A RSV F protein mutant and its preparation method and application

Abstract

The present invention provides a RSV F protein mutant and its preparation method and application. Specifically, the present invention provides a mutant protein, the mutant protein is in monomeric form or trimer form, wherein the trimer is formed by three mutant proteins in monomeric form; wherein the mutant protein in monomeric form is a fusion protein of the first polypeptide element and the trimerization domain element derived from RSV F protein; wherein, in the first polypeptide element, the Pep27 fragment of RSV F protein is replaced by a linker sequence; and the mutant proteins in monomeric and trimer forms are both bound by antibodies that specifically recognize RSV pre-F. The mutant protein of the present invention can be stably in a pre-fusion state, can induce the production of specific IgG antibodies and neutralizing antibodies, has excellent immunogenicity, and can be used to develop RSV vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a RSV F protein mutant and a preparation method and application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is a pathogen that mainly causes respiratory infections in infants and adults over 65 years old. Infected people will experience clinical symptoms such as fever, cough and dyspnea. Vaccination is an important means to prevent the disease, but there is currently no safe and effective RSV vaccine on the market in China.

[0003] RSV belongs to the family Paramyxoviridae and the genus Pneumovirus. It is an enveloped single-stranded negative-sense RNA virus. The RSV genome is about 15 kb long and can encode 11 proteins, including nine structural proteins and two non-structural proteins. The structural proteins include three transmembrane surface glycoproteins: small hydrophobic protein (SH), attachment protein (G) and fusion protein (F). The SH protein promotes host cell membrane permeability by forming ion channels. The G protein interacts with target cell receptors to attach virus particles to respiratory epithelial cells and immune cells. The F protein mediates the fusion of the viral membrane with the host cell membrane and has been shown to interact with nucleolin. In addition, the F protein expressed on the surface of infected cells also mediates the mutual fusion between neighboring cells, leading to the formation of syncytia. F protein and G protein are currently known RSV antigens that can induce the production of neutralizing antibodies. However, unlike the G protein, the F protein plays a key role in infection, has high neutralizing properties, and is highly conserved between virus subtypes, making it the main target of RSV vaccine research.

[0004] The F protein is a type I transmembrane protein with a total length of 574 amino acids, including a signal peptide (aa2-20), a signal peptide cleavage site (aa21-25), an F2 region (aa26-109), a polypeptide cleavage region (aa110-136), and an F1 region (aa137-574). The initial form of the F protein is an inactive precursor F0. In order to exercise its fusion function, the F0 precursor will be double-cleaved by a furin-like protease at sites 109 and 136, and after removing the 27-aa peptide (p27), two subunits F1 and F2 are produced. The F1 and F2 subunits are connected by two disulfide bonds to form an F1-F2 heterodimer. The F0 protein without the signal peptide sequence and the uncut P27 or the F1-F2 heterodimer is the RSV F protomer, and the RSV F protomer trimerizes to form the final RSV F protein complex.

[0005] The mature F protein is expressed as a metastable prefusion conformation on the virus particle or cell membrane, and can be easily rearranged into a low-energy postfusion conformation. During viral fusion and entry, the F protein changes from a metastable prefusion conformation (pre-F) to a stable postfusion conformation (post-F). The prefusion protein conformation consists of six potential antigenic sites (Ø, IV). In the process of rearrangement from the prefusion conformation to the postfusion conformation, the antigenic epitopes Ø and V are lost, and the antigenic epitope Ø is a highly neutralization-sensitive epitope. Studies have shown that according to different forms of RSV vaccines designed based on the two conformations, the neutralizing antibodies induced by the prefusion conformation F protein can prevent the fusion between the virus and the cell; while the antibodies induced by the postfusion conformation F protein are not very effective. However, compared with the postfusion conformation, the prefusion conformation is extremely unstable, and the artificially synthesized wild-type sequence RSV F subunit vaccine spontaneously or quickly changes conformation to the postfusion conformation.

[0006] Therefore, there is an urgent need in the art to develop an immunogen that maintains the pre-fusion conformation of the F protein and improves its stability, as well as a composition comprising such an immunogen, such as a vaccine. Summary of the invention

[0007] The object of the present invention is to provide a RSV F protein mutant, by optimizing the amino acid sequence of the wild-type RSV F protein, to obtain a pre-F mutant protein sequence that can stably express the pre-fusion conformation trimer protein; the mutant protein sequence can be used to express RSV pre-F mutant protein; the mutant protein sequence can be applied to the development of RSV vaccines including but not limited to subunit vaccines or mRNA vaccines.

[0008] In a first aspect of the present invention, a mutant protein is provided, wherein the mutant protein is in a monomeric form or a trimer form, wherein the trimer is formed by three mutant proteins in the monomeric form;

[0009] Wherein, the mutant protein in monomeric form is a fusion protein derived from the first polypeptide element and the trimerization domain element of the RSV F protein;

[0010] wherein, in the first polypeptide element, the Pep27 fragment of the RSV F protein is replaced by a linker sequence;

[0011] Furthermore, both the monomeric and trimeric mutant proteins are bound by antibodies that specifically recognize RSV pre-F.

[0012] In another preferred example, the affinity A1 of the mutant protein in monomeric or trimeric form to the first antibody is significantly higher than the affinity A0 of the wild-type RSV F protein (as shown in SEQ ID NO: 1) or the protein shown in SEQ ID NO: 2 to the first antibody; wherein the first antibody is an antibody that specifically recognizes RSV pre-F.

[0013] In another preferred embodiment, the significantly higher refers to: A1 / A0≥150%, preferably ≥200%, such as 150%-500%.

[0014] In another preferred embodiment, compared with the wild-type RSV F protein (as shown in SEQ ID NO: 1), in the first polypeptide element, the amino acid sequence of SCSISN (SEQ ID NO: 3) at position 211 to position 216 mutates to SCSPPN, SCSISP, SPSISN, SCPPSN, PCSISN or SPPISN.

[0015] In another preferred embodiment, the antibody that specifically recognizes RSV pre-F is selected from the following group: AM14 antibody, D25 antibody, or a combination thereof.

[0016] In another preferred embodiment, the mutant protein has an amino acid mutation that reduces helix fusion in the Loop region of the RSV F protein.

[0017] In another preferred embodiment, the Loop region is positions 211-216 of the amino acid sequence of the wild-type RSV F protein (as shown in SEQ ID NO: 1).

[0018] In another preferred embodiment, the amino acid mutation that reduces helix fusion is the introduction of 1-2 proline (P) mutations in the Loop region.

[0019] In another preferred embodiment, relative to the amino acid sequence of the wild-type RSV F protein (such as SEQ ID NO: 1), the mutant protein has a mutation selected from the following group A:

[0020] (Z1) loop region mutation, wherein the mutation is an amino acid mutation that reduces helix fusion;

[0021] (Z2) optional disulfide bond mutation;

[0022] (Z3) The combination of Z1 and Z2.

[0023] In another preferred embodiment, the mutant protein further contains a mutation selected from the following group B: cavity filling mutation, electrostatic mutation, or a combination thereof.

[0024] In another preferred embodiment, the mutant protein comprises at least one amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein (such as SEQ ID NO: 1), and wherein the amino acid mutation is selected from one or more of the following groups:

[0025] (1) Disulfide bond mutation;

[0026] (2) loop region mutations;

[0027] (3) cavity filling mutation;

[0028] (4) Electrostatic mutation.

[0029] In another preferred embodiment, the mutant protein contains the following mutations:

[0030] (1) Disulfide bond mutation;

[0031] (2) loop region mutations;

[0032] (3) cavity-filling mutations; and

[0033] (4) Electrostatic mutation.

[0034] In another preferred embodiment, the amino acid mutation comprises at least one disulfide bond mutation, at least one cavity filling mutation and at least one electrostatic mutation.

[0035] In another preferred embodiment, the amino acid mutation comprises at least one disulfide bond mutation, at least one loop region mutation, at least one cavity filling mutation and at least one electrostatic mutation.

[0036] In another preferred embodiment, the disulfide bond mutation is selected from one or more combinations of the following groups:

[0037] A170C and V179C; V154C and V300C; S155C and I288C; S155C and V300C; L158C and S290C; L158C and I291C; L158C and A298C; V164C and V296C; K168C and V296C; A170C and A177C; Y198C and K226C; F237C and M289C; V243C and I288C; T244C and S287C; L257C and V278C; L258C and S275C; I280C and K359C; I280C and F366C; I288C and G151C; V300C and G15 1C; Q302C and G151C; F32C and Y441C; K75C and V220C; L78C and V220C; E82C and F223C; E82C and Q224C; E82C and N227C; K85C and L231C; Y86C and N227C; A89C and L230C; A89C and L231C; A89C and T234C; E92C and T234C; E92C and S238C; L93C and T234C; L93C and I292C; Q94C and I292C; L96C and F237C; L96C and M289C; M97C and I291C; and M97C and I292C.

[0038] In another preferred embodiment, the loop region includes the amino acid sequence of SCSISN (SEQ ID NO: 3) from position 211 to position 216.

[0039] In another preferred embodiment, the loop region mutation is selected from one or more combinations of the following groups:

[0040] (1) S at position 211, 213 or 215 is substituted by G, A, V, L, I, P or F;

[0041] (2) C at position 212 is substituted by G, A, V, L, I, P, or F;

[0042] (3) I at position 214 is substituted with G, A, V, L, P, or F;

[0043] (4) The N at position 216 is substituted by G, A, V, L, I, P or F.

[0044] In another preferred embodiment, the loop region mutation is selected from one or more combinations of the following groups:

[0045] (1) S at position 211, 213 or 215 is substituted by P;

[0046] (2) C at position 212 is replaced by P;

[0047] (3) I at position 214 was replaced by P;

[0048] (4) The N at position 216 is substituted by P.

[0049] In another preferred embodiment, the loop region mutation is selected from the following group:

[0050] SCSPPN, SCSISP, SPSISN, SCPPSN, PCSISN or SPPISN.

[0051] In another preferred embodiment, the cavity filling mutation is selected from one or more combinations of the following groups:

[0052] (1) S at position 55, 62 or 290 is substituted by I, Y, L or W;

[0053] (2) T at position 54, 58, 189, 219 or 397 is replaced by I, Y, L or W;

[0054] (3) A at position 147 or 298 is substituted by I, Y, L or W;

[0055] (4) V at position 164, 187, 192, 207, 220, 296, 300 or 495 is substituted with I, Y, L or W;

[0056] (5) R at position 106 is substituted by I, Y, L or W.

[0057] In another preferred embodiment, the electrostatic mutation is selected from one or more combinations of the following groups:

[0058] (1) E at position 82, 92 or 487 is replaced by F, T, H or W;

[0059] (2) K at position 315, 394, or 399 is substituted by F, T, H, or W;

[0060] (3) D at position 392, 486, or 489 is substituted by F, T, H, or W;

[0061] (4) R at position 106 or 339 is substituted by F, T, H or W;

[0062] (5) E at position 161 is substituted by F, T, H, or W;

[0063] (6) The S at position 182 or 173 is substituted by F, T, H or W.

