RSV f protein mutant, and preparation method therefor and use thereof

Abstract

Provided are an RSV F protein mutant, and a preparation method therefor and a use thereof. Provided is a mutant protein, the mutant protein being in the form of a monomer or a trimer, wherein the trimer is formed by three monomeric mutant proteins; the monomeric mutant protein is a fusion protein comprising a first polypeptide element derived from an RSV F protein and a trimerization domain element; in the first polypeptide element, a Pep27 fragment of the RSV F protein is replaced with a linker sequence; and the monomeric and trimeric mutant proteins are both bound by an antibody that specifically recognizes RSV pre-F. The mutant protein can stably remain in a pre-fusion state, can induce the production of specific IgG antibodies and neutralizing antibodies, has excellent immunogenicity, and can be used for the development of RSV vaccines.

Description

RSV F protein mutant, preparation method and application thereof TECHNICAL FIELD

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

[0002] Respiratory syncytial virus (RSV) is a pathogen that mainly causes respiratory tract infectious diseases in infants and adults over 65 years old. Infected individuals may experience fever, cough, and difficulty breathing, among other clinical symptoms. Vaccination is an important means of preventing the disease, but there is currently no safe and effective RSV vaccine on the market in China.

[0003] RSV belongs to the Paramyxoviridae family and Pneumovirus genus, and is a single-stranded negative-sense RNA virus with a membrane. The RSV genome is about 15 kb in length and can encode 11 proteins, including nine structural proteins and two non-structural proteins. Among the structural proteins, there are 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 fusion of the viral membrane with the host cell membrane and has been shown to interact with nucleolin. In addition, F protein expressed on the surface of infected cells also mediates the fusion of adjacent cells, leading to the formation of syncytia. F protein and G protein are currently known RSV antigens that can induce neutralizing antibodies. However, unlike G protein, F protein exhibits a key role in infection, high neutralization characteristics, and high conservation among virus subtypes, making it the primary target for RSV vaccine research.

[0004] F protein is a type I transmembrane protein, 574 amino acids in length, containing 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 F protein is the inactive precursor F0. To perform its fusion function, the F0 precursor is double-cleaved at positions 109 and 136 by a furin-like protease, removing a 27-aa peptide (p27) to produce F1 and F2 subunits. The F1 and F2 subunits are connected by two disulfide bonds to form a F1-F2 heterodimer. The uncut P27 F0 protein without a signal peptide sequence or the F1-F2 heterodimer is the RSV F protomer, which trimerizes to form the final RSV F protein complex.

[0005] Mature F protein is expressed on the virion or cell membrane in a metastable pre-fusion conformation that can readily rearrange into a lower energy post-fusion conformation. During viral fusion and entry, the F protein transitions from a metastable pre-fusion (pre-F) to a stable post-fusion (post-F) conformation. The pre-fusion protein conformation is composed of 6 potential antigenic sites During the rearrangement from the pre-fusion to the post-fusion conformation, the antigenic sites and V are lost, while the antigenic site is a highly neutralization-sensitive antigenic site. Studies have shown that different versions of RSV vaccines designed based on the two conformations, the pre-fusion F protein induces neutralizing antibodies that prevent viral fusion between the virus and the cell; while the post-fusion F protein induces antibodies that are not very effective. However, compared to the post-fusion conformation, the pre-fusion conformation is extremely unstable, and artificial wild-type sequence RSV F subunit vaccines are spontaneously or quickly rearranged into the post-fusion 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, and a composition, such as a vaccine, comprising the immunogen. SUMMARY

[0007] The purpose of the present application is to provide a RSV F protein mutant, by optimizing the amino acid sequence of the wild-type RSV F protein, to obtain a mutant protein sequence capable of stably expressing a pre-fusion conformation trimer protein pre-F; the mutant protein sequence can be used to express the 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 application, a mutant protein is provided, which is in a monomeric form or a trimeric form, wherein the trimer is formed by three monomeric forms of the mutant protein;

[0009] wherein the monomeric form of the mutant protein is a fusion protein derived from a first polypeptide element and a 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] and both the monomer and the trimer form of the mutant protein are specifically recognized by antibodies binding to RSV pre-F.

[0012] In another preferred embodiment, the affinity of the mutein in monomeric or trimeric form to the first antibody A1 is significantly higher than the affinity 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 A0; wherein the first antibody is an antibody specifically recognizing 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, the first polypeptide element has a mutation of the amino acid sequence SCSISN (SEQ ID NO: 3) at positions 211 to 216 to SCSPPN, SCSISP, SPSISN, SCPPSN, PCSISN or SPPISN, compared to the wild-type RSV F protein (as shown in SEQ ID NO: 1).

[0015] In another preferred embodiment, the antibody specifically recognizing RSV pre-F is selected from the group consisting of the AM14 antibody, the D25 antibody, or a combination thereof.

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

[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 a mutation of 1-2 prolines (P) in the Loop region.

[0019] In another preferred embodiment, the mutein has a mutation selected from the group A:

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

[0021] (Z2) an optional disulfide bond mutation;

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

[0023] In another preferred embodiment, the mutein further comprises a mutation selected from the group B: a cavity filling mutation, an 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 a 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:

[0025] (1) a disulfide bond mutation;

[0026] (2) a loop region mutation;

[0027] (3) a cavity-filling mutation;

[0028] (4) an electrostatic mutation.

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

[0030] (1) a disulfide bond mutation;

[0031] (2) a loop region mutation;

[0032] (3) a cavity-filling mutation; and

[0033] (4) an 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 of the following combinations:

[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 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.

[0038] In another preferred embodiment, the loop region comprises the SCSISN (SEQ ID NO: 3) amino acid sequence at positions 211 to 216.

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

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

[0041] (2) C at position 212 is substituted with 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) N at position 216 is substituted with 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:

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

[0046] (2) C at position 212 is substituted with P;

[0047] (3) I at position 214 is substituted with P;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0082] In another preferred embodiment, the amino acid mutations are one or more combinations selected from the group consisting of:

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

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

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

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

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

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

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

[0090] In another preferred embodiment, the mutein comprises a F1 polypeptide and a F2 polypeptide connected by a linker sequence, with deletion of the pep27 sequence.

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

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

[0093] In another preferred embodiment, the F2 polypeptide is selected from the sequences set forth 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 set forth in any one of SEQ ID NO: 19-28.

[0095] In another preferred embodiment, the mutein comprises a F1 polypeptide and a F2 polypeptide selected from the group consisting of:

[0096] (1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and a 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 Fl 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 group consisting of:

[0117] (1) an amino acid sequence as set forth 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 as set forth 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 with a signal peptide, an epitope, or a heterologous multimerization element.

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

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

[0122] In another preferred embodiment, the sequence of the T4 fibritin foldon is as set forth in SEQ ID NO: 16.

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

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

[0125] In a third aspect of the present application, a vector comprising the polynucleotide of the second aspect of the present application is provided.

[0126] In a fourth aspect of the present application, a host cell expressing the mutant protein of the first aspect of the present application, or comprising the polynucleotide of the second aspect of the present application, or comprising the vector of the third aspect of the present application is provided.

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

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

[0129] In a fifth aspect of the application, there is provided a method of producing the mutein of the first aspect of the application, comprising the steps of:

[0130] (a) culturing the host cell of the fourth aspect of the application under conditions suitable for expression, thereby producing the mutein of the first aspect of the application.

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

[0132] In a sixth aspect of the application, there is provided a pharmaceutical composition comprising (a) the mutein of the first aspect of the application, a polynucleotide encoding the mutein, or a vector expressing the mutein; and (b) a pharmaceutically acceptable carrier.

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

[0134] In a seventh aspect of the application, there is provided the use of the mutein of the first aspect of the application or a polynucleotide encoding the mutein or a vector expressing the mutein, for the manufacture of a composition or formulation for:

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

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

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

[0138] In another preferred embodiment, the composition is an 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 formulation is a laboratory formulation.

[0141] In another preferred embodiment, the composition is administered to a patient infected with RSV having a bias towards RSV pre-F protein or pre-fusion protein conformation and / or a bias towards RSV post-F protein or post-fusion protein conformation.

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

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

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

[0145] In a ninth aspect of the present application, 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 of the sixth aspect of the present application.

[0146] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0147] Figure 1 shows the schematic diagram of F protein Loop structure and amino acid sequence, wherein Figure A shows the schematic diagram of F protein Loop structure; Figure B shows that the three LOOP regions (160aa-161aa, 172aa-175aa, 211aa-216aa) colored based on computational structural biology can be seen as the key positions for preventing Helix fusion.

[0148] Figure 2 shows the plasmid identification result in the embodiment of the present application, wherein Band 1: DNA Ladder 5000; Band 2: SVR79 plasmid; Band 3: EcoRI / HindIII enzyme-digested plasmid 4.3K / 1.6K, wherein the plasmid backbone size is 4.3K and the pre-F size is 1.6K.

[0149] Figure 3 shows the SDS-PAGE result in the embodiment of the present application, wherein MK: marker; R: SVR79 protein.

[0150] Figure 4 shows the HPLC result in the embodiment of the present application, wherein Figure A shows the HPLC result of SVR79, wherein 10.997 represents the peak time of SVR79 F protein trimer; Figure B shows the HPLC result of BSA, wherein 11.444 and 12.557 respectively represent the peak time of BSA monomer and dimer.

