Recombinant respiratory syncytial virus pre-fusion (f) protein mutant and use thereof

Abstract

Provided is a recombinant respiratory syncytial virus (RSV) fusion (RSV F) protein, comprising F2 and F1 polypeptides, wherein the recombinant RSV F protein contains cysteines (C) at positions corresponding to the amino acid positions 72 and 76 of SEQ ID NO: 1, and independently contains, relative to SEQ ID NO: 1, amino acid replacements at one or more positions selected from positions corresponding to the amino acid positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489 and 492 of SEQ ID NO: 1. Further provided are an immunogenic composition comprising said protein, and the use thereof in the prevention and / or treatment of RSV infections.

Description

Recombinant respiratory syncytial virus prefusion F protein mutant and its use Technical Field

[0001] The present invention relates to the field of medicine and particularly to recombinant prefusion RSV F proteins, nucleic acid molecules encoding these RSV F proteins, and their use in preventing and treating RSV infection. Background Art

[0002] Respiratory syncytial virus (RSV) is an enveloped non-segmented negative-strand RNA virus in the genus Pneumovirus of the family Paramyxoviridae. It is the most common cause of bronchiolitis and pneumonia in children in their first year of life. RSV also causes recurrent infections, including serious lower respiratory tract diseases, which may occur at any age, especially in the elderly or those with impaired heart, lung or immune systems. Passive immunity is currently used to prevent the serious illness caused by RSV infection, especially in infants with premature birth, bronchopulmonary dysplasia or congenital heart disease. Current treatment includes the use of the RSV neutralizing antibody Palivizumab (MedImmune, Inc.), which combines the linear conformational epitopes of 24 amino acids on the RSV fusion (F) protein.

[0003] In nature, the RSV F protein is initially expressed as a single polypeptide precursor, called F0. F0 trimerizes in the endoplasmic reticulum and is processed by cellular furin-like proteases at two conserved sites to produce F1, F2, and Pep27 polypeptides. The Pep27 polypeptide is cleaved and does not constitute part of the mature F protein. The F2 polypeptide is derived from the N-terminal portion of the F0 precursor and is linked to the F1 polypeptide via two disulfide bonds. The F1 polypeptide is derived from the C-terminal portion of the F0 precursor and anchors the mature F protein in the membrane via a transmembrane domain that is linked to a cytoplasmic tail of approximately 24 amino acids. The three protomers of the F2-F1 heterodimer assemble to form the mature F protein, which adopts a metastable prefusion conformation that is triggered to undergo conformational changes that fuse the viral and target cell membranes. Due to its mandatory role in RSV invasion, the RSV F protein is a target for neutralizing antibodies and a subject of vaccine development. McLellan, et al. Science 342, 592-598 (2013); McLellan, et al. Nat Struct Mol Biol 17, 248-250 (2010); McLellan, et al. Science 340, 1113-1117 (2013).

[0004] Therefore, there is a need for effective vaccines against RSV, in particular vaccines comprising RSV F protein in a prefusion conformation. It is an object of the present invention to provide means for obtaining such stable prefusion RSV F protein for use in vaccination against RSV. Summary of the Invention

[0005] The present invention provides a recombinant respiratory syncytial virus (RSV) fusion (F) protein that is capable of being stably maintained in a prefusion conformation.

[0006] In one embodiment, the recombinant RSV F protein of the invention is a soluble multimeric protein, such as a trimeric protein.

[0007] The present invention also provides a nucleic acid molecule encoding the recombinant RSV F protein, and a vector comprising the nucleic acid molecule.

[0008] The present invention also relates to methods for stabilizing RSV F proteins in a prefusion conformation, and to prefusion RSV F proteins obtained or obtainable by said methods.

[0009] The present invention also relates to pharmaceutical compositions comprising the recombinant RSV F protein of the present invention, a nucleic acid molecule encoding the recombinant RSV F protein, and / or a vector comprising the nucleic acid molecule, and optionally a pharmaceutically acceptable excipient.

[0010] The present invention also relates to an RSV vaccine comprising the recombinant RSV F protein of the present invention, a nucleic acid molecule encoding the recombinant RSV F protein, and / or a vector comprising the nucleic acid molecule, and optionally an adjuvant.

[0011] The present invention also relates to a method for inducing an anti-respiratory syncytial virus (RSV) immune response in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant RSV F protein as described herein, a nucleic acid molecule encoding the recombinant RSV F protein, and / or a vector comprising the nucleic acid molecule. In particular, the present invention relates to a method for inducing anti-respiratory syncytial virus (RSV) F antibodies in a subject, comprising administering to the subject a therapeutically effective amount of an immunogenic composition comprising a recombinant RSV F protein as described herein, a nucleic acid molecule encoding the recombinant RSV F protein, and / or a vector comprising the nucleic acid molecule.

[0012] The present invention also relates to the recombinant RSV F protein of the present invention, a nucleic acid molecule encoding the recombinant RSV F protein, and / or a vector comprising the nucleic acid molecule, for use in preventing and / or treating RSV infection.

[0013] The present invention also relates to the use of the recombinant RSV F protein of the present invention, the nucleic acid molecule encoding the recombinant RSV F protein, and / or the vector comprising the nucleic acid molecule in the preparation of a medicament or vaccine for inducing an immune response against the RSV F protein, preventing and / or treating RSV infection.

[0014] The present invention also relates to a method for inducing an immune response against RSV F protein in a subject, preventing and / or treating RSV infection, comprising administering to the subject a therapeutically effective amount of a recombinant RSV F protein of the present invention, a nucleic acid molecule encoding the recombinant RSV F protein, and / or a vector comprising the nucleic acid molecule.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1: Screening of amino acid lengths of F2 and F1 polypeptides (ELISA method). Shows the relative expression levels of C1-1 to C1-5 polypeptides in Expi293F cells as detected by ELISA. PreF: prefusion F protein.

[0017] Figure 2: Screening of F2 polypeptide and F1 polypeptide linkers (ELISA method). The relative expression levels of LM1 to LM35 polypeptides in Expi293F cells detected by ELISA are shown. PreF: prefusion F protein.

[0018] Figure 3: C-terminal trimer screening (polyacrylamide gel electrophoresis). Shows the relative expression levels of T1 to T10 polypeptides in Expi293F cells detected by polyacrylamide gel electrophoresis. Marker: Molecular weight standard.

[0019] Figure 4: Cysteine ​​mutation screening (ELISA method). Shows the relative expression levels of CM1 to CM79 polypeptides in Expi293F cells detected by ELISA. PreF: prefusion F protein.

[0020] Figure 5: Proline mutation screening (ELISA method). Shows the relative expression levels of PM1 to PM44 polypeptides in Expi293F cells detected by ELISA. PreF: prefusion F protein.

[0021] Figure 6: Cavity hydrophobicity mutation screening (ELISA method). Shows the relative expression levels of V1 to V68 polypeptides in Expi293F cells detected by ELISA. PreF: prefusion F protein.

[0022] Figure 7: RSV F protein double-antibody sandwich ELISA. Shown are RSV F protein double-antibody sandwich ELISAs using motavizumab and 101F monoclonal antibodies, and motavizumab and a His antibody. Mota: motavizumab monoclonal antibody; HRP: horseradish peroxidase; Q1.5.10-Pre: prefusion RSV F protein; Q1.5.9-Post: postfusion RSV F protein; culture supernatant Pre: prefusion F protein culture supernatant; culture supernatant Post: postfusion F protein culture supernatant.

[0023] Figure 8: Double-antibody sandwich ELISA method for specific recognition of RSV PreF protein and RSV F protein trimer. Shown are double-antibody sandwich ELISA methods for specific recognition of RSV PreF protein and RSV F protein trimer, established using motavizumab monoclonal antibody and D25 monoclonal antibody, and motavizumab monoclonal antibody and AM14 antibody. Mota: motavizumab monoclonal antibody; HRP: horseradish peroxidase; Q1.5.10-Pre: prefusion conformation RSV F protein; Q1.5.9-Post: postfusion conformation RSV F protein; culture supernatant Pre: culture supernatant of prefusion F protein; culture supernatant Post: culture supernatant of postfusion F protein.

[0024] Figure 9: Serum binding antibody IgG titer 5 weeks after immunization (GMT represents geometric mean titer, grouping is consistent with neutralizing antibody).

[0025] Figure 10: Antigen immune binding antibody detection, where: 1 represents Group 1; 2 represents Group 2; 3 represents Group 3; 4 represents Group 4; GMT, geometric mean titer; first immunization, primary immunization; second immunization, booster immunization; second immunization challenge, challenge experiment after second immunization.

[0026] Figure 11: Antigen immune neutralizing antibody detection, where: 1 represents Group 1; 2 represents Group 2; 3 represents Group 3; 4 represents Group 4; 5 represents blank group; First immunization, primary immunization; Second immunization, booster immunization; Second immunization and challenge, challenge experiment after second immunization; Type A, RSV virus subtype A; Type B, RSV virus subtype B. DETAILED DESCRIPTION

[0027] Unless otherwise noted, the scientific and technical terms used herein should have the meanings commonly known to those skilled in the art. In addition, unless otherwise required, singular terms should include plural terms, and plural terms should include singular terms. The aforementioned techniques and methods are generally carried out according to conventional methods well known in the art and described in the references cited in this specification. See, for example, Sambrook et al.Molecular Cloning:A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)) and Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J.Wiley & Sons (New York, NY 1994), which are incorporated by reference; Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, are hereby incorporated by reference in their entirety.

[0028] The fusion protein (F protein) of respiratory syncytial virus (RSV) participates in the fusion of viral membrane and the host cell membrane of infection. RSV F mRNA is translated into a precursor protein called F0, which contains a signal peptide sequence (such as amino acid residues 1-25 of SEQ ID NO:1) at the N-terminus, which is removed in the endoplasmic reticulum. F0 is cut by cellular proteases (particularly furin or furin-like proteases) at two sites (between amino acid residues 109 / 110 and 136 / 137), removing short glycosylation intervening sequences (pep27 polypeptide, amino acid residues 110 to 136 of SEQ ID NO:1) to produce two polypeptides called F1 and F2. The F1 polypeptide contains a hydrophobic fusion peptide at its N-terminus, and the C-terminus contains a transmembrane (TM) and cytoplasmic region. The F2 polypeptide is covalently linked to F1 by two disulfide bonds. The F1-F2 heterodimer is assembled into a homotrimer in the virion.

[0029] The potential method of producing the vaccine that resists RSV infects hopes that the RSV F protein of purification is in stable prefusion conformation.For soluble protein vaccine, RSV F protein needs to carry out brachymembrane to produce soluble secretory F protein by disappearance transmembrane (TM) and cytoplasmic region.The soluble F protein in disappearance TM region is obviously more unstable than full-length protein.In order to obtain being in stable prefusion conformation and demonstrating the soluble F protein of high expression level and high stability, need to carry out stabilization by this prefusion conformation.

[0030] To stabilize soluble RSV F in a prefusion conformation and cleaved into F1 and F2 polypeptides, a minor fibritin-based trimerization domain was fused to the C-terminus of soluble RSV-F (McLellan, et al. Science 342, 592-598 (2013); McLellan, et al. Nat Struct Mol Biol 17, 248-250 (2010); McLellan, et al. Science 340, 1113-1117 (2013)).

[0031] The present invention provides a novel recombinant respiratory syncytial virus (RSV) fusion (F) protein comprising F2 and F1 polypeptides and comprising cysteine ​​(C) at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1, and amino acid substitutions at one or more (e.g., 2, 3, 4, 5 or more) positions selected from the group consisting of amino acid positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489, and 492, relative to SEQ ID NO: 1. Preferably, the amino acid substitutions are selected from the group consisting of isoleucine (I), valine (V), leucine (L), phenylalanine (F), and proline (P).

[0032] According to the present invention, a specific stabilizing amino acid (e.g., I, V, L, F, or P) at one or more positions corresponding to amino acid positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489, and / or 492 of SEQ ID NO: 1 (numbered according to SEQ ID NO: 1), in combination with cysteine ​​(C) at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1, stabilizes the RSV F protein in a prefusion conformation.

[0033] In one embodiment, the recombinant RSV F protein comprises cysteine ​​(C) at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 and an amino acid replacement at one or more (e.g., 2, 3, 4, 5, or more) positions selected from the group consisting of amino acid positions 67, 68, 80, 84, 87, 198, 202, 207, 210, 219, and 489 corresponding to SEQ ID NO: 1 relative to SEQ ID NO: 1. Preferably, the amino acid replacement is selected from I, V, L, F, and P.

[0034] When describing amino acid positions herein, the numbers refer to the positions in a reference sequence, such as SEQ ID NO: 1. Positions in a sequence of interest that correspond to positions in a reference sequence can be determined by methods known in the art.

[0035] The position corresponding to the amino acid position of a reference sequence (e.g., SEQ ID NO: 1) refers to the amino acid position identified when aligned with the reference sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved / identical amino acid residues as a guide. Typically, to identify corresponding positions, amino acid sequences are aligned to obtain the highest order match (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J Applied Math 48:1073).

[0036] As used herein, sequence alignment refers to the use of homology to align two or more amino acid sequences. Typically, two or more sequences with 50% or higher identity are aligned. Related or variant polypeptide molecules can be aligned by any method known to those skilled in the art. Such methods typically require maximal matching and include the use of a variety of available algorithms (e.g., BLASTP) and other methods known to those skilled in the art or manual alignment. By alignment, those skilled in the art can identify the corresponding amino acid residues between a sequence and a reference sequence. Corresponding positions can also be aligned based on structural alignments, such as using computer simulations of molecular structure.

[0037] As used herein, relative to SEQ ID NO: 1 refers to comparing a sequence of interest to the sequence shown in SEQ ID NO: 1 to determine corresponding amino acid positions.

[0038] In one embodiment, in the recombinant RSV F protein, the amino acids at positions corresponding to amino acid positions 67, 68, 80, 84, 87, 198, 202, 207, 210, 219, and / or 489 of SEQ ID NO: 1, preferably amino acid positions 67, 68, 87, and / or 198, are independently I, V, L, or F. For example, the amino acid at the position corresponding to amino acid position 67 of SEQ ID NO: 1 is V, the amino acid at the position corresponding to amino acid position 68 of SEQ ID NO: 1 is I, the amino acid at the position corresponding to amino acid position 68 of SEQ ID NO: 1 is V, the amino acid at the position corresponding to amino acid position 87 of SEQ ID NO: 1 is L, and / or the amino acid at the position corresponding to amino acid position 198 of SEQ ID NO: 1 is F.