[0064] In another preferred embodiment, the amino acid mutation is one or more combinations selected from the following groups:

[0065] (1) Combination of I288C, G151C, I214P and S215P;

[0066] (2) combination of L158C, A298C, and N216P;

[0067] (3) Combination of F237C, M289C and C212P;

[0068] (4) combination of K168C, V296C, I214P and S215P;

[0069] (5) A170C, V179C, S213P and I214P combination;

[0070] (6) Combination of E82C, Q224C and S211P;

[0071] (7) A170C, V179C, I214P and S215P combination;

[0072] (8) Combination of A170C, A177C, I214P and S215P;

[0073] (9) Combination of L158C, A298C, C212P and S213P;

[0074] (10) combination of Y198C, K226C, I214P and S215P;

[0075] (11) a combination of Y198C, K226C, I214P, S215P and R106F;

[0076] (12) combination of Y198C, K226C, I214P, S215P and K399T;

[0077] (13) combination of Y198C, K226C, I214P, S215P, R106F and K399T;

[0078] (14) a combination of I288C, G151C, I214P, S215P, R106F, and K399T;

[0079] (15) A170C, V179C, S213P, I214P and R106F combination;

[0080] (16) A170C, V179C, S213P, I214P and K399T combination;

[0081] (17) Combination of A170C, V179C, S213P, I214P, R106F and K399T.

[0082] In another preferred embodiment, the amino acid mutation is one or more combinations selected from the following groups:

[0083] (1) Combination of Y198C, K226C, I214P and S215P;

[0084] (2) combination of Y198C, K226C, I214P, S215P, and K399T;

[0085] (3) combination of Y198C, K226C, I214P, S215P, R106F, and K399T;

[0086] (4) Combination of I288C, G151C, I214P and S215P;

[0087] (5) a combination of I288C, G151C, I214P, S215P, R106F, and K399T;

[0088] (6) Combination of A170C, V179C, S213P and I214P.

[0089] In another preferred embodiment, the mutant protein is in the form of a trimer.

[0090] In another preferred embodiment, the mutant protein comprises a pep27 sequence deleted and an F1 polypeptide and an F2 polypeptide connected by a linker sequence.

[0091] In another preferred example, the pep27 sequence refers to the amino acid sequence of ELPRFMNYTLNNAKKTNVTLSKKRKRR (SEQ ID NO: 4) from position 110 to 136 in the wild-type protein.

[0092] In another preferred example, the linker sequence is selected from the amino acid sequence of SGSGR (SEQ ID NO: 5).

[0093] In another preferred example, the F2 polypeptide is selected from the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0094] In another preferred embodiment, the F1 polypeptide is selected from the amino acid sequence shown in any one of SEQ ID NOs: 19-28.

[0095] In another preferred embodiment, the mutant protein comprises an F1 polypeptide and an F2 polypeptide selected from the following group:

[0096] (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 19;

[0097] (2) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19;

[0098] (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 20;

[0099] (4) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20;

[0100] (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 21;

[0101] (6) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21;

[0102] (7) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 22;

[0103] (8) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22;

[0104] (9) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 23;

[0105] (10) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 23;

[0106] (11) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 24;

[0107] (12) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24;

[0108] (13) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 25;

[0109] (14) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25;

[0110] (15) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 26;

[0111] (16) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26;

[0112] (17) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 27;

[0113] (18) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27;

[0114] (19) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 18 and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 28;

[0115] (20) an F2 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18 and an F1 polypeptide comprising an amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.

[0116] In another preferred embodiment, the mutant protein is selected from the following group:

[0117] (1) the amino acid sequence shown in any one of SEQ ID NOs: 6-15;

[0118] (2) An amino acid sequence that is at least 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 6-15.

[0119] In another preferred embodiment, the amino acid sequence of the mutant protein is expressed by fusion via a signal peptide, an epitope or a heterologous multimerization element.

[0120] In another preferred embodiment, the heterologous multimerization element is a trimer element.

[0121] In another preferred embodiment, the source of the trimer element is selected from the following group: influenza virus HA, coronavirus spike, HIV gp41, NadA, GCN4, GCN4 mutant, ATCase, T4 fibritin fold, TNC, or a combination thereof.

[0122] In another preferred example, the sequence of the T4 fibritin foldon is shown as SEQ ID NO:16.

[0123] In another preferred embodiment, the wild-type RSV F protein is subtype A, subtype B, strain A2, strain Ontario or strain Buenos Aires.

[0124] In the second aspect of the present invention, a polynucleotide encoding the mutant protein described in the first aspect of the present invention is provided.

[0125] In the third aspect of the present invention, a vector is provided, wherein the vector comprises the polynucleotide described in the second aspect of the present invention.

[0126] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell expresses the mutant protein described in the first aspect of the present invention, or comprises the polynucleotide described in the second aspect of the present invention, or comprises the vector described in the third aspect of the present invention.

[0127] In another preferred embodiment, the cell is a prokaryotic or eukaryotic cell (such as Escherichia coli, yeast, plant cell, CHO cell, etc.).

[0128] In another preferred embodiment, the cell has the polynucleotide described in the second aspect of the present invention integrated into its genome.

[0129] In a fifth aspect of the present invention, a method for preparing the mutant protein according to the first aspect of the present invention is provided, comprising the steps of:

[0130] (a) culturing the host cell according to the fourth aspect of the present invention under conditions suitable for expression, thereby producing the mutant protein according to the first aspect of the present invention.

[0131] In another preferred embodiment, the method further comprises: (b) isolating or purifying the mutant protein from the culture system.

[0132] In the sixth aspect of the present invention, a pharmaceutical composition is provided, comprising (a) the mutant protein according to the first aspect of the present invention, a polynucleotide encoding the mutant protein, or a vector expressing the mutant protein; and (b) a pharmaceutically acceptable carrier.

[0133] In another preferred embodiment, the pharmaceutical composition includes a vaccine composition.

[0134] In the seventh aspect of the present invention, there is provided a use of the mutant protein or the polynucleotide encoding the mutant protein or the vector expressing the mutant protein as described in the first aspect of the present invention for preparing a composition or preparation, wherein the composition or preparation is used for:

[0135] (a) preventing and / or treating RSV infection; and / or

[0136] (b) increasing the conformation of RSV pre-F protein or pre-fusion protein and / or decreasing the conformation of RSV post-F protein or post-fusion protein in the cell.

[0137] In another preferred embodiment, the composition is a pharmaceutical composition.

[0138] In another preferred embodiment, the composition is a RSV vaccine.

[0139] In another preferred embodiment, the RSV vaccine includes but is not limited to a subunit vaccine or an mRNA vaccine.

[0140] In another preferred embodiment, the preparation is a laboratory preparation.

[0141] In another preferred embodiment, the composition is administered to RSV-infected patients whose RSV pre-F protein or pre-fusion protein conformation is low and / or whose RSV post-F protein or post-fusion protein conformation is high.

[0142] In an eighth aspect of the present invention, a kit is provided, comprising the mutant protein described in the first aspect of the present invention.

[0143] In another preferred embodiment, the kit further comprises instructions.

[0144] In another preferred embodiment, the kit further comprises a detection reagent for detecting the content of RSV pre-F protein.

[0145] In the ninth aspect of the present invention, a method for preventing or treating RSV infection in an individual is provided, comprising administering to the individual an effective amount of the pharmaceutical composition according to the sixth aspect of the present invention.

[0146] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] Figure 1 The schematic diagram of the F protein Loop structure and the amino acid sequence are shown, where Figure A shows the schematic diagram of the F protein Loop structure; Figure B shows that based on computational structural biology, it can be seen that the three colored LOOP regions (160aa~161aa, 172aa~175aa, 211aa~216 aa) are the key positions to prevent Helix fusion.

[0148] Figure 2 The plasmid identification result diagram in the embodiment of the present invention is shown, wherein, band 1: DNA Ladder 5000; band 2: SVR79 plasmid; band 3: EcoRI / HindIII restriction plasmid 4.3K / 1.6K, wherein the plasmid backbone size is 4.3K and the pre-F size is 1.6K.

[0149] Figure 3 The SDS-PAGE result diagram in the embodiment of the present invention is shown, wherein MK: marker; R: SVR79 protein.

[0150] Figure 4 The HPLC result graphs in the examples of the present invention are shown, wherein graph A represents the HPLC result of SVR79, wherein 10.997 represents the peak time of the SVR79 F protein trimer; and graph B represents the HPLC result of BSA, wherein 11.444 and 12.557 represent the peak times of the BSA monomer and dimer, respectively.

[0151] Figure 5The figure shows the result of sandwich ELISA antigen detection of the pre-fusion F mutant (SVR79) provided by the present invention, wherein the supernatant refers to the cell supernatant after SVR79 plasmid transfection.

[0152] Figure 6 The statistical analysis result graph of proline mutation, affinity and expression in the present invention is shown, wherein the horizontal axis is the mutation site relative to the wild-type RSV F protein amino acid sequence as shown in SEQ ID NO: 1, for example, 211P indicates that the 211th position relative to the wild-type RSV F protein amino acid sequence as shown in SEQ ID NO: 1 is mutated to proline (P), and so on; the vertical axis is the frequency of occurrence of each mutation site in all mutants, and RSVFC represents the control protein (its sequence is shown in SEQ ID NO.2). Among all mutants, statistics are performed on mutants with affinity>RSVFC (a total of 67 mutants), expression>1.0 ng / mL (a total of 63 mutants), and expression>2.0 ng / mL (a total of 45 mutants). Among them, the mutant with high affinity: 214P-215P has the highest frequency of occurrence (14 / 67), and the mutant with high expression: 214P-215P has the highest frequency of occurrence (10 / 45).

[0153] Figure 7 The statistical analysis result graph of disulfide bond mutation, affinity and expression in the present invention is shown, wherein the horizontal axis is the mutation site relative to the wild-type RSV F protein amino acid sequence as shown in SEQ ID NO: 1, for example, 154C-300C means that the 154th position and the 300th position relative to the wild-type RSV F protein amino acid sequence as shown in SEQ ID NO: 1 are mutated to cysteine ​​(C) to form a disulfide bond, and the same applies to the others; the vertical axis is the frequency of occurrence of each mutation site in all mutants, and RSVFC represents the control protein (sequence as shown in SEQ ID NO.2). Among all mutants, statistics were performed on mutants with affinity>RSVFC (a total of 67 mutants), expression>1.0 ng / mL (a total of 63 mutants), and expression>2.0ng / mL (a total of 45 mutants). Among them, the mutants with high affinity: 258C-275C and 288C-151C appeared most frequently (5 / 67), and the mutants with high expression level: 198C-226C and 97C-291C appeared most frequently (6 / 45), where "C" represents.

[0154] Figure 8The figure shows the RSV pre-F specific IgG antibody titer of the first round of screening in the mouse serum in the embodiment of the present invention; wherein A401 is an adjuvant; compared with the positive control group (DS-Cav1), the mutant SVR79 can induce the mouse body to produce a higher titer of RSV pre-F specific binding antibody (10 7 ), the immunogenicity is optimal.

[0155] Fig. 9 The figure shows the RSV pre-F specific IgG antibody titer of the second round of screening in mouse serum in the embodiment of the present invention; wherein MF59 is an adjuvant; compared with the positive control group (DS-Cav1), the mutant SVR79 can induce the mouse body to produce a higher titer of RSV pre-F specific binding antibody (10 6 ), the immunogenicity is better.

[0156] Fig.10 The results of the second round of screening of mouse serum neutralizing antibody levels in the examples of the present invention are shown, wherein A401 and MF59 are adjuvants; DS-Cav1 is used as a positive control.