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

[0152] Figure 6 shows the statistical analysis results of proline mutation and affinity, expression amount in the application, wherein the abscissa is the mutation site relative to the wild-type RSV F protein amino acid sequence shown in SEQ ID NO: 1, for example, 211P represents the mutation to proline (P) at the 211th position relative to the wild-type RSV F protein amino acid sequence shown in SEQ ID NO: 1, and the like; the ordinate is the frequency of each mutation site appearing in all mutants, and RSVFC represents the control protein (the sequence is shown in SEQ ID NO. 2). The statistics are performed on the mutants with affinity > RSVFC (a total of 67 mutants), expression amount > 1.0 ng / mL (a total of 63 mutants), and expression amount > 2.0 ng / mL (a total of 45 mutants) in all mutants. Among them, the mutants with high affinity: 214P-215P have the highest frequency (14 / 67), and the mutants with high expression amount: 214P-215P have the highest frequency (10 / 45).

[0153] Figure 7 shows the statistical analysis results of disulfide bond mutation and affinity, expression amount in the application, wherein the abscissa is the mutation site relative to the wild-type RSV F protein amino acid sequence shown in SEQ ID NO: 1, for example, 154C-300C represents the mutation to cysteine (C) at the 154th and 300th positions relative to the wild-type RSV F protein amino acid sequence shown in SEQ ID NO: 1 to form a disulfide bond, and the like; the ordinate is the frequency of each mutation site appearing in all mutants, and RSVFC represents the control protein (the sequence is shown in SEQ ID NO. 2). The statistics are performed on the mutants with affinity > RSVFC (a total of 67 mutants), expression amount > 1.0 ng / mL (a total of 63 mutants), and expression amount > 2.0 ng / mL (a total of 45 mutants) in all mutants. Among them, the mutants with high affinity: 258C-275C and 288C-151C have the highest frequency (5 / 67), and the mutants with high expression amount: 198C-226C and 97C-291C have the highest frequency (6 / 45), wherein “C” represents.

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

[0155] Figure 9 shows the RSV pre-F specific IgG antibody titers in the serum of mice immunized with the mutant SVR79 in the second round of screening in the embodiments of the present application; MF59 is the adjuvant; compared with the positive control group (DS-Cav1), the mutant SVR79 can induce the production of higher titers of RSV pre-F specific binding antibodies in the mice (10 6 ), and has better immunogenicity.

[0156] Figure 10 shows the results of detecting the neutralizing antibody levels in the serum of mice in the second round of screening in the embodiments of the present application; A401 and MF59 are the adjuvants; DS-Cav1 is used as the positive control.

[0157] Figure 11 shows the results of cotton rat immunization experiments in the embodiments of the present application; A shows the results of RSV pre-F specific IgG antibody titers, wherein D0 represents the day of immunization, D21 represents the 21st day after immunization, and D49 represents the 49th day after immunization; B shows the results of neutralizing antibody detection 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, and mRNA-1345 represents the commercial RSV prevention vaccine as the positive control of the mRNA vaccine. DETAILED DESCRIPTION

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

[0159] TERMS

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

[0161] As used herein, the terms "mutant protein," "mutant," "protein mutant" can be used interchangeably and all refer to a protein having one or more alterations in the amino acid sequence or protein structure that constitutes it as compared to a wild-type protein. The alterations can include deletion, insertion, substitution, truncation and / or deletion of one or more amino acids, processing or cleavage of the protein structure. In the present application, the protein mutant refers to a respiratory syncytial virus (RSV) pre-F protein mutant.

[0162] As used herein, the term "amino acid mutation" generally refers to a modification made to an amino acid in a parent amino acid sequence. For example, the modification can include substitution, insertion and / or deletion of one or more amino acids. In the present application, the amino acid mutation can include deletion or substitution of at least one amino acid residue at a specified position of an amino acid sequence. In certain embodiments, the amino acid mutation can enable conformational optimization of a protein constituted by the amino acid sequence. The amino acid mutation can be generated using genetic methods or chemical methods well known in the art. For example, the genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like.

[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 together, and the protein trimer can be linked together by a special chemical structure. In the present application, the protein trimer can be an 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 can include a change in chemical bonds in a protein molecule, a change in the folding pattern 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 for transmembrane. For example, the transmembrane protein can include a secreted protein or a cell membrane protein. For example, the signal peptide can be synthesized in the form of a precursor substance polypeptide at the N-terminus of the transmembrane protein.

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

[0167] As used herein, the term "pre-fusion conformation" generally refers to the conformation of the structural proteins of a virus prior to membrane fusion when the virus has not yet infected a host cell. Generally, the pre-fusion of wild-type RSV F protein is a metastable state of the protein that undergoes a discontinuous, stepwise, and irreversible conformational change to a lower energy stable state conformation (post-fusion conformation) after the virus has infected a host cell and undergone membrane fusion.

[0168] In the present application, the expression of protein mutation site is generally expressed by "amino acid + amino acid position number + mutated amino acid". In the present application, the mutation can include, but is not limited to, addition, substitution, deletion and / or deletion of amino acid. For example, the term "E218C" generally refers to the mutation of glutamic acid (E) at position 218 to cysteine (C).

[0169] As used herein, the term "polynucleotide" generally refers to an isolated form of nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analog thereof, either synthesized artificially or isolated from its natural environment.

[0170] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed. The vector can be transformed, transduced or transfected into a host cell, so that the genetic material elements carried by the vector can be expressed in the host cell. For example, the vector includes: a plasmid; a phagemid; a cosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC); a bacteriophage such as a lambda phage or a M13 phage, and an animal virus, etc. The types of animal viruses used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site. The vector can also include components that facilitate its entry into cells or facilitate the integration of the desired element into the host cell, such as viral particles, liposomes, protein coats or integrases, but not only these substances.

[0171] As used herein, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or disorder. The pharmaceutical composition can comprise the RSV F protein mutant described herein, the polynucleotide molecule described herein, the vector described herein, and / or the cell described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition can further comprise one or more suitable formulations of (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable ingredients of the composition are generally non-toxic to the recipient at the doses and concentrations employed.

[0172] As used herein, the term "pharmaceutically acceptable carrier" generally includes a pharmaceutically acceptable carrier, excipient, or stabilizer, which is nontoxic to the cells or mammals being exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers can 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, but not excluding other elements.

[0174] The present application is a new type of protective vaccine with high safety, extensive protection, and reliable preparation process, which is designed based on structural biology and combined with immunological principles. The stability of the recombinant protein vaccine provided in the present application is significantly improved. The RSV F protein mutant can be stably maintained in the pre-fusion state, and can induce a higher level of neutralizing antibodies in animal immunization tests alone or supplemented with a suitable adjuvant.

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

[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 "prefusion specific antibody", "first antibody", "antibody that specifically recognizes RSV pre-F" are used interchangeably to refer to an antibody that specifically binds to RSV F glycoprotein in the prefusion conformation, but does not bind to RSV F protein in the postfusion conformation. Exemplary prefusion specific antibodies include, but are not limited to: D25 and AM14 antibodies.

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

[0179] As used herein, the term "DSCavI" refers to the molecule described in the literature "McLellan, J. S., et al. (2013). Structure based design of a fusion glycoprotein vaccine for respiratory syncytial virus." Science 342(6158): 592 598.

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

[0181] As used herein, the term "AM14" refers to the antibody described in the literature "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 binds only to the trimeric and simultaneously prefusion conformation of the F protein of respiratory syncytial virus, and not to the monomeric form of the fusion F protein.

[0182] RSV pre-F protein

[0183] The F protein is a type I transmembrane protein, 574 amino acids in length, comprising a signal peptide (aa 2-20), a signal peptide cleavage site (aa 21-25), an F2 region (aa 26-109), a polypeptide cleavage region (aa 110-136), and an Fl region (aa 137-574). The initial form of the F protein is the inactive precursor F0, which is cleaved twice by a furin-like protease at positions 109 and 136 to generate the Fl and F2 subunits after removal of a 27-aa peptide (p27). The Fl and F2 subunits associate through two disulfide bonds to form the Fl-F2 heterodimer. The uncleaved P27 of F0 protein or the Fl-F2 heterodimer without the signal peptide sequence is the RSV F protomer, which trimerizes to form the final RSV F protein complex.

[0184] The mature F protein is expressed on the virion or cell membrane in a metastable prefusion conformation that can readily rearrange to a lower-energy postfusion conformation. The prefusion protein conformation is composed of six potential antigenic sites . During the rearrangement from the prefusion to the postfusion conformation, the antigenic sites and V are lost, while of which the antigenic sites is a highly neutralization-sensitive antigenic site. Studies have shown that different forms of RSV vaccines designed based on the two conformations, the prefusion F protein induces neutralizing antibodies that prevent fusion between the virus and the cell, while the postfusion F protein induces antibodies that are not very effective.

[0185] RSV F protein mutants

[0186] In some aspects, the present disclosure provides mutants of wild-type RSV F protein, wherein the mutants exhibit mutations introduced in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type RSV F protein and have immunogenicity against the wild-type RSV F protein or against a virus comprising the wild-type F protein. In certain embodiments, the RSV F mutants have certain beneficial features, such as enhanced immunogenicity properties compared to the corresponding wild-type F protein or improved stability of the prefusion conformation of the mutant or the prefusion trimer conformation of the mutant. In yet other embodiments, the present disclosure provides RSV F mutants that exhibit one or more mutations introduced as described herein and bind a prefusion-specific antibody selected from antibody D25 or antibody AM14.

