WO2025201243A1 - Recombinant respiratory syncytial virus pre-fusion (f) protein mutant and use thereof - Google Patents

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

The present invention relates to the field of medicine. In particular, the present invention relates 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

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.

In nature, the RSV F protein is initially expressed as a single polypeptide precursor, designated F0. F0 trimerizes in the endoplasmic reticulum and is processed by cellular furin-like proteases at two conserved sites to produce the F1, F2, and Pep27 polypeptides. The Pep27 polypeptide is cleaved off and does not constitute a 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 to the membrane via a transmembrane domain connected 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 entry, the RSV F protein is a target for neutralizing antibodies and the 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).

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

Summary of the Invention

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

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

The present invention also provides a nucleic acid molecule encoding the recombinant RSV F protein, and a vector containing the nucleic acid molecule.

The present invention also relates to a method for stabilizing the prefusion conformation of RSV F protein, and to a prefusion RSV F protein obtained or obtainable by the method.

The present invention also relates to a pharmaceutical composition 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.

The present invention also relates to an RSV vaccine, which comprises 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 comprises an adjuvant.

The present invention also relates to methods 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.

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

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 containing the nucleic acid molecule in the preparation of a drug or vaccine for inducing an immune response against RSV F protein, preventing and/or treating RSV infection.

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 the recombinant RSV F protein of the present invention, a nucleic acid molecule encoding the recombinant RSV F protein, and/or a vector containing the nucleic acid molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

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.

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.

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.

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.

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.

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 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.

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, respectively, 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.

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

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.

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

Unless otherwise indicated, scientific and technical terms used herein shall have the meanings generally known to those skilled in the art. In addition, unless otherwise required, singular terms shall include the plural and plural terms shall include the singular. The aforementioned techniques and methods are generally performed 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, N.Y. (2001)) and Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989), which are incorporated by reference. All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, are hereby incorporated by reference in their entirety.

The fusion protein (F protein) of respiratory syncytial virus (RSV) is involved in the fusion of the viral membrane with the infected host cell membrane. RSV F mRNA is translated into a precursor protein, designated F0, which contains a signal peptide sequence at its N-terminus (e.g., amino acid residues 1-25 of SEQ ID NO:1), which is removed in the endoplasmic reticulum. F0 is cleaved by cellular proteases (specifically furin or furin-like proteases) at two sites (between amino acid residues 109/110 and 136/137), removing a short glycosylated intervening sequence (the pep27 polypeptide, amino acid residues 110 to 136 of SEQ ID NO:1), yielding two polypeptides, designated F1 and F2. The F1 polypeptide contains a hydrophobic fusion peptide at its N-terminus and a transmembrane (TM) and cytoplasmic region at its C-terminus. The F2 polypeptide is covalently linked to F1 via two disulfide bonds. F1-F2 heterodimers assemble as homotrimers within the virion.

Potential approaches to producing vaccines against RSV infection rely on purified RSV F protein in a stable prefusion conformation. For soluble protein vaccines, RSV F protein is truncated by deleting the transmembrane (TM) and cytoplasmic regions to produce a soluble, secreted F protein. Soluble F protein lacking the TM region is significantly less stable than the full-length protein. To obtain a soluble F protein in a stable prefusion conformation that exhibits high expression levels and stability, this prefusion conformation needs to be stabilized.

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)).

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 isoleucine (I), valine (V), leucine (L), phenylalanine (F) and proline (P) relative to SEQ ID NO: 1.

According to the present invention, specific stabilizing amino acids (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 (numbered according to SEQ ID NO: 1) of SEQ ID NO: 1, combined with cysteine (C) at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1, stabilize the RSV F protein in the prefusion conformation.

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 substitution at one or more (e.g., 2, 3, 4, 5, or more) positions selected from 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 substitution is selected from I, V, L, F, and P.

When describing amino acid positions herein, the numbers indicate 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.

The position corresponding to an amino acid position of a reference sequence (e.g., SEQ ID NO: 1) refers to the amino acid position identified when aligned to 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, the amino acid sequences are aligned to obtain the highest order match (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin,H.G.,eds.,Humana Press,New Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;Sequenc e 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).