[0157] Fig.11 The results of the cotton rat immunization experiment in the embodiments of the present invention are shown, wherein Figure A represents the RSV pre-F specific IgG antibody titer results, wherein D0 represents the day of immunization, D21 represents the 21st day after the first immunization, D49 represents the 49th day after the first immunization, and Figure B represents the neutralizing antibody test results of the serum sample on the 49th day, wherein DS-Cav1 is used as the positive control of the antibody; AS01E represents the adjuvant, mRNA-SVR79 represents the mRNA vaccine of SVR79; mRNA-1345 represents a commercial vaccine for preventing RSV as a positive control of the mRNA vaccine. DETAILED DESCRIPTION

[0158] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly obtained a class of RSV mutant proteins with unique structures (referred to as "mutant proteins of the present invention"). On the one hand, the mutant proteins of the present invention can be stably in a fusion precursor state, can form a trimer form similar to the RSV pre-fusion conformation trimer protein pre-F, and have significantly improved affinity with antibodies that specifically recognize RSV pre-F. Animal experiments have verified that the mutant proteins of the present invention can induce the production of specific IgG antibodies and neutralizing antibodies, have excellent immunogenicity, and can be used to develop RSV vaccines. On this basis, the inventors have completed the present invention.

[0159] the term

[0160] As used herein, the term "respiratory syncytial virus" or "RSV" belongs to the genus Pneumovirus of the family Paramyxoviridae. The virus is usually an important viral pathogen causing acute lower respiratory tract infection (ALRI) in infants, the elderly and immunocompromised adults, and can cause interstitial pneumonia and bronchiolitis.

[0161] As used herein, the terms "mutant protein", "mutant", and "protein mutant" are used interchangeably and refer to proteins whose amino acid sequence or protein structure has one or more changes compared to the wild-type protein. The changes may include: deletion, insertion, substitution, truncation and / or deletion of one or more amino acids, processing or cutting of protein structure. In the present application, protein mutant refers to respiratory syncytial virus (RSV) pre-F protein mutant.

[0162] As used herein, the term "amino acid mutation" generally refers to the modification of an amino acid in a parent amino acid sequence. For example, the modification may include replacement, insertion and / or deletion of one or more amino acids. In the present application, the amino acid mutation may include deletion or replacement of at least one amino acid residue at a specified position of an amino acid sequence. In certain embodiments, the amino acid mutation can optimize the conformation of a protein composed of the amino acid sequence. Amino acid mutations may be produced using genetic or chemical methods well known in the art. For example, genetic methods may include site-directed mutagenesis, PCR, and gene synthesis, etc.

[0163] As used herein, the term "trimer" generally refers to a protein structure composed of three protein subunits of the same type or different types, and the protein trimer can be connected together by a special chemical structure. In the present application, the protein trimer can be the F protein of respiratory syncytial virus.

[0164] As used herein, the term "conformational change" generally refers to a change in the spatial structure of a protein molecule. For example, the conformational change may include a change in a chemical bond in a protein molecule, or a change in the folding mode of a polypeptide.

[0165] As used herein, the term "signal peptide" generally refers to an amino acid sequence present at the N-terminus of a transmembrane protein as a signal when it crosses the membrane. For example, the transmembrane protein may include a secretory protein or a cell membrane protein. For example, the signal peptide can be synthesized at the N-terminus of the transmembrane protein in the form of a precursor polypeptide.

[0166] As used herein, the term "structural protein" generally refers to a protein that constitutes a component of a virus particle. The structural protein may include a structural protein of a respiratory syncytial virus. The structural protein described in the present application may include an F protein, a G protein, and an SH protein.

[0167] As used herein, the term "pre-fusion conformation" generally refers to the conformation of the structural protein of the virus before it infects the host cell and membrane fusion has not yet occurred. Generally speaking, the wild-type RSV F protein is a metastable protein before fusion, and its conformation undergoes discontinuous, gradual and irreversible conformational changes to a lower energy stable state conformation (post-fusion conformation) after the virus infects the host cell and membrane fusion occurs.

[0168] In this application, the expression of protein mutation sites is usually expressed as "amino acid + amino acid position + amino acid after mutation". In this application, the mutation may include but is not limited to the addition, replacement, deletion and / or deletion of amino acids. For example, the term "E218C" usually refers to the mutation of glutamic acid (E) at position 218 to cysteine ​​(C).

[0169] As used herein, the term "polynucleotide" generally refers to isolated forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either isolated from their natural environment or artificially synthesized.

[0170] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it to enter cells or assist the target element to be integrated into host cells, such as virus particles, liposomes, protein coats or integrase, but not limited to these substances.

[0171] As used herein, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or illness. The pharmaceutical composition may include RSV F protein mutants described herein, polynucleotide molecules described herein, carriers described herein and / or cells described herein, and optionally pharmaceutically acceptable carriers. In addition, the pharmaceutical composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are usually nontoxic to the recipient at the dosage and concentration used.

[0172] As used herein, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium, and / or nonionic surfactants.

[0173] As used herein, the term "comprising" generally means including the features specifically specified, but not excluding other elements.

[0174] This application is based on structural biology and combined with immunological principles to design a new type of protective vaccine with high safety, wide protection and reliable preparation process. The stability of the recombinant protein vaccine provided in this application is significantly improved. The RSV F protein mutant can be stably maintained in a fusion precursor state, and can induce higher neutralizing antibody levels in animal immune tests when used alone or with a suitable adjuvant.

[0175] As used herein, the term "adjuvant" generally refers to any substance that assists or regulates the action of a drug, including but not limited to immunological adjuvants, which enhance the immune response to an antigen or diversify the immune response. In the present application, the adjuvant can be used to enhance the antigenicity of the RSV F protein mutant. In some embodiments, the adjuvant can include a mineral (e.g., alum, aluminum hydroxide, or phosphate) suspension. In some embodiments, the adjuvant can include an oil-in-water emulsion. In some embodiments, the adjuvant can include a liposome. In some embodiments, the adjuvant can include an immunostimulant such as A401, MF59.

[0176] As used herein, the term "vaccine" refers to a pharmaceutical composition comprising an immunogen capable of eliciting a prophylactic or therapeutic immune response in an individual. Typically, a vaccine elicits an antigen-specific immune response against a pathogen, such as a viral pathogen.

[0177] As used herein, the terms "pre-fusion specific antibody", "first antibody", "antibody that specifically recognizes RSV pre-F" are used interchangeably and refer to antibodies that specifically bind to RSV F glycoprotein in a pre-fusion conformation but do not bind to RSV F protein in a post-fusion conformation. Exemplary pre-fusion specific antibodies include (but are not limited to): D25 and AM14 antibodies.

[0178] In the present application, in the case of an antibody binding to a given target molecule, the term "specific binding" means that the antibody binds to the target molecule with a higher affinity than it binds to other substances tested. For example, an antibody that specifically binds to the RSV F protein in a prefusion conformation is an antibody that binds to the RSV F protein in a prefusion conformation with a higher affinity than it binds to the RSV F protein in a postfusion conformation.

[0179] As used herein, the term "DSCavI" is a molecule described in the document "McLellan, JS, et al. (2013). "Structurebased design of a fusion glycoprotein vaccine for respiratory syncytialvirus." Science 342(6158): 592 598".

[0180] As used herein, the term "D25" refers to the antibody described in WO 2008 / 147196 A2, which is capable of specifically recognizing the F protein of the respiratory syncytial virus in the prefusion conformation.

[0181] As used herein, the term "AM14" refers to the antibody described in the document "Wayne Harshbarger., et al. (2021) Improved epitope resolution of the prefusion trimer specific antibody AM14 bound to the RSV F glycoprotein, mAbs, 13:1, DOI: 10.1080 / 19420862.2021.1955812", which only binds to the respiratory syncytial virus F protein that is trimerized and in the prefusion conformation, and does not bind to the monomeric fusion F protein.

[0182] RSV pre-F protein

[0183] The F protein is a type I transmembrane protein with a total length of 574 amino acids, including a signal peptide (aa2-20), a signal peptide cleavage site (aa21-25), an F2 region (aa26-109), a polypeptide cleavage region (aa110-136), and an F1 region (aa137-574). The initial form of the F protein is an inactive precursor F0. In order to exercise its fusion function, the F0 precursor will be double-cleaved by a furin-like protease at sites 109 and 136, and after removing the 27-aa peptide (p27), two subunits F1 and F2 are produced. The F1 and F2 subunits are connected by two disulfide bonds to form an F1-F2 heterodimer. The F0 protein without the signal peptide sequence and the uncut P27 or the F1-F2 heterodimer is the RSV F protomer, and the RSV F protomer trimerizes to form the final RSV F protein complex.

[0184] The mature F protein is expressed as a metastable prefusion conformation on viral particles or cell membranes, and can be easily rearranged into a low-energy postfusion conformation. The prefusion protein conformation consists of 6 potential antigenic sites (Ø, IV). In the process of rearrangement of the prefusion conformation to the postfusion conformation, the epitopes Ø and V are lost, and the epitope Ø is a highly neutralization-sensitive epitope. Studies have shown that according to different forms of RSV vaccines designed based on the two conformations, the neutralizing antibodies induced by the prefusion conformation F protein can prevent the fusion between the virus and cells, while the antibodies induced by the postfusion conformation F protein are not very effective.

[0185] RSV F protein mutants

[0186] In some aspects, the present invention provides a mutant of a wild-type RSV F protein, wherein the mutant exhibits a mutation introduced in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type RSV F protein and has immunogenicity for a wild-type RSV F protein or for a virus comprising a wild-type F protein. In certain embodiments, the RSV F mutant has certain beneficial features, such as an enhanced immunogenicity characteristic compared to the corresponding wild-type F protein or an improved stability of a pre-fusion configuration of a mutant or a pre-fusion trimer configuration of a mutant. In other embodiments, the present disclosure provides a RSV F mutant, which exhibits one or more mutations introduced as described herein and is combined with a pre-fusion specific antibody selected from antibody D25 or antibody AM14.

[0187] The amino acid mutation introduced in RSV F protein mutants includes amino acid substitution, deletion or addition.In some embodiments, the only mutation in the amino acid sequence of the mutant is an amino acid substitution relative to wild-type RSV F protein.

[0188] Natural RSV F protein shows significant sequence conservation between RSV subtypes. For example, RSV subtypes A and B share 90% sequence identity, and between F0 precursor molecules, RSV subtypes A and B each share 81% sequence identity with cattle RSV F protein. Within RSV subtypes, F0 sequence identity is even higher; For example, in RSV A, B and cattle subtypes, RSV F0 precursor protein has about 98% sequence identity. Almost all identified RSV F0 precursor sequences are composed of 574 amino acid lengths, and the length is usually slightly different due to the length of the C-terminal cytoplasmic tail region. The sequence identity between various natural RSV F proteins is known in the art (see, for example, WO2014 / 160463).

[0189] 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 almost 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 proteins.For purposes of this disclosure (unless otherwise indicated by context), RSV F protein amino acid position is with reference to the sequence (Gene bank: ACO83301.1) of F0 precursor polypeptide described in SEQ ID NO:1.However, it should be noted that, and it will be appreciated by those skilled in the art that, for example, if additional amino acid residues are added or removed compared to SEQ ID NO:1, different RSV F0 sequences can have different numbering systems. Thus, it should be understood that when referring to a particular amino acid residue by its number, the description is not limited to the amino acid at exactly 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 the residue is not at the same exact numbered position, for example if the RSV sequence is shorter or longer than SEQ ID NO: 1, or has insertions or deletions compared to SEQ ID NO: 1.