[0187] The amino acid mutations introduced in the RSV F protein mutants include amino acid substitutions, deletions, or additions. In some embodiments, the only mutations in the amino acid sequence of the mutant are amino acid substitutions relative to the wild-type RSV F protein.

[0188] Natural RSV F proteins exhibit substantial sequence conservation among RSV subtypes. For example, RSV subtypes A and B share 90% sequence identity, and each of RSV subtypes A and B shares 81% sequence identity with the bovine RSV F protein among the F0 precursor molecules. Within RSV subtypes, F0 sequence identity is even higher; for example, the RSV F0 precursor proteins have about 98% sequence identity within each of RSV subtypes A, B, and bovine. Almost all identified RSV F0 precursor sequences consist of 574 amino acids in length, and the length is typically slightly different due to the length of the C-terminal cytoplasmic tail. Sequence identity among various natural RSV F proteins is known in the art (see, e.g., WO 2014 / 160463).

[0189] In view of the substantial conservation of RSV F sequences, one of ordinary skill in the art can readily compare amino acid positions between different natural RSV F sequences to identify corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, the furin cleavage site falls in the same amino acid position among almost all identified natural RSV F0 precursor proteins. Thus, the conservation of natural RSV F protein sequences among strains and subtypes permits the use of a reference RSV F sequence to compare the amino acid at a particular position in a RSV F protein. For the purposes of the present disclosure (unless the context indicates otherwise), RSV F protein amino acid positions refer to the sequence of the F0 precursor polypeptide set forth in SEQ ID NO: 1 (Gene bank: ACO83301.1). However, it should be noted, and one of skill in the art will appreciate, that different RSV F0 sequences can have different numbering systems, e.g., if additional amino acid residues are added or removed as compared to SEQ ID NO: 1. Thus, it should be understood that where a particular amino acid residue is referred to by its number, the description is not limited only to the amino acid that is precisely at that numbered position when counting from a given amino acid sequence, but also refers to the equivalent / corresponding amino acid residue in any and all RSV F sequences, even if that residue is not at the same precise numbered position, e.g., if the RSV sequence is shorter or longer than SEQ ID NO: 1, or has insertions or deletions as compared to SEQ ID NO: 1.

[0190] Structure of RSV F protein mutants

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

[0192] F1 and F2 polypeptides of RSV F mutants

[0193] In some embodiments, the mutant is in the mature form of the RSV F protein, which comprises two separate polypeptide chains, an F1 polypeptide and an F2 polypeptide. In some other embodiments, the F2 polypeptide is linked 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 a single chain protein form, in which the F2 polypeptide is linked to the F1 polypeptide by a peptide bond or a peptide linker. Any suitable peptide linker that links the two polypeptides 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. Relevant sequences are shown in Table 1:

[0194] Table 1

[0195] The F1 polypeptide chain of the mutant can 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 can 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 native 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). Note that amino acid residue 514 forward in the native F1 polypeptide sequence is an optional sequence in the F1 polypeptide of the RSV F mutants provided herein, and thus can not be present in the F1 polypeptide of the mutant. Relevant sequences are shown in Table 1 and Table 2:

[0196] Table 2

[0197] In some embodiments, the F1 polypeptide of the 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 particular embodiments, the mutant comprises an F1 polypeptide in which amino acid residues from positions 510, 511, 512, 513, 514, 515, 520, 525, or 530 to 574 are absent. Typically, for mutants linked to a trimerization domain, such as a foldon, amino acids 514 to 754 can be absent. Thus, in some particular embodiments, amino acid residues 514 to 574 are absent in the F1 polypeptide of the mutant. In yet other particular embodiments, the F1 polypeptide of the RSV F mutant comprises or consists of amino acid residues 137-513 of the native F0 polypeptide sequence, such as the amino acid sequence set forth in SEQ ID No: 1.

[0198] In other aspects, the F1 polypeptide of the RSV F mutant can include a linkage to the C-terminus of a trimerization domain, such as a foldon. Many of the sequences of the RSV F mutants disclosed herein include sequences that are not essential to the function of the RSV F protein, such as inducing an immune response, such as a protease cleavage site sequence, such as a thrombin cleavage site (LVPRGS); a protein tag, such as a 6xHis-tag (HHHHHH) and a streptavidin tag II (WSHPGFEK); or a linker sequence, such as GG and GS. Those skilled in the art will recognize such sequences, and, where appropriate, understand that these sequences are not included in the disclosed RSV F mutants.

[0199] In the RSV F mutants provided herein, the F2 polypeptide chain can have the same length as the full-length F2 polypeptide of the corresponding wild-type RSV F protein; it can also have a deletion, 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.

[0200] The mutants can form a monomeric form in the F0 form (i.e., a single chain polypeptide comprising a F2 polypeptide joined to an F1 polypeptide with or without a portion or full-length pep 27) or a F1-F2 heterodimeric form. The mutants can also be in a trimeric form, comprising three identical monomeric forms.

[0201] The mutants of the present application can be glycosylated proteins (i.e., glycoproteins) or non-glycosylated proteins.

[0202] The F1 polypeptide and the F2 polypeptide of the RSV F protein mutants into which one or more mutations are introduced can be from any wild-type RSV F protein known in the art or discovered in the future, including but not limited to the F protein amino acid sequences of RSV subtypes A and subtype B strains (including A2 Ontario and Buenos Aires) or any other subtypes.

[0203] trimerization domain

[0204] As used herein, the terms "domain," "motif" can be used interchangeably.

[0205] In some embodiments, the RSV F mutants provided by the disclosure are linked to a trimerization domain. In some embodiments, the trimerization domain promotes the formation of a trimer of 3 F protein monomers.

[0206] 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 linked to the mutants provided by the disclosure include: (1) GCN4 leucine zippers (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 linked to the F mutant at the C-terminus of the Fl polypeptide. In particular embodiments, the foldon domain is a T4 fibritin foldon domain, such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 16).

[0207] Generally, the multimerization domain is positioned at the C-terminus of the Fl polypeptide. It can be directly joined to the Fl polypeptide chain. Optionally, the multimerization domain is linked to the Fl polypeptide via a linker, such as an amino acid linker, e.g., the sequence GG, GS, or SAIG. The linker can also be a longer linker (e.g., including the repeat sequence GG). A variety of configuration neutral linkers are known in the art that can be used in the mutants provided by the disclosure. In some embodiments, the F mutant comprising a foldon domain includes a protease cleavage site for removal of the foldon domain from the Fl polypeptide, such as a thrombin site between the Fl polypeptide and the foldon domain.

[0208] Introduction of mutations in RSV F protein mutants

[0209] The RSV F mutants of the application comprise a F1 polypeptide and a F2 polypeptide, wherein (1) the F1 polypeptide or (2) the F2 polypeptide or (3) both the F1 polypeptide and the F2 polypeptide comprise 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 the RSV F mutants confers beneficial properties to the mutants, such as enhanced immunogenicity, improved stability, increased affinity, or formation or improved stability of a particular desired physical form or conformation of the mutant. Such introduced amino acid mutations are referred to as "disulfide mutations," "cavity-filling mutations," or "electrostatic mutations," and are described in detail below. RSV F mutants comprising any additional mutations are also encompassed by the application, provided that the immunogenic properties of the mutant are not significantly adversely affected by the additional mutations.

[0210] Conservative amino acid substitutions can be made according to, for example, the following Table 3 to produce conservatively variant mutants.

[0211] Table 3

[0212] Disulfide mutations

[0213] 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 mutation of a pair of amino acid residues in a wild-type RSV F protein to a pair of cysteine residues. The introduced pair of cysteine residues are permissive for the formation of a disulfide bond between the introduced cysteine residues, which serves to stabilize the conformation or oligomeric state of the protein, such as the prefusion conformation. For stabilizing the prefusion conformation of the mutant, the pair of residues to be mutated to cysteine should be in close proximity in the prefusion conformation, but distal in the postfusion conformation. 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 conformation, 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 the pair of residues (e.g., beta carbons) in the prefusion conformation is less than 5 A, and in the postfusion conformation is greater than 5 A. but in the postfusion conformation is greater than

[0214] In some embodiments, the disclosure provides mutants of wild-type RSV F protein in which the introduced amino acid mutation is a mutation of a pair of amino acid residues in the wild-type RSV F protein to a pair of cysteines ("disulfide bond mutations"). The introduced pair of cysteine residues permits the formation of a disulfide bond between the cysteine residues that stabilizes a conformation or oligomeric state of the protein, such as the prefusion conformation. Examples of particular 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; V300C 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.