[0039] As used herein, "independently" means that the amino acid at each position can be a different amino acid residue. For example, the amino acid at positions 68, 87, and / or 198 can be I, V, L, or F independently, meaning that the amino acid at positions 68, 87, and 198 can be I, V, L, or F, and they can be the same amino acid residue or different amino acid residues, and there is no specific relationship between them unless otherwise indicated.

[0040] In one embodiment, in the recombinant RSV F protein, the amino acid corresponding to amino acid position 161, 211, 213, 214, 217, 459, 460, 463, 470, 472, 479, 481, 485 and / or 492 of SEQ ID NO: 1, preferably amino acid position 161, 214, 460 and / or 463, is P.

[0041] In one embodiment, in the recombinant RSV F protein, the amino acid corresponding to amino acid position 102 of SEQ ID NO: 1 is A, the amino acid corresponding to amino acid position 379 of SEQ ID NO: 1 is V, and / or the amino acid corresponding to amino acid position 447 of SEQ ID NO: 1 is V.

[0042] In one embodiment, the present invention provides a novel recombinant respiratory syncytial virus (RSV) fusion (F) protein comprising F2 and F1 polypeptides and comprising cysteine ​​(C) at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1, and amino acid substitutions at one or more positions selected from amino acid positions 67, 68, 87, 98, 161, 214, 460 and 463 corresponding to SEQ ID NO: 1 relative to SEQ ID NO: 1.

[0043] According to the present invention, the specific stabilizing amino acid present at said position increases the stability of the protein in the pre-fusion conformation. According to the present invention, the specific amino acid may already be present in the amino acid sequence (e.g. 102A, 379V and / or 447V), or it may be introduced into the specific amino acid according to the present invention by substitution (mutation) of the amino acid at said position.

[0044] In certain embodiments, the recombinant RSV F protein further comprises a C at at least one position, preferably at least two positions, selected from the group consisting of amino acid positions 26, 38, 41, 55, 67, 71, 75, 76, 89, 101, 148, 155, 159, 171, 188, 191, 207, 215, 231, 232, 242, 250, 286, 290, 291, 318, 324, 327, 330, 332, 345, 350, 363, 387, 389, 392, 399, 403, 409, 410, 420, 437, 443, 464, 466, 480, 484, 485, 498, 491, 492, and 493 corresponding to SEQ ID NO: 1.

[0045] In certain embodiments, the recombinant RSV F protein further comprises a C at at least one position, preferably at at least two positions, selected from amino acid positions 55, 71, 76, 89, 101, 155, 159, 188, 231, 232, 242, 250, 290, 291, 324, 327, 330, 332, 389, 399, 410, 437, 443, 464, 466, 480, 485, and 493 corresponding to SEQ ID NO: 1.

[0046] In certain preferred embodiments, the recombinant RSV F protein comprises amino acids at positions corresponding to SEQ ID NO: 1: (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 485; (xiii) 410 and 464; (xiv) 443 and 466; (xv) 26 and 363; (xvi) 38 and 318; (xvii) 41 and 409; (xviii) 67 and 207; (xix) 75 and 215; (xx) 148 and 286; (xxi) 171 and 191; (xxii) 345 and 350; (xxiii) 387 and 492; (xxiv) 392 and 491; (xxv) 403 and 420; and (xxvi) 484 and 498, wherein one or more of the groups of positions comprises a C.

[0047] In certain preferred embodiments, the recombinant RSV F protein comprises a C at one or more groups of positions corresponding to amino acid positions (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 485; (xiii) 410 and 464; and (xiv) 443 and 466 of SEQ ID NO: 1.

[0048] In certain preferred embodiments, the recombinant RSV F protein comprises a C at one or more of the groups of positions corresponding to amino acid positions (i) 55 and 188; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (ix) 327 and 330; and (xii) 399 and 485 of SEQ ID NO: 1.

[0049] In certain embodiments, the recombinant RSV F protein comprises a C at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 and a C at a group of positions corresponding to amino acid positions (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 485; (xiii) 410 and 464; and (xiv) 443 and 466 of SEQ ID NO: 1, and a C at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1, relative to SEQ ID NO: 1. One or more (e.g., 2, 3, 4, 5 or more) of amino acid positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489, and 492 of NO: 1 comprises an amino acid substitution (e.g., selected from I, V, L, F, and P). For example, the recombinant RSV F protein comprises Cs at positions corresponding to amino acid positions 72, 76, 89 and 231, 72, 76, 89 and 231, 72, 76, 327 and 330, 72, 76, 399 and 485, 72, 76, 101 and 242, 72, 76, 155 and 290, or 72, 76, 55 and 188 of SEQ ID NO: 1.

[0050] In certain embodiments, the recombinant RSV F protein comprises the following amino acids at positions corresponding to the amino acid positions of SEQ ID NO: 1 below:

[0051] (a)72C / 76C / 89C / 231C / 68I / 198F;

[0052] (b)72C / 76C / 89C / 231C / 68I / 198F / 161P;

[0053] (c)72C / 76C / 89C / 231C / 68I / 198F / 161P / 463P;

[0054] (d)72C / 76C / 89C / 231C / 68I / 198F / 463P

[0055] (e)72C / 76C / 89C / 231C / 68I / 198F / 214P / 463P;

[0056] (f)72C / 76C / 89C / 231C / 68I / 198F / 214P / 460P;

[0057] (g)72C / 76C / 89C / 231C / 67V / 214P / 460P;

[0058] (h)72C / 76C / 89C / 231C / 67V / 214P / 198F;

[0059] (i)72C / 76C / 327C / 330C / 67V / 214P / 460P;

[0060] (j)72C / 76C / 327C / 330C / 67V / 87L / 198F;

[0061] (k)72C / 76C / 399C / 485C / 68I / 198F / 214P / 460P;

[0062] (l)72C / 76C / 399C / 485C / 68V / 87L / 198F / 214P / 460P;

[0063] (m)72C / 76C / 399C / 485C / 87L;

[0064] (n)72C / 76C / 399C / 485C / 87L / 214P;

[0065] (o)72C / 76C / 399C / 485C / 87L / 161P;

[0066] (p)72C / 76C / 399C / 485C / 87L / 460P;

[0067] (q)72C / 76C / 399C / 485C / 67V / 460P;

[0068] (r)72C / 76C / 399C / 485C / 67V / 198F / 463P;

[0069] (s)72C / 76C / 68V / 87L / 198F / 214P / 460P;

[0070] (t)72C / 76C / 67V / 198F / 463P;

[0071] (u)72C / 76C / 67V / 198F / 214P;

[0072] (v)72C / 76C / 101C / 242C / 214P;

[0073] (w)72C / 76C / 101C / 242C / 214P / 463P;

[0074] (x)72C / 76C / 101C / 242C / 214P / 463P / 87L / 198F;

[0075] (y)72C / 76C / 101C / 242C / 214P / 463P / 67V;

[0076] (z)72C / 76C / 101C / 242C / 214P / 463P / 198F;

[0077] (aa)72C / 76C / 101C / 242C / 214P / 67V / 87L / 198F;

[0078] (bb)72C / 76C / 101C / 242C / 214P / 67V / 198F;

[0079] (cc)72C / 76C / 101C / 242C / 68I / 198F / 214P / 460P;

[0080] (dd)72C / 76C / 101C / 242C / 67V;

[0081] (ee)72C / 76C / 101C / 242C / 67V / 214P;

[0082] (ff)72C / 76C / 101C / 242C / 67V / 214P / 463P;

[0083] (gg)72C / 76C / 101C / 242C / 67V / 214P / 460P;

[0084] (hh)72C / 76C / 155C / 290C / 68I / 87L / 198F;

[0085] (ii)72C / 76C / 155C / 290C / 68I / 87L / 198F / 214P;

[0086] (jj)72C / 76C / 155C / 290C / 68I / 87L / 198F / 463P;

[0087] (kk)72C / 76C / 155C / 290C / 68I / 87L / 198F / 460P;

[0088] (ll)72C / 76C / 155C / 290C / 67V / 198F;

[0089] (mm)72C / 76C / 155C / 290C / 67V / 198F / 460P, or

[0090] (nn)72C / 76C / 55C / 188C / 68I / 198F.

[0091] As used herein, when an amino acid mutation is referred to in a number-letter format (e.g., 72C), the number indicates the position of the amino acid (unless otherwise specified, it indicates the position number of the reference sequence, such as SEQ ID NO: 1), and the second letter indicates the amino acid residue after the mutation.

[0092] As used herein, when referring to amino acid mutations, “ / ” indicates that the mutations exist at the same time, for example, 72C / 76C indicates that the mutations 72C and 76C exist at positions 72 and 76 at the same time.

[0093] In further embodiments, the recombinant RSV F protein comprises, except for the amino acids at the specified positions, the F2 polypeptide comprising amino acids corresponding to positions 26-109 or 26-105 of SEQ ID NO: 1 (i.e., except for the amino acid residues at the specified positions, the amino acid residues at the remaining positions are identical to amino acids 26-109 or 26-105 of SEQ ID NO: 1) and / or the Fi polypeptide comprising amino acids corresponding to positions 137-513 or 147-513 of SEQ ID NO: 1 (i.e., except for the amino acid residues at the specified positions, the amino acid residues at the remaining positions are identical to amino acids 137-513 or 147-513 of SEQ ID NO: 1).

[0094] In one embodiment, the F2 and F1 polypeptides are connected by a linker. The linker can be any linker known in the art that is suitable for connecting polypeptides. In one embodiment, the linker is selected from AAGAATAA, GSPAG, GGASPAGG, GGASPAAPAPAG, AEAAAKEAAAKA, PAPAP, GPPPG, GSGS and GGSGGSGGS.

[0095] In a further embodiment, the recombinant RSV F protein includes a trimerization domain.The trimerization domain can be any suitable trimerization domain known in the art, for example, including but not limited to the trimerization domain (such as SEQ ID NO:7 and 16-24) derived from T4 fibritin Foldon, hCorla corona protein-1, T3XV, leucine zipper GCN4-1, leucine zipper GCN4-2, chloramphenicol acetyltransferase, hCorla corona protein-2, mCorla corona protein, Langerhans protein or maternal protein 1 cartilage matrix protein.In one embodiment, the trimerization domain can be selected from the trimerization domain (such as SEQ ID NO:7) derived from T4 fibritin and the trimerization domain (such as SEQ ID NO:18 or 19) derived from leucine zipper GCN4.

[0096] In one embodiment, the trimerization domain is linked to the C-terminus of the F1 polypeptide, preferably linked to the F1 polypeptide via a linker sequence such as SAIG, GG or SA.

[0097] In certain embodiments, the recombinant RSV F protein, in addition to the amino acid residues at the specified positions, the F2 polypeptide comprises amino acids 26-105 corresponding to SEQ ID NO: 1, and the Fi polypeptide comprises amino acids 137-513 or 147-513 corresponding to SEQ ID NO: 1, optionally connected by a linker such as GSGS or GGSGGSGGS.

[0098] In certain embodiments, the recombinant RSV F protein, in addition to the amino acid residues at the specified positions, the F2 polypeptide comprises amino acids 26-105 corresponding to SEQ ID NO: 1, and the Fi polypeptide comprises amino acids 147-513 corresponding to SEQ ID NO: 1, connected by the linker GGSGGSGGS.

[0099] In certain embodiments, the recombinant RSV F protein further comprises a trimerization domain, such as a T4 fibritin-derived trimerization domain, optionally linked to the C-terminus of the F1 polypeptide (e.g., residue 513L), preferably linked to the F1 polypeptide via a linker sequence such as SAIG, GG, or SA.

[0100] In certain embodiments, the recombinant RSV F protein further comprises the following parts: (i) a thrombin cleavage site, such as LVPRGS, (ii) a purification tag, such as a HIS tag, such as HHHHHH, and / or (iii) a streptomycin tag, such as WSHPQFEK. Optionally, the parts are connected to the Fi polypeptide or trimerization domain via a linker sequence, such as SAIG, GG, or SA. When the recombinant RSV F protein comprises two or three parts, the parts can also be connected via a linker sequence, such as SAIG, GG, or SA.

[0101] In certain embodiments, the recombinant RSV F protein comprises an F2 polypeptide, an Fi polypeptide, a linker (e.g., SAIG), a trimerization domain (e.g., SEQ ID NO: 7), a linker (e.g., GG), a thrombin cleavage site (e.g., LVPRGS), a purification tag (e.g., a HIS tag, e.g., HHHHHH), a linker (SA), and a streptomycin tag (e.g., WSHPQFEK).

[0102] In certain embodiments, the recombinant RSV F protein comprises an F2 polypeptide, an Fi polypeptide, a linker (e.g., SAIG), a trimerization domain (e.g., SEQ ID NO: 7), a linker (e.g., GG), a thrombin cleavage site (e.g., LVPRGS), a purification tag (e.g., a HIS tag, e.g., HHHHHH), a linker (SA), and a streptomycin tag (e.g., WSHPQFEK).

[0103] In certain embodiments, the recombinant RSV F protein comprises one or more, preferably all, amino acid substitutions selected from P102A, I379V, and M447V relative to SEQ ID NO: 1.

[0104] In one embodiment, the recombinant RSV F protein is from RSV subtype A.

[0105] In one embodiment, the recombinant RSV F protein is from RSV subtype B.

[0106] In one embodiment, the recombinant RSV F protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 25-64.

[0107] The invention provides new recombinant RSV F protein, it is stable in pre-fusion conformation, namely comprises (display) and pre-fusion conformation F protein has at least one specific epi-position.Pre-fusion conformation F protein has specific epi-position and is the non-existent epi-position in conformation after fusion.Do not wish to be bound by any particular theory, believe that the pre-fusion conformation of described recombinant RSV F protein can contain those identical epi-positions on the RSV F protein expressed on natural RSV virion, and therefore can provide the advantage for causing protective neutralizing antibodies.

[0108] In certain embodiments, the recombinant RSV F protein of the invention comprises at least one epitope that can be recognized by a specific monoclonal antibody specific for the prefusion RSV F protein.