[0190] Structures of RSV F protein mutants

[0191] The RSV F protein mutant provided by the present disclosure includes F1 polypeptide and F2 polypeptide. In several embodiments, the mutant further includes a trimerization domain. In some embodiments, F1 polypeptide or F2 polypeptide includes at least one introduced modification (e.g., amino acid substitution) as described in detail below. In some other embodiments, F1 polypeptide and F2 polypeptide each include at least one introduced modification (e.g., amino acid substitution) as described in detail below.

[0192] F1 polypeptide and F2 polypeptide of RSV F mutant

[0193] In some embodiments, the mutant is a mature form of the RSV F protein, which comprises two separate polypeptide chains, namely the F1 polypeptide and the F2 polypeptide. In some other embodiments, the F2 polypeptide is connected to the F1 polypeptide by one or two disulfide bonds to form an F2-F1 polypeptide heterodimer. In yet other embodiments, the RSV F mutant is in the form of a single-chain protein, in which the F2 polypeptide is connected to the F1 polypeptide by a peptide bond or a peptide linker. Any suitable peptide linker that links the two polypeptide chains together can be used. Examples of such linkers include G, GG, GGG, GS, and SAIG ​​linker sequences. The linker can also be a full-length pep27 sequence or a fragment thereof. The relevant sequences are shown in Table 1:

[0194] Table 1

[0195]

[0196] The F1 polypeptide chain of the mutant may have the same length as the full-length F1 polypeptide of the corresponding wild-type RSV F protein (shown in SEQ ID NO: 1); however, it may also have deletions, such as up to 60 amino acid residues deleted from the C-terminus of the full-length F1 polypeptide. The full-length F1 polypeptide of the RSV F mutant corresponds to amino acid positions 137-574 of the natural RSV F0 precursor, and includes (from N-terminus to C-terminus) an extracellular region (residues 137-524), a transmembrane domain (residues 525-550), and a cytoplasmic domain (residues 551-574). It should be noted that amino acid residues 514 forward in the natural F1 polypeptide sequence are optional sequences in the F1 polypeptide of the RSV F mutant provided herein, and therefore may not be present in the F1 polypeptide of the mutant. The relevant sequences are shown in Tables 1 and 2:

[0197] Table 2

[0198]

[0199] In some embodiments, the F1 polypeptide of RSV F mutant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks the cytoplasmic domain and a portion or all of the transmembrane domain. In some specific embodiments, the mutant comprises the F1 polypeptide, wherein the amino acid residues from positions 510, 511, 512, 513, 514, 515, 520, 525 or 530 to 574 do not exist. Typically, for mutants connected to trimerization domains (such as foldons), amino acids 514 to 754 may not exist. Therefore, in some specific embodiments, amino acid residues 514 to 574 do not exist in the F1 polypeptide of the mutant. In other specific embodiments, the F1 polypeptide of RSV F mutant comprises amino acid residues 137-513 of a natural F0 polypeptide sequence, such as the amino acid sequence described in SEQ ID No: 1, or is composed of it.

[0200] On the other hand, the F1 polypeptide of the RSV F mutant may include a C-terminal link to a trimerization domain (such as a foldon). Many sequences of the RSV F mutant disclosed herein include protease cleavage site sequences, such as a thrombin cleavage site (LVPRGS); protein tags, such as 6×His-tags (HHHHHH) and streptomycin tag II (WSHPGFEK); or linker sequences (such as GG and GS), which are not required for the function of the RSV F protein, such as inducing an immune response. Those skilled in the art will recognize such sequences, and understand, when appropriate, that these sequences are not included in the disclosed RSV F mutants.

[0201] In the RSV F mutants provided herein, the F2 polypeptide chain may have the same length as the full-length F2 polypeptide of the corresponding wild-type RSV F protein; it may also have deletions, such as 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residues from the N-terminus or C-terminus of the F2 polypeptide.

[0202] The mutants in the form of F0 (i.e., a single polypeptide comprising an F2 polypeptide joined to an F1 polypeptide with or without partial or full-length pep 27) or F1-F2 heterodimers can form monomeric forms. The mutants can also be in the form of trimers, which comprise three identical monomeric forms.

[0203] The mutants of the present invention may be glycosylated proteins (ie, glycoproteins) or non-glycosylated proteins.

[0204] The F1 polypeptide and F2 polypeptide of the RSV F protein mutant 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 (including A2 Ontario and Buenos Aires) or any other subtype F protein amino acid sequences.

[0205] Trimerization domain

[0206] As used herein, the terms "domain", "motif" are used interchangeably.

[0207] In some embodiments, the RSV F mutants provided by the present disclosure are linked to a trimerization domain. In some embodiments, the trimerization domain promotes the formation of trimers of 3 F protein monomers.

[0208] Several exogenous multimerization domains that promote the formation of stable trimers of soluble proteins are known in the art. Examples of such multimerization domains that can be connected to mutants provided in the present disclosure include: (1) GCN4 leucine zipper (Harbury et al. 1993 Science 262: 1401-1407); (2) trimerization motif from lung surfactant protein (Hoppe et al. 1994 FEB S Lett 344: 191-195); (3) collagen (McAlinden et al. 2003 Biol Chem 278: 42200-42207); and (4) bacteriophage T4 fibritin foldon (Miroshnikov et al. 1998 Protein Eng 11: 329-414). In some embodiments, the foldon domain is connected to the F mutant at the C-terminus of the F1 polypeptide. In specific embodiments, the foldon domain is a T4 fibritin foldon domain, such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 16).

[0209] Typically, the multimerization domain is located at the C-terminus of the F1 polypeptide. It can be directly attached to the F1 polypeptide chain. Optionally, the multimerization domain is connected to the F1 polypeptide via a linker (such as an amino acid linker, for example, the sequence GG, GS or SAIG). The linker can also be a longer linker (for example, including a repeating sequence GG). A variety of configuration neutral linkers are known in the art, which can be used in the mutants provided by the present disclosure. In some embodiments, the F mutant comprising the foldon domain includes a protease cleavage site for removing the foldon domain from the F1 polypeptide, such as a thrombin site between the F1 polypeptide and the foldon domain.

[0210] Introduced mutations in RSV F protein mutants

[0211] RSV F mutants of the present invention include F1 polypeptides and F2 polypeptides, wherein (1) F1 polypeptides or (2) F2 polypeptides or (3) F1 polypeptides and F2 polypeptides both include one or more introduced amino acid mutations relative to the amino acid sequence of the corresponding native F protein. The introduction of such amino acid mutations in RSV F mutants imparts beneficial properties to the mutants, such as enhanced immunogenicity, improved stability, increased affinity, or the formation of a specific desired physical form or configuration of the mutant or improved stability. Such introduced amino acid mutations are referred to as "disulfide bond mutations", "cavity filling mutations" or "electrostatic mutations", and are described in detail below. RSV F mutants including any additional mutations are also encompassed by the present invention, as long as the immunogenic properties of the mutants are not significantly adversely affected by the additional mutations.

[0212] Those skilled in the art can generate conservative variant mutants by performing conservative amino acid substitutions, for example, as shown in Table 3 below.

[0213] Table 3

[0214]

[0215] Disulfide bond mutation

[0216] In some embodiments, the RSV F mutants provided by the present disclosure include one or more disulfide bond mutations. The term "disulfide bond mutation" refers to a pair of amino acid residues in the wild-type RSV F protein that mutates into a pair of cysteine ​​residues. The introduced cysteine ​​residues allow the formation of a disulfide bond between the introduced cysteine ​​residues, which is used to stabilize the configuration or oligomeric state of the protein, such as the pre-fusion configuration. For the pre-fusion configuration of the stabilizing mutant, the residue pair for mutating into cysteine ​​should be very close in the pre-fusion configuration, but away from the post-fusion configuration. Such residues can be identified by suitable methods known in the art, such as by visual inspection of the crystal structure of RSV F in the prefusion configuration, or more quantitative selection using computational protein design software (such as BioLuminate™ [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio™ [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015], and Rosetta™ [Rosetta, University of Washington, Seattle, 2015]). Preferably, the distance between residue pairs (e.g., beta carbons) in the prefusion configuration is less than 8 Å, but greater than 20 Å in the postfusion configuration.

[0217] In some embodiments, the disclosure provides a mutant of a wild-type RSV F protein, wherein the amino acid mutation introduced is a mutation of a pair of amino acid residues in the wild-type RSV F protein to a pair of cysteines ("disulfide bond mutation"). The introduced cysteine ​​residues allow for formation of a disulfide bond between the cysteine ​​residues that stabilizes the protein's configuration or oligomeric state, such as a pre-fusion configuration. Examples of specific pairs of such mutations include A170C and V179C; V154C and V300C; S155C and I288C; S155C and V300C; L158C and S290C; L158C and I291C; L158C and A298C; V164C and V296C; K168C and V296C; A170C and A177C; Y198C and K226C; F237C and M289C; V243C and I288C; T244C and S287C; L257C and V278C; L258C and S275C; I280C and K359C; I280C and F366C; I288C and G151C; V3 00C and G151C; Q302C and G151C; F32C and Y441C; K75C and V220C; L78C and V220C; E82C and F223C; E82C and Q224C; E82C and N227C; K85C and L231C; Y86C and N227C; A89C and L230C; A89C and L231C; A89C and T234C; E92C and T234C; E92C and S238C; L93C and T234C; L93C and I292C; Q94C and I292C; L96C and F237C; L96C and M289C; M97C and I291C; and M97C and I292C.

[0218] Cavity-filling mutation

[0219] In other embodiments, the disclosure provides RSV F mutants comprising one or more cavity filling mutations. The term "cavity filling mutation" refers to the amino acid replacement of the amino acid residues in the wild-type RSV F protein by the expected filling of the internal cavity of the mature RSV F protein. In one application, such cavity filling mutations contribute to the pre-fusion configuration of the stabilizing RSV F protein mutant. The cavity in the pre-fusion configuration of the RSV F protein can be identified by methods known in the art, such as by visual inspection of the crystal structure of the RSV F in the pre-fusion configuration, or by using computational protein design software. The amino acid to be substituted for the cavity filling mutation generally includes small aliphatic amino acids (e.g., Arg, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). It may also include an amino acid embedded in the pre-fusion configuration but exposed to the solvent in the post-fusion configuration. The example of the substituted amino acid includes large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp). In some specific embodiments, the RSV F protein mutant includes a cavity filling mutation selected from the following:

[0220] (1) S at position 55, 62 or 290 is substituted by I, Y, L or W;

[0221] (2) T at position 54, 58, 189, 219 or 397 is replaced by I, Y, L or W;

[0222] (3) A at position 147 or 298 is substituted by I, Y, L or W;

[0223] (4) V at position 164, 187, 192, 207, 220, 296, 300 or 495 is substituted with I, Y, L or W;

[0224] (5) R at position 106 is substituted by I, Y, L or W.