[0215] Cavity-filling mutations

[0216] In other embodiments, the disclosure provides RSV F mutants comprising one or more cavity-filling mutations. The term "cavity-filling mutation" refers to an amino acid substitution in a wild-type RSV F protein that is expected to fill an internal cavity of the mature RSV F protein. In one application, such cavity-filling mutations help to stabilize the prefusion conformation of the RSV F protein mutant. Cavities in the prefusion conformation of the RSV F protein can be identified by methods known in the art, such as by visual inspection of the crystal structure of RSV F in the prefusion conformation, or by using computational protein design software such as BioLuminate TM [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio TMDiscovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE TM Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015], and Rosetta TM [Rosetta, University of Washington, Seattle, 2015]). The amino acids to be replaced for cavity- filling mutations typically include small aliphatic amino acids (e.g., Arg, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). It can also include amino acids that are buried in the pre-fusion conformation but are solvent exposed in the post-fusion conformation. Examples of replacement amino acids include large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp). In some embodiments, the RSV F protein mutant comprises a cavity-filling mutation selected from the group consisting of:

[0217] (1) S at position 55, 62, or 290 is replaced with I, Y, L, or W;

[0218] (2) T at position 54, 58, 189, 219, or 397 is replaced with I, Y, L, or W;

[0219] (3) A at position 147 or 298 is replaced with I, Y, L, or W;

[0220] (4) V at position 164, 187, 192, 207, 220, 296, 300, or 495 is replaced with I, Y, L, or W;

[0221] (5) R at position 106 is replaced with I, Y, L, or W.

[0222] Electrostatic mutations

[0223] In some embodiments, the present disclosure provides RSV F protein mutants comprising one or more electrostatic mutations. The term "electrostatic mutation" refers to an amino acid mutation introduced into a wild-type RSV F protein that reduces the ionic repulsion between or increases the ionic attraction between residues in the protein that are in close proximity to one another in the folded structure. Since hydrogen bond bonding is a special case of ionic attraction, an electrostatic mutation can increase hydrogen bond bonding between such proximal residues. In one example, an electrostatic mutation can be introduced to improve trimer stability. In some embodiments, an electrostatic mutation is introduced to reduce repulsive ionic interactions or increase attractive ionic interactions (potentially including hydrogen bonds) between residues in close proximity in the prefusion conformation of the RSV F glycoprotein, but not in its postfusion conformation. For example, in the prefusion conformation, the acidic side chain of Asp486 from one protomer of the 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 postfusion conformation, the acidic side chain of Asp486 is located on the surface of the trimer and is exposed to solvent. In several embodiments, the RSV F protein mutants comprise the electrostatic D486T substitution, which reduces repulsive ionic interactions or increases attractive ionic interactions with the acidic residues of Glu487 and Asp489 from another protomer of the RSV F trimer. In general, introducing an electrostatic mutation will increase the melting temperature (Tm) of the prefusion conformation or prefusion trimer conformation of the RSV F protein.

[0224] 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 such as BioLuminate TM [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio TM [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE TM [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015.] and Rosetta TM [Rosetta, University of Washington, Seattle, 2015.]).

[0225] In some embodiments, the RSV F protein mutant comprises an electrostatic mutation selected from the group consisting of:

[0226] (1) E at position 82, 92, or 487 is substituted with F, T, H, or W;

[0227] (2) K at position 315, 394, or 399 is substituted with F, T, H, or W;

[0228] (3) D at position 392, 486, or 489 is substituted with F, T, H, or W;

[0229] (4) R at position 106 or 339 is substituted with F, T, H, or W;

[0230] (5) E at position 161 is substituted with F, T, H, or W;

[0231] (6) S at position 182 or 173 is substituted with F, T, H, or W.

[0232] Loop region proline mutations

[0233] Loop structures are structural elements in proteins that have high irregularity and flexibility, usually containing 5 to 20 amino acid residues, and their main role is to connect different functional regions of the protein, playing a key role in many important biological functions. In an alpha helix, the hydrogen on the backbone nitrogen atom of an amino acid binds to the carbonyl oxygen atom of the fourth amino acid in front through a hydrogen bond. However, since the backbone nitrogen atom of proline has been combined with 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 alpha helix. The side chain of proline forms a ring structure with its amino nitrogen, making its backbone nitrogen atom a secondary amine. This ring structure limits the rotational freedom of the backbone dihedral angle of proline, making it unable to adapt to the specific dihedral angle required for the alpha helix. Therefore, mutating the amino acids in the Loop region of the F protein to proline can make the F protein more rigid and more likely to maintain the prefusion conformation.

[0234] The Loop structure of RSV F protein is shown in the schematic diagram (Figure 1A) and the amino acid sequence, and based on computational structural biology, it can be seen that the three LOOP regions (160aa-161aa, 172aa-175aa, 211aa-216aa) colored are key positions to prevent Helix fusion (Figure 1B).

[0235] Coding sequence

[0236] The present application also relates to polynucleotides encoding the RSV F protein mutants of the present application. The polynucleotides of the present application can be in the form of DNA or RNA. The 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 thereof. The full-length sequence of the nucleotide of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the RSV F protein mutants of the present application (or a fragment thereof, or a derivative thereof) can be obtained by complete chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or as a vector) and cells known in the art.

[0237] The present application also relates to vectors comprising the polynucleotides of the present application, and host cells genetically engineered with the vectors of the present application or the coding sequences of the present application.

[0238] Vectors and host cells

[0239] The coding polynucleotide sequence can be inserted into a recombinant expression vector or genome. The term "recombinant expression vector" refers to a plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector known in the art. In general, any plasmid and vector can be used as long as it can replicate in the host and be stable. An important feature of the expression vector is that it usually contains an origin of replication, a promoter, a marker gene and a translation control element.

[0240] The skilled person can use well-known methods to construct an expression vector containing the "pyruvate carboxylase mutant protein and / or malate transporter mutant protein" coding DNA sequence and suitable transcription / translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be operably linked to a suitable promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0241] The host cells described herein include host cells containing the above-mentioned expression vector or the host cells in which the coding sequence of the mutant protein of the present application is integrated on the genome. The host cells described herein are prokaryotic cells, eukaryotic cells. In a preferred embodiment, the prokaryotic cells are Escherichia coli.

[0242] Preparation method

[0243] The RSV F protein mutants of the present application can be chemically synthesized or recombinant.

[0244] A preferred method is to produce the RSV F protein mutants of the present application using recombinant technology. The polynucleotides of the present application can be used to express or produce recombinant RSV F protein mutants by conventional recombinant DNA technology. In general, the following steps are involved:

[0245] (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a RSV F protein mutant of the application, or with a recombinant expression vector containing the polynucleotide;

[0246] (2) culturing the host cell in a suitable culture medium;

[0247] (3) isolating, purifying the protein from the culture medium or the cell.

[0248] The recombinant RSV F protein mutant can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. 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 precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0249] Pharmaceutical composition

[0250] The present application also provides a pharmaceutical composition (e.g. a vaccine composition) comprising (ii) a pharmaceutically acceptable carrier and (ii) a RSV F protein mutant of the first aspect of the application, or a polynucleotide encoding the mutant, or a vector (e.g. a viral vector) containing the polynucleotide.

[0251] The pharmaceutical composition can be in any suitable form, depending on the method of administration desired for the patient. It can be presented in unit dosage form, generally in a sealed container, and can be provided as part of a kit. Such kits will generally, but not necessarily, include instructions for use. They can include a plurality of the unit dosage forms.

[0252] 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, or nasal, or anal, etc. The composition can be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0253] Applications

[0254] The present application provides the use of a RSV F protein mutant of the application, a polynucleotide molecule encoding a RSV F protein mutant of the application, for example, for the preparation of a pharmaceutical composition, in particular for the prevention or treatment of RSV infection.

[0255] The main advantage of the present application is that:

[0256] (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 fusion pre-conformation trimer protein is obtained. The mutant protein sequence can be used to express the RSV pre-F mutant protein.

[0257] (2) The RSV F protein mutant screened by the present application can induce the production of specific IgG antibodies and neutralizing antibodies, and has good immunogenicity.

[0258] (3) The mutant of the present application can be used as a component of RSV vaccine, and has better safety.

[0259] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, and are generally carried out according to 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 specified, percentages and parts are weight percentages and weight parts.

[0260] The experimental materials involved in the present application can be obtained from commercial channels unless otherwise specified.

[0261] Example 1 Gene synthesis and construction of expression plasmid

[0262] The full-length sequence of RSV F protein (Gene bank: ACO83301.1) was searched in GeneBank database, and the natural sequence of RSV F was designed, named as wild-type RSV F (wRSV F), and the protein sequence is shown in SEQ ID NO: 1. Based on the sequence of wRSV F, disulfide bond mutation and proline mutation in the LOOP region were carried out, and the mutant protein was designed and named as SVR+, and the amino acid sequence of the mutant protein of the present application is shown in Table 1.

[0263] The amino acid sequences of wRSV F protein and the mutant protein of the present application were optimized by DNA2.0 for mammalian codons to obtain gene sequences, and the specific sequences are shown in Table 4 below:

[0264] Table 4

[0265] The plasmid was then sent to Jiangsu Saixiao Fei Biotechnology Co., Ltd. for synthesis and homologous recombination with PTT5 (EcoRI / HindIII). The recombinant plasmid was transformed into DH5a competent cells to obtain SVR79 plates, which were placed in a 37°C incubator overnight. The next day, three clones were selected, and the plasmid was extracted. After EcoRI / HindIII digestion, a plasmid backbone of about 4.3 kb and a pre-F band of 1.6 kb were generated (Figure 2), which was consistent with the expected result. The plasmid was sent to Goldengene for sequencing, and the plasmid with correct sequencing results was selected for subsequent post-transcriptional transfection. The plasmid DNA used for transfection was purified using the Gold Hi EndoFree Plasmid Maxi Kit kit (CWBIO). For all commercial kits or reagents, procedures were performed according to the manufacturer's protocol.