[0109] The present invention also provides a method for stabilizing the prefusion conformation of the RSV F protein that protects the F2 and F1 polypeptides, the method comprising: introducing into the RSV F protein one or more mutations selected from the group consisting of:

[0110] The amino acids at positions 72 and 76 corresponding to amino acid positions of SEQ ID NO: 1 are mutated to C, and the amino acid at one or more positions selected from the group consisting of amino acid positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489 and 492, preferably 67, 68, 87, 98, 161, 214, 460 and 463, corresponding to SEQ ID NO: 1 are mutated (e.g., to I, V, L, F or P).

[0111] In certain embodiments, the method further comprises mutating an amino acid at one or more positions selected from the group consisting of amino acid positions 26, 38, 41, 55, 67, 71, 75, 76, 89, 101, 148, 155, 159, 171, 188, 191, 207, 215, 231, 232, 242, 250, 286, 290, 291, 318, 324, 327, 330, 332, 345, 350, 363, 387, 389, 392, 399, 403, 409, 410, 420, 437, 443, 464, 466, 480, 484, 485, 498, 491, 492, and 493 of SEQ ID NO: 1 to a C.

[0112] In certain embodiments, the method further comprises mutating an amino acid at one or more positions selected from the group consisting of amino acid positions 55, 71, 76, 89, 101, 155, 159, 188, 231, 232, 242, 250, 290, 291, 324, 327, 330, 332, 389, 399, 410, 437, 443, 464, 466, 480, 485, and 493 corresponding to SEQ ID NO: 1 to a C.

[0113] Preferably, the method comprises replacing the amino acid positions (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 485; (xiii) 410 and 464; (xiv) 443 and 465. The amino acid at one or more groups of positions in the sequence of (i) 47 and (ii) 50 is mutated to C; (ii) 6; (iii) 26 and 363; (iv) 392 and 491; (v) 403 and 420; and (v) 484 and 498 is mutated to C.

[0114] Preferably, the method comprises mutating the amino acid at one or more groups of positions corresponding to amino acid positions (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 485; (xiii) 410 and 464; and (xiv) 443 and 466 of SEQ ID NO: 1 to C.

[0115] More preferably, the method comprises mutating the amino acid at one or more groups of positions corresponding to amino acid positions (i) 55 and 188; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (ix) 327 and 330; and (xii) 399 and 485 of SEQ ID NO: 1 to C.

[0116] In one embodiment, the method includes mutating the amino acid at the position corresponding to amino acid position 67, 68, 80, 84, 87, 198, 202, 207, 210, 219 and / or 489, preferably 67, 68, 87 and / or 198 of SEQ ID NO: 1 in the RSV F protein independently to I, V, L or F. For example, the amino acid at the position corresponding to amino acid position 67 of SEQ ID NO: 1 is mutated to V, the amino acid at the position corresponding to amino acid position 68 of SEQ ID NO: 1 is mutated to I, the amino acid at the position corresponding to amino acid position 68 of SEQ ID NO: 1 is mutated to V, the amino acid at the position corresponding to amino acid position 87 of SEQ ID NO: 1 is mutated to L, and / or the amino acid at the position corresponding to amino acid position 198 of SEQ ID NO: 1 is mutated to F.

[0117] In one embodiment, the method comprises mutating the amino acid corresponding to amino acid position 161, 211, 213, 214, 217, 459, 460, 463, 470, 472, 479, 481, 485, and / or 492 of SEQ ID NO: 1, preferably 161, 214, 460, and / or 463, in the RSV F protein to P.

[0118] In one embodiment, the method includes mutating the amino acid at the position corresponding to amino acid position 102 of SEQ ID NO: 1 to A, the amino acid at the position corresponding to amino acid position 379 of SEQ ID NO: 1 to V, and / or the amino acid at the position corresponding to amino acid position 447 of SEQ ID NO: 1 to V in the RSV F protein.

[0119] In one embodiment, the method includes mutating the amino acids at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 in an RSV F protein comprising F2 and Fi polypeptides to C, and mutating the amino acid at one or more positions selected from the group consisting of amino acid positions 67, 68, 87, 98, 161, 214, 460, and 463 of SEQ ID NO: 1. For example, the amino acids at positions corresponding to amino acid positions 67, 68, 87, and / or 98 of SEQ ID NO: 1 are independently mutated to I, V, L, or F. Preferably, the amino acid at the position corresponding to amino acid position 67 of SEQ ID NO: 1 is mutated to V; and / or the amino acid at the position corresponding to amino acid position 68 of SEQ ID NO: 1 is mutated to I; and / or the amino acid at the position corresponding to amino acid position 87 of SEQ ID NO: 1 is mutated to L; and / or the amino acid at the position corresponding to amino acid position 198 of SEQ ID NO: 1 is mutated to F. In certain embodiments, the method comprises mutating the amino acid at positions corresponding to amino acid positions 161, 214, 460, and / or 463 of SEQ ID NO: 1 to P.

[0120] According to the present invention, the specific stabilizing amino acid present at said position increases the stability of the protein in the pre-fusion conformation. According to the present invention, the specific amino acid may already be present in the amino acid sequence (e.g. corresponding to 102A, 379V and / or 447V of SEQ ID NO: 1), or it may be introduced into the specific amino acid position according to the present invention by mutation (substitution) of the amino acid at that position.

[0121] In certain embodiments, the method comprises mutating the amino acids at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 to C and mutating the amino acids at two or more other positions corresponding to the amino acid positions of SEQ ID NO: 1. In certain embodiments, the method performs the following mutations:

[0122] (a) mutating the amino acids at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 to C;

[0123] (b) selected from the group corresponding to SEQ ID NO:1 mutation S55C / L188C; G71C / V76C; A89C / L231C; P101C / G242C; S155C / S290C; H159C / I291C; E232C / Y250C; T324C / N437C; K327C / S330C; I332C / P480C; P389C / S493C; K399C / S485C; L410C / G464C; and one of S443C / S466C, preferably selected from one of S55C / L188C, A89C / L231C, P101C / G242C, S155C / S290C, K327C / S330C, K399C / S485C, and

[0124] (c) an amino acid mutation at at least one position selected from the group consisting of amino acid positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489, and 492, preferably 67, 68, 87, 198, 161, 214, 460, and 463, of SEQ ID NO: 1, preferably independently to I, V, L, F, or P, more preferably to I, V, L, F, or P, and / or to P, e.g., to P, e.g., to I, V, L, F, or P, The amino acid at position 67 of SEQ ID NO: 1 is mutated to V, the amino acid at position corresponding to amino acid position 68 of SEQ ID NO: 1 is mutated to I, the amino acid at position corresponding to amino acid position 68 of SEQ ID NO: 1 is mutated to V, the amino acid at position corresponding to amino acid position 87 of SEQ ID NO: 1 is mutated to L, and / or the amino acid at position corresponding to amino acid position 198 of SEQ ID NO: 1 is mutated to F.

[0125] As used herein, a mutation corresponding to SEQ ID NO: 1 means that the sequence of interest comprises the mutation at a position corresponding to the amino acid position of SEQ ID NO: 1 relative to SEQ ID NO: 1. For example, a mutation S55C corresponding to SEQ ID NO: 1 means that the sequence of interest comprises a C at a position corresponding to amino acid position 55 of SEQ ID NO: 1.

[0126] In certain embodiments, the method comprises introducing mutations (a) corresponding to amino acid mutations T72C, V76C of SEQ ID NO: 1 and another group of mutations selected from (b) and at least one selected from (c), for example, two or more, three or more, four or more, five or more of said amino acid mutations.

[0127] As used herein, when referring to an amino acid mutation in the form of a letter-number-letter (e.g., T72C), the first letter represents the original amino acid residue, the number represents the position of the amino acid (unless otherwise specified, it represents the position number of the reference sequence, such as SEQ ID NO: 1), and the second letter represents the amino acid residue after the mutation. Therefore, since the sequence before the mutation may be different, the first letter may not be the amino acid represented. As long as the amino acid after the mutation at the position is the same, it is considered to be a mutation described in the present invention. For example, when referring to T72C, it covers the case where the amino acid at position 72 (including T, as well as other amino acid residues) is mutated to C. Therefore, T72C and 72C represent the same mutation results.

[0128] In certain embodiments, the method comprises introducing an amino acid mutation selected from the group consisting of:

[0129] (a)T72C / V76C / A89C / L231C / K68I / Y198F

[0130] (b)T72C / V76C / A89C / L231C / K68I / Y198F / E161P

[0131] (c)T72C / V76C / A89C / L231C / K68I / Y198F / E161P / E463P

[0132] (d)T72C / V76C / A89C / L231C / K68I / Y198F / E463P

[0133] (e)T72C / V76C / A89C / L231C / K68I / Y198F / I214P / E463P

[0134] (f)T72C / V76C / A89C / L231C / K68I / Y198F / I214P / N460P

[0135] (g)T72C / V76C / A89C / L231C / N67V / I214P / N460P

[0136] (h)T72C / V76C / A89C / L231C / N67V / I214P / Y198F

[0137] (i)T72C / V76C / K327C / S330C / N67V / I214P / N460P

[0138] (j)T72C / V76C / K327C / S330C / N67V / K87L / Y198F

[0139] (k)T72C / V76C / K399C / S485C / K68I / Y198F / I214P / N460P

[0140] (l)T72C / V76C / K399C / S485C / K68V / K87L / Y198F / I214P / N460P

[0141] (m)T72C / V76C / K399C / S485C / K87L

[0142] (n)T72C / V76C / K399C / S485C / K87L / I214P

[0143] (o)T72C / V76C / K399C / S485C / K87L / E161P

[0144] (p)T72C / V76C / K399C / S485C / K87L / N460P

[0145] (q)T72C / V76C / K399C / S485C / N67V / N460P

[0146] (r)T72C / V76C / K399C / S485C / N67V / Y198F / E463P

[0147] (s)T72C / V76C / K68V / K87L / Y198F / I214P / N460P

[0148] (t)T72C / V76C / N67V / Y198F / E463P

[0149] (u)T72C / V76C / N67V / Y198F / I214P

[0150] (v)T72C / V76C / P101C / G242C / I214P

[0151] (w)T72C / V76C / P101C / G242C / I214P / E463P

[0152] (x)T72C / V76C / P101C / G242C / I214P / E463P / K87L / Y198F

[0153] (y)T72C / V76C / P101C / G242C / I214P / E463P / N67V

[0154] (z)T72C / V76C / P101C / G242C / I214P / E463P / Y198F

[0155] (aa)T72C / V76C / P101C / G242C / I214P / N67V / K87L / Y198F

[0156] (bb)T72C / V76C / P101C / G242C / I214P / N67V / Y198F

[0157] (cc)T72C / V76C / P101C / G242C / K68I / Y198F / I214P / N460P

[0158] (dd)T72C / V76C / P101C / G242C / N67V

[0159] (ee)T72C / V76C / P101C / G242C / N67V / I214P

[0160] (ff)T72C / V76C / P101C / G242C / N67V / I214P / E463P

[0161] (gg)T72C / V76C / P101C / G242C / N67V / I214P / N460P

[0162] (hh)T72C / V76C / S155C / S290C / K68I / K87L / Y198F

[0163] (ii)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / I214P

[0164] (jj)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / E463P

[0165] (kk)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / N460P

[0166] (ll)T72C / V76C / S155C / S290C / N67V / Y198F

[0167] (mm)T72C / V76C / S155C / S290C / N67V / Y198F / N460P, or

[0168] (nn)T72C / V76C / S55C / L188C / K68I / Y198F.

[0169] In certain embodiments, the method does not introduce any other mutations in the RSV F protein except for the amino acid mutations introduced above. For example, the recombinant RSV F protein obtained by the method comprises, in addition to the amino acid mutations introduced above, an F2 polypeptide comprising amino acids corresponding to amino acids 26-109 or 26-105 of SEQ ID NO: 1 (or identical to amino acids 26-109 or 26-105 of SEQ ID NO: 1), and / or an F1 polypeptide comprising amino acids corresponding to amino acids 137-513 or 147-513 of SEQ ID NO: 1 (or identical to amino acids 137-513 or 147-513 of SEQ ID NO: 1), optionally connected by a linker such as GSGS or GGSGGSGGS.

[0170] In one embodiment, the F2 and F1 polypeptides are connected via a linker. The linker can be any linker known in the art suitable for connecting polypeptides. In one embodiment, the linker is selected from the group consisting of AAGAATAA, GSPAG, GGASPAGG, GGASPAAPAPAG, AEAAAKEAAAKA, PAPAP, GPPPG, GSGS, and GGSGGSGGS.

[0171] In a further embodiment, the method includes connecting RSV F albumen with a trimerization domain.The trimerization domain can be any suitable trimerization domain known in the art, for example, including but not limited to the trimerization domain (such as SEQ ID NO:7 and 16-24) derived from T4fibritin Foldon, hCor1a crown protein-1, T3XV, leucine zipper GCN4-1, leucine zipper GCN4-2, chloramphenicol acetyltransferase, hCor1a crown protein-2, mCor1a crown protein, Langerhans protein or maternal protein 1 cartilage matrix protein.In one embodiment, the trimerization domain can be selected from the trimerization domain (such as SEQ ID NO:7) derived from T4 fibritin and the trimerization domain (such as SEQ ID NO:18 or 19) derived from leucine zipper GCN4.In one embodiment, the trimerization domain is connected to the C-end of Fi polypeptide, preferably connected with Fi polypeptide by linker sequence such as SAIG, GG or SA.

[0172] In one embodiment, the RSV F protein is from RSV A subtype.

[0173] In one embodiment, the RSV F protein is from RSV subtype B.

[0174] The present invention further provides recombinant RSV F proteins obtained or obtainable by the method and uses thereof.

[0175] In certain embodiments, the recombinant RSV F protein of the invention is a trimeric protein.

[0176] As mentioned above, the recombinant RSV F protein of the present invention also encompasses a fragment of RSV F protein before fusion. The fragment can be from one or both of the aminoterminal (for example, by cutting a signal sequence) and the carboxyl terminal deletion (for example, by lacking a transmembrane region and / or a cytoplasmic tail region). Fragment can be selected to comprise an immunologically active fragment of the F protein, i.e., the part causing an immune response in a subject. This can be determined using a computer, in vitro and / or in vivo methods routinely.