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.

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

In one embodiment, in the recombinant RSV F protein, the amino acids 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 corresponding to amino acid position 67 of SEQ ID NO: 1 is V, the amino acid corresponding to amino acid position 68 of SEQ ID NO: 1 is I, the amino acid corresponding to amino acid position 68 of SEQ ID NO: 1 is V, the amino acid corresponding to amino acid position 87 of SEQ ID NO: 1 is L, and/or the amino acid corresponding to amino acid position 198 of SEQ ID NO: 1 is F.

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.

In one embodiment, in the recombinant RSV F protein, the amino acid 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 amino acid positions 161, 214, 460 and/or 463 is P.

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.

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 72 and 76 corresponding to amino acid positions of SEQ ID NO: 1, and comprising an amino acid replacement 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.

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.

In some 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 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.

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.

In certain preferred embodiments, the recombinant RSV F protein comprises amino acids at 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) 399 and 486; (xiv) 399 and 487; (xv) 399 and 488; (xv) 399 and 489 ... )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 contain a C in one or more of the following positions:

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.

In certain preferred embodiments, the recombinant RSV F protein comprises C 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.

In certain embodiments, the recombinant RSV F protein comprises C at positions corresponding to amino acid positions 72 and 76 of SEQ ID NO: 1 and C at 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; (xii i) 410 and 464; and (xiv) a group of positions among 443 and 466 comprise C, and relative to SEQ ID NO:1, one or more (e.g., 2, 3, 4, 5 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 492 of SEQ ID NO:1 comprise an amino acid substitution (e.g., selected from I, V, L, F and P). For example, the recombinant RSV F protein comprises C at 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 corresponding to SEQ ID NO:1.

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:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

As used herein, when referring to an amino acid mutation 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.

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.

In a further embodiment, the recombinant RSV F protein, in addition to the amino acids at the specified positions, the F2 polypeptide comprises 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 the same as the amino acids at positions 26-109 or 26-105 of SEQ ID NO: 1) and/or the F1 polypeptide comprises 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 the same as the amino acids at positions 137-513 or 147-513 of SEQ ID NO: 1).

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.

In further embodiments, the recombinant RSV F protein comprises a trimerization domain. The trimerization domain can be any suitable trimerization domain known in the art, including, but not limited to, a trimerization domain 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 (e.g., as shown in SEQ ID NOs: 7 and 16-24). In one embodiment, the trimerization domain can be selected from a trimerization domain derived from T4 fibritin (e.g., SEQ ID NO: 7) and a trimerization domain derived from leucine zipper GCN4 (e.g., SEQ ID NOs: 18 or 19).

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.

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 F1 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.

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 F1 polypeptide comprises amino acids 147-513 corresponding to SEQ ID NO: 1, which are connected by the linker GGSGGSGGS.

In certain embodiments, the recombinant RSV F protein further comprises a trimerization domain, such as a T4 fibritin-derived trimerization domain, which is 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.

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.

In certain embodiments, the recombinant RSV F protein comprises an F2 polypeptide, an F1 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).

In certain embodiments, the recombinant RSV F protein comprises an F2 polypeptide, an F1 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).

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.

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

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

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

The present invention provides novel recombinant RSV F proteins that are stabilized in a prefusion conformation, i.e., contain (display) at least one epitope specific for the prefusion conformation F protein. The epitope specific for the prefusion conformation F protein is an epitope that is not present in the postfusion conformation. Without wishing to be bound by any particular theory, it is believed that the prefusion conformation of the recombinant RSV F protein may contain the same epitopes as those on the RSV F protein expressed on native RSV virions and, therefore, may provide advantages for eliciting protective neutralizing antibodies.

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 pre-fusion RSV F protein.

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:

The amino acids at positions 72 and 76 corresponding to the amino acid positions of SEQ ID NO: 1 are mutated to C, and the amino acids 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 corresponding to SEQ ID NO: 1, preferably 67, 68, 87, 98, 161, 214, 460 and 463 are mutated (for example, to I, V, L, F or P).