[0266] Example 2 Protein expression

[0267] HEK 293F cells (purchased from Thermo Fisher Scientific) were passaged at a density of 0.5-0.6 x 10 6 / ml one day before transfection. On the day of transfection, the cell density was counted, and when the density was 1-1.4 x 10 6 / ml and the viability was >90%, it could be used for plasmid transfection. Transfection complex preparation: for a transfection volume of 300 ml, two centrifuge tubes / culture bottles were prepared and placed separately. The recombinant plasmid prepared in Example 1 was taken:

[0268] 7.5 ml PBS and 300 μg recombinant plasmid were added to tube ① and mixed well;

[0269] 7.5 ml PBS and 300 μl FreeStyle TMMAX Transfection Reagent (purchased from Thermo Fisher Scientific) were added to tube ② and mixed well; the diluted transfection reagent was added to the diluted recombinant plasmid and mixed well to prepare the transfection complex.

[0270] After the transfection complex was incubated for 15-20 min, it was added to the cell culture at a uniform speed. The transfection was carried out at 37°C, CO2 8% concentration, 130 rpm shaking speed. After 5 days of culture, the culture supernatant was collected for detection of the expression of the target protein.

[0271] Example 3 Protein purification

[0272] Take the above-mentioned example 2 transfected cell culture supernatant 300ml, culture supernatant supplemented with 200mM NaCl, using 5ml Ni Smart Beads filling (purchased from Changzhou Tianremanhe Biotechnology Co., Ltd.) for subsequent enrichment and purification: the specific chromatography steps are as follows:

[0273] 1) Column equilibration: equilibration buffer (20mM PB (phosphate buffer), 300mM NaCl, pH 7.4) equilibration column, buffer and sample can not have EDTA, Arg and other substances, until the pH reaches 8.0, the UV detection reading is stable, generally 5-10 column volumes, detection zero can be loaded;

[0274] 2) Loading: culture supernatant is filtered with 0.22μm filter, and the supernatant is loaded onto the chromatography column at a flow rate of 2.5ml / min;

[0275] 3) Equilibrium after loading: after loading, equilibrate the chromatography column with equilibration buffer, generally 5-10 column volumes, remove unbound proteins, until the UV detection reading returns to baseline or relatively stable;

[0276] 4) Washing: wash with washing buffer (20mM PB, 300mM NaCl, 20mM Imidazole, pH 7.4), (20mM PB, 300mM NaCl, 50mM Imidazole, pH 7.4), generally 4-6 column volumes, respectively, collect the elution fractions;

[0277] 5) Elution: wash the chromatography column with elution buffer (20mM PB, 300mM NaCl, 500mM Imidazole, pH 7.4), generally 4-6 column volumes, collect the elution fractions;

[0278] 6) Column cleaning and preservation: use 0.2M NaOH to wash the chromatography column, generally 5-10 column volumes, then use purified water to wash the chromatography column, generally 5-10 column volumes, finally use 20% ethanol to wash the chromatography column, generally 5-10 column volumes, and finally store the chromatography column in 20% ethanol solution.

[0279] The elution fractions were identified by SDS-PAGE as shown in Figure 3, and the SVR79 protein had a theoretical molecular weight of 56kda. Due to the influence of glycosylation, the SDS-PAGE electrophoresis showed a band position of 60kda, which was consistent with the theory.

[0280] 7) Preparation of mobile phase PBS system: 100mM phosphate buffer solution 150mM sodium chloride (pH 6.8), vacuum filtration and ultrasonic with 0.22μm membrane.

[0281] 8) Equilibration: The column (BioCore SEC-300 4.6x300mm column, 5μm) was equilibrated with the mobile phase at a low flow rate (0.1 mL / min) until the baseline was stable, then the flow rate was increased to 0.265 mL / min, and the sample was analyzed after the baseline was stable for at least 30 min

[0282] 9) Injection: The sample was centrifuged at 12000 r for 5 min, the supernatant was taken, and 40μg of BSA and the sample were injected

[0283] The results are shown in Figure 4. The peak time of the BSA monomer (67 kda) and the BSA dimer (134 kda) was used to infer that the purified F protein was a trimer structure. In Figure 4A, 10.997 represents the peak time of the SVR79 F protein trimer, and in Figure 4B, 11.444 and 12.557 represent the peak times of the BSA monomer and the BSA dimer, respectively. The peak times of the BSA monomer (67 kda) and the BSA dimer (134 kda) were used to infer that the purified F protein was about 200KD and was a trimer structure.

[0284] The results of the detection experiment using the RSV pre-F sandwich ELISA antigen detection kit are shown in Figure 5. The purified protein was a pre-F conformation trimer.

[0285] Example 4: Antigen screening

[0286] The present application carried out two rounds of RSV F protein mutant optimization. Among them,

[0287] 1. For the first round of RSV F protein mutant optimization, the inventors optimized the sequence from the following two aspects:

[0288] (1) Disulfide bond mutation: increase disulfide bond crosslinking (C-C), a total of 41 pairs of mutations;

[0289] (2) Proline mutation: modify proline, fix the angle between adjacent secondary structures, prevent transition to post F conformation, a total of 8 mutations; A total of 328 mutants were detected by the method described below.

[0290] 2. For the second round of RSV F protein mutant optimization, the inventors optimized the sequence from the following two aspects:

[0291] (1) Cavity filling mutation: enhance stability, single point mutation, a total of 44 mutations;

[0292] (2) Electrostatic mutation: promote expression and solubility, single point mutation, a total of 65 mutations; A total of 109 mutants were detected by the method described below, and each mutant was made in duplicate.

[0293] 4.1 Mutant expression detection

[0294] 4.1.1 Experimental reagents:

[0295] Coating solution: Take sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, and place them in a 2 L beaker. Add 600 ml of purified water, stir to dissolve, adjust the pH to 9.6 with NaOH solution, and dilute with water to 1 L. Mix well.

[0296] Washing solution: Take 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, and add 600 ml of purified water to a 2 L beaker. Stir to dissolve, and add 0.5 ml of Tween 20. Dilute with water to 1 L, and mix well.

[0297] Blocking solution: Take a 50 ml centrifuge tube, weigh 2 g of skimmed milk powder, and add 40 ml of PBS to prepare 40 ml of blocking solution. Mix well on a vortex mixer and store at 4°C for later use.

[0298] Dilution solution: Take a 50 ml centrifuge tube, measure 10 ml of blocking solution, and add 40 ml of PBS to prepare 50 ml of sample dilution solution. Mix well on a vortex mixer and store at 4°C for later use.

[0299] Color developing solution: Take a 50 ml centrifuge tube, measure 15 ml of PB solution, and add 5.6 μl of hydrogen peroxide. Mix well, then add 150 μl of TMB mother liquor. Mix well, wrap with tin foil, and store at room temperature in the dark. Prepare fresh each time.

[0300] Termination solution: Measure 300 ml of 3 mol HCL, and add 600 ml of purified water to prepare 900 ml of 1 mol HCL.

[0301] Secondary antibody: HRP anti-His Tag Antibody, 652504, biolegend.

[0302] Plate coating protein: RSV F Antibody (11A9), Epitope II, DA091, Suzhou Jinan Protein Technology Co., Ltd.

[0303] Standard: Recombinant RSV Prefusion F-trimer Protein (strain A2), DRA230, Suzhou Jinan Protein Technology Co., Ltd.

[0304] Detection sample: Mutant cell supernatant after transfection

[0305] 4.1.2 Experimental steps:

[0306] (1) Coating: Dilute the coating liquid to 10 ug / ml, then add to the enzyme-labeled plate, 100ul / well, 37°C incubation for 1h;

[0307] (2) Washing: Wash 3 times with washing liquid, 300ul / time, 5min each time, after washing the plate, dry the enzyme-labeled plate on the water-absorbing paper, and there is no obvious washing liquid residue in the plate hole;

[0308] (3) Blocking: Add blocking solution, 200ul per well, then incubate at 37°C for 1h;

[0309] (4) Washing: Repeat step (2);

[0310] (5) Binding: Dilute the detection sample with diluent, dilute by 0, 10, 20 times respectively, then add to the enzyme-labeled plate, 100ul per well, 37°C incubation for 1h;

[0311] (6) Washing: Repeat step (2);

[0312] (7) Incubation of secondary antibody: Add secondary antibody, 100ul per well, 37°C incubation for 1h;

[0313] (8) Washing: Repeat step (2);

[0314] (9) Color development: Add color developing solution, 100ul / well, avoid light, 37°C incubation for 15min;

[0315] (10) Termination: Add termination solution, 100ul / well;

[0316] (11) Reading: Place the enzyme-labeled plate in the enzyme-labeled instrument, set the wavelength to 450nm, then read the value.

[0317] Use RSVFC to make a standard curve, and calculate the concentration of the target protein in the cell supernatant by substituting the OD450 reading value of the detection sample diluted by different times into the standard curve.