[0177] In certain embodiments, the recombinant protein according to the invention comprises a signal sequence corresponding to amino acids 1-25 of SEQ ID NO: 1. Signal sequences are present at the N-terminus of most newly synthesized proteins and are typically cleaved by a signal peptidase to produce a free signal peptide and the mature protein.

[0178] In certain embodiments, the recombinant protein according to the present invention does not comprise a signal sequence.

[0179] In certain embodiments, the recombinant RSV F protein according to the present invention is a soluble protein (i.e., non-membrane bound). In certain embodiments, the recombinant RSV F protein according to the present invention comprises a truncated F1 domain and comprises a heterologous trimerization domain connected to the truncated F1 polypeptide. According to the present invention, by connecting the heterologous trimerization domain to the C-terminal amino acid residue of the truncated F1 polypeptide, in combination with one or more mutations as described herein, a soluble RSV F protein that exhibits high expression and binds to pre-fusion specific antibodies is provided.

[0180] In certain embodiments, the heterotrimerization domain comprises the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 7).

[0181] In certain embodiments, the Fi polypeptide in the recombinant protein of the present invention comprises a truncated form thereof. As used herein, a "truncated form" refers to a Fi polypeptide that is not a full-length Fi polypeptide, i.e., one or more amino acid residues are deleted at the N-terminus or at the C-terminus. According to the present invention, at least the transmembrane domain and the cytoplasmic tail have been deleted to express a soluble extracellular domain.

[0182] In certain embodiments, the trimerization domain is linked to amino acid residue 513 of the RSV Fi polypeptide.In certain embodiments, the trimerization domain comprises SEQ ID NO: 7 and is linked to amino acid residue 513 of the RSV Fi polypeptide directly or through a linker (eg, SAIG, GG, or SA).

[0183] In certain embodiments, compared with wild-type RSV F protein, the expression level of recombinant RSV F protein of the present invention is improved. In certain embodiments, compared with the wild-type F protein not containing the replacement described in the present invention, after 5 to 10 days of harvesting protein, the content before fusion (defined as the fraction of the F protein bound by the specific antibody before fusion) is significantly higher.

[0184] The recombinant RSV F protein according to the present invention is stabilized in the prefusion conformation due to the presence of one or more stabilizing amino acids (already present or introduced by mutation), i.e., it is less likely to change to the postfusion conformation when the protein is processed (e.g., such as purification, freeze-thaw cycles, and / or storage, etc.).

[0185] In certain embodiments, the recombinant RSV F protein according to the present invention has increased stability when stored at 4°C compared to an RSV F protein without the one or more mutations. In certain embodiments, these recombinant proteins are stable for at least 15 days, preferably at least 18 days, preferably at least 30 days, preferably at least 60 days, preferably at least 6 months, and even more preferably at least 1 year when stored at 4°C. "Stable upon storage" means that after the protein is stored in a solution (e.g., culture medium) at 4°C for at least 30 days, the proteins still exhibit at least one epitope specific for a pre-fusion specific antibody. In certain embodiments, when the recombinant RSV F protein is stored at 4°C, the proteins exhibit this at least one pre-fusion specific epitope for at least 6 months, preferably for at least 1 year.

[0186] In certain embodiments, a recombinant RSV F protein according to the invention has increased stability, as determined by measuring the melting temperature as described in Example 2, compared to an RSV F protein without the one or more mutations.

[0187] In certain embodiments, these proteins display at least one prefusion-specific epitope after 1 to 6 freeze-thaw cycles in a suitable formulation buffer.

[0188] In certain embodiments, these proteins contain a His-tag or a Strep II tag. A His-tag or polyhistidine-tag is an amino acid motif in a protein that consists of at least five histidine (H) residues; a Strep II tag is an amino acid sequence consisting of 10 residues (SAWSHPQFEK (SEQ ID NO: 10)). These tags are often at the N- or C-terminus of a protein and are typically used for purification purposes.

[0189] RSV is known to exist as a single serotype with two antigenic subtypes A and B. The amino acid sequence of the mature processed F protein of these two types is approximately 93% identical. As used herein, amino acid position is given with reference to the sequence of SEQ ID NO:1. As used herein, "amino acid at position " x " of RSV F protein" means the amino acid corresponding to the amino acid at position " x " in the RSV F protein of SEQ ID NO:1. Note that in the numbering system used herein, 1 refers to the N-terminal amino acid (SEQ ID NO:1) of immature F0 protein. When using the F protein of another RSV strain, the amino acid position of the F protein will be numbered with reference to the numbering of SEQ ID NO:1 by making the sequence of other RSV strains compare with the F protein of SEQ ID NO:1 by inserting room when necessary. Sequence alignment can be performed using methods well known in the art, such as CLUSTALW, Bioedit or CLC Workbench.

[0190] As used herein, nucleotide sequences are provided from the 5' to 3' direction, and amino acid sequences are provided from N-terminus to C-terminus, as is customary in the art.

[0191] The amino acid according to the present invention may be any of the 20 naturally occurring (or "standard") amino acids. Table 1 below shows the abbreviations and properties of the standard amino acids.

[0192] Table 1. Standard amino acids, abbreviations and properties

[0193] Those skilled in the art will appreciate that proteins can be mutated by conventional molecular biology procedures.Compared with RSV F proteins that do not comprise the one or more mutations, mutations according to the present invention preferably result in increased expression levels and / or stabilization of the recombinant RSV F protein.

[0194] The present invention further provides nucleic acid molecules encoding the recombinant RSV F protein according to the present invention.

[0195] In a preferred embodiment, the nucleic acid molecules encoding the protein according to the present invention are codon-optimized for expression in mammalian cells (e.g., human cells). Codon-optimized methods are known in the art and have been described in WO 96 / 09378. If at least one non-preferred codon is replaced by a more preferred codon compared to the wild-type sequence, the sequence is considered to be codon-optimized. In this article, a non-preferred codon is a codon that is less frequently used than another codon encoding the same amino acid in an organism, and a more preferred codon is a codon that is more frequently used than a non-preferred codon in an organism. Codon usage frequencies for specific organisms can be found in codon frequency tables, such as http: / / www.kazusa.or.jp / codon. Preferably, more than one non-preferred codon, preferably all non-preferred codons, are replaced by preferred codons. Replacement by preferred codons generally results in higher expression.

[0196] Those skilled in the art will appreciate that due to the degeneracy of the genetic code, many different polynucleotides and nucleic acid molecules can encode the same protein. Therefore, unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may or may not include introns.

[0197] Nucleic acid sequences can be generated using conventional molecular biology techniques, or can be generated de novo by DNA synthesis.

[0198] The present invention also provides vectors comprising the nucleic acid molecules described above. In certain embodiments, the nucleic acid molecules according to the present invention are therefore part of a vector. Vectors can be easily manipulated by methods well known to those skilled in the art and can, for example, be designed to replicate in prokaryotic and / or eukaryotic cells. In addition, many vectors can be used for the transformation of eukaryotic cells and can be integrated, in whole or in part, into the genomes of these cells to produce stable host cells containing the desired nucleic acid in their genomes. The vector used can be any vector suitable for cloning DNA and for transcribing the target nucleic acid. Suitable vectors according to the present invention are, for example, adenoviruses, alphaviruses, paramyxoviruses, vaccinia viruses, herpes viruses, retroviral vectors, etc. Those skilled in the art can select suitable expression vectors and insert them into the nucleotide sequence of the present invention in a functional manner.

[0199] The present invention further relates to host cells comprising nucleic acid molecules encoding the recombinant RSV F protein. Recombinant RSV F protein can be produced by recombinant DNA techniques, which relate to expressing these molecules in host cells (e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines (e.g., HEK293 cells, PER.C6 cells) or yeast, fungi, insect cells, etc.), or transgenic animals or plants. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. Typically, the generation of recombinant proteins (e.g., recombinant RSV F proteins of the present invention) in host cells comprises introducing heterologous nucleic acid molecules encoding proteins in an expressible form into the host cells, cultivating these cells under conditions that contribute to the expression of the nucleic acid molecules, and allowing the protein to be expressed in the cells. Nucleic acid molecules encoding proteins in an expressible form can be the form of an expression cassette, and typically comprise sequences that need to cause expression of nucleic acids, such as one or more enhancers, promoters, polyadenylation signals, etc.

[0200] Cell culture media are available from various suppliers, and suitable media can be routinely selected for host cells expressing a protein of interest, such as recombinant RSV F protein. Suitable media may or may not contain serum.

[0201] The present invention further provides a composition comprising a recombinant RSV F protein and / or nucleic acid molecules and / or a carrier as described above. The present invention therefore provides a composition comprising a recombinant RSV F protein, which is presented in a pre-fusion conformation of the RSV F protein but does not exist in the post-fusion conformation. The present invention also provides a composition comprising a nucleic acid molecule and / or a carrier encoding this recombinant RSV F protein. The present invention further provides an immunogenic composition comprising a recombinant RSV F protein and / or nucleic acid molecules and / or a carrier as described above. The present invention further provides a pharmaceutical composition comprising a recombinant RSV F protein and / or nucleic acid molecules and / or a carrier as described above and one or more pharmaceutically acceptable excipients.

[0202] The present invention also provides the use of recombinant RSV F proteins, nucleic acid molecules, and / or vectors according to the present invention for inducing an immune response against RSV F proteins in a subject. Further provided are methods for inducing an immune response against RSV F proteins in a subject, comprising administering to the subject a recombinant RSV F protein, and / or nucleic acid molecules, and / or vector according to the present invention. Further provided are the use of recombinant RSV F proteins, and / or nucleic acid molecules, and / or vectors according to the present invention for the manufacture of a medicament for inducing an immune response against RSV F proteins in a subject.

[0203] The recombinant RSV F proteins, nucleic acid molecules, or vectors of the invention can be used to prevent and / or treat RSV infection in a subject in need of such prevention and / or treatment.

[0204] Recombinant RSV F proteins, nucleic acid molecules, and / or vectors according to the invention can be used, for example, for the independent treatment and / or prevention of diseases or conditions caused by RSV, or in combination with other preventive and / or therapeutic measures (such as vaccines, antiviral agents, and / or monoclonal antibodies).

[0205] The present invention further provides for utilizing recombinant RSV F protein according to the present invention, nucleic acid molecules, and / or vector in the method for preventing and / or treating RSV infection in a subject, comprising administering to a subject in need thereof a recombinant RSV F protein of the present invention, a nucleic acid molecule encoding it, and / or a vector comprising this nucleic acid molecule. A therapeutically effective amount refers to that protein, nucleic acid molecule or vector are effectively used to prevent, alleviate and / or treat the amount of the disease or illness caused by being infected by RSV. Prevention encompasses the propagation of suppressing or reducing RSV or suppressing or reducing the outbreak, development or progress of one or more symptoms relevant to being infected by RSV. As used herein, alleviation can refer to reducing visible or perceptible symptoms of disease, viremia or any other measurable manifestation of influenza infection.

[0206] For use to a subject (e.g., the human race), the present invention can adopt a pharmaceutical composition comprising a recombinant RSV F protein of the present invention, a nucleic acid molecule encoding the same and / or a carrier comprising the nucleic acid molecule, and a pharmaceutically acceptable excipient. In this article, "pharmaceutically acceptable" means that the excipient will not cause any unnecessary or adverse effects in the subject they use at the dosage and concentration employed. Such pharmaceutically acceptable excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by A.R. Gennaro, Mack Publishing Company, 1990). Although lyophilized preparations can be used, RSV F protein or nucleic acid molecules are preferably prepared and used as sterile solutions. In certain embodiments, RSV F protein can be formulated into an injectable formulation.

[0207] In certain embodiments, the compositions according to the present invention further comprise one or more adjuvants. Adjuvants are known in the art to further enhance the immune response to the applied antigenic determinants. The term "adjuvant" herein refers to one or more substances that cause immune system stimulation. In other embodiments, these compositions do not comprise an adjuvant.

[0208] In certain embodiments, the present invention provides methods for producing vaccines against respiratory syncytial virus (RSV), comprising providing a recombinant RSV F protein, nucleic acid, or vector according to the present invention and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to a medicament or composition containing an active ingredient that effectively induces a certain degree of immunity against a certain pathogen or disease in a subject, which will at least cause a reduction (to no extent) in the severity, duration, or other manifestations of symptoms associated with being infected by the pathogen or the disease. In the present invention, the vaccine comprises an effective amount of a recombinant RSV F protein of the present invention and / or a nucleic acid molecule encoding the recombinant RSV F protein and / or a vector comprising the nucleic acid molecule, which results in an immune response against the RSV F protein. This provides a method for preventing the frequency of severe lower respiratory tract diseases that cause hospitalization in a subject and reducing complications (such as pneumonia and bronchiolitis) caused by RSV infection and replication. The term "vaccine" according to the present invention represents that it is a pharmaceutical composition, and therefore typically includes a pharmaceutically acceptable diluent, carrier, or excipient. It may or may not include another active ingredient. In certain embodiments, the vaccine further comprises other components that induce an immune response, for example, against other proteins of RSV and / or against other infectious agents.

[0209] The composition can be administered to a subject, such as a human subject. The total dose of RSV F protein in a composition for single administration can be, for example, about 0.01 μg to about 10 mg, such as 1 μg-1 mg, such as 10 μg-100 μg. In a composition for single administration, the total dose of a vector comprising DNA encoding a recombinant RSV F protein of the invention can be, for example, about 0.1×10 10 vp / ml to 2x10 11 , preferably between about 1x10 10 vp / ml and 2x10 11 vp / ml, preferably between 5x10 10 vp / ml and 1x10 11 Between vp / ml.

[0210] Administration of the compositions according to the present invention can be performed using standard routes of administration, including but not limited to parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, or mucosal administration, e.g., intranasal, oral, etc. In one embodiment, the composition is administered by intramuscular injection.

[0211] As used herein, a subject is preferably a mammal, such as a rodent, such as a mouse, a cotton rat, or a non-human primate or a human. Preferably, the subject is a human.

[0212] Protein, nucleic acid molecule, carrier and / or composition can also be used as the first time in the initial immunity-boosting scheme or as reinforcement.Typically, booster vaccination will be used for the first time to the subject ("primary vaccination") after one week and one year, preferably at a time between two weeks and four months to the same subject.In certain embodiments, use includes the first use and at least one booster administration.