[0225] Electrostatic mutation

[0226] In some embodiments, the disclosure provides RSV F protein mutants including one or more electrostatic mutations.Term " electrostatic mutation " refers to the amino acid mutation introduced into wild-type RSV F protein, and the amino acid mutation reduces the ion repulsion between the residues close to each other in the folded structure in the protein or increases the ion attraction between the residues.Because hydrogen bonding is a special case of ion attraction, electrostatic mutation can increase the hydrogen bonding between such approaching residues.In one example, electrostatic mutation can be introduced to improve trimer stability.In some embodiments, in the pre-fusion configuration of RSV F glycoprotein but not in the configuration after its fusion, electrostatic mutation is introduced to reduce the repulsive ion interaction between the residues very close in RSV F glycoprotein or increase the attractive ion interaction (potentially including hydrogen bond).For example, in the pre-fusion configuration, the acidic side chain of Asp486 from a protomer of RSV F glycoprotein trimer is located at the trimer interface and is structurally sandwiched between two other acidic side chains of Glu487 and Asp489 from another protomer. On the other hand, in the configuration after fusion, the acidic side chain of Asp486 is located on the trimer surface and is exposed to solvent.In several embodiments, RSV F protein mutant includes electrostatic D486T replacement, which reduces the repulsive ion interaction with the acidic residues of Glu487 and Asp489 from another protosome of RSV F trimer or increases attractive ion interaction.Usually, introducing electrostatic mutation will increase the melting temperature (Tm) of the pre-fusion configuration or pre-fusion trimer configuration of RSV F albumen.

[0227] Unfavorable electrostatic interactions in the prefusion or prefusion trimer conformation can be identified by methods known in the art, such as by visual inspection of the crystal structure of RSV F in the prefusion or prefusion trimer conformation, or by using computational protein design software.

[0228] In some embodiments, the RSV F protein mutant comprises an electrostatic mutation selected from the group consisting of:

[0229] (1) E at position 82, 92 or 487 is replaced by F, T, H or W;

[0230] (2) K at position 315, 394, or 399 is substituted by F, T, H, or W;

[0231] (3) D at position 392, 486, or 489 is substituted by F, T, H, or W;

[0232] (4) R at position 106 or 339 is substituted by F, T, H or W;

[0233] (5) E at position 161 is substituted by F, T, H, or W;

[0234] (6) The S at position 182 or 173 is substituted by F, T, H or W.

[0235] Proline mutation in the loop region

[0236] The loop structure is a highly irregular and flexible structural unit in proteins, usually containing 5 to 20 amino acid residues. Its main function is to connect different functional regions of proteins and plays a key role in many important biological functions. In the α-helix, the hydrogen on the main chain nitrogen atom of the amino acid is bonded to the carbonyl oxygen atom of the fourth amino acid in front through a hydrogen bond. However, since the main chain nitrogen atom of proline has been bound to its side chain to form a ring, the nitrogen atom of proline cannot provide a hydrogen bond donor and therefore cannot participate in the hydrogen bond network in the α-helix. The side chain of proline forms a ring structure with its amino nitrogen, making its main chain nitrogen atom a secondary amine. This ring structure limits the rotational freedom of the main chain dihedral angle of proline, resulting in its inability to adapt to the specific dihedral angle required by the α-helix. Therefore, proline mutation of the amino acids in the loop region of the F protein can make the F protein more rigid and easier to maintain the pre-fusion conformation.

[0237] Schematic diagram of the RSV F protein loop structure ( Figure 1 Based on computational structural biology, it can be seen that the three colored LOOP regions (160aa~161aa, 172aa~175aa, 211aa~216 aa) are the key positions to prevent Helix fusion ( Figure 1 Middle B).

[0238] Coding sequence

[0239] The present invention also relates to polynucleotides encoding RSV F protein mutants of the present invention. The polynucleotides of the present invention can be in DNA form or RNA form. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence or a degenerate variant. The full-length nucleotide sequence of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the RSV F protein mutant of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.

[0240] The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the present invention.

[0241] Vectors and host cells

[0242] The coding polynucleotide sequence can be inserted into a recombinant expression vector or genome. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0243] Those skilled in the art can use well-known methods to construct expression vectors containing "pyruvate carboxylase mutant protein and / or malate transporter mutant protein" encoding DNA sequence and suitable transcription / translation control signals, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to a suitable promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation.

[0244] The host cells described herein include host cells containing the above expression vector or the host cells in which the mutant protein coding sequence of the present invention is integrated into the genome. The host cells described herein are prokaryotic cells and eukaryotic cells. In a preferred embodiment, the prokaryotic cells are Escherichia coli.

[0245] Preparation method

[0246] The RSV F protein mutants of the present invention can be chemically synthesized or recombinant.

[0247] A preferred method is to produce RSV F protein mutants of the present invention using recombinant technology. Through conventional recombinant DNA technology, polynucleotides of the present invention can be used to express or produce recombinant RSV F protein mutants. Generally speaking, the following steps are included:

[0248] (1) using a polynucleotide (or variant) encoding a RSV F protein mutant of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

[0249] (2) culturing host cells in a suitable culture medium;

[0250] (3) Isolate and purify proteins from culture medium or cells.

[0251] The RSV F protein mutant of the recombinant can be expressed in the cell or on the cell membrane or secreted into the cell. If necessary, the protein of the recombinant can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and the combination of these methods.

[0252] Pharmaceutical composition

[0253] The present invention also provides a pharmaceutical composition (such as a vaccine composition), which contains (ii) a pharmaceutically acceptable carrier and (ii) the RSV F protein mutant described in the first aspect of the present invention, or a polynucleotide encoding the mutant, or a vector containing the polynucleotide (such as a viral vector, etc.).

[0254] The pharmaceutical composition can be in any suitable form, depending on the method of administration required by the patient. It can be provided in the form of a unit dosage form, usually placed in a sealed container, and can be provided as part of a kit. Such a kit usually (but not necessarily) includes instructions for use. It can include a plurality of the unit dosage forms.

[0255] The pharmaceutical composition is suitable for any appropriate route of administration, such as injection (including subcutaneous, intradermal, intramuscular, intraperitoneal, microneedle or intravenous injection), inhalation or oral, nasal, or anal routes. The composition can be prepared by any method known in the pharmaceutical field, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0256] application

[0257] The present invention provides uses of the RSV F protein mutants of the present invention and polynucleotide molecules encoding the RSV F protein mutants of the present invention, for example, for preparing pharmaceutical compositions, especially pharmaceutical compositions for preventing or treating RSV infection.

[0258] The main advantages of the present invention are:

[0259] (1) By optimizing the amino acid sequence of the wild-type RSV F protein, a pre-F mutant protein sequence capable of stably expressing a pre-fusion conformational trimer protein is obtained; the mutant protein sequence can be used to express the RSV pre-F mutant protein.

[0260] (2) The RSV F protein mutants screened by the present invention can induce the production of specific IgG antibodies and neutralizing antibodies and have good immunogenicity.

[0261] (3) The mutant of the present invention can be used as a component of RSV vaccine and has better safety.

[0262] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0263] Unless otherwise specified, the experimental materials involved in the present invention can be obtained from commercial channels.

[0264] Example 1 Gene synthesis and expression plasmid construction

[0265] The full-length sequence of RSV F protein was found in the GeneBank database (Gene bank: ), a natural sequence RSV F was designed and named wild-type RSV F (wRSV F), and the protein sequence is shown in SEQ ID NO: 1. Disulfide bond mutation and LOOP region proline mutation were performed on the basis of the wRSV F sequence, and a mutant protein was designed and named SVR+ number. The amino acid sequence of the mutant protein of the present invention is shown in Table 1.

[0266] The amino acid sequences of the wRSV F protein and the mutant protein of the present invention were optimized by mammalian codons using DNA2.0 to obtain gene sequences. The specific sequences are shown in Table 4 below:

[0267] Table 4

[0268]

[0269] It was then handed over to Jiangsu Saisuofei Biotechnology Co., Ltd. for synthesis and homologous recombination with PTT5 (EcoRI / HindIII). The recombinant plasmid was transformed into DH5α competent medium to obtain SVR79 plates, which were placed in a 37-degree incubator for overnight culture. On the second day, three clones were selected and the plasmid was extracted. After the plasmid was digested with EcoRI / HindIII, a plasmid backbone of about 4.3kb and a pre-F band of 1.6kb were produced ( Figure 2 ), which was consistent with expectations. The plasmid was sent to GoldWeiZhi for sequencing, and the plasmid with the correct sequencing results was selected for subsequent post-transcription transfection. The plasmid DNA for transfection was purified using the GoldHi EndoFree Plasmid Maxi Kit (CWBIO). For all commercial kits or reagents, the procedures were performed according to the manufacturer's protocol.

[0270] Example 2 Protein Expression

[0271] HEK 293F cells (purchased from Thermo Fisher Scientific) were cultured at a density of 0.5-0.6×10 cells / mL one day before transfection. 6 / ml. On the day of transfection, the cell density was counted and when the density was 1-1.4×10 6 / ml, and activity>90%, it can be used for plasmid transfection. Transfection complex preparation: With 300ml as the transfection volume, two centrifuge tubes / culture bottles need to be prepared and placed separately. Take the recombinant plasmid prepared in Example 1:

[0272] Add 7.5 ml PBS and 300 μg recombinant plasmid to tube ① and mix well;

[0273] Add 7.5 ml PBS and 300 μl FreeStyle TMMAX Transfection Reagent (purchased from Thermo Fisher Scientific) to tube ② and mix well; add the diluted transfection reagent to the diluted recombinant plasmid and mix well to prepare a transfection complex.

[0274] After the transfection complex was allowed to stand for 15 to 20 minutes, a single drop was added to the cell culture at a constant speed.

[0275] CO2

[0276] The transfected cells were cultured at a concentration of 8% and a shaking speed of 130 rpm. After 5 days, the culture supernatant was collected for target protein expression detection.

[0277] Example 3 Protein Purification

[0278] Take 300 ml of the cell culture supernatant after transfection in Example 2 above, add 200 mM NaCl to the culture supernatant, and then use 5 ml Ni Smart Beads filler (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) for subsequent enrichment and purification: The specific chromatography steps are as follows:

[0279] 1) Column balancing: balance the column with balancing buffer (20mM PB (phosphate buffer), 300mM NaCl, pH 7.4). Do not contain EDTA, Arg and other substances in the buffer and sample until the pH reaches 8.0 and the UV detection reading is stable, which is generally 5 to 10 column volumes. Samples can be loaded after the detection is zeroed;

[0280] 2) Sample loading: The culture supernatant was filtered using a 0.22 μm filter, and the supernatant was loaded onto a chromatography column at a flow rate of 2.5 ml / min;

[0281] 3) Equilibration after loading: After loading, equilibrate the column with equilibration buffer, usually for 5 to 10 column volumes, to remove unbound proteins until the UV detection reading returns to the baseline or is relatively stable;

[0282] 4) Washing: Wash with washing buffer (20mM PB, 300mM NaCl, 20mM Imidazole, pH7.4) and (20mM PB, 300mM NaCl, 50mM Imidazole, pH7.4), respectively, generally washing 4 to 6 column volumes each, and collecting the eluted fractions respectively;

[0283] 5) Elution: Wash the column with column washing buffer (20mM PB, 300mM NaCl, 500mM Imidazole, pH7.4), usually 4 to 6 column volumes, and collect the eluted fractions;

[0284] 6) Column cleaning and storage: Rinse the chromatography column with 0.2M NaOH, generally for 5 to 10 column volumes, then rinse the chromatography column with purified water, generally for 5 to 10 column volumes, and finally rinse the chromatography column with 20% ethanol, generally for 5 to 10 column volumes. Finally, store the chromatography column in 20% ethanol solution.