[0318] 4.2 Affinity test

[0319] 4.2.1 BLI experiment

[0320] The purpose of this experiment is to detect the stability and affinity of RSV protein under different conditions by BLI experiment. The instrument emits white light to the sensor surface and collects the reflected light. The reflection spectrum of different frequencies is affected by the thickness of the optical film layer of the biosensor and forms interference. Therefore, once the number of molecules combined 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 changes in the thickness and density of the biological film on the sensor surface, so as to accurately quantify the interaction process between the molecules to be measured. It can reflect the binding and dissociation information of the interacting molecules in real time, and also can quantify the concentration.

[0321] (a) Main instruments and materials:

[0322] Fortebio (Sartorius, Octet Red 96e);

[0323] 96-well black plate (Molecular Devices, 655209)

[0324] (b) Main reagents and materials:

[0325] Pro-A Biosensors (Sartorius, 18-5013)

[0326] Running buffer: 1×PBST (phosphate buffer), pH 7.4

[0327] Sample diluent: 1×PBST, pH 7.4

[0328] Regeneration solution: 10 mM Glycine-HCl (Glycine-HCl regeneration solution), pH 1.5

[0329] (c) Detection process

[0330] Equilibrium: immerse the sensor in the running buffer for 60 s

[0331] Capture: dilute the antigen to 10 ug / mL with the sample diluent, and the capture height is about 1 nm

[0332] Equilibrium: immerse the sensor in the running buffer for 60 s

[0333] Binding: gradient dilution of SVR protein, combined with Motavizumab or D25 or MPE8 or DI-14359 or AM14 antibody sensor for 180 s

[0334] Dissociation: dissociate the sensor in the running diluent for 300 s

[0335] Regeneration: regenerate in 10 mM Glycine-HCl for 5 s each time

[0336] Neutralization: Immediately after each regeneration, immerse in running buffer for 5 s.

[0337] (d) Data analysis

[0338] Binding experiment buffer is 0.02% PBST buffer, and 0.02% PBST is used as a negative control. After subtracting the negative control, the sample signal (the solidified signal represents the case where the sensor captures the antigen, and the sample signal represents the case where the sensor that captures the antigen binds the SVR protein, and after subtraction, the binding of the antigen and the SVR protein is obtained) is fitted using Octet analysis software Data Analysis HT 12.0.

[0339] 4.2.2 ELISA experiment

[0340] (a) Reagent preparation:

[0341] Coating solution: Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate into a 2 L beaker, add 600 ml of purified water, stir to dissolve, adjust the pH to 9.6 with NaOH solution, dilute with water to 1 L, and mix well.

[0342] 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 into a 2 L beaker, add 600 ml of purified water, stir to dissolve, measure 0.5 ml of Tween 20, dilute with water to 1 L, and mix well.

[0343] Blocking solution: Take a 50 ml centrifuge tube, weigh 2 g of skimmed milk powder, add 40 ml of PBS, prepare 40 ml of blocking solution, mix well on a vortex mixer, and store at 4°C for standby.

[0344] Dilution solution: Take a 50 ml centrifuge tube, measure 10 ml of blocking solution, add 40 ml of PBS, prepare 50 ml of sample dilution solution, mix well on a vortex mixer, and store at 4°C for standby.

[0345] Color developing solution: Take a 50 ml centrifuge tube, measure 15 ml of PB solution, add 5.6 μl of hydrogen peroxide, mix well, then add 150 μl of TMB mother liquor, mix well, wrap with tin foil paper, store at room temperature in the dark, and prepare it for use.

[0346] Termination solution: Measure 300 ml of 3 mol HCL, then add 600 ml of purified water, prepare 900 ml of 1 mol HCL.

[0347] Secondary antibody: HRP anti-His Tag Antibody, 652504, biolegend.

[0348] Coating protein: RSV Pre-F Antibody (3C12), Epitope Φ, DA101, Suzhou Dongkang Protein Technology Co., Ltd.

[0349] Standard: Recombinant RSV Prefusion F-trimer Protein (strain A2), DRA230, Suzhou Dongkang Protein Technology Co., Ltd.

[0350] Detection sample: supernatant of mutant transfected cells

[0351] (b) Experimental method:

[0352] (1) Coating: dilute the above coating protein to 10 ug / ml with coating solution, then add to the enzyme-labeled plate, 100ul / well, 37℃ incubate for 1h;

[0353] (2) Washing: wash 3 times with washing solution, 300ul / time, 5min each time, after the plate washing is completed, dry the enzyme-labeled plate on the absorbent paper, and there is no obvious washing solution residue in the plate hole;

[0354] (3) Blocking: add blocking solution, 200ul per well, then 37℃ incubate for 1h;

[0355] (4) Washing: repeat step 2;

[0356] (5) Binding: dilute the detection sample with diluent by half to 12 gradients, then add to the enzyme-labeled plate, 100ul per well, 37℃ incubate for 1h;

[0357] (6) Washing: repeat step 2;

[0358] (7) Incubate secondary antibody: add secondary antibody, 100ul per well, 37℃ incubate for 1h;

[0359] (8) Washing: repeat step 2;

[0360] (9) Color development: add color developing solution, 100ul / well, avoid light, 37℃ incubate for 15min;

[0361] (10) Termination: add termination solution, 100ul / well;

[0362] (11) Reading: place the enzyme-labeled plate in the enzyme-labeled instrument, set the wavelength to 450nm, then read.

[0363] The OD450 reading value of the sample to be tested is fitted by four parameters, and the EC50 is calculated. The standard curve is prepared using the RSV FC protein, and the OD450 reading value of the sample to be tested is substituted into the standard curve for back calculation to calculate the concentration of the target protein in the cell supernatant.

[0364] 4.3 Stability experiment

[0365] The stability of the sequence-optimized RSV F protein mutants was evaluated by stress test and storage stability experiment.

[0366] (1) During the heat stress test, the crude culture supernatant of the sequence-optimized mutants, i.e. 293T cell culture supernatant, was incubated at 50°C or 70°C or pH 3.5 for 1 hour, and detected by pre-fusion specific monoclonal antibody D25 in an ELISA assay. The detection method was affinity detection according to the experimental method of Example 4.2.

[0367] (2) During the storage stability test, the pre-fusion antibody reactivity in the crude culture supernatant before and after freeze-thawing 10 times was compared, and detected by pre-fusion specific monoclonal antibody D25 in an ELISA assay. The detection method was affinity detection according to the experimental method of Example 4.2.

[0368] The results of the first round of screening of disulfide bond and proline mutations showed that:

[0369] (1) A total of 67 mutants with affinity > RSV FC control, among which the disulfide bond mutations 258C-275C, 288C-151C and the proline mutations 214P-215P appeared most frequently in each high-affinity mutant, with frequencies of 5 / 67, 5 / 67 and 14 / 67, respectively (Figure 7).

[0370] (2) A total of 63 mutants with expression > 1.0 ng / mL, and 45 mutants with expression > 2.0 ng / mL, among which the disulfide bond mutations 198C-226C, 97C-291C and the proline mutations 214P-215P appeared most frequently in each mutant with expression > 2.0 ng / mL, with frequencies of 6 / 45, 6 / 45 and 10 / 45, respectively (Figure 6).

[0371] According to the affinity ranking, 10 mutants with expression > 2.0 ng / mL were selected from the top 40 for expression and purification screening, and the results showed that the SVR88 / SVR79 / SVR72 / SVR46 mutants had the highest expression, with expression > 85 ng / ml, as shown in Table 5:

[0372] Table 5

[0373] The affinity detection results with different antibodies showed that the D25 and AM14 antibody affinity values of the two mutants SVR88 and SVR152 were in the same order of magnitude as the RSV FC standard protein, as shown in Table 6:

[0374] Table 6

[0375] Note: "-" means no binding to the antibody.

[0376] The results of the affinity detection of the 10 priority mutants obtained in the first round of screening with different antibodies in Table 6 show that the affinity of the SVR45, SVR46, SVR90, SVR64 and SVR201 mutants to the antibodies is poor, and therefore, the five mutants are no longer subjected to subsequent stability screening.

[0377] The stability of the sequence-optimized RSV F protein mutants is evaluated by stress test and storage stability experiment. During the stress test, the crude culture supernatant of the sequence-optimized mutants is incubated at 50°C or 70°C or pH 3.5 for 1 hour and detected in an ELISA assay with the pre-fusion specific monoclonal antibody D25. The detection method detects the affinity according to the experimental method of Example 4.2. The more stable mutants are expected to have higher stress resistance. During the storage stability assay, the pre-fusion antibody reactivity in the crude culture supernatant after freeze-thawing for 10 times is compared with the reactivity of the fresh culture supernatant. The results are shown in Table 7:

[0378] Table 7

[0379] The results in Table 7 show that the stability of the SVR152, SVR79 and SVR88 mutants is good, and the above three mutants are used for subsequent immune evaluation.

[0380] For the second round of RSV F protein mutant optimization, the inventors sequence-optimized from the following two aspects:

[0381] 1. Cavity filling mutation, enhancing stability, single point mutation, a total of 44 mutations;

[0382] 2. Electrostatic mutation, promoting expression and solubility, single point mutation, a total of 65 mutations.