[0213] In addition, the proteins of the present invention can be used as diagnostic tools, for example, to test the immune status of a subject by determining the presence of antibodies capable of binding to the recombinant proteins of the present invention in the serum of the subject. Therefore, the present invention also relates to an in vitro diagnostic method for detecting the presence of RSV infection in a subject, the method comprising: a) contacting a biological sample obtained from the subject with a protein according to the present invention; and b) detecting the presence of an antibody-protein complex.

[0214] As used herein, a biological sample is any biological sample from a test subject, in particular a sample comprising nucleic acids or polypeptides (e.g., antibodies), such as blood, plasma, platelets, saliva, sputum, urine, and the like. In one embodiment, the biological sample of the present invention is a body fluid sample, including but not limited to blood, saliva, tissue fluid samples, urine, lymph, and cerebrospinal fluid. Liquid samples from various sources can be used directly for detection or can be pre-treated by centrifugation, precipitation, filtration, and the like before detection. In one embodiment, the sample can be a blood sample, such as a serum sample, and other types of samples. In one embodiment, the sample is a sample derived from blood, such as whole blood or serum. The sample used for detection in the method of the present invention should generally be collected in a clinically acceptable manner, such as by collecting in a manner that protects nucleic acids or proteins. The sample can also be pre-treated to increase the accessibility of the target molecule, such as by cleavage (mechanical, chemical, enzymatic cleavage, etc.), purification, centrifugation, separation, and the like. The sample can also be labeled to facilitate detection of the presence of the target molecule (fluorescence, radioactivity, luminescence, chemical, enzymatic labeling, etc.). As used herein, the term "sample" also encompasses that tissue and / or cells and / or body fluids of a subject have been taken from the subject and, for example, have been placed on a microscope slide and the claimed method is performed on the slide.

[0215] It is well known to those skilled in the art that the presence of antibody-complex formed in a sample can be detected by any suitable technique, such as immunological techniques, including but not limited to enzyme-linked immunosorbent assay (ELISA), AlphaLISA, immunoblotting, dot blotting, immunoprecipitation, colloidal gold immunochromatography, etc.

[0216] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

[0217] Example

[0218] In order to better illustrate the purpose, technical route and effect of the present invention, the present invention is further explained below in conjunction with examples. It should be noted that the following examples are only used to explain the present invention and cannot be used to limit the scope of protection of the present invention. All technical solutions that are the same or similar to the present invention, as well as solutions obtained by transforming the technical parameters in the present invention, all fall within the scope of protection of the present invention.

[0219] Example 1: Preparation of stable prefusion RSV F protein

[0220] The RSV A2F protein sequence (GeneBank: P03420.1) containing three naturally occurring mutations (P102A, I379V, and M447V) was modified as follows:

[0221] T4 fibritin-derived trimerization domain (Foldon domain, residues 518-544, SEQ ID NO: 7);

[0222] Thrombin cleavage site (residues 547-552): LVPRGS (SEQ ID NO: 8);

[0223] Purification HIS tag (residues 553-558): HHHHHH (SEQ ID NO: 9);

[0224] Streptag II (561-568): WSHPQFEK (SEQ ID NO: 10).

[0225] The above sequences were codon-optimized for mammalian cells (293F, CHO, and other cell lines) and sent to biological companies (General Biotech, Shanghai Bioengineering, GeneWeizhi, and GenScript, etc.) for synthesis.

[0226] RSV A2 representative sequence: P03420.1, containing three naturally occurring substitutions (P102A, I379V, and M447V)

[0227] ●Signal sequence (residues 1-25)

[0228] F2 polypeptide (residues 26-109)

[0229] pep27 polypeptide (residues 110-136)

[0230] F1 polypeptide (residues 137-513)

[0231] ● Furin cleavage sites are referred to as RARR (SEQ ID NO: 11) and KKRKRR (SEQ ID NO: 12)

[0232] A: Fusion polypeptide of F2 polypeptide and F1 polypeptide

[0233] T4 fibritin-derived trimerization domain (foldon domain, residues 518-544)

[0234] Thrombin cleavage site (residues 547-552): LVPRGS

[0235] ● Purification HIS tag (residues 553-558): HHHHHH

[0236] Streptag II (561-568): WSHPQFEK

[0237] Linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG), and 559-560 (SA))

[0238] C1-1: F2 polypeptide amino acids 26-105 + F1 polypeptide amino acids 137-513 + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + Streptag II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG), and 559-560 (SA))

[0239] C1-2: F2 polypeptide amino acids including 26-105 + GSGS linker (SEQ ID NO: 14) + F1 polypeptide amino acids 137-513 + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + Streptag II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG), and 559-560 (SA))

[0240] C1-3: F2 polypeptide amino acids 26-105 + GGSGGSGGS linker (SEQ ID NO: 15) + F1 polypeptide amino acids 137-513 + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + Streptag II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG), and 559-560 (SA))

[0241] C1-4: F2 polypeptide amino acids 26-105 + F1 polypeptide amino acids 147-513 + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + Streptag II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG), and 559-560 (SA))

[0242] C1-5: F2 polypeptide amino acids 26-105 + GGSGGSGGS linker (SEQ ID NO: 15) + F1 polypeptide amino acids 147-513 + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + Streptag II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG), and 559-560 (SA))

[0243] C1-1 (enzyme cleavage site-pep27 peptide deletion)

[0244] C1-2 (restriction site-pep27 peptide deletion-GSGS)

[0245] C1-3 (restriction site-pep27 peptide deletion-GGSGGSGGS)

[0246] C1-4 (restriction site-pep27 peptide-fusion peptide 10aa deleted)

[0247] C1-5 (restriction site-pep27 peptide-fusion peptide 10aa deletion-GGSGGSGGS)

[0248] B: Preparation of Stable Prefusion RSV F Peptide—Linker

[0249] F2 polypeptide amino acids 26-105 + linker + F1 polypeptide amino acids 147-513 + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + Streptag II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG), and 559-560 (SA))

[0250] There are 21 designs of flexible GS connectors, 3 designs of semi-flexible GSPA connectors, 9 designs of rigid EAK / PA / PG / PT connectors, and 2 designs of mixed connectors.

[0251] Table 2: Different linker types and sequences linking F2 and F1 polypeptides

[0252] C: Preparation of Stable Prefusion RSV F Peptide—Trimerization Domain

[0253] Using C1-5 (F2 polypeptide amino acids 26-105 + GGSGGSGGS linker (SEQ ID NO: 15) + F1 polypeptide amino acids 147-513 + residues 514-517 (SAIG, SEQ ID NO: 13)) + trimerization domain + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + streptomycin tag (Streptag) II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG) and 559-560 (SA)) as the starting peptide, the amino acid sequence shown in Table 3 was connected to the C-terminus of the F1 peptide.

[0254] Table 3: Trimerization domain origin and sequence

[0255] D: Preparation of Stable Prefusion RSV F Peptide - Stabilizing Mutations - Cysteine ​​Mutations

[0256] Using C1-5 (F2 polypeptide amino acids 26-105 + GGSGGSGGS linker (SEQ ID NO: 15) + F1 polypeptide amino acids 147-513) + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + streptomycin tag (Streptag) II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG) and 559-560 (SA)) as the starting peptide, further mutations as shown in Table 4 were performed.

[0257] Table 4

[0258] E: Preparation of stable prefusion RSV F peptide - stabilizing mutations - proline mutations

[0259] Using C1-5 (F2 polypeptide amino acids 26-105 + GGSGGSGGS linker (SEQ ID NO: 15) + F1 polypeptide amino acids 147-513) + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + streptomycin tag (Streptag) II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG) and 559-560 (SA)) as the starting peptide, further mutations as shown in Table 5 were performed.

[0260] Table 5

[0261] F: Preparation of stable prefusion RSV F peptides - stabilizing mutations - cavity hydrophobic filling mutations

[0262] Using C1-5 (F2 polypeptide amino acids 26-105 + GGSGGSGGS linker (SEQ ID NO: 15) + F1 polypeptide amino acids 147-513) + T4 fibritin-derived trimerization domain (foldon domain, residues 518-544) + thrombin cleavage site (residues 547-552): LVPRGS + purification HIS tag (residues 553-558): HHHHHH + streptomycin tag (Streptag) II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG) and 559-560 (SA)) as the starting peptide, further mutations as shown in Table 6 were performed.

[0263] Table 6

[0264] The above sequences were codon-optimized for mammalian cells (293F, CHO, and other cell lines) and sent to biological companies (General Biotech, Shanghai Bioengineering, GeneWeizhi, and GenScript, etc.) for synthesis.

[0265] I: Sequence expression plasmid preparation

[0266] Plasmid construction involves ligating the codon-optimized sequence described above to plasmids such as pcDNA3.1(+) and pCAGGS via the HindIII and NotI restriction sites. The method involves amplifying the target fragment via PCR, digesting the vector with HindIII and NotI, recovering the PCR product and digested vector gel, and inserting the target fragment into the vector using an infusion enzyme. Transformation, sequencing, and plasmid extraction are then performed. The specific steps are as follows:

[0267] 1. Target fragment PCR

[0268] The RSV F protein (SEQ ID NO: 1) nucleotide sequence was used as a template and primers were used for amplification. The PCR reaction system was as follows:

[0269] Table 7: PCR reaction system

[0270] PCR reaction conditions: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 15 s, extension at 68°C for 3 min, for a total of 30 cycles; 72°C for 20 min; and storage at 4°C.

[0271] 2. Vector Enzyme Digestion

[0272] The vector was digested with HindIII and NotI restriction enzymes. The digestion reaction system was as follows:

[0273] Table 8: Enzyme digestion reaction system

[0274] Digest at 37°C for 6 hours or overnight.

[0275] 3. Glue recycling

[0276] Use a DNA gel recovery kit to recover the PCR target product and vector enzyme digestion product. For specific steps, refer to the Tiangen Biochemical DP219 kit instructions.

[0277] 4. Connect the vector and the target fragment

[0278] The ligation was performed using Infusion enzyme, with a vector to target fragment mass ratio of 1:3, as follows:

[0279] Table 9: Ligation reaction system

[0280] The prepared reaction system was centrifuged instantaneously, placed on a PCR instrument, and connected at 50°C for 15 minutes.

[0281] 5. Conversion

[0282] Transform the ligation product using the Trans5α Chemically Competent Cell Kit. Refer to the instructions for the Quanshijin Biotech CD201 Kit for specific steps. Spread the transformed cells onto the corresponding resistant solid culture medium and incubate inverted at 37°C for 14–16 hours. Pick a single colony and inoculate it into 5 mL of LB medium (100 μg / mL Amp). Incubate the culture in a shaker at 37°C at 220 rpm for 14–16 hours.

[0283] 6. Plasmid Miniprep

[0284] Use a high-purity plasmid mini-extraction kit for small-scale plasmid extraction. The specific steps are as per the instructions of the Tiangen Biochemical DP104 kit.

[0285] 7. Identification and Sequencing

[0286] 1) Can be identified by PCR of single clones, bacterial suspension, or extracted plasmids;

[0287] 2) Digest the extracted plasmid with enzymes (refer to the vector enzyme digestion system and conditions), using 10 μl of the enzyme digestion system;

[0288] 3) The plasmid identified as correct by enzyme digestion was sent to a sequencing company for sequencing.

[0289] 8. Large-scale extraction of plasmids

[0290] Plasmid extraction was performed using the QIAGEN Plasmid Maxi Kit 12965. Specific steps were performed according to the Qiagen 12965 kit instructions. The plasmid concentration was determined by spectrophotometry. If the OD 260 / OD 280 =1.8-2.0, A 260 / A 230 >2.0, A 260 >0.1, A 320 If the value is ≤0.01, the plasmid meets the standard and can be used for the next experiment.

[0291] II: Sequence site mutation plasmid preparation

[0292] Sequences with site mutations were obtained through point mutagenesis, while plasmids with more mutation sites were obtained through synthesis. The specific operation of point mutagenesis is as follows: using the RSV F protein nucleotide sequence as a template, primers are used to amplify point mutations and construct mutant plasmids.

[0293] 1. Mutation Primer Synthesis

[0294] The forward and reverse primers of the single-site mutation primers are two complementary paired primers, synthesized by Sangon Biotech (Shanghai) Co., Ltd., Suzhou Jinweizhi Co., Ltd. and other companies, and used for the next step of plasmid PCR amplification.

[0295] 2. Plasmid Circular PCR Amplification

[0296] The point mutation PCR reaction system is as follows:

[0297] Table 10: PCR reaction system

[0298] PCR reaction conditions: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 15 s, extension at 68°C for 8 min, for a total of 20 cycles; 72°C for 20 min; and storage at 4°C.

[0299] 3. DpnI digestion of amplified products

[0300] Digest the PCR product with Dpn I to increase the success rate of single point mutation colonies. Digest at 37°C for 6 hours or overnight. The digestion reaction system is as follows:

[0301] Table 11: Enzyme digestion reaction system

[0302] 4. Conversion

[0303] Transform the DpnI digested product using the Trans5α Chemically Competent Cell Kit. Refer to the instructions for the Quanshijin Biotech CD201 Kit. Spread the transformed cells onto a solid culture medium of the appropriate resistance and incubate inverted at 37°C for 14–16 hours. Select a single colony and inoculate it into 5 mL of LB medium (100 μg / mL Amp). Incubate the culture in a shaker at 37°C at 220 rpm for 14–16 hours.

[0304] 5. Plasmid Sequencing Identification

[0305] Extract a small amount of plasmid from a single colony or bacterial suspension and send it to a sequencing company for sequencing and verification. Compare the sequencing results with the original sequence, and the correct plasmid can be prepared in large quantities for subsequent experiments.

[0306] III: RSV F protein expression

[0307] The above mutant plasmids are expressed by transfecting mammalian cells (CHO, 293F, etc.) as follows:

[0308] 1. Cell Culture

[0309] Expi293F cells (Thermo) were cultured and passaged on a shaker at 120 rpm and 80% relative humidity and 8% CO2 at 37°C. The viable cell density and viability were determined before transfection.