[0285] The eluted fractions were identified by SDS-PAGE. Figure 3 As shown, the theoretical molecular weight of SVR79 protein is 56 kda. Due to the influence of glycosylation, SDS-PAGE electrophoresis shows that the band position is 60 kda, which is consistent with the theory.

[0286] 7) Preparation of mobile phase PBS system: 100 mM phosphate buffered saline solution 150 mM sodium chloride (pH 6.8), vacuum filtration with 0.22 μm membrane, and ultrasonication.

[0287] 8) Equilibration: The nanospectral column (BioCore SEC-300 4.6×300mm column, 5μm) was flushed with the required mobile phase at a low flow rate (0.1mL / min) until the baseline was stable, then the flow rate was increased to 0.265mL / min. After equilibration for at least 30 minutes, the sample can be analyzed after the baseline is stable.

[0288] 9) Sample injection: Centrifuge the sample at 12000r / 5min, take the supernatant, and inject 40μg of BSA and sample

[0289] The results are as follows Figure 4 As shown, based on the peak times of BSA monomer (molecular weight 67 kda) and dimer (molecular weight 134 kda), it was inferred that the purified F protein was a trimer structure. Figure 4 In Figure A, 10.997 represents the peak time of SVR79 F protein trimer. Figure 4 In B, 11.444 and 12.557 represent the elution time of BSA monomer and dimer, respectively. Combined with the elution time of BSA monomer (molecular weight 67kda) and dimer (molecular weight 134kda), it is inferred that the size of the purified F protein is about 200KD and is a trimer structure.

[0290] The results were confirmed by RSV pre-F sandwich ELISA antigen detection kit. Figure 5 As shown, the purified protein is a pre-fusion conformation trimer pre-F.

[0291] Example 4 Antigen Screening

[0292] The present invention performed two rounds of RSV F protein mutant optimization. Among them,

[0293] 1. For the first round of RSV F protein mutant optimization, the inventors performed sequence optimization from the following two aspects:

[0294] (1) Disulfide bond mutation: increase disulfide cross-linking (CC), a total of 41 pairs of mutations;

[0295] (2) Proline mutation: Proline was modified to fix the turn angle between adjacent secondary structures and prevent the transition to the post F conformation. There were 8 mutations in total. A total of 328 mutants were tested using the method described below.

[0296] 2. For the second round of RSV F protein mutant optimization, the inventors performed sequence optimization from the following two aspects:

[0297] (1) Cavity-filling mutations: enhancing stability, single-point mutations, a total of 44 mutations;

[0298] (2) Electrostatic mutation: promoting expression and solubilization, single-point mutation, a total of 65 mutations; a total of 109 mutants were tested using the method described below, with 2 replicate wells for each mutant.

[0299] 4.1 Detection of mutant expression

[0300] 4.1.1 Experimental reagents:

[0301] Coating solution: Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, place in a 2L beaker, add 600 ml of depurified water, stir to dissolve, adjust the pH to 9.6 with NaOH solution, dilute to 1L with water, and mix well.

[0302] Washing solution: Weigh 3.581 g of disodium hydrogen phosphate dodecahydrate, 0.245 g of potassium dihydrogen phosphate, 8.19 g of sodium chloride, and 0.201 g of potassium chloride, add 600 ml of depurified water, place in a 2 L beaker and stir to dissolve, measure 0.5 ml of Tween 20, dilute to 1 L with water, and mix well.

[0303] Blocking solution: Take a 50ml centrifuge tube, weigh 2g of skimmed milk powder, add 40ml of PBS to prepare 40ml of blocking solution, mix well on a vortex mixer and store at 4℃ for later use.

[0304] Dilution solution: Take a 50ml centrifuge tube, measure 10ml of blocking solution, add 40ml PBS to prepare 50ml sample dilution solution, mix well on a vortex mixer and store at 4℃ for later use.

[0305] Color development solution: Take a 50ml centrifuge tube, measure 15ml of PB solution, add 5.6µl of hydrogen peroxide, mix well, then add 150µl of TMB stock solution, mix well, wrap in tin foil and store at room temperature away from light, prepare and use immediately.

[0306] Stop solution: Measure 300ml of 3mol HCL and add 600ml of depurified water to make 900ml of 1 mol HCL.

[0307] Secondary antibody: HRP anti-His Tag Antibody, 652504, biolegend.

[0308] Coating protein: RSV F Antibody (11A9), Epitope II, DA091, Suzhou Nearshore Protein Technology Co., Ltd.

[0309] Standard: Recombinant RSV Prefusion F-trimer Protein (strain A2), DRA230, Suzhou Nearshore Protein Technology Co., Ltd.

[0310] Test sample: Cell supernatant after mutant transfection

[0311] 4.1.2 Experimental steps:

[0312] (1) Coating: Dilute the above-mentioned coating protein to a concentration of 10 ug / ml with coating solution, then add it to the ELISA plate, 100 ul / well, and incubate at 37°C for 1 h;

[0313] (2) Washing: Wash with washing solution three times, 300 μl / time, 5 min each time. After washing, pat the plate dry on absorbent paper. No obvious washing solution residue should remain in the wells.

[0314] (3) Blocking: Add 200 μl of blocking solution to each well and incubate at 37°C for 1 h.

[0315] (4) Washing: repeat step (2);

[0316] (5) Binding: dilute the test sample with diluent, dilute 0, 10, and 20 times respectively, then add 100 μl to each well of the ELISA plate and incubate at 37°C for 1 h;

[0317] (6) Washing: repeat step (2);

[0318] (7) Incubation with secondary antibody: add 100 μl of secondary antibody to each well and incubate at 37°C for 1 h.

[0319] (8) Washing: repeat step (2);

[0320] (9) Color development: Add 100 μl of color development solution per well and incubate at 37°C for 15 min in the dark.

[0321] (10) Stop: Add stop solution, 100 μl / well;

[0322] (11) Reading: Place the ELISA plate in the ELISA reader, set the wavelength to 450 nm, and then read the results.

[0323] RSVFC was used to prepare a standard curve, and the OD450 readings of the test samples diluted at different times were substituted into the standard curve for back calculation to calculate the concentration of the target protein in the cell supernatant.

[0324] 4.2 Affinity test

[0325] 4.2.1 BLI experiment

[0326] The purpose of this experiment is to detect the stability and affinity of RSV protein under different conditions through BLI experiment. The instrument emits white light to the sensor surface and collects the reflected light. The reflection spectra of different frequencies are affected by the thickness of the optical film layer of the biosensor and form interference. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement can directly reflect the thickness and density changes of the biofilm on the sensor surface, so as to accurately quantitatively measure the interaction process between the molecules to be tested, and can reflect the binding and dissociation information of the interacting molecules in real time, and can also quantify the concentration.

[0327] (a) Main instruments and materials:

[0328] Fortebio (Sartorius, Octet Red 96e);

[0329] 96-well black board (Molecular Devices, 655209)

[0330] (b) Main reagent materials:

[0331] Pro-A Biosensors (Sartorius, 18-5013)

[0332] Running buffer: 1× PBST (phosphate buffered saline), pH 7.4

[0333] Sample diluent: 1×PBST, pH 7.4

[0334] Regeneration solution: 10 mM Glycine-HCl, pH 1.5

[0335] (c) Detection process

[0336] Equilibration: Sensor immersed in running buffer for 60 seconds

[0337] Capture: Dilute the antigen to 10ug / mL with sample diluent, capture height is about 1nm

[0338] Equilibration: Sensor immersed in running buffer for 60 s

[0339] Binding: SVR protein was serially diluted and bound to the capture Motavizumab or D25 or MPE8 or DI-14359 or AM14 antibody sensor for 180 s

[0340] Dissociation: The sensor is dissociated in the running diluent for 300 seconds

[0341] Regeneration: Regeneration in 10 mM Glycine-HCl three times, 5 s each time

[0342] Neutralization: Immerse in running buffer for 5 seconds immediately after each regeneration.

[0343] (d) Data analysis

[0344] The binding experiment buffer was 0.02% PBST buffer, and 0.02% PBST was used as a negative control. The sample signal (the solidified signal represents the situation of the sensor capturing the antigen, and the sample signal represents the situation of the sensor capturing the antigen binding to the SVR protein, and the binding of the antigen and the SVR protein is obtained after subtraction) was fitted using Octet analysis software Data AnalysisHT 12.0 after deducting the negative control.

[0345] 4.2.2 ELISA experiment

[0346] (a) Reagent preparation:

[0347] Coating solution: Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, place in a 2L beaker, add 600 ml of depurified water, stir to dissolve, adjust the pH to 9.6 with NaOH solution, dilute to 1L with water, and mix well.

[0348] Washing solution: Weigh 3.581 g of disodium hydrogen phosphate dodecahydrate, 0.245 g of potassium dihydrogen phosphate, 8.19 g of sodium chloride, and 0.201 g of potassium chloride, add 600 ml of depurified water, place in a 2 L beaker and stir to dissolve, measure 0.5 ml of Tween 20, dilute to 1 L with water, and mix well.

[0349] Blocking solution: Take a 50ml centrifuge tube, weigh 2g of skimmed milk powder, add 40ml of PBS to prepare 40ml of blocking solution, mix well on a vortex mixer and store at 4℃ for later use.

[0350] Dilution solution: Take a 50ml centrifuge tube, measure 10ml of blocking solution, add 40ml PBS to prepare 50ml sample dilution solution, mix well on a vortex mixer and store at 4℃ for later use.

[0351] Color development solution: Take a 50ml centrifuge tube, measure 15ml of PB solution, add 5.6µl of hydrogen peroxide, mix well, then add 150µl of TMB stock solution, mix well, wrap in tin foil and store at room temperature away from light, prepare and use immediately.

[0352] Stop solution: Measure 300ml of 3mol HCL and add 600ml of depurified water to make 900ml of 1 mol HCL.

[0353] Secondary antibody: HRP anti-His Tag Antibody, 652504, biolegend.

[0354] Coating proteins: RSV Pre-F Antibody (3C12), Epitope Φ, DA101, Suzhou Nearshore Protein Technology Co., Ltd.

[0355] Standard: Recombinant RSV Prefusion F-trimer Protein (strain A2), DRA230, Suzhou Nearshore Protein Technology Co., Ltd.

[0356] Test sample: cell supernatant after mutant transfection.

[0357] (b) Experimental methods:

[0358] (1) Coating: Dilute the above-mentioned coating protein to a concentration of 10 ug / ml with coating solution, then add it to the ELISA plate, 100 ul / well, and incubate at 37°C for 1 h;

[0359] (2) Washing: Use washing solution. Wash 3 times, 300ul / time, 5min each time. After washing, pat the ELISA plate dry on absorbent paper. There should be no obvious washing solution residue in the wells.

[0360] (3) Blocking: Add blocking solution, 200ul per well, and then incubate at 37℃ for 1h;

[0361] (4) Washing: repeat step 2;

[0362] (5) Binding: dilute the test sample by half in 12 gradients with diluent, then add 100 μl to each well of the ELISA plate and incubate at 37°C for 1 h.