In some embodiments, the method further includes mutating an amino acid at one or more positions selected from 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 to C.

In certain embodiments, the method further comprises mutating an amino acid at one or more 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 to C.

Preferably, the method comprises modifying the amino acid 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; The amino acid at one or more groups of positions among (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 is mutated to a C.

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.

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.

In one embodiment, the method includes mutating the amino acids at positions 67, 68, 80, 84, 87, 198, 202, 207, 210, 219, and/or 489, preferably 67, 68, 87, and/or 198, of 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.

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

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

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 F1 polypeptides to C, and mutating the amino acid at one or more positions selected from 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 corresponding to amino acid positions 161, 214, 460 and/or 463 of SEQ ID NO:1 to P.

According to the present invention, the presence of a specific stabilizing amino acid 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.

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:

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

(b) selected from the mutations corresponding to SEQ ID NO: 1: S55C/L188C; G71C/V76C; A89C/L231C; P101C/G242C; S155C/S290C; H159C/I291C; E232C/Y250C; T324C/N437C; K327C/S330C; I332C/P 480C; 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

(c) an amino acid mutation selected from at least one of the 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 492 of SEQ ID NO: 1, preferably 67, 68, 87, 198, 161, 214, 460 and 463, preferably independently mutated to I, V, L, F or P, more preferably to amino acid mutations at positions 67, 68, 87 and/or 198. to I, V, L or F, and/or, the amino acid at position 161, 214, 460 and/or 463 is mutated to P, 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.

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.

In certain embodiments, the method comprises introducing mutations (a) corresponding to the 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.

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 according to 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.

In certain embodiments, the method comprises introducing an amino acid 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.

In certain embodiments, the method does not introduce any other mutations into the RSV F protein in addition to the amino acid mutations described above. For example, the recombinant RSV F protein obtained by the method comprises, in addition to the amino acid mutations introduced above, the 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 the 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 linked by a linker such as GSGS or GGSGGSGGS.

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.

In a further embodiment, the method comprises linking the RSV F protein to a trimerization domain. The trimerization domain can be any suitable trimerization domain known in the art, including, but not limited to, a trimerization domain 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 (e.g., as shown in SEQ ID NOs: 7 and 16-24). In one embodiment, the trimerization domain can be selected from a trimerization domain derived from T4 fibritin (e.g., SEQ ID NO: 7) and a trimerization domain derived from leucine zipper GCN4 (e.g., SEQ ID NOs: 18 or 19). 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.

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

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

The present invention further provides recombinant RSV F protein obtained or obtainable by the method and its use.

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

As described above, the recombinant RSV F proteins of the present invention also encompass fragments of pre-fusion RSV F proteins. The fragments can be derived from either or both of the amino-terminus (e.g., by cleavage of the signal sequence) and the carboxyl-terminus (e.g., by deletion of the transmembrane region and/or the cytoplasmic tail). The fragments can be selected to comprise immunologically active fragments of the F protein, i.e., portions that elicit an immune response in a subject. This can be routinely determined using computer, in vitro, and/or in vivo methods.

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

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

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 linked to the truncated F1 polypeptide. According to the present invention, by linking 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 binding to prefusion-specific antibodies is provided.

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

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.

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 directly or through a linker (e.g., SAIG, GG, or SA) to amino acid residue 513 of the RSV Fi polypeptide.

In certain embodiments, the expression level of the recombinant RSV F protein of the present invention is increased compared to wild-type RSV F protein. In certain embodiments, the prefusion content (defined as the fraction of F protein bound to prefusion-specific antibodies) is significantly higher 5 to 10 days after harvesting the protein compared to wild-type F protein without the substitutions described herein.

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), that is, it is not easy to change into the postfusion conformation during protein processing (for example, purification, freeze-thaw cycles, and/or storage, etc.).