[0310] 2. Transfection

[0311] The viable cell density and viability should reach 4.5-5.5×10 6 The viable cells / mL and 95%, the plasmid usage is 2μg / 10 6 Cells, PEI / DNA ratio of 3:1. Incubate the plasmid DNA and PEI transfection reagent complex at room temperature for 10-20 minutes, then add the PEI-DNA complex to the transfection flask while shaking the flask. Continue shaking the flask for 5-7 days.

[0312] 3. Protein Expression Identification

[0313] The cell culture medium was collected, and the supernatant was centrifuged and analyzed by 10% denaturing non-reducing SDS-PAGE. The membrane was then electrophoresed onto a PVDF membrane and blocked with 5% skim milk powder for 1 hour at room temperature. HRP-labeled mouse anti-His monoclonal antibody (1:4000 dilution) was added and incubated at 25°C for 1 hour. The membrane was washed four times with PBST for 10 minutes each time, and the membrane was developed by chemiluminescence and exposed.

[0314] IV: RSV F protein purification

[0315] Proteins with His tags are purified by tagging, while proteins without tags are purified by two-step chromatography. The specific operation process is as follows:

[0316] 1. RSV F protein purification (with His tag)

[0317] (1) Sample processing: centrifuge the protein expression solution at 2000 g at 4°C for 10 min and collect the supernatant; centrifuge at 7000 rpm at 4°C for 40 min and collect the supernatant;

[0318] (2) Affinity chromatography: The chromatography buffers used were Buffer A (10 mM PBS + 0.5 M NaCl pH 7.4) and Buffer B (Buffer A + 0.5 M imidazole);

[0319] (3) The chromatography column was Cytiva HisTrap HP, CV = 5 mL, flow rate = 5 mL / min;

[0320] (4) Chromatography: First, equilibrate with Buffer A for 10 CV, then load the sample and equilibrate with Buffer A for 10 CV, then elute with Buffer B for 5 CV; 3 mL / tube, collect in separate tubes, and combine the target protein;

[0321] (5) Buffer exchange: using a Cytiva HiPrep 20 / 10 Desalting column, CV = 48 mL, flow rate = 10 mL / min, the protein was exchanged into 10 mM PBS pH 7.4 buffer;

[0322] (6) Concentration: Use a 10 kDa ultrafiltration centrifuge tube to concentrate the purified protein at 4 °C and 5000 g for 15 min, 3-4 times.

[0323] 2. RSV F protein purification (without tag)

[0324] (1) Sample processing: centrifuge the protein expression solution at 2000 g at 4°C for 10 min and collect the supernatant; centrifuge at 7000 rpm at 4°C for 40 min and collect the supernatant;

[0325] (2) Anion chromatography: The chromatography buffers were Buffer A: 20 mM Tris-HCl and Buffer B: Buffer A + 0.5 M NaCl. First, an anion chromatography column (Cytiva) was used for chromatography. The specific procedures were as follows: 1) Buffer A was equilibrated for 10 CV; 2) Sample was loaded; 3) Buffer A was equilibrated for 10 CV; 4) 20 CV, 100% Buffer B; and the target protein eluate was harvested.

[0326] (3) Cationic chromatography: The chromatography buffers were Buffer A: 20 mM citrate buffer and Buffer B: Buffer A + 0.5 M NaCl. The protein eluate was chromatographed on a cationic chromatography column (Cytiva) with the following steps: 1) 10 CV equilibration with Buffer A; 2) sample loading; 3) 10 CV equilibration with Buffer A; 4) 20 CV, 100% Buffer B; and the target protein eluate was harvested.

[0327] (4) Concentration: Use TFF ultrafiltration, liquid exchange and concentration.

[0328] 3. Protein Concentration Determination

[0329] (1) Preparation of standard: Take 20 μl of 25 mg / ml standard protein and add 980 μl of diluent (the diluent should be the same as the solvent used for the test protein) to prepare a 0.5 mg / ml standard protein;

[0330] (2) Sample preparation: dilute the protein to be tested by 2-fold, 4-fold, 8-fold, and 16-fold gradients. Perform gradient dilutions based on the approximate concentration of the protein to be tested. If the concentration of the protein to be tested is low, the dilution gradient can be reduced accordingly.

[0331] (3) Preparation of BCA working solution: Prepare the working solution according to the volume ratio of A solution: B solution = 50:1 (BCA working solution was purchased from Biyuntian Company);

[0332] (4) Add standard: add 0, 1, 2, 4, 8, 12, 16, and 20 μl of standard to a 96-well assay plate, and add diluent to make up to 20 μl. The standard assay concentrations are: 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml;

[0333] (5) Sample addition: 20 μl of samples with different dilution ratios were added to a 96-well plate and tested in duplicate;

[0334] (6) Add working solution: Add 200 μl of BCA working solution to each well and incubate at 37°C for 20–30 minutes.

[0335] (7) Reading: Detect absorbance at 562 nm using a microplate reader;

[0336] (8) Calculation: Calculate the detection protein concentration based on the standard curve and the sample dilution multiple.

[0337] V: RSV F protein expression detection - ELISA method

[0338] The expression of F protein was detected using a double antibody sandwich ELISA method established with different monoclonal antibodies. The specific procedures are as follows:

[0339] 1. RSV F protein expression detection (used to detect the total F protein content, including pre-fusion conformation protein and post-fusion conformation protein)

[0340] (1) RSV F monoclonal antibody Motavizumab (Corning) was used for plating at a concentration of 2 μg / ml, 100 μl / well, and incubated at 4°C overnight;

[0341] (2) Wash the plate twice with washing buffer (PBS + 0.05% Tween 20) and pat dry;

[0342] (3) Add blocking solution (1% BSA, PBS) to a 96-well plate, 300 μl / well, incubate at 37°C for 1 hour; wash the plate once with washing buffer and pat dry;

[0343] (4) Add antigen diluted with diluent (0.1% BSA, PBST, Tween-20, 0.05%) (8 dilutions each), positive control (2), negative control (diluent), 100 μl / well;

[0344] (5) Oscillating at 350 rpm for 2 h at room temperature;

[0345] (6) Wash the plate 5 times with washing buffer and pat dry;

[0346] (7) Add enzyme-labeled secondary antibody (RSV monoclonal antibody 101F-HRP or His monoclonal antibody-HRP), 100 μl / well, 37°C, 1 hour, antibody concentration 4 μg / ml;

[0347] (8) Wash the plate 5 times with washing buffer and pat dry.

[0348] (9) Add TMB substrate (Solarbio, CAT NO 1200), 75 μl / well, and develop color for 10 minutes;

[0349] (10) Terminate the reaction by adding 2M H2SO4 (75 μl / well) and read the OD450 optical density using a microplate reader.

[0350] The results are shown in Figure 7.

[0351] 2. RSV PreF conformational protein expression detection (used for pre-fusion conformational protein content detection, only identifies pre-fusion conformational protein)

[0352] (1) RSV F monoclonal antibody Motavizumab (Corning) was used for plating at a concentration of 2 μg / ml, 100 μl / well, and incubated at 4°C overnight;

[0353] (2) Wash the plate twice with washing buffer (PBS + 0.05% Tween 20) and pat dry;

[0354] (3) Add blocking solution (1% BSA, PBS) to a 96-well plate, 300 μl / well, incubate at 37°C for 1 hour; wash the plate once with washing buffer and pat dry;

[0355] (4) Add antigen diluted with diluent (0.1% BSA, PBST, Tween-20, 0.05%) (8 dilutions each), positive control (2), negative control (diluent), 100 μl / well;

[0356] (5) Shake at 350 rpm for 2 hours at room temperature;

[0357] (6) Wash the plate 5 times with washing buffer and pat dry;

[0358] (7) Add enzyme-labeled secondary antibody (RSV monoclonal antibody D25-HRP or monoclonal antibody AM22-HRP or AM14-HRP), 100 μl / well, 37°C, 1 hour, antibody concentration 4 μg / ml;

[0359] (8) Wash the plate 5 times with washing buffer and pat dry.

[0360] (9) Add TMB substrate (Solarbio, CAT NO 1200), 75 μl / well, and develop color for 10 minutes;

[0361] (10) Add 2M H2SO4 to terminate the reaction, 75μl / well, and read the OD on a microplate reader. 450 Optical density value.

[0362] The results are shown in Figure 8.

[0363] VI: Detection of RSV F protein expression—Octet method

[0364] Octet was used to detect the binding ability of 293F expressed pre-fusion protein concentration and RSV monoclonal antibody. The detected monoclonal antibody mainly included RSV F protein epitopes. Specific monoclonal antibodies D25, 8897, 5C4; RSV F protein epitope II specific monoclonal antibodies Palivizumab and Motavizumab; RSV F protein epitope IV specific monoclonal antibody 101F; RSV F protein trimer specific monoclonal antibody AM14.

[0365] 1. Equipment and Materials

[0366] Octet R4 antigen-antibody analysis system, 96-well black PP microtiter plate, His1K sensor, PBST buffer (containing 0.02% Tween-20 and 2% BSA), regeneration solution (10 mM Glycine-HCL, pH 1.7)

[0367] 2. Sample and sensor pretreatment

[0368] Sample pretreatment: First, centrifuge the cell expression supernatant at 200g for 10 minutes. Aspirate the supernatant to obtain a crude pre-fusion protein sample. Next, dilute 100 μl of the crude pre-fusion protein sample twice with 100 μl of buffer. Simultaneously, treat 100 μl of the crude protein sample at 60°C for 1 hour and dilute with 100 μl of buffer.

[0369] Sensor pretreatment: Add one row of buffer solution to the pre-wet plate, 200ul per well, and pre-wet one row of His 1K sensors in the buffer solution for 10 minutes.

[0370] 3. Program Settings

[0371] (1) Curing step settings: time 120s, vibration frequency 1000rpm.

[0372] (2) Combined step settings: time 120s, vibration frequency 1000rpm.

[0373] (3) Baseline step setting: time 60s, vibration frequency 1000rpm.

[0374] (4) Regeneration step setting: time 30 s, 3 cycles, each cycle including 5 s of sensor treatment with regeneration solution and 5 s of buffer solution, vibration frequency 1000 rpm.

[0375] 4. Experimental Procedure

[0376] Set up the experimental program based on the number of specific analytes, with each cycle proceeding sequentially through the "curing-baseline-binding-regeneration" sequence. For example, add analyte to the first and second rows, buffer to the third row, 3.6μg / ml monoclonal antibody to the fourth row, and regeneration solution and buffer to the final two rows, respectively. Set up the experimental program "1 curing-3 baseline-4 binding-11 regeneration-2 curing-3 baseline-4 binding-11 regeneration." After two cycles, the concentration trends of the analytes in the first and second rows, as well as their binding capacity to the monoclonal antibody, can be determined. Place the pre-wetted plate loaded with sensors and the 96-well ELISA plate in the corresponding positions of the instrument, incubate at 30°C for 10 minutes, and then start the program.

[0377] VII: Results Analysis

[0378] After the experiment is completed, enter the Octet Analysis Studio data analysis software for data processing. During the experiment, it is necessary to set up a well of 200μl buffer as a blank control, and all results need to deduct the signal value of the blank well. Enter the Epitope Binning window, set the immobilized analyte Pre-F step to antigen, and the monoclonal antibody binding step to antibody. The concentration trend of different immobilized substances can be preliminarily judged by the antigen binding signal value, but its concentration value cannot be accurately understood. The binding ability between different analytes and monoclonal antibodies can be judged by the antibody binding signal value. If the analytes added above are untreated Pre-F protein crude samples and Pre-F protein crude samples after 60℃ treatment for 1 hour, the changes in binding ability can also be observed by comparing the antibody signal values.

[0379] The expression results are shown in Figures 1 to 6. As shown in Figure 1, C1-5 was the best expressed. As shown in Figure 2, the flexible GS linker LM18, the semi-flexible GSPA linker LM23, the rigid PA linker LM29, and the rigid PG linker LM31 were the best. As shown in Figure 3, the T4 fibritin Foldon and the leucine zipper GCN4 were the best. As shown in Figure 4, the cysteine ​​mutations S55C / L188C, G71C / V76C, T72C / V76C, A89C / L231C, P101C / G242C, H159C / I291C, E232C / Y250C, T324C / N437C, K327C / S330C, I332C / P480C, P389C / S493C, K399C / S485C, L410C / G464C, S 443C / S466C (14) are the best, and Q26C / N363C, S38C / T318C, S41C / S409C, N67C / V207C, K75C / S215C, I148C / Y286C, L171C / K191C, N345C / S350C, F387C / I492C, D392C / S491C, S403C / T420C, and P484C / K498C (12) are the second best. As shown in Figure 5, E161P, S211P, S213P, I214P, I217P, V459P, N460P, E463P, K470P, E472P, D479P, L481P, S485P, and I492P (14 mutations) are optimal. As shown in Figure 6, the optimal mutations for N67, K68, K80, D84, K87, Y198, Q202, V207, Q210, T219, and D489 are I, V, L, and F.

[0380] Example 2: Stability Analysis of Pre-fusion F Protein with Stable Combination Mutations

[0381] The mutant RSV F protein designed and prepared in Example 1 was used for the following stability analysis.

[0382] I: Temperature stability: Treat at 50℃, 60℃, 70℃ for 1 hour, freeze and thaw three times

[0383] (1) Place the protein sample in a PCR instrument and incubate at 50°C for 60 minutes;

[0384] (2) Place the protein sample in a PCR instrument and incubate at 60°C for 60 minutes;

[0385] (3) Place the protein sample in a PCR instrument and incubate at 70°C for 60 minutes;

[0386] (4) The protein sample was frozen and thawed three times in a liquid nitrogen incubator at 37°C.

[0387] II: pH stability: 1 hour treatment at pH 3.5 and pH 10.0

[0388] (1) The protein solution was adjusted to pH 10 using NaOH buffer and incubated at room temperature for 1 hour. The protein solution was then neutralized back to pH 7.5 using HCl buffer.

[0389] (2) The protein solution was adjusted to pH 3.5 using HCl buffer and incubated at room temperature for 1 hour. The protein solution was then neutralized back to pH 7.5 using NaOH buffer.

[0390] III: Salt concentration stability: treated under high salt concentration 3.5M for 1 hour

[0391] The salt concentration of the protein solution was adjusted to 3.0 M osmotic pressure using 4.5 M MgCl2 solution, incubated at room temperature for 60 min, and the protein was replaced in PBS buffer for subsequent experiments.