[0383] A total of 109 mutants are detected by the above method, and each mutant is made into 2 duplicate wells. The mutation site is 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:

[0384] Table 8

[0385] Note: * means that at least one duplicate well of the mutant meets the screening conditions in this column; blank indicates that the detection results of 2 duplicate wells of the mutant do not meet the screening conditions in this column, and are no longer subjected to subsequent detection.

[0386] The results of the second round of RSV F protein mutant optimization shown in Table 8 indicate that:

[0387] (1) A total of 36 mutants have an expression amount of at least 1 complex hole > 2.0 ng / mL, and 33 mutants have an expression amount of 2 complex holes > 2.0 ng / mL;

[0388] (2) The number of mutants with high expression amounts (> 2.0 ng / mL) in the electrostatic filling mutation (33 mutants) is greater than the number of mutants with high expression amounts in the space filling mutation (3 mutants);

[0389] (3) The protein expression amount of the second round of RSV F protein mutant optimization is greatly improved compared to the protein expression amount of the first round of optimization: 34 mutants have an expression amount > 10.0 ng / mL, and 20 mutants have an expression amount > 80.0 ng / mL;

[0390] (4) Among the 36 mutants with high expression amounts, the EC50 of at least 1 complex hole in 21 mutants is < EC50 RSVFC (That is, the affinity (D25) in Table 8 > RSV FC, and the smaller the EC50 value, the tighter the binding of the antibody and the target molecule, and the stronger the affinity), and the EC50 of 2 complex holes in 15 mutants is < EC50 RSVFC .

[0391] The 21 mutants with high expression amounts and good affinity were selected for the above stability test, and the mutants SVRA99 (R106F) and SVR83 (K399T) passed the stability test, and the results are shown in Table 9:

[0392] Table 9

[0393] Note: “-” indicates no binding.

[0394] According to the sites R106F and K399T screened by the second round of RSV F protein mutant high-throughput hole plate screening, combined with the results of the first round of screening (SVR152, SVR88, SVR79), a combination mutant was constructed, and protein expression and purification and antibody affinity detection were performed, as shown in Table 10:

[0395] Table 10

[0396] The results of Table 10 show that R106F and K399T have different effects on the expression amount and antibody affinity of different mutant proteins: (1) the expression amount of the SVR152 mutant protein is increased; (2) the expression amount of the SVR88 and SVR79 mutant proteins is decreased; (3) the affinity of the SVR79 mutant protein to the D25 antibody is greatly reduced; and (4) the affinity of the SVR152 and SVR79 mutant proteins to the ADI-14359 antibody is increased.

[0397] Based on the above results, SVR88B, SVR88C, SVRA99, SVRA83, and SVR152A were screened for stability tests, and the results are shown in Table 11.

[0398] Table 11

[0399] Based on the antibody affinity data, expression amount data, and stability test data shown in Table 10 and Table 11, in the combination of the first and second rounds of RSV F protein mutant optimization, the SVR88C and SVRA99 mutants were screened for subsequent immunization experiments.

[0400] Example 5 Immunization Experiment

[0401] This example relates to two rounds of immunization experiments. In the first round of immunization experiment, serum samples were collected on day 28 after immunizing mice twice with the mutants screened in the first round (SVR152, SVR88, and SVR79), and preF-specific binding antibodies were detected by ELISA.

[0402] In the second round of immunization experiment, serum samples were collected on day 42 after immunizing mice twice with the SVR88C and SVRA99 mutants and the SVR79 mutant screened in the combination of the first and second rounds of RSV F protein mutant optimization, and preF-specific binding antibodies were detected by ELISA. The specific experimental process is as follows:

[0403] (1) Vaccine preparation: 5 ug of purified RSV pre-F protein was mixed and emulsified with 5 ug of A401 or MF59 adjuvant (purchased from Shangkang Xinuo), and no antigen precipitation or layering was observed after the emulsified vaccine was placed at 4 degrees overnight.

[0404] (2) Animal immunization: 6-8 week old female BALB / c mice were selected, weighed, and subjected to two rounds of RSV F protein mutant immunization experiments, with the immunization mode being intramuscular injection.

[0405] The specific grouping and immunization scheme of the first round of RSV F protein mutant immunization experiment are shown in Table 12.

[0406] Table 12

[0407] The specific groupings and immunization protocols for the second round of RSV F protein mutant immunization experiments are shown in Table 13:

[0408] Table 13

[0409] DS-Cav1 was used as a positive control. The sampling and testing time after the first round of immunization with RSV F protein mutants was day 28, and the sampling and testing time after the second round of immunization with RSV F protein mutants was day 42.

[0410] (3) Specific IgG antibody detection: ELISA plates were coated with 5 μg / ml of purified pre-F protein and incubated overnight. The plates were blocked with PBS / 3% bovine serum albumin, and then control and mouse serum samples were added (the serum samples collected in (2) above were diluted sequentially to 10 μL). 2 10 3 10 4 10 5 10 6 10 7 (Times), and incubated at 37°C for 2 hours. Wash the plate and incubate with rabbit anti-mouse IgG for 30 minutes at room temperature. Wash the plate again, incubate with anti-rabbit IgG-horseradish peroxidase conjugate for 30 minutes at room temperature, wash again, and incubate with 2,2′-azinobis-(3-ethylbenzothiazoline sulfonic acid), with an optical reading of 405 nm.

[0411] (4) Neutralizing antibody detection: HEp-2 cells were resuscitated and cultured before the experiment, and the cells were pre-digested and seeded into 96-well plates at 2.5 × 10⁻⁶. 5 cells / mL, 0.1mL / well, incubated at 37℃, 5% CO2 Incubate overnight in an incubator; dilute the serum samples collected in (2) above by 40 times, and then dilute them by 3 times in sequence, for a total of 8 dilution gradients, and set up negative and positive controls at the same time; add RSV A2 virus with a titer of 5000 PFU / mL, incubate for 1 hour in an incubator, transfer to cell culture plate, infect HEp-2 cells, and incubate for 1 day; fix and stain the cells after 1 day, and calculate the neutralizing antibody titer.

[0412] (5) The method for detecting neutralizing antibodies is as follows:

[0413] 1) Cell preparation and plate seeding: HEp-2 cells (purchased from Shanghai Yuchi Biotechnology) were revived and cultured before the experiment. The passage number used for detection was 3-20 passages after revival. Cells were pre-digested and seeded into 96-well plates at 2.5 × 10⁵ cells / mL, 0.1 mL / well, and incubated at 37℃ with 5% humidity. CO2 Incubate overnight in an incubator;

[0414] 2) Sample dilution: confirm that the cells are normal, take serum samples, dilute;

[0415] 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 replicates for VC and 8 replicates for CC;

[0416] 4) Virus dilution: after thawing the virus solution, dilute the virus to 5000 PFU / mL with diluent according to the virus titration titer;

[0417] 5) Virus addition: take the virus solution in step 4), add 60 μL / well to the sample wells and VC wells of the 96-well plate and mix well;

[0418] 6) Neutralization reaction: place the 96-well plate in a 37°C, 5% CO2 incubator for 1 h; CO2

[0419] 7) Cell preparation and infection: take the 96-well plate with cells inoculated in advance, discard the original culture medium, and add 100 μL of the mixed solution per well to the 96-well plate with cells;

[0420] 8) Incubation: place the 96-well plate in a 37°C, 5% CO2 incubator for 1 day; CO2

[0421] 9) Fixation: after incubation, discard the original culture medium, add 100 μL of fixing solution per well, and fix for 20 min or overnight;

[0422] 10) Staining: after fixing the cell plate, discard the fixing solution, wash twice with 1x PBS, add 300 μL / well of blocking solution, and block for 30 min; discard the blocking solution, wash twice with 1x PBS, add 50 μL / well of primary antibody working solution, and incubate at 37°C for 1 h; discard the antibody, wash twice with 1x PBS, add 50 μL / well of HRP secondary antibody working solution, and incubate at 37°C for 1 h; discard the antibody, wash twice with 1x PBS, add 30 μL / well of KPL TrueBlue Peroxidase Substrate developing solution, and incubate at room temperature for 5-20 min; discard the developing solution, and count the spots using an enzyme-linked immunosorbent spot analyzer.

[0423] 11) Result calculation: neutralizing antibody titer: calculate the inhibition rate of each gradient of the sample:

[0424] Inhibition rate = 1 - (sample replicate spot number / VC well average spot number),

[0425] ​​The sample dilution ratio-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.

[0426] After two immunizations of the mutants (SVR152, SVR88 and SVR79) in the first round of screening, serum samples were collected on day 28, and RSV pre-F specific binding antibodies were detected by ELISA. The results are shown in Figure 8. Compared with the control group DS-Cav-1 group, the SVR79 mutant protein can induce the mouse body to produce high titer of RSV pre-F specific binding antibodies (10 7 ), with statistical difference; compared with other mutant proteins, the immunogenicity of the SVR79 mutant protein is optimal.

[0427] The mutants (SVR88C and SVRA99) in the second round of screening and the SVR79 mutant with optimal immunization evaluation in the first round were subjected to immunization evaluation again. After two immunizations of the mice, serum samples were collected on day 42, and RSV pre-F specific binding antibodies were detected by ELISA. The results are shown in Figure 9. The mutant SVR79 can reach the same level of RSV pre-F binding antibody titer (10 6 ) as the control DS-Cav1, however, the high-quality mutants SVR88C and SVRA99 screened on the basis of SVR79 through cavity filling and electrostatic mutation do not currently show better humoral immune effect.