In certain embodiments, the recombinant RSV F proteins according to the present invention have increased stability when stored at 4°C compared to RSV F proteins without the one or more mutations. In certain embodiments, the recombinant proteins are stable when stored at 4°C 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. "Stable when stored" means that after the proteins are stored in a solution (e.g., culture medium) at 4°C for at least 30 days, the proteins still display at least one epitope specific for a prefusion-specific antibody. In certain embodiments, the proteins display the at least one prefusion-specific epitope for at least 6 months, preferably for at least 1 year, when the recombinant RSV F proteins are stored at 4°C.

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

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

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 consisting 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 located at the N- or C-terminus of a protein and are typically used for purification purposes.

RSV is known to exist as a single serotype with two antigenic subtypes, A and B. The amino acid sequences of the mature, processed F proteins of these two types are approximately 93% identical. As used herein, amino acid positions are given with reference to the sequence of SEQ ID NO: 1. As used herein, "the amino acid at position "x" of the 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 of the immature F0 protein (SEQ ID NO: 1). When an F protein from another RSV strain is used, the amino acid positions of that F protein will be numbered with reference to the numbering of SEQ ID NO: 1 by inserting gaps where necessary to align the sequence of the other RSV strain with the F protein of SEQ ID NO: 1. Sequence alignment can be performed using methods well known in the art, such as CLUSTALW, Bioedit, or CLC Workbench.

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.

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.

Table 1. Standard amino acids, abbreviations and properties

Those skilled in the art will appreciate that proteins can be mutated using conventional molecular biology procedures. Mutations according to the present invention preferably result in increased expression levels and/or increased stabilization of the recombinant RSV F protein compared to an RSV F protein that does not contain the one or more mutations.

The present invention further provides a nucleic acid molecule encoding the recombinant RSV F protein according to the present invention.

In a preferred embodiment, the nucleic acid molecules encoding the proteins according to the present invention are codon-optimized for expression in mammalian cells (e.g., human cells). Methods for codon optimization are known in the art and have been described, for example, in WO 96/09378. A sequence is considered to be codon-optimized if at least one non-preferred codon is replaced by a more preferred codon compared to the wild-type sequence. In this article, a non-preferred codon is a codon that is not used as frequently in an organism as another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a particular organism 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.

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.

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

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.

The present invention also relates to host cells containing nucleic acid molecules encoding the recombinant RSV F protein. Recombinant RSV F protein can be produced by recombinant DNA technology, which involves expressing these molecules in host cells (e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines (such as HEK293 cells, PER.C6 cells) or yeast, fungi, insect cells, etc.), or transgenic animals or plants. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. Generally, the production of recombinant proteins (such as the recombinant RSV F protein of the present invention) in host cells includes introducing a heterologous nucleic acid molecule encoding the protein in an expressible form into the host cells, culturing the cells under conditions conducive to the expression of the nucleic acid molecule, and allowing the protein to be expressed in the cells. The nucleic acid molecule encoding the protein in an expressible form can be in the form of an expression cassette and generally contains sequences necessary to cause expression of the nucleic acid, such as one or more enhancers, promoters, polyadenylation signals, etc.

Cell culture media are available from various suppliers, and one can routinely select an appropriate culture medium for host cells expressing a protein of interest, such as recombinant RSV F protein. Suitable culture media may or may not contain serum.

The present invention further provides compositions comprising a recombinant RSV F protein and/or a nucleic acid molecule and/or a vector as described above. The present invention therefore provides a composition comprising a recombinant RSV F protein that exhibits an epitope that is present in the prefusion conformation of the RSV F protein but absent in the postfusion conformation. The present invention also provides a composition comprising a nucleic acid molecule and/or a vector encoding such a recombinant RSV F protein. The present invention further provides an immunogenic composition comprising a recombinant RSV F protein, and/or a nucleic acid molecule, and/or a vector as described above. The present invention further provides a pharmaceutical composition comprising a recombinant RSV F protein, and/or a nucleic acid molecule, and/or a vector as described above, and one or more pharmaceutically acceptable excipients.

The present invention also provides uses 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 molecule, and/or vector according to the present invention. Further provided are uses 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.