[0392] The protein solution treated with high temperature, repeated freezing and thawing, extreme pH value and high osmotic pressure was diluted to 50 μg / ml with PBS, and the amount of RSV F protein before fusion was detected by ELISA (method). Comparisons were made to evaluate the effects of different treatment conditions on the conformation of the pre-fusion protein. The smaller the change, the more stable the conformation of the pre-fusion protein.

[0393] IV: RSV F protein Tm value determination

[0394] The PR.NT.48 Protein Stability Analyzer utilizes NanoDSF technology, leveraging changes in tryptophan and tyrosine autofluorescence during protein unfolding to assess properties such as thermal stability, colloidal stability, chemical stability, isothermal stability, and protein renaturation ability. It can provide data such as protein Tm, Cm, and ΔG. Equipped with a backscattering module, the instrument can simultaneously measure protein aggregation and thermal stability, providing data such as Tagg. The assay requires no additional dye, is not restricted by buffer conditions, and can be used over a wide range of protein sample concentrations. Therefore, it is widely applicable for stability studies of various proteins and biomacromolecules.

[0395] The specific operations are as follows:

[0396] (1) Centrifuge the purified F protein at 20,000 g for 15 minutes and load the protein into a capillary tube suitable for PR.NT.48 (siphon effect);

[0397] (2) The excitation power was set to 80% or 100%. The measurement temperature was increased from 20°C to 95°C at a rate of 2°C per minute.

[0398] (3) The tryptophan residues of the protein are excited at 280 nm, and the fluorescence intensity is recorded at 330 and 350 nm. The thermal denaturation curve is determined by measuring the intrinsic fluorescence of the protein, and the protein Tm value is calculated using instrument analysis software.

[0399] Table 12: Stability results of RSV F proteins containing different combinations of mutations.

[0400] Table 13: Amino acid sequences of RSV F proteins containing different mutation combinations shown in Table 12

[0401] Example 3: Immunogenicity Analysis of Stable Combination Mutation Pre-fusion F Protein

[0402] I: RSV F protein immunogenicity experimental protocol (mouse)

[0403] (1) Female BALB / c mice aged 6-8 weeks were randomly divided into groups, with 6-8 mice in each group.

[0404] (2) Immunization with purified and quantified prefusion F protein at a protein dose of 10 μg / mouse, using aluminum adjuvant in a volume of 100 μl / mouse, injected intramuscularly into the left hind limb;

[0405] (3) Vaccinate once at week 0 and week 3, and set up a negative control group (normal saline).

[0406] (4) Blood was collected at 3 weeks (3 weeks after the first vaccination) and 5 weeks (2 weeks after the second vaccination), and serum was separated for subsequent testing.

[0407] II: RSV F protein immunogenicity testing - neutralizing antibody testing

[0408] Principle: RSV fusion protein (F) and attachment protein (G) are the primary antigenic determinants that induce protective neutralizing antibodies in the host. Evaluating RSV-specific neutralizing antibodies is crucial for evaluating the immune efficacy of RSV vaccines and for developing anti-RSV monoclonal antibodies.

[0409] (1) This method uses RSV A2 and B subtype strains as research objects, and uses HEp-2 cells to establish the RSV virus microcytopathic effect assay (CPENT method).

[0410] (2) This method adds serially diluted samples containing anti-RSV antibodies and controls to a 96-well plate, then dilutes the RSV virus solution to the corresponding infection titer, adds it to the 96-well plate, and incubates for 1 hour.

[0411] (3) One hour later, HEp-2 cells were inoculated and cultured in a 37°C, 5% CO2 incubator for 4-6 days. The cytopathic effect (CPE) was observed and the presence of CPE in each well was recorded.

[0412] (4) The neutralizing antibody titer was calculated by accumulating the number of positive and negative wells using the Reed-Muench method.

[0413] Table 14: Neutralizing antibody titers of some recombinant RSV F proteins containing different mutation combinations in Table 12 (animal results)

[0414] Note: The sequence of 28-control is:

[0415] III: F protein immunogenicity detection - combined antibody detection

[0416] The indirect ELISA method was used to detect the IgG antibody titers against pre-fusion F and post-fusion F proteins in mouse serum.

[0417] (1) The serum to be tested was diluted 200-fold in a 96-well plate, and then serially diluted 2-fold. An equal amount of negative control serum was mixed and diluted 200-fold;

[0418] (2) Add the diluted serum at a volume of 100 μl / well to an ELISA plate coated with pre-fusion F or post-fusion F protein, incubate at room temperature for 1 hour, and then wash the plate four times;

[0419] (3) Add 100 μl / well of 1:10,000 diluted goat anti-mouse IgG-HRP secondary antibody, incubate at room temperature for 1 hour, and then wash the plate four times;

[0420] (4) Add TMB colorimetric solution at a volume of 100 μl / well and develop color at room temperature for 5-10 min;

[0421] (5) Add 50 μl / well of stop solution to terminate the reaction and read the plate on a microplate reader (OD450-OD630).

[0422] (6) The cutoff value was 2.1 times the OD value of the negative control group. The dilution factor ≥ the cutoff value was the antibody titer. The geometric mean titer (GMT) of the serum titer of each group of mice was taken as the antibody titer.

[0423] As shown in FIG9 , the serum binding antibody IgG titers of the partial recombinant RSV F proteins shown in Table 14 were 5 weeks after immunization of the animals.

[0424] IV: RSV F protein immunogenicity assay - cellular immunity assay

[0425] 1. Isolation of Mouse Splenic Lymphocytes

[0426] Mouse splenic lymphocytes were isolated using mouse lymphocyte separation medium (Dakoway, China) and the operation was performed according to the instructions. After the mice were killed by cervical dislocation, the spleen was isolated and ground into a cell suspension in 4 mL of mouse lymphocyte separation medium. The suspension was transferred to a 15 mL centrifuge tube and covered with 1 mL of RPMI 1640 medium (HYclone, USA) to keep the interface clear. Centrifuge at room temperature with a slow acceleration and deceleration of 800g for 30 minutes. After the cells were layered, the middle white membrane lymphocyte layer was aspirated, 10 mL of RPMI 1640 medium was added for washing, and the cells were centrifuged at 300g for 10 minutes. After discarding the supernatant, the cells were resuspended in ELISpot-specific serum-free medium (Dakoway, China) and counted for later use.

[0427] 2. IFN-γ, IL-2, and IL-4 ELISpot Assays

[0428] Mouse spleen lymphocytes were analyzed using the Mouse IFN-γ ELISpotPLUS (ALP) kit, Mouse IL-2 ELISpotPLUS (ALP) kit, and Mouse IL-4 ELISpotPLUS (ALP) kit (Mabtech, Sweden) according to the manufacturer's instructions. The steps are as follows:

[0429] (1) Wash the pre-coated ELISpot 96-well plate 4 times with sterile PBS at a volume of 200 μL / well, add 200 μL of ELISpot-specific serum-free medium to each well, incubate at room temperature for at least 30 minutes, and then discard the medium. Dilute the stimulant with ELISpot-specific serum-free medium to make the final concentration as follows: the protein concentration is 20 μg / mL. In the ELISpot 96-well plate, first add 50 μL of diluted stimulant to each well, or add 50 μL of medium as a negative control, and then add 50 μL of cell suspension respectively. Cell usage: 3×10 cells for IFN-γ and IL-2 detection plates 5 IL-4 detection plate is 7×10 5 The cells were cultured in a 37°C 5% CO2 incubator for 40 hours.

[0430] (2) Discard the cell suspension and add 200 μL of pre-cooled sterile deionized water to each well and incubate for 2 minutes. After discarding the deionized water, wash the wells 5 times with 200 μL of PBS per well, incubating for 2 minutes each time. Dilute the primary antibody with PBS containing 0.5% calf serum and add 100 μL to the corresponding ELISpot plate. Incubate at room temperature for 2 hours. The antibody dilution ratio is as follows: biotin-labeled anti-mouse IFN-γ antibody 1:1000, biotin-labeled anti-mouse IL-2 antibody 1:1000, biotin-labeled anti-mouse IL-4 antibody 1:1000.

[0431] (3) Discard the antibody and wash the ELISpot plate five times with 200 μL / well PBS, incubating for 2 minutes each time.

[0432] (4) Dilute the secondary antibody in PBS containing 0.5% calf serum and add 100 μL per well to the corresponding ELISpot plate. Incubate at room temperature for 1 hour. The antibody dilution ratio is as follows: ALP-labeled streptavidin 1:1000.

[0433] (5) Discard the antibody and wash the ELISpot plate five times with 200 μL / well PBS, incubating for 2 minutes each time.

[0434] (6) Filter the BCIP / NPT-plus colorimetric solution through a 0.45 μm filter, add 100 μL of colorimetric solution to each well, incubate at 37°C for about 2-5 minutes in the dark, or until obvious spots appear in the positive wells, discard the colorimetric solution, rinse the front and back of the membrane with plenty of deionized water, and read the plate after drying.

[0435] (7) After the ELISpot was dried overnight, the spots were counted using an ELISpot plate reader (CTL, USA). The final data was presented as 10 6 The number of spot-forming units (SFUs) of IFN-γ, IL-4, or IL-2 in each lymphocyte was recorded. The plates were stored in the dark after counting.

[0436] V: RSV F protein immune protection experiment - challenge experiment (cotton rat)

[0437] Female cotton rats aged 6-8 weeks were injected intramuscularly with 5 μg RSV F on day 0 (primary immunization) and day 21 (secondary immunization, booster immunization). Within 14-28 days after the second immunization, 10 5 -10 6 The RSV virus (A subtype and B subtype) of PFU is infected (challenging poison), and mice are put to death 5 days after infection. Serum samples are collected 3 weeks (W3) after initial immunity, 3 weeks (W6) after booster immunization, and 5 days (W6+5) after attacking poison respectively, and serum neutralizing antibody titer is detected using RSV virus microcytopathic method, and the neutralizing antibody titration of serum is detected using ELISA method. In addition, the lung and nasal tissue of the rear mice of separation execution are observed, and histopathological changes are observed, and the load of RSV virus in the detection tissue homogenate is detected.

[0438] The cotton rat experiment was divided into 5 groups (recombinant RSV F protein mutant antigens were selected from Table 14), among which:

[0439] The first group of antigen mutations are T72C-V76C, K399C-S485C and K87L,

[0440] The second group of antigen mutations are T72C-V76C, K327C-S330C, N67V, I214P and N460P,

[0441] The third group of antigen mutations are T72C-V76C, K399C-S485C, N67V, Y198F and E463P,

[0442] The fourth group of antigen mutations were S155C, S290C, S190F and V207L ​​in the positive control group.

[0443] The fifth group was the blank group.

[0444] The results showed that the antigen-immunized group induced higher levels of binding and neutralizing antibodies, and the viral load in the antigen-immunized group was lower than that in the virus-infected group, demonstrating that the antigen has an immune protective effect. Binding antibody detection is shown in Figure 10, neutralizing antibody detection is shown in Figure 11, and tissue viral load detection is shown in Table 15.

[0445] Table 15: Tissue viral load

[0446] Although the present invention is described and illustrated by the above embodiments, the scope of protection of the present invention is not limited to the above embodiments. Persons skilled in the art should understand that, based on the basic inventive concept of the present invention, any changes and modifications to the present invention, equivalent structures or equivalent process transformations, or direct or indirect applications in other related technical fields, all fall within the scope of protection and disclosure of the present invention.

Claims

1. A recombinant respiratory syncytial virus (RSV) fusion (RSV F) protein comprising F2 and F1 polypeptides, wherein: The recombinant RSV F protein comprises cysteine ​​(C) at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 and an amino acid replacement independently relative to SEQ ID NO: 1 at one or more positions selected from the group consisting of amino acid positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489 and 492, preferably 67, 68, 87, 198, 161, 214, 460 and 463, of SEQ ID NO: 1, preferably, the amino acid replacement is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F) and proline (P).

2. The recombinant RSV F protein of claim 1 , further comprising a residue selected from the group consisting of amino acid positions 26, 38, 41, 55, 67, 71, 75, 76, 89, 101, 148, 155, 159, 171, 188, 191, 207, 215, 231, 232, 242, 250, 286, 290, 291, 318, 324, 327, 330, 332, 345, 350, 363, 387, 389, 392, 399, 403, 409, 410, 420, 437, , 443, 464, 466, 480, 484, 485, 498, 491, 492 and 493, preferably one of positions 55, 71, 76, 89, 101, 155, 159, 188, 231, 232, 242, 250, 290, 291, 324, 327, 330, 332, 389, 399, 410, 437, 443, 464, 466, 480, 485 and 493, preferably two positions contain C, Preferably, at positions corresponding to amino acids 1 and 2 of SEQ ID NO: 1, (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 485; (xiii) 410 and 464; (xiv) 443 and 466; (xv) 26 and 363; (xvi) 38 and 318; (xvii) 41 and 409; (xviii) 67 and 207; (xix) 75 and 215; (xx) 148 and 286; (xxi) 171 and 191; (xxii) 345 and 350; (xxiii) 387 and 492; (x xiv) 392 and 491; (xxv) 403 and 420; and (xxvi) 484 and 498, preferably (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 485; (xiii) 410 and 464; and (xiv) 443 and 466, more preferably (i) 55 and 188; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (ix) 327 and 330; and (xii) 399 and 485 contain C at one or more groups of positions.

3. The recombinant RSV F protein of claim 1 or 2, wherein: (i) the amino acids at positions corresponding to amino acid positions 67, 68, 80, 84, 87, 198, 202, 207, 210, 219 and / or 489, preferably 67, 68, 87 and / or 198 of SEQ ID NO: 1 are independently I, V, L or F, preferably, one or more selected from 67V, 68I, 68V, 87L and 198F; and / or (ii) the amino acid at the position corresponding to amino acid position 161, 211, 213, 214, 217, 459, 460, 463, 470, 472, 479, 481, 485 and / or 492 of SEQ ID NO: 1, preferably 161, 214, 460 and / or 463 is P.