[0363] (6) Washing: Repeat step 2;

[0364] (7) Incubation with secondary antibody: add 100 μl of secondary antibody to each well and incubate at 37°C for 1 h.

[0365] (8) Washing: Repeat step 2;

[0366] (9) Color development: Add 100 μl of color development solution per well and incubate at 37°C for 15 min in the dark.

[0367] (10) Stop: Add stop solution, 100 μl / well;

[0368] (11) Reading: Place the ELISA plate in the ELISA reader, set the wavelength to 450 nm, and then read the results.

[0369] The OD450 readings of the samples to be tested were subjected to four-parameter fitting to calculate EC50. RSVFC protein was used to prepare a standard curve, and the OD450 readings of the samples to be tested were substituted into the standard curve for back calculation to calculate the concentration of the target protein in the cell supernatant.

[0370] 4.3 Stability test

[0371] The stability of the sequence-optimized RSV F protein mutants was evaluated by stress testing and storage stability experiments.

[0372] (1) During the heat stress test, the crude culture supernatant of the sequence-optimized mutant, i.e., the supernatant of 293T cell culture fluid, was incubated at 50°C or 70°C or pH 3.5 for 1 hour and detected by the pre-fusion specific monoclonal antibody D25 in an ELISA assay. The affinity was detected according to the experimental method in 4.2 of this Example.

[0373] (2) During the storage stability test, the reactivity of the prefusion antibody in the crude culture supernatant was compared before and after 10 freeze-thaw cycles, and detected in an ELISA assay using the prefusion-specific monoclonal antibody D25. The affinity was detected according to the experimental method in 4.2 of this Example.

[0374] The first round of screening of disulfide bond and proline mutations showed:

[0375] (1) There were 67 mutants with affinity > RSVFC control, among which disulfide bond mutations 258C-275C, 288C-151C and proline mutations 214P-215P appeared most frequently in all high-affinity mutants, 5 / 67, 5 / 67, and 14 / 67, respectively. Figure 7 ).

[0376] (2) There were 63 mutants with expression levels >1.0 ng / mL and 45 mutants with expression levels >2.0 ng / mL. Among them, disulfide bond mutations 198C-226C, 97C-291C and proline mutations 214P-215P appeared most frequently in the mutants with expression levels >2.0 ng / mL, accounting for 6 / 45, 6 / 45 and 10 / 45, respectively. Figure 6 ).

[0377] According to affinity ranking, 10 mutants with expression levels >2.0 ng / mL among the top 40 were selected for expression purification screening. The results showed that the SVR88 / SVR79 / SVR72 / SVR46 mutants had the highest expression levels, all >85 ng / ml, as shown in Table 5:

[0378] Table 5

[0379]

[0380] The results of affinity tests with different antibodies showed that the affinity values ​​of D25 and AM14 antibodies of the two mutants SVR88 and SVR152 were in the same order of magnitude as the RSVFC standard protein, as shown in Table 6:

[0381] Table 6

[0382]

[0383] Note: “-” indicates no binding with the antibody.

[0384] The affinity test results of the 10 priority mutants obtained in the first round of screening with different antibodies in Table 6 showed that the SVR45, SVR46, SVR90, SVR64 and SVR201 mutants had poor affinity with antibodies. Therefore, these five mutants were no longer subject to subsequent stability screening.

[0385] The stability of the RSV F protein mutants after sequence optimization was evaluated by stress testing and storage stability experiments. During the stress test, the crude culture supernatant of the mutant after sequence optimization was incubated at 50°C or 70°C or pH 3.5 for 1 hour and detected with pre-fusion specific monoclonal antibody D25 in an ELISA assay. The detection method detects affinity according to the experimental method of Example 4.2. It is expected that more stable mutants have higher stress resistance. During the storage stability assay, the reactivity of pre-fusion antibodies in the crude culture supernatant after 10 freeze-thaw cycles was compared with the reactivity of fresh culture supernatant. The results are shown in Table 7:

[0386] Table 7

[0387]

[0388] The results in Table 7 show that the stability of SVR152, SVR79 and SVR88 mutants was good, and the above three mutants were used for subsequent immune evaluation.

[0389] For the second round of RSV F protein mutant optimization, the inventors performed sequence optimization from the following two aspects:

[0390] 1. Cavity filling mutations, enhancing stability, single point mutations, a total of 44 mutations;

[0391] 2. Electrostatic mutation, promoting expression and solubility, single point mutation, a total of 65 mutations.

[0392] A total of 109 mutants were tested by the above method, and 2 duplicate wells were made for each mutant. Among them, the mutation site is mutated relative to the site shown in the wild-type RSV F protein (SEQ ID NO: 1), and the screening results of the second round of RSV F protein mutants are shown in Table 8:

[0393] Table 8

[0394]

[0395] Note: * indicates that at least one replicate well of the mutant meets the screening criteria of this column; a blank cell indicates that the test results of the two replicate wells of the mutant do not meet the screening criteria of this column and no further testing is performed.

[0396] The results of the second round of RSV F protein mutant optimization shown in Table 8 show that:

[0397] (1) The expression level of at least one replicate well of 36 mutants was >2.0 ng / mL, and the expression levels of two replicate wells of 33 mutants were both >2.0 ng / mL;

[0398] (2) the number of mutants with high expression levels (>2.0 ng / mL) in the electrostatic filling mutations (33) was greater than the number of mutants with high expression levels in the spatial filling mutations (3);

[0399] (3) The protein expression levels of the second round of RSV F protein mutant optimization were significantly improved compared with those of the first round of optimization: the expression levels of 34 mutants were >10.0 ng / mL, and the expression levels of 20 mutants were >80.0 ng / mL;

[0400] (4) Among the 36 mutants with high expression levels, at least one of the replicates in 21 mutants had an EC50 <EC50 RSVFC (referring to affinity (D25)>RSVFC in Table 8, the smaller the EC50 value, the tighter the binding between the antibody and the target molecule, and the stronger the affinity). Among them, the EC50 of 2 duplicate wells of 15 mutants <EC50 RSVFC .

[0401] 21 mutants with high expression levels and good affinity were selected for the above stability test, among which mutants SVRA99 (R106F) and SVR83 (K399T) passed the stability test, and the results are shown in Table 9:

[0402] Table 9

[0403]

[0404] Note: “-” indicates no combination.

[0405] Based on the sites R106F and K399T screened by the high-throughput plate of the second round of RSV F protein mutants, combined with the results of the first round of screening (SVR152, SVR88, SVR79), combined mutants were constructed, and protein expression purification and antibody affinity testing were performed, as shown in Table 10:

[0406] Table 10

[0407]

[0408] The results in Table 10 show that R106F and K399T have different effects on the expression levels of different mutant proteins and antibody affinity: (1) increased the expression level of SVR152 mutant protein; (2) decreased the expression levels of SVR88 and SVR79 mutant proteins; (3) greatly reduced the affinity of SVR79 mutant protein for D25 antibody; (4) increased the affinity of SVR152 and SVR79 mutant proteins for ADI-14359 antibody.

[0409] Based on the above results, SVR88B, SVR88C, SVRA99, SVRA83, and SVR152A were screened for stability testing. The results are shown in Table 11:

[0410] Table 11

[0411]

[0412] According to the binding antibody affinity data, expression data and stability test data shown in Tables 10 and 11, SVR88C and SVRA99 mutants were selected from the first and second rounds of RSV F protein mutant optimization combinations for subsequent immunization experiments.

[0413] Example 5 Immunity Experiment

[0414] This embodiment involves two rounds of immunization experiments, wherein the first round of immunization experiments is to immunize mice twice with mutants (SVR152, SVR88 and SVR79) screened in the first round, collect serum samples on the 28th day, and detect preF-specific binding antibodies by ELISA;

[0415] The second round of immunization experiment was to immunize mice twice with the SVR88C and SVRA99 mutants and SVR79 mutants selected from the first and second rounds of RSV F protein mutant optimization combinations. Serum samples were collected on the 42nd day and preF-specific binding antibodies were detected by ELISA. The specific experimental process is as follows:

[0416] (1) Vaccine preparation: 5 μg of purified RSV pre-F protein was mixed with 5 μg of A401 or MF59 adjuvant (purchased from CanSino Biologics) and emulsified. After emulsification, the vaccine was left to stand overnight at 4 degrees without antigen precipitation or stratification.

[0417] (2) Animal immunization: Female BALB / c mice aged 6-8 weeks were selected and weighed. Two rounds of RSV F protein mutant immunization experiments were performed by intramuscular injection.

[0418] Among them, the specific grouping and immunization scheme of the first round of RSV F protein mutant immunization experiment are shown in Table 12.

[0419] Table 12

[0420]

[0421] The specific grouping and immunization scheme of the second round of RSV F protein mutant immunization experiment are shown in Table 13:

[0422] Table 13

[0423]

[0424] Among them, DS-Cav1 was used as a positive control. The sampling and detection time was the 28th day after the first round of RSV F protein mutant immunization, and the sampling and detection time was the 42nd day after the second round of RSV F protein mutant immunization.

[0425] (3) Specific IgG antibody detection: ELISA plates were coated with 5ug / ml purified pre-F protein and incubated overnight. The plates were blocked with PBS / 3% bovine serum albumin, and then control and mouse serum samples (serum samples collected in (2) above were diluted to 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 The plates were washed and incubated with rabbit anti-mouse IgG for 30 min at room temperature. The plates were washed, incubated with anti-rabbit IgG-horseradish peroxidase conjugate for 30 min at room temperature, washed again, and incubated with 2,2′-azinobis-(3-ethylbenzothiazoline sulfonic acid) with an optical readout of 405 nm.

[0426] (4) Neutralizing antibody detection: HEp-2 cells were revived and cultured before the experiment. The cells were digested and inoculated into 96-well plates in advance. 2.5×10 5 cells / mL, 0.1 mL / well, incubate at 37°C, 5% CO2Incubate overnight in an incubator; dilute the serum sample collected in (2) above by 40 times, and then dilute it by 3 times in sequence, with a total of 8 dilution gradients, and set up negative and positive controls; add 5000 PFU / mL RSV A2 virus, incubate in an incubator for 1 hour, transfer to a cell culture plate, infect HEp-2 cells, and incubate in an incubator for 1 day; fix and stain the cells after 1 day, and calculate the neutralizing antibody titer.

[0427] (5) The neutralizing antibody detection method is as follows:

[0428] 1) Cell preparation and plate seeding: HEp-2 cells (purchased from Shanghai Yuchi Biotechnology) were revived and cultured before the experiment. The passages used in the test were 3 to 20 after revivification. The cells were digested and inoculated into 96-well plates in advance, 2.5×105 cells / mL, 0.1 mL / well, and placed at 37°C and 5% CO2 Incubate overnight in an incubator;

[0429] 2) Sample dilution: After confirming that the cells are normal, take a serum sample and dilute it;

[0430] 3) Set up controls: Set up controls in the 96-well plate (neutralization plate) in step 2), add 120 μL of diluent to the cell control (CC), and add 60 μL of diluent to the virus control (VC); set up 8 replicate wells for VC and 8 replicate wells for CC;

[0431] 4) Virus dilution: After the virus solution is melted, dilute the virus with diluent to 5000 PFU / mL according to the virus titration titer;

[0432] 5) Virus addition: Take the virus solution from step 4), add 60 μL / well to the sample wells and VC wells of the 96-well plate and mix well;

[0433] 6) Neutralization reaction: Place the 96-well plate in a 37°C, 5% CO2 Incubate in the incubator for 1 h;

[0434] 7) Cell preparation and infection: Take the 96-well plate inoculated with cells in advance, discard the original culture medium, and add 100 μL / well of the mixed solution to the corresponding 96-well plate with cells;

[0435] 8) Culture: Place the 96-well plate in a 37°C, 5% CO2 Incubate in an incubator for 1 day;

[0436] 9) Fixation: After incubation, discard the original culture medium and add 100 μL of fixative to each well for 20 min or overnight.