The recombinant RSV F protein, nucleic acid molecule or vector of the present invention can be used to prevent and/or treat RSV infection in a subject in need of such prevention and/or treatment.

The recombinant RSV F protein, nucleic acid molecule and/or vector according to the present 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).

The present invention further provides methods for preventing and/or treating RSV infection in a subject using recombinant RSV F proteins, nucleic acid molecules, and/or vectors according to the present invention, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant RSV F protein, a nucleic acid molecule encoding the same, and/or a vector comprising the nucleic acid molecule. A therapeutically effective amount refers to an amount of a protein, nucleic acid molecule, or vector that is effective for preventing, alleviating, and/or treating a disease or condition caused by infection with RSV. Prevention encompasses inhibiting or reducing the spread of RSV or inhibiting or reducing the onset, development, or progression of one or more symptoms associated with infection with RSV. As used herein, alleviating can refer to reducing visible or perceptible disease symptoms, viremia, or any other measurable manifestation of influenza infection.

For administration to a subject (e.g., a human), the present invention can employ a pharmaceutical composition comprising a recombinant RSV F protein of the present invention, a nucleic acid molecule encoding the same, and/or a vector comprising the nucleic acid molecule, and a pharmaceutically acceptable excipient. As used herein, "pharmaceutically acceptable" means that the excipient does not cause any unnecessary or adverse effects in the subject to which it is administered at the dosage and concentration employed. Such pharmaceutically acceptable excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th ed., A.R. Gennaro, ed., Mack Publishing Company, 1990). Although available as a lyophilized formulation, the RSV F protein or nucleic acid molecule is preferably formulated and administered as a sterile solution. In certain embodiments, the RSV F protein can be formulated as an injectable formulation.

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.

In certain embodiments, the present invention provides methods for producing a vaccine 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 an agent or composition containing active ingredients effective to induce a degree of immunity against a pathogen or disease in a subject, resulting in at least a reduction in the severity, duration, or other manifestations of symptoms associated with infection by that pathogen or disease (or even complete absence thereof). 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 severe lower respiratory tract disease leading to hospitalization in a subject and reducing the frequency of complications caused by RSV infection and replication, such as pneumonia and bronchiolitis. The term "vaccine" according to the present invention indicates that it is a pharmaceutical composition and, therefore, typically includes a pharmaceutically acceptable diluent, carrier, or excipient. It may or may not contain additional active ingredients. 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.

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

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.

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.

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.

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.

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.

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.

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.

Example

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.

Example 1: Preparation of stable prefusion RSV F protein

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

T4 fibritin-derived trimerization domain (foldon domain, residues 518-544, SEQ ID NO: 7);

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

Purified HIS tag (residues 553-558): HHHHHH (SEQ ID NO: 9);

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

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.

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

●Signal sequence (residues 1-25)

F2 polypeptide (residues 26-109)

pep27 polypeptide (residues 110-136)

F1 polypeptide (residues 137-513)

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

A: Fusion polypeptide of F2 polypeptide and F1 polypeptide

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))

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))

C1-2: F2 polypeptide amino acids 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 + streptomycin tag (Streptag) II (561-568): WSHPQFEK + linker sequence (residues 514-517 (SAIG, SEQ ID NO: 13), 545-546 (GG) and 559-560 (SA))

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))

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))

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))

C1-1 (enzyme cleavage site-pep27 peptide deletion)

C1-2 (restriction site-pep27 peptide deletion-GSGS)

C1-3 (restriction site-pep27 peptide deletion-GGSGGSGGS)

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

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

B: Preparation of stable prefusion RSV F peptides—linkers

●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))

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.

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

C: Preparation of stable prefusion RSV F peptide—trimerization domain

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.

Table 3: Trimerization domain origin and sequence

D: Preparation of stable prefusion RSV F peptides - stabilizing mutations - cysteine mutations

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.

Table 4

E: Preparation of stable prefusion RSV F peptides - stabilizing mutations - proline mutations

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.

Table 5

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

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.

Table 6

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.

I: Sequence expression plasmid preparation

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:

1. Target fragment PCR

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:

Table 7: PCR reaction system

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.