4. The recombinant RSV F protein of any one of claims 1-3, comprising the following amino acids at positions corresponding to the amino acid positions of SEQ ID NO: 1: (a)T72C / V76C / A89C / L231C / K68I / Y198F, (b)T72C / V76C / A89C / L231C / K68I / Y198F / E161P, (c)T72C / V76C / A89C / L231C / K68I / Y198F / E161P / E463P, (d)T72C / V76C / A89C / L231C / K68I / Y198F / E463P, (e)T72C / V76C / A89C / L231C / K68I / Y198F / I214P / E463P, (f)T72C / V76C / A89C / L231C / K68I / Y198F / I214P / N460P, (g)T72C / V76C / A89C / L231C / N67V / I214P / N460P, (h)T72C / V76C / A89C / L231C / N67V / I214P / Y198F, (i)T72C / V76C / K327C / S330C / N67V / I214P / N460P, (j)T72C / V76C / K327C / S330C / N67V / K87L / Y198F, (k)T72C / V76C / K399C / S485C / K68I / Y198F / I214P / N460P, (l)T72C / V76C / K399C / S485C / K68V / K87L / Y198F / I214P / N460P, (m)T72C / V76C / K399C / S485C / K87L, (n)T72C / V76C / K399C / S485C / K87L / I214P, (o)T72C / V76C / K399C / S485C / K87L / E161P, (p)T72C / V76C / K399C / S485C / K87L / N460P, (q)T72C / V76C / K399C / S485C / N67V / N460P, (r)T72C / V76C / K399C / S485C / N67V / Y198F / E463P, (s)T72C / V76C / K68V / K87L / Y198F / I214P / N460P, (t)T72C / V76C / N67V / Y198F / E463P, (u)T72C / V76C / N67V / Y198F / I214P, (v)T72C / V76C / P101C / G242C / I214P, (w)T72C / V76C / P101C / G242C / I214P / E463P, (x)T72C / V76C / P101C / G242C / I214P / E463P / K87L / Y198F, (y)T72C / V76C / P101C / G242C / I214P / E463P / N67V, (z)T72C / V76C / P101C / G242C / I214P / E463P / Y198F, (aa)T72C / V76C / P101C / G242C / I214P / N67V / K87L / Y198F, (bb)T72C / V76C / P101C / G242C / I214P / N67V / Y198F, (cc)T72C / V76C / P101C / G242C / K68I / Y198F / I214P / N460P, (dd)T72C / V76C / P101C / G242C / N67V, (ee)T72C / V76C / P101C / G242C / N67V / I214P, (ff)T72C / V76C / P101C / G242C / N67V / I214P / E463P, (gg)T72C / V76C / P101C / G242C / N67V / I214P / N460P, (hh)T72C / V76C / S155C / S290C / K68I / K87L / Y198F, (ii)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / I214P, (jj)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / E463P, (kk)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / N460P, (ll)T72C / V76C / S155C / S290C / N67V / Y198F, (mm)T72C / V76C / S155C / S290C / N67V / Y198F / N460P, or (nn)T72C / V76C / S55C / L188C / K68I / Y198F.

5. The recombinant RSV F protein of any one of claims 1-4, wherein, in addition to the above amino acid substitutions, the F2 polypeptide comprises amino acids 26-109 or 26-105 corresponding to SEQ ID NO: 1 and / or the Fi polypeptide comprises amino acids 137-513 or 147-513 corresponding to SEQ ID NO: 1, optionally the F2 and Fi polypeptides are connected by a linker, preferably the linker is selected from AAGAATAA, GSPAG, GGASPAGG, GGASPAAPAPAG, AEAAAKEAAAKA, PAPAP, GPPPG, GSGS, or GGSGGSGGS; Preferably, the recombinant RSV F protein further comprises a trimerization domain, such as a trimerization domain of T4 fibritin Foldon, hCorla corona protein-1, T3XV, leucine zipper GCN4-1, leucine zipper GCN4-2, chloramphenicol acetyltransferase, hCorla corona protein-2, mCorla corona protein, Langerhans protein, or maternal protein 1 cartilage matrix protein (e.g., as shown in SEQ ID NOs: 7 and 16-24), preferably a trimerization domain derived from T4 fibritin or leucine zipper GCN4, as shown in any one of SEQ ID NOs: 7 and 18-19, optionally linked to the C-terminus of the F1 polypeptide, preferably linked to the F1 polypeptide via a linker sequence such as SAIG, GG, or SA.

6. The recombinant RSV F protein of any one of claims 1-5, comprising an amino acid sequence selected from any one of SEQ ID NOs: 25-64.

7. A method for stabilizing the prefusion conformation of an RSV F protein comprising F2 and F1 polypeptides, preferably, the F2 polypeptide comprises amino acids 26-109 or 26-105 corresponding to SEQ ID NO: 1 and / or the F1 polypeptide comprises amino acids 137-513 or 147-513 corresponding to SEQ ID NO: 1, the method comprising: Introducing one or more of the following mutations into the RSV F protein: The amino acids at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 are mutated to C, and The mutation is selected from the amino acid corresponding to one or more positions 67, 68, 80, 84, 87, 102, 161, 198, 202, 207, 210, 211, 213, 214, 217, 219, 379, 447, 459, 460, 463, 470, 472, 479, 481, 485, 489 and 492 of SEQ ID NO: 1, preferably 67, 68, 87, 98, 161, 214, 460 and 463, preferably, the amino acid at said position is independently mutated to I, V, L, F or P.

8. The method of claim 7, further comprising modifying the amino acid positions 26, 38, 41, 55, 67, 71, 75, 76, 89, 101, 148, 155, 159, 171, 188, 191, 207, 215, 231, 232, 242, 250, 286, 290, 291, 318, 324, 327, 330, 332, 345, 350, 363, 387, 389, 392, 399, 403, 409, 410, 420, 437, 440, 451, 460, 470, 480, 490, 501, 512, 521, 523, 524, 536, 541, 542, 543, 544, 555 43, 464, 466, 480, 484, 485, 498, 491, 492 and 493, preferably 55, 71, 76, 89, 101, 155, 159, 188, 231, 232, 242, 250, 290, 291, 324, 327, 330, 332, 389, 399, 410, 437, 443, 464, 466, 480, 485 and 493, at one, preferably two, positions are mutated to C, Preferably, the amino acid residues selected from the group consisting of amino acid positions (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) 332 and 480; (xi) 389 and 493; (xii) 399 and 437. 85; (xiii) 410 and 464; (xiv) 443 and 466; (xv) 26 and 363; (xvi) 38 and 318; (xvii) 41 and 409; (xviii) 67 and 207; (xix) 75 and 215; (xx) 148 and 286; (xxi) 171 and 191; (xxii) 345 and 350; (xxiii) 387 and 492; (xxi v) 392 and 491; (xxv) 403 and 420; and (xxvi) 484 and 498, preferably (i) 55 and 188; (ii) 71 and 76; (iii) 89 and 231; (iv) 101 and 242; (v) 155 and 290; (vi) 159 and 291; (vii) 232 and 250; (viii) 324 and 437; (ix) 327 and 330; (x) (iv) 101 and 242; (v) 155 and 290; (ix) 327 and 330; and (xii) 399 and 485, wherein the amino acid in one or more groups of positions is mutated to C.

9. The method of claim 7 or 8, comprising: (i) independently mutating an amino acid selected from positions corresponding to amino acid positions 67, 68, 80, 84, 87, 198, 202, 207, 210, 219 and / or 489, preferably 67, 68, 87 and / or 198 of SEQ ID NO: 1, to I, V, L or F, preferably, the amino acid mutation is selected from one or more of 67V, 68I, 68V, 87L and 198F; and / or (ii) mutating an amino acid selected from the positions corresponding to amino acid positions 161, 211, 213, 214, 217, 459, 460, 463, 470, 472, 479, 481, 485 and / or 492 of SEQ ID NO: 1, preferably 161, 214, 460 and / or 463, to P.

10. The method of any one of claims 7-9, comprising introducing into the RSV F protein a mutation selected from the group consisting of: (a)T72C / V76C / A89C / L231C / K68I / Y198F, (b)T72C / V76C / A89C / L231C / K68I / Y198F / E161P, (c)T72C / V76C / A89C / L231C / K68I / Y198F / E161P / E463P, (d)T72C / V76C / A89C / L231C / K68I / Y198F / E463P, (e)T72C / V76C / A89C / L231C / K68I / Y198F / I214P / E463P, (f)T72C / V76C / A89C / L231C / K68I / Y198F / I214P / N460P, (g)T72C / V76C / A89C / L231C / N67V / I214P / N460P, (h)T72C / V76C / A89C / L231C / N67V / I214P / Y198F, (i)T72C / V76C / K327C / S330C / N67V / I214P / N460P, (j)T72C / V76C / K327C / S330C / N67V / K87L / Y198F, (k)T72C / V76C / K399C / S485C / K68I / Y198F / I214P / N460P, (l)T72C / V76C / K399C / S485C / K68V / K87L / Y198F / I214P / N460P, (m)T72C / V76C / K399C / S485C / K87L, (n)T72C / V76C / K399C / S485C / K87L / I214P, (o)T72C / V76C / K399C / S485C / K87L / E161P, (p)T72C / V76C / K399C / S485C / K87L / N460P, (q)T72C / V76C / K399C / S485C / N67V / N460P, (r)T72C / V76C / K399C / S485C / N67V / Y198F / E463P, (s)T72C / V76C / K68V / K87L / Y198F / I214P / N460P, (t)T72C / V76C / N67V / Y198F / E463P, (u)T72C / V76C / N67V / Y198F / I214P, (v)T72C / V76C / P101C / G242C / I214P, (w)T72C / V76C / P101C / G242C / I214P / E463P, (x)T72C / V76C / P101C / G242C / I214P / E463P / K87L / Y198F, (y)T72C / V76C / P101C / G242C / I214P / E463P / N67V, (z)T72C / V76C / P101C / G242C / I214P / E463P / Y198F, (aa)T72C / V76C / P101C / G242C / I214P / N67V / K87L / Y198F, (bb)T72C / V76C / P101C / G242C / I214P / N67V / Y198F, (cc)T72C / V76C / P101C / G242C / K68I / Y198F / I214P / N460P, (dd)T72C / V76C / P101C / G242C / N67V, (ee)T72C / V76C / P101C / G242C / N67V / I214P, (ff)T72C / V76C / P101C / G242C / N67V / I214P / E463P, (gg)T72C / V76C / P101C / G242C / N67V / I214P / N460P, (hh)T72C / V76C / S155C / S290C / K68I / K87L / Y198F, (ii)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / I214P, (jj)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / E463P, (kk)T72C / V76C / S155C / S290C / K68I / K87L / Y198F / N460P, (ll)T72C / V76C / S155C / S290C / N67V / Y198F, (mm)T72C / V76C / S155C / S290C / N67V / Y198F / N460P, or (nn)T72C / V76C / S55C / L188C / K68I / Y198F.

11. The method of any one of claims 7 to 10, comprising linking the F2 and F1 polypeptides via a linker, preferably, the linker is selected from the group consisting of AAGAATAA, GSPAG, GGASPAGG, GGASPAAPAPAG, AEAAAKEAAAKA, PAPAP, GPPPG, GSGS and GGSGGSGGS; and / or, The method comprises linking the RSV F protein to a trimerization domain, preferably, the trimerization domain is derived from the trimerization domain of T4 fibritin Foldon, hCorla corona protein-1, T3XV, leucine zipper GCN4-1, leucine zipper GCN4-2, chloramphenicol acetyltransferase, hCorla corona protein-2, mCorla corona protein, Langerhans protein or maternal protein 1 cartilage matrix protein (e.g., as shown in SEQ ID NOs: 7 and 16-24), more preferably, the trimerization domain is selected from the trimerization domain derived from T4 fibritin (e.g., SEQ ID NO: 7) and the trimerization domain derived from leucine zipper GCN4 (e.g., SEQ ID NO: 18 or 19); optionally, the trimerization domain is linked to the C-terminus of the F1 polypeptide, preferably linked to the F1 polypeptide via a linker sequence such as SAIG, GG or SA.

12. A nucleic acid molecule encoding the recombinant RSV F protein of any one of claims 1-6.

13. The nucleic acid molecule of claim 12 which has been codon-optimized for expression in mammalian cells.

14. A vector comprising the nucleic acid molecule according to claim 12 or claim 13.

15. A pharmaceutical composition comprising the recombinant RSV F protein of any one of claims 1-6, a stabilized RSV F protein obtained by the method of any one of claims 7-11, a nucleic acid molecule of claim 12 or 13, and / or a vector of claim 14, and optionally a pharmaceutically acceptable excipient.

16. An RSV vaccine comprising the recombinant RSV F protein of any one of claims 1-6, a stabilized RSV F protein obtained by the method of any one of claims 7-11, the nucleic acid molecule of claim 12 or 13, and / or the vector of claim 14, and optionally an adjuvant.

17. The recombinant RSV F protein of any one of claims 1-6, the stabilized RSV F protein obtained by the method of any one of claims 7-11, the nucleic acid molecule of claim 12 or 13, and / or the vector of claim 14, for use in inducing an immune response against the RSV F protein.

18. The recombinant RSV F protein of any one of claims 1 to 6, the stabilized RSV F protein obtained by the method of any one of claims 7 to 11, the nucleic acid molecule of claim 12 or 13, the vector of claim 14, for use in preventing and / or treating RSV infection.

19. Use of the recombinant RSV F protein of any one of claims 1-6, the stabilized RSV F protein obtained by the method of any one of claims 7-11, the nucleic acid molecule of claim 12 or 13, and / or the vector of claim 14 in the preparation of a medicament or vaccine for inducing an immune response against the RSV F protein, preventing and / or treating RSV infection.

20. A method for inducing an immune response against RSV F protein, preventing and / or treating RSV infection in a subject, comprising administering to the subject an effective amount of the recombinant RSV F protein of any one of claims 1-6, the stabilized RSV F protein obtained by the method of any one of claims 7-11, the nucleic acid molecule of claim 12 or 13, the vector of claim 14, the pharmaceutical composition of claim 15, and / or the RSV vaccine of claim 16.

21. An in vitro diagnostic method for detecting the presence of RSV infection in a subject, comprising: a) contacting a biological sample from the subject with the recombinant RSV F protein of any one of claims 1-6 or a stabilized RSV F protein obtained by the method of any one of claims 7-11; and b) detecting the presence of the antibody-protein complex.

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