[0437] 10) Staining: After the cell plate was fixed, the fixative was discarded, and the cells were washed twice with 1×PBS. 300 μL / well of blocking solution was added and blocked for 30 min; the blocking solution was discarded, and the cells were washed twice with 1×PBS. 50 μL / well of primary antibody working solution was added and incubated at 37°C for 1 h; the antibody was discarded, and the cells were washed twice with 1×PBS. 50 μL / well of HRP secondary antibody working solution was added and incubated at 37°C for 1 h; the antibody was discarded, and the cells were washed twice with 1×PBS. 30 μL / well of KPL TrueBlue Peroxidase Substrate colorimetric solution was added and incubated at room temperature for 5-20 min; the colorimetric solution was discarded, and the spots were counted using an ELISA spot analyzer.

[0438] 11) Result calculation: Neutralizing antibody titer: Calculate the inhibition rate of each gradient of the sample:

[0439] Inhibition rate = 1-(number of spots in sample duplicate wells / average number of spots in VC wells),

[0440] The sample dilution multiple-inhibition rate was input into the software for fitting and the neutralizing antibody titer of the sample was calculated; the Reed-Muench method was used to calculate the 50% neutralizing antibody titer of the sample.

[0441] After the mutants (SVR152, SVR88, and SVR79) selected in the first round were immunized twice with mice, serum samples were collected on the 28th day and RSV pre-F specific binding antibodies were detected by ELISA. Figure 8 The results showed that compared with the control group DS-Cav-1 group, the SVR79 mutant protein could induce the mouse body to produce high titer RSV pre-F specific binding antibodies (10 7 ), with statistically significant difference; compared with other mutant proteins, SVR79 mutant protein has the best immunogenicity.

[0442] The mutants selected in the second round (SVR88C and SVRA99) were again evaluated for immunity with the SVR79 mutant that was the best in the first round of immunity evaluation. After two immunizations of mice, serum samples were collected on the 42nd day and RSV pre-F specific binding antibodies were detected by ELISA. The results are shown in Fig. 9 As shown, mutant SVR79 can achieve the same level of RSV pre-F binding antibody titer as the control DS-Cav1 (10 6 ), however, the high-quality mutants SVR88C and SVRA99 selected by cavity filling and electrostatic mutation on the basis of SVR79 have not shown better humoral immune effects.

[0443] The inventors then continued to analyze from the perspective of neutralizing antibodies through the above neutralization test. The results are as follows Fig.10As shown, the SVR79 mutant induced the highest neutralizing antibody titer (EC 50 =6677), which was significantly different from other mutants (P<0.0001) and much higher than the control group DS-Cav1+MF59 (EC 50 =961) about 7 times.

[0444] Therefore, the SVR79 mutant was finally selected for subsequent immune challenge experiments.

[0445] Example 6 Cotton rat immunization experiment

[0446] Female cotton rats aged 6-8 weeks were selected and weighed. Two rounds of RSV F protein mutant immunization experiments were performed on day 0 and day 21, respectively, and the immunization method was intramuscular injection. mRNA-1345 is a commercial vaccine for the prevention of RSV. The specific grouping and immunization scheme of the immunization experiment are shown in Table 14 below, where DS-Cav1 was used as a positive control and AS01E was used as an adjuvant.

[0447] Table 14

[0448]

[0449] (1) Immunological experiments

[0450] Experimental animals: 15 female cotton rats were randomly divided into 5 groups, 3 rats in each group, 6-8 weeks old.

[0451] The experimental steps are as follows:

[0452] Isolation observation: 15 cotton rats were randomly divided into 5 groups, 3 rats in each group, and 3 rats in each cage for 3 days.

[0453] Blood was collected at day 0 and serum was frozen at -80℃ for subsequent detection of specific antibodies; first immunization.

[0454] On the 21st day, blood was collected and serum was frozen at -80°C for subsequent detection of specific antibodies; a second immunization was performed on the 21st day.

[0455] On the 49th day, blood was collected and serum was frozen at -80°C for subsequent detection of specific IgG antibodies and neutralizing antibodies.

[0456] (2) Specific IgG antibody detection:

[0457] (a) Reagent preparation:

[0458] Coating solution: Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, place in a 2L beaker, add 600 ml of depurified water, stir to dissolve, adjust the pH to 9.6 with NaOH solution, dilute to 1L with water, and mix well.

[0459] Washing solution: Weigh 3.581 g of disodium hydrogen phosphate dodecahydrate, 0.245 g of potassium dihydrogen phosphate, 8.19 g of sodium chloride, and 0.201 g of potassium chloride, add 600 ml of depurified water, place in a 2L beaker and stir to dissolve, measure 0.5 ml of Tween 20, dilute to 1L with water, and mix well.

[0460] Blocking solution: Take a 50ml centrifuge tube, weigh 2g of skimmed milk powder, add 40ml of PBS to prepare 40ml of blocking solution, mix well on a vortex mixer and store at 4℃ for later use.

[0461] Dilution solution: Take a 50ml centrifuge tube, measure 10ml of blocking solution, add 40ml PBS to prepare 50ml sample dilution solution, mix well on a vortex mixer and store at 4℃ for later use.

[0462] Color development solution: Take a 50ml centrifuge tube, measure 15ml of PB solution, add 5.6µl of hydrogen peroxide, mix well, then add 150µl of TMB stock solution, mix well, wrap in tin foil and store at room temperature away from light, prepare and use immediately.

[0463] Stop solution: Measure 300 ml of 3M HCL and add 600 ml of depurified water to make 900 ml of 1 M HCL.

[0464] Secondary antibody: Horseradish peroxidase-conjugated chicken anti-cotton rat secondary antibody, PAB29753, Abnova.

[0465] Encapsid proteins: RSV preF protein, DRA230, nearshore protein.

[0466] (b) Experimental methods:

[0467] 1) Coating: Dilute preF protein to 5 μg / ml and add to a 96-well plate, 100 μL / well. Seal the plate with a sealing film and incubate at 4°C overnight.

[0468] 2) Wash the plate: 300 μL / well PBST buffer, wash five times, and spin dry.

[0469] 3) Blocking: Add 200 μL of PBS containing 3% BSA to each well and incubate at 37˚C for 2 h.

[0470] 4) Wash the plate: 300 μL / well PBST buffer, wash five times, and spin dry.

[0471] 5) Sample addition: Add 100 μL of serially diluted serum to each well (serum starts at 100-fold dilution and is diluted 10-fold to 107) and incubate at 37˚C for 1.5 h.

[0472] 6) Wash the plate: 300 μL / well PBST buffer, wash five times, and spin dry.

[0473] 7) Add secondary antibody: Add 100 μL of 0.05 μg / ml horseradish peroxidase-labeled chicken anti-cotton rat secondary antibody to each well and incubate at 37˚C for 1 hour.

[0474] 8) Wash the plate: 300 μL / well PBST buffer, wash five times, and spin dry.

[0475] 9) Color development: Add 100 μL of TMB (tetramethylbenzidine) color development solution to each well and incubate at 37˚C for 15 min.

[0476] 10) Stop: Add 50 μL of stop solution to each well to stop color development.

[0477] 11) Reading: Measure the absorbance (OD value) of each well using an ELISA reader (450 nm wavelength)

[0478] (3) The neutralizing antibody detection method is the same as the neutralizing antibody detection method in Example 5.

[0479] The results of specific antibody and neutralizing antibody tests are as follows Fig.11 As shown. RSV pre-F specific binding antibodies were detected by ELISA. The results are shown Fig.11 As shown in A, compared with the control group DS-Cav1, the SVR79 mutant protein can induce cotton rats to produce similar levels of RSV pre-F specific binding antibodies, among which the specific antibodies of D21 are significantly higher than those of the DS-Cav1 control. The serum samples on day 49 were tested for neutralizing antibodies, and the results are as follows Fig.11 As shown in B, the SVR79 mutant protein can produce higher neutralizing antibody titers than the control DS-Cav1. The subunit vaccine neutralizing antibody titer can reach 28859, which is about 4.9 times that of the control DS-Cav1 (neutralizing antibody titer is 5904), and the SVR79 mRNA vaccine neutralizing antibody titer can reach 72000, which is about 5.3 times that of the control mRNA-1345 (neutralizing antibody titer is 13619). In summary, the SVR79 mutant protein has the potential to be developed into a candidate vaccine for the prevention of RSV disease.

[0480] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A mutant protein, characterized in that The mutant protein is in monomeric form or trimer form, wherein the trimer is formed by three mutant proteins in monomeric form; Wherein, the mutant protein in monomeric form is a fusion protein derived from the first polypeptide element and the trimerization domain element of the RSV F protein; wherein, in the first polypeptide element, the Pep27 fragment of the RSV F protein is replaced by a linker sequence; The mutant protein consists of an F2 polypeptide, an F1 polypeptide and a linker sequence: the amino acid sequence of the F2 polypeptide is shown in SEQ ID NO: 17, and the amino acid sequence of the F1 polypeptide is selected from any one of SEQ ID NO: 19, 23, 24, 25, and 27.

2. The mutant protein according to claim 1, characterized in that The linker sequence is selected from the group consisting of G, GG, GGG, GS, SAIG ​​and SGSGR.

3. The mutant protein according to claim 1, characterized in that The amino acid sequence of the mutant protein is selected from the group consisting of SEQ ID NOs: 6, 10, 12, 13 and 15.

4. The mutant protein according to claim 1, characterized in that The amino acid sequence of the mutant protein is based on SEQ ID NO: 6, and only has R106F mutation and K399T mutation relative to the amino acid sequence of the wild-type RSV F protein shown in SEQ ID NO:

1.

5. The mutant protein according to claim 1, characterized in that The amino acid sequence of the mutant protein is based on SEQ ID NO: 15, with only the K399T mutation relative to the amino acid sequence of the wild-type RSV F protein shown in SEQ ID NO:

1.

6. The mutant protein according to claim 1, characterized in that The amino acid sequence of the mutant protein is based on SEQ ID NO: 15, and only has R106F mutation and K399T mutation relative to the amino acid sequence of the wild-type RSV F protein shown in SEQ ID NO:

1.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises (a) the mutant protein according to claim 1, a polynucleotide encoding the mutant protein, or a vector expressing the mutant protein; and (b) a pharmaceutically acceptable carrier.

8. Use of the mutant protein or the polynucleotide encoding the mutant protein or the vector expressing the mutant protein according to claim 1, characterized in that: For preparing a composition or preparation for: (a) Prevention and / or treatment of RSV infection.

9. A kit, characterized in that: It comprises the mutant protein according to claim 1, and also comprises a detection reagent for detecting the content of RSV pre-F protein.

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