2. Vector Enzyme Digestion

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

Table 8: Enzyme digestion reaction system

Digest at 37°C for 6 hours or overnight.

3. Glue recycling

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.

4. Connect the vector and the target fragment

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

Table 9: Ligation reaction system

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

5. Conversion

Transform the ligation product using the Trans5α Chemically Competent Cell Kit, following the instructions for the Quanshijin Biotech CD201 Kit. Spread the transformed cells onto a suitable resistant solid culture medium 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) in a shaker at 37°C at 220 rpm for 14–16 hours.

6. Plasmid Miniprep

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.

7. Identification and Sequencing

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

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

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

8. Large-scale extraction of plasmids

Plasmid extraction was performed using the QIAGEN Plasmid Maxi Kit 12965. Specific steps were followed according to the Qiagen 12965 kit instructions. Plasmid concentration was determined spectrophotometrically. If _OD260 / _OD280 = 1.8-2.0, _A260 / _A230 > 2.0, _A260 > 0.1, and _A320 ≤ 0.01, the plasmid met the standards and could be used in the next step.

II: Sequence site mutation plasmid preparation

Sequences with site mutations were obtained through point mutagenesis, while plasmids with more mutation sites were synthesized. The point mutagenesis procedure was as follows: using the RSV F protein nucleotide sequence as a template, primers were used to amplify the point mutations and construct mutant plasmids.

1. Mutation Primer Synthesis

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.

2. Plasmid Circular PCR Amplification

The point mutation PCR reaction system is as follows:

Table 10: PCR reaction system

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.

3. DpnI digestion of amplified products

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:

Table 11: Enzyme digestion reaction system

4. Conversion

Transform the DpnI digested product using the Trans5α Chemically Competent Cell Kit, following the instructions for the Genbio CD201 kit. Spread the transformed cells onto a suitable resistant solid culture medium 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) in a shaker at 37°C and 220 rpm for 14–16 hours.

5. Plasmid Sequencing Identification

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.

III: RSV F protein expression

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

1. Cell Culture

Expi293F cells (Thermo) were cultured and passaged at 80% relative humidity, 8% CO ₂ , 37° C., and 120 rpm on a shaker. The viable cell density and viability were determined before transfection.

2. Transfection

The viable cell density and viability should reach 4.5-5.5 × ^10⁶ viable cells/mL and 95%, respectively. The plasmid dosage should be 2 μg/ ^10⁶ cells, and the PEI/DNA ratio should be 3:1. Incubate the plasmid DNA and PEI transfection reagent complex at room temperature for 10-20 minutes. Then, while shaking the flask, add the PEI-DNA complex to the transfection flask and continue shaking for 5-7 days.

3. Protein Expression Identification

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.

IV: RSV F protein purification

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:

1. RSV F protein purification (with His tag)

(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;

(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);

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

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

(5) Buffer exchange: Use Cytiva HiPrep 20/10 Desalting column, CV = 48 mL, flow rate = 10 mL/min, and exchange the protein into 10 mM PBS pH 7.4 buffer;

(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.

2. RSV F protein purification (without tag)

(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;

(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 steps were as follows: 1) Buffer A was equilibrated for 10 CV; 2) Sample loading; 3) Buffer A was equilibrated for 10 CV; 4) 20 CV, 100% Buffer B; and the target protein eluate was harvested.

(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.

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

3. Protein Concentration Determination

(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;

(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.

(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);

(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;

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

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

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

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

V: RSV F protein expression detection - ELISA method

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:

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

(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.

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

(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;

(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;

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

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

(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;

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

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

(10) Terminate the reaction by adding 2M H ₂ SO ₄ (75 μl/well) and read the OD450 optical density using a microplate reader.

The results are shown in Figure 7.

2. RSV PreF conformational protein expression detection (used for prefusion conformational protein content detection, only identifies prefusion conformational protein)

(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.

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

(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;

(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;

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

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

(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;

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

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

(10) Add 2M H ₂ SO ₄ to terminate the reaction, 75 μl/well, and read the OD ₄₅₀ optical density value with a microplate reader.

