; and / or, the DNA sequence corresponding to the 3'-UTR is shown in SEQ ID NO: 210; And / or, the nucleotides constituting the poly(A) tail contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides in succession; preferably, the nucleotides constituting the poly(A) tail contain one or more nucleotides other than A nucleotides; more preferably, the DNA sequence corresponding to the poly(A) tail is as shown in SEQ ID NO:

211.

15. The nucleic acid according to any one of claims 1 to 14, comprising modified nucleotides; Preferably, the nucleic acid contains modified nucleosides; preferably, the modified nucleosides include at least one of modified uridine, modified cytidine, modified adenosine and modified guanosine.

16. The nucleic acid of any one of claims 1 to 15, comprising a polynucleotide encoding one or more mutants of the RSV F protein.

17. The nucleic acid according to any one of claims 1 to 16, further comprising a polynucleotide encoding other proteins or polypeptides other than the mutant of RSV F protein.

18. The nucleic acid according to any one of claims 1 to 17, further comprising one or more of the following: a polynucleotide encoding a transmembrane domain of an RSV F protein, a polynucleotide encoding a pep27 polypeptide, a polynucleotide encoding a signal peptide, and a polynucleotide encoding a trimerization domain.

19. A genetic engineering vector, comprising the nucleic acid according to any one of claims 1 to 18, or comprising a polynucleotide capable of being transcribed into the nucleic acid according to any one of claims 1 to 18.

20. A host cell comprising the genetic engineering vector according to claim 19.

21. A mutant of the RSV F protein encoded by the nucleic acid of any one of claims 1 to 18; Preferably, the mutant is a trimer.

22. A delivery vector comprising a nucleic acid composition, wherein the nucleic acid composition comprises a first nucleic acid or a first genetic engineering vector, wherein the first nucleic acid is the nucleic acid according to any one of claims 1 to 18, and the first genetic engineering vector is the genetic engineering vector according to claim 19; Alternatively, the nucleic acid composition includes multiple first nucleic acids or multiple first genetic engineering vectors, the first nucleic acid is the nucleic acid described in any one of claims 1 to 18, the first genetic engineering vector is the genetic engineering vector described in claim 19, and the multiple first nucleic acids are independent of each other or the multiple first genetic engineering vectors are independent of each other.

23. The delivery vector of claim 22, wherein the nucleic acid composition further comprises a second nucleic acid or a second vector, wherein the second nucleic acid comprises a polynucleotide encoding other proteins or polypeptides other than the mutant of the RSV F protein, and the second vector comprises a polynucleotide encoding other proteins or polypeptides other than the mutant of the RSV F protein; And / or, the first nucleic acid and the second nucleic acid are RNA; more preferably, the first nucleic acid and the second nucleic acid are mRNA.

24. The delivery vector according to any one of claims 22 to 23, wherein the delivery vector is lipid nanoparticles (LNPs), cationic liposomes, cationic proteins or lipid polymers (LPP); Preferably, the delivery vehicle is a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid, wherein the cationic lipid comprises the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof: in: L 3 and L 4 are the same or different, and are each independently C1-C12 alkylene, C2-C12 alkenylene or C2-C12 alkynylene; preferably L 3 and L 4 are the same or different, and are each independently C3-C10 alkylene, C3-C10 alkenylene or C3-C10 alkynylene; further preferably, L 3 and L 4 The same or different, each independently is C3-C10 alkylene; most preferably, L 3 and L 4 The same or different, each independently a C5-C8 alkylene group; G 4 and G 5 are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S- or -SC(=O)-; preferably, G 4 and G 5 are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)- or -O-; most preferably, G 4 and G 5 are the same or different, each independently selected from -O-(C=O)- or -(C=O)-O-; R 18 and R 19 are the same or different, and are each independently a C5-C27 alkyl group, or a C5-C27 alkenyl group containing one or more double bonds; preferably, R 18 and R 19 are the same or different, and are each independently a C8-C20 alkyl group or a C8-C20 alkenyl group containing one or more double bonds; further preferably, R 18 and R 19 are the same or different, and are each independently a C9-C17 alkyl group or a C9-C18 alkenyl group containing one or two double bonds; most preferably, R 18 and R 19 the same or different, each independently R 20 is halogen, hydroxyl, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; preferably, R 20 is halogen, hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; further preferably, R 20 is halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; most preferably, R 20 is fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl or acetamido; z is 1, 2 or 3.

25. The delivery vector according to claim 24, wherein the cationic lipid is the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof: Alternatively, the cationic lipid is the following compound, or a pharmaceutically acceptable salt thereof:

26. A pharmaceutical composition comprising the nucleic acid of any one of claims 1 to 18, the genetically engineered vector of claim 19, the host cell of claim 20, the mutant of the RSV F protein of claim 21 or the delivery vector of any one of claims 22 to 25, and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition according to claim 26, comprising a plurality of said delivery vectors; Alternatively, the pharmaceutical composition comprises a plurality of said nucleic acids.

28. Use of the nucleic acid according to any one of claims 1 to 18, the genetic engineering vector according to claim 19, the host cell according to claim 20, the mutant of RSV F protein according to claim 21, the delivery vector according to any one of claims 22 to 25, or the pharmaceutical composition according to any one of claims 26 to 27 in the preparation of a drug; Preferably, the medicament is used to prevent or treat RSV infection or a disease caused by RSV infection; Preferably, the drug is a vaccine.

29. A vaccine comprising the nucleic acid of any one of claims 1 to 18, the genetic engineering vector of claim 19, the mutant of RSV F protein of claim 21, or the delivery vector of any one of claims 22 to 25.

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