[0428] Then the inventors continued to analyze from the perspective of neutralizing antibodies through the above neutralization test, and the results are shown in Figure 10. The SVR79 mutant can induce the highest neutralizing antibody titer (EC 50 = 6677), which is extremely significantly different (P < 0.0001) compared with other mutants, and is about 7 times higher than the control group DS-Cav1+MF59 (EC 50 = 961).

[0429] Therefore, the SVR79 mutant is finally selected for subsequent immunization and challenge experiments.

[0430] Example 6 Cotton rat immunization experiment

[0431] 6-8 week old female cotton rats were selected, and the body weight was weighed. Two rounds of RSV F protein mutant immunization experiments were performed on day 0 and day 21, and the immunization method was intramuscular injection. mRNA-1345 is a commercial vaccine for preventing RSV. The specific grouping and immunization scheme of the immunization experiment are shown in Table 14, wherein DS-Cav1 is used as a positive control, and AS01E is an adjuvant.

[0432] Table 14

[0433] (1) Immunization experiment

[0434] Experimental animals: 15 guinea pigs, female; randomly divided into 5 groups, 3 in each group, 6-8 weeks old.

[0435] The experimental steps are as follows:

[0436] Isolation and observation: 15 guinea pigs were randomly divided into 5 groups, 3 in each group, 3 in each cage for 3 days of feeding and observation.

[0437] Blood collection on day 0, serum collection at -80°C for storage, for subsequent detection of specific antibodies; first immunization.

[0438] Blood collection on day 21, serum collection at -80°C for storage, for subsequent detection of specific antibodies; second immunization on day 21.

[0439] Blood collection on day 49, serum collection at -80°C for storage, for subsequent detection of specific IgG antibodies and neutralizing antibodies.

[0440] (2) Detection of specific IgG antibodies:

[0441] (a) Reagent preparation:

[0442] Coating solution: weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, place in a 2 L beaker, add 600 ml of purified water, stir to dissolve, adjust pH to 9.6 with NaOH solution, dilute with water to 1 L, and mix well.

[0443] 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 purified water, place in a 2 L beaker, stir to dissolve, measure 0.5 ml of Tween 20, dilute with water to 1 L, and mix well.

[0444] Blocking solution: take a 50 ml centrifuge tube, weigh 2 g of skimmed milk powder, add 40 ml of PBS, prepare 40 ml of blocking solution, mix well on a vortex mixer, and store at 4°C for later use.

[0445] Dilution solution: take a 50 ml centrifuge tube, measure 10 ml of blocking solution, add 40 ml of PBS, prepare 50 ml of sample dilution solution, mix well on a vortex mixer, and store at 4°C for later use.

[0446] Color developing solution: take a 50 ml centrifuge tube, measure 15 ml of PB solution, add 5.6 μl of hydrogen peroxide, mix well, then add 150 μl of TMB mother liquor, mix well, wrap with tin foil paper, store at room temperature in the dark, and prepare fresh for use.

[0447] Stop solution: take 3M HCL 300ml, add 600ml of purified water, make 900ml 1M HCL.

[0448] Secondary antibody: horseradish peroxidase labeled chicken anti-mouse secondary antibody, PAB29753, Abnova.

[0449] Coat plate protein: RSV preF protein, DRA230, Coastal proteins.

[0450] (b) Experimental method:

[0451] 1) Coating: dilute preF protein to 5 μg / ml, add to 96-well plates, 100 μL / well, after sealing with plate sealing film, incubate at 4°C overnight.

[0452] 2) Wash plate: 300 μL / well PBST buffer, wash five times, spin dry.

[0453] 3) Blocking: add 200 μL of PBS containing 3% BSA to each well, incubate at 37°C for 2h.

[0454] 4) Wash plate: 300 μL / well PBST buffer, wash five times, spin dry.

[0455] 5) Add sample: add 100 μL of gradient-diluted serum to each well (dilute serum 100 times, dilute to 107 by 10 times gradient), incubate at 37°C for 1.5h.

[0456] 6) Wash plate: 300 μL / well PBST buffer, wash five times, spin dry.

[0457] 7) Add secondary antibody: add 100 μL of 0.05 μg / ml horseradish enzyme labeled chicken anti-mouse secondary antibody to each well, incubate at 37°C for 1h.

[0458] 8) Wash plate: 300 μL / well PBST buffer, wash five times, spin dry.

[0459] 9) Color development: add 100 μL of TMB (tetramethylbenzidine) color developing solution to each well, incubate at 37°C for 15min.

[0460] 10) Stop: add 50 μL of stop solution to each well to stop color development.

[0461] 11) Read: measure the absorbance (OD value) of each well with a microplate reader (wavelength 450nm)

[0462] (3) The neutralizing antibody detection method is the same as that in Example 5.

[0463] The results of specific antibody and neutralizing antibody detection are shown in Figure 11. RSV pre-F specific binding antibodies were detected by ELISA. The results are shown in Figure 11A. 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, and the specific antibodies of D21 are significantly higher than the DS-Cav1 control. The serum samples on day 49 were detected for neutralizing antibodies, and the results are shown in Figure 11B. The SVR79 mutant protein can produce higher neutralizing antibody titers than the control DS-Cav1. The neutralizing antibody titers of the subunit vaccine can reach 28859, which is about 4.9 times that of the control DS-Cav1 (neutralizing antibody titer of 5904). The neutralizing antibody titers of the mRNA vaccine of SVR79 can reach 72000, which is about 5.3 times that of the control mRNA-1345 (neutralizing antibody titer of 13619). In summary, the SVR79 mutant protein has the potential to develop into a candidate vaccine for preventing RSV diseases.

[0464] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that various modifications or alterations can be made to the above-described disclosure without departing from the scope of the application.

Claims

1. A mutein, characterized in that, the mutant protein is in monomeric form or in trimeric form, wherein the trimer is formed by three monomeric forms of the mutant protein; the mutant protein is in monomeric form or in trimeric form, wherein the trimer is formed by three monomeric forms of the mutant protein; the first polypeptide element is derived from a RSV F protein, wherein the Pep27 fragment of the RSV F protein is replaced by a linker sequence; and the mutant protein in monomeric and trimeric form is specifically recognized by an antibody binding to RSV pre-F.

2. The mutein according to claim 1, characterized in that, the mutant protein comprises at least one amino acid mutation relative to the amino acid sequence of a wild-type RSV F protein of sequence as set forth in SEQ ID NO: 1, and wherein the amino acid mutation is selected from one or more of the following groups: (1) a disulfide bond mutation; (2) a loop region mutation; (3) a cavity filling mutation; (4) an electrostatic mutation.

3. The mutein of claim 2, characterized in that, the disulfide bond mutation is selected from one or more of the following combinations: 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 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.

4. The mutein of claim 2, characterized in that, the loop region mutation is selected from the following groups: SCSPPN, SCSISP, SPSISN, SCPPSN, PCSISN, or SPPISN.

5. The mutein of claim 2, wherein the cavity filling mutation is selected from one or more of the following combinations: (1) S at position 55, 62, or 290 is substituted with I, Y, L, or W; (2) T at position 54, 58, 189, 219, or 397 is substituted with I, Y, L, or W; (3) A at position 147 or 298 is substituted with I, Y, L, or W; (4) V at position 164, 187, 192, 207, 220, 296, 300, or 495 is substituted with I, Y, L, or W; (5) the R at position 106 is substituted with I, Y, L, or W.

6. The mutein of claim 2, wherein the electrostatic mutations are selected from one or more combinations of the group consisting of: (1) the E at position 82, 92, or 487 is substituted with F, T, H, or W; (2) the K at position 315, 394, or 399 is substituted with F, T, H, or W; (3) the D at position 392, 486, or 489 is substituted with F, T, H, or W; (4) the R at position 106 or 339 is substituted with F, T, H, or W; (5) the E at position 161 is substituted with F, T, H, or W; (6) the S at position 182 or 173 is substituted with F, T, H, or W.

7. The mutein of claim 1, wherein the mutant protein comprises an F2 polypeptide and an Fl polypeptide selected from the group consisting of: (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 19; (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 Fl 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; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 20; (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 Fl 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; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 21; (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 Fl 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; (7) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 22; (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 Fl 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; (9) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (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 Fl 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; (11) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 24; (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 Fl 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; (13) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 25; (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 Fl 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; (15) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 26; (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 Fl 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; (17) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 17 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 27; (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 Fl 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; (19) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 18 and an Fl polypeptide comprising the amino acid sequence of SEQ ID NO: 28; or (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 Fl 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.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises (a) the mutein of claim 1, a polynucleotide encoding the mutein, or a vector expressing the mutein; and (b) a pharmaceutically acceptable carrier.

9. Use of the mutein according to claim 1 or a polynucleotide encoding the mutein or a vector expressing the mutein, characterized in that, For use in the preparation of a composition or formulation for: (a) preventing and / or treating RSV infection; and / or (b) increasing RSV pre-F protein or pre-fusion protein conformation and / or decreasing RSV post-F protein or post-fusion protein conformation in a cell.

10. A kit characterized in that, The mutein of claim 1, further comprising a detection reagent for detecting the amount of RSV pre-F protein.

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