The results are shown in Figure 8.

VI: Detection of RSV F protein expression—Octet method

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.

1. Equipment and Materials

Octet R4 Antigen-Antibody Assay 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)

2. Sample and sensor pretreatment

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.

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

3. Program Settings

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

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

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

(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.

4. Experimental Procedure

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.

VII: Results Analysis

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 immobilization analyte Pre-F step to antigen, and the binding monoclonal antibody step to antibody. The concentration trend of different solids 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 force can also be observed by comparing the antibody signal values.

The expression results are shown in Figures 1 to 6. As shown in Figure 1, C1-5 expressed optimally. As shown in Figure 2, flexible GS linker LM18, semi-flexible GSPA linker LM23, rigid PA linker LM29, and rigid PG linker LM31 were optimal. As shown in Figure 3, T4 fibritin Foldon and leucine zipper GCN4 were optimal. As shown in Figure 4, 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.

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

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

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

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

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

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

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

II: pH stability: 1 hour at pH 3.5 and pH 10.0

(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.

(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.

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

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 with PBS buffer for subsequent experiments.

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.

IV: RSV F protein Tm value determination

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.

The specific operations are as follows:

(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);

(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.

(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.

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

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

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

I: RSV F protein immunogenicity protocol (mouse)

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

(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;

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

(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.

II: RSV F protein immunogenicity testing - neutralizing antibody testing

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.

(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).

(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.

(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.

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

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

Note: The sequence of 28-control is:

III: F protein immunogenicity detection - combined antibody detection

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

(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;

(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;

(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;

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

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

(6) The cutoff value was 2.1 times the OD value of the negative control group, and 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.

As shown in Figure 9, the serum binding antibody IgG titers of some recombinant RSV F proteins shown in Table 14 were 5 weeks after immunization of animals.

IV: RSV F protein immunogenicity testing - cellular immunity testing

1. Isolation of Mouse Splenic Lymphocytes

Mouse spleen lymphocytes were isolated using mouse lymphocyte separation medium (Dakoway, China) and the operation was performed according to the instructions. After the mouse was killed by cervical dislocation, the spleen was separated 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 culture 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 stratified, the middle white membrane lymphocyte layer was aspirated, 10 mL of RPMI 1640 culture medium was added for washing, and centrifuged at 300g for 10 minutes. After discarding the supernatant, the cells were resuspended in ELISpot-specific serum-free culture medium (Dakoway, China) and counted for later use.

2. IFN-γ, IL-2, and IL-4 ELISpot Assays

Mouse splenic 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:

(1) Wash the pre-coated ELISpot 96-well plate four times with sterile PBS at a volume of 200 μL/well. Add 200 μL of ELISpot-specific serum-free medium to each well and incubate at room temperature for at least 30 minutes before discarding the medium. Dilute the stimulant with ELISpot-specific serum-free medium to the following final concentration: 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 to each well. Cell usage: 3×10 ⁵ cells/well for IFN-γ and IL-2 detection plates, and 7×10 ⁵ cells/well for IL-4 detection plates. Incubate in a 37°C 5% CO ₂ incubator for 40 hours.

(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 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 for 2 hours at room temperature. 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.

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

(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.

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

(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.

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

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

Female cotton rats aged 6-8 weeks were injected intramuscularly with 5 μg RSV F, respectively, on day 0 (one immunization, primary immunization) and day 21 immunization (two immunizations, booster immunization). Within 14-28 days after the two immunizations, 10 ⁵ -10 ⁶ PFU of RSV virus (subtype A and subtype B) were used to infect (challenge) the mice. Serum samples were collected 3 weeks after the primary immunization (W3), 3 weeks after the booster immunization (W6), and 5 days after the challenge (W6+5). Serum neutralizing antibody titers were detected using the RSV virus microcytopathic assay, and serum neutralizing antibody titration was detected using an ELISA method. In addition, lung and nasal tissues of the mice were separated and sacrificed to observe histopathological changes and detect the RSV virus load in the tissue homogenate.

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

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

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

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

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

The fifth group was the blank group.

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.

Table 15: Tissue viral load

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 (21)

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 independently comprises an amino acid replacement at one or more positions selected from 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 492 of SEQ ID NO: 1, preferably 67, 68, 87, 198, 161, 214, 460 and 463, relative to SEQ ID NO: 1, preferably, the amino acid replacement is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F) and proline (P). 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, 40 3, 409, 410, 420, 437, 443, 464, 466, 480, 484, 485, 498, 491, 492, and 493, preferably one, preferably two, 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 contain a C, Preferably, at amino acid 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 49 3; (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) 38 7 and 492; (xxiv) 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 and 485; (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. The recombinant RSV F protein of claim 1 or 2, wherein: (i) the amino acids at 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 position 161, 211, 213, 214, 217, 459, 460, 463, 470, 472, 479, 481, 485 and/or 492, preferably 161, 214, 460 and/or 463, corresponding to SEQ ID NO: 1 is P. The recombinant RSV F protein of any one of claims 1-3, comprising the following amino acids at the 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. 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 F1 polypeptide comprises amino acids 137-513 or 147-513 corresponding to SEQ ID NO: 1, optionally the F2 and F1 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 NO: 7 and 16-24), preferably a trimerization domain derived from T4 fibritin or leucine zipper GCN4, as shown in any one of SEQ ID NO: 7 and 18-19, optionally connected to the C-terminus of the F1 polypeptide, preferably connected to the F1 polypeptide via a linker sequence such as SAIG, GG or SA. 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. A method for stabilizing the prefusion conformation of a RSV F protein comprising F2 and F1 polypeptides, preferably, the F2 polypeptide comprising amino acids 26-109 or 26-105 corresponding to SEQ ID NO: 1 and/or the F1 polypeptide comprising amino acids 137-513 or 147-513 corresponding to SEQ ID NO: 1, the method comprising: introducing into the RSV F protein one or more of the following mutations: mutating the amino acids at positions 72 and 76 of SEQ ID NO: 1 to C, and The mutation is selected from the amino acids 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 492 of SEQ ID NO: 1, preferably 67, 68, 87, 98, 161, 214, 460 and 463, preferably, the amino acids at said positions are independently mutated to I, V, L, F or P. 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, 411, 428, 430, 431, 432, 443, 458, 460, 471, 480, 490, 501, 511, 520, 521, 522, 523, 524, 525 , 410, 420, 437, 443, 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, wherein one, preferably two, amino acids 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; and (xi) 389 and 49 3; (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, 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, and more preferably, the amino acid in one or more groups of 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 is mutated to C. 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 positions 161, 211, 213, 214, 217, 459, 460, 463, 470, 472, 479, 481, 485 and/or 492, preferably 161, 214, 460 and/or 463, corresponding to amino acid positions of SEQ ID NO: 1 to P. The method of any one of claims 7 to 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. 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 includes connecting 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 (for example, as shown in SEQ ID NO: 7 and 16-24), more preferably, the trimerization domain is selected from the trimerization domain derived from T4 fibritin (for example, SEQ ID NO: 7) and the trimerization domain derived from leucine zipper GCN4 (for example, SEQ ID NO: 18 or 19); optionally, the trimerization domain is connected to the C-terminus of the F1 polypeptide, preferably connected to the F1 polypeptide via a linker sequence such as SAIG, GG or SA. A nucleic acid molecule encoding the recombinant RSV F protein of any one of claims 1-6. The nucleic acid molecule of claim 12, which has been codon-optimized for expression in mammalian cells. A vector comprising the nucleic acid molecule according to claim 12 or claim 13. A pharmaceutical composition comprising 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, and optionally comprising a pharmaceutically acceptable excipient. A RSV vaccine comprising 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, and optionally an adjuvant. 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 RSV F protein. 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 the vector of claim 14 are used for preventing and/or treating RSV infection. 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 RSV F protein, preventing and/or treating RSV infection. 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. 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 a 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 an antibody-protein complex.