Mutants of rsv f protein and uses thereof

Abstract

The present invention relates to mutants of RSV F protein comprising amino acid mutations relative to the amino acid sequence of RSV wild-type F protein, including a mutation at position 176 from K to P, R, H, S, T, N, Q, V, I, L or M; and / or a mutation at position 186 from S to P, R, H, K, D, E, T, N, Q, C, A, V, I, L, M, F, Y, or W. The present invention also provides related DNA coding sequences, recombinant plasmids and mRNA sequences and corresponding RSV vaccines. The present invention significantly improves the expression and stability of F protein with pre- and post-fusion conformation and trimeric conformation by introducing specific amino acid substitutions at specific amino acid positions, in combination with cavity filling, electrostatic mutations, proline mutations, etc., and significantly inhibits the transition of F protein to post-fusion conformation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to mutants of the respiratory syncytial virus (RSV) F protein and their application in RSV vaccines and diagnostics. Background Technology

[0002] Respiratory syncytial virus (RSV) was isolated from the respiratory tract of chimpanzees in 1956. It is named for the syncytial-like structure formed by cell fusion during tissue culture. RSV belongs to the family Pneumoviridae, genus Orthopneumovirus, and is an RNA virus. Its G and F proteins are the main glycoproteins on the surface of the syncytial cell membrane, serving as important viral antigens and the primary viral antigens that stimulate the body to produce neutralizing antibodies. RSV has only one serotype, with two subtypes: A and B. RSV primarily causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months of age, and upper respiratory tract infections such as rhinitis and colds in older children and adults. Long-acting monoclonal antibodies can effectively prevent RSV infection in infants. Nicevirumab injection is currently the first and only long-acting monoclonal antibody for the prevention of all infants. It has been approved in several countries for the prevention of respiratory syncytial virus-related lower respiratory tract infections in infants before or during the first respiratory syncytial virus infection season, as well as in children under 2 years of age who are still susceptible to severe respiratory syncytial virus infection.

[0003] Currently, there are no approved safe and effective vaccines for preventing RSV infection in my country, highlighting the urgent need for RSV vaccines that can induce or provide a protective immune response.

[0004] The RSV genome is approximately 15 kb in length, containing 10 genes encoding 11 proteins, including 8 structural proteins (F, G, M2-1, M2-2, SH, N, P, L) and 3 non-structural proteins (NS1, NS2, NS3). Fusion glycoprotein (F) and attachment glycoprotein (G) are two major envelope glycoproteins. The F protein is a type I viral fusion protein that mediates viral-host cell membrane fusion, enabling the viral envelope to fuse with the host cell membrane to form multinucleated giant cells. Neutralizing antibodies against the F protein effectively inhibit RSV infection; naturally occurring RSV infection in humans primarily induces neutralizing antibodies against the F protein. The F protein's amino acid sequence exhibits over 90% conservation across RSV subtypes, demonstrating significantly higher genetic stability than the G protein. Furthermore, the F protein is a crucial target for inducing the production of CD8+ T cells. Therefore, the RSV F protein is an important target for RSV vaccine development.

[0005] The RSV F protein has a trimer structure and mediates the fusion of the viral envelope and host cell through conformational changes. Before fusion initiation, the F protein adopts a pre-fusion conformation, which is unstable and has a low energy barrier. When very close to the host, the fusion peptide inserts into the host cell membrane, allowing the F protein to cross both the viral envelope and the host cell membrane. Subsequently, the F protein forms a hairpin structure in the trimer, connecting the two membranes together and mediating viral fusion with the host cell membrane. The post-fusion conformation of the F protein is very stable. The pre-fusion and post-fusion conformations of the F protein differ significantly in structure and have different antigenic epitopes. The pre-fusion conformation of the F protein contains key neutralizing epitopes against RSV infection. When the F protein transitions to the post-fusion conformation, it loses most of these key neutralizing epitopes. Therefore, maintaining the stability of the pre-fusion trimer conformation of the F protein is crucial.

[0006] To address the structural instability of the pre-fusion conformation F protein, numerous research and development attempts have been made to increase stability by substituting amino acids at specific amino acid sites while preserving the neutralizing epitopes of the pre-F conformation. However, due to the complexity of clinical applications, research in various aspects is still far from sufficient, and it is necessary to continuously search for a stable pre-fusion RSV F peptide that is ultimately suitable for drug development. Summary of the Invention

[0007] The purpose of this invention is to provide an amino acid mutation that promotes the pre-fusion conformation of RSV F protein and its application. The mutant of RSV F protein can improve the expression level and stability of the pre-fusion conformation and pre-fusion trimer conformation of F protein.

[0008] The technical solutions for achieving the above objectives include the following.

[0009] A first aspect of the present invention is to provide a mutant of RSV F protein, said mutant comprising an amino acid mutation relative to the amino acid sequence of RSV wild-type F protein, said amino acid mutation comprising: the amino acid at position 176 being replaced by P (proline), or R (arginine), or H (histidine), or S (serine), or T (threonine), or N (asparagine), or Q (glutamine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine); and / or

[0010] The amino acid at position 186 is substituted with P (proline), or R (arginine), or H (histidine), or K (lysine), or D (aspartic acid), or E (glutamic acid), or T (threonine), or N (asparagine), or Q (glutamine), or C (cysteine), or A (alanine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine), or F (phenylalanine), or Y (tyrosine), or W (tryptophan). In some embodiments, the amino acid sequence of the RSV wild-type F protein is as shown in SEQ ID NO:1.

[0011] In some preferred embodiments, the amino acid at position 176 is replaced with proline.

[0012] In some preferred embodiments, the amino acid at position 186 is replaced by proline. In some embodiments, the mutant also introduces a disulfide bond mutation selected from at least one of the following: S155C-S290C, S466C-S443C, T103C-I148C, S55C-L188C, D392C-S491C, D392C-S493C, or an amino acid near the above mutation site is replaced by cysteine.

[0013] In some embodiments, the mutant also introduces a cavity-filling mutation and / or an electrostatic mutation, the cavity-filling mutation being selected from at least one of the following: T54H, N67I, K80M, K87Y, K87M, S190F, and V207L.

[0014] In some preferred embodiments, the mutation comprises the substitution of proline for the amino acid at position 176, and comprises any one of the following cavity-filling mutations: K80M, K87Y, or K87M. In some preferred embodiments, the mutation comprises the substitution of proline for the amino acid at position 186, and comprises any one of the following cavity-filling mutations: K80M, K87Y, or K87M.

[0015] In some embodiments, the mutant also introduces an electrostatic mutation, which is D486S.

[0016] In some embodiments, the mutant also introduces a linker mutation between F1 and F2, wherein the linker is at least one of the following: replacing the amino acid sequence from position 109 to position 135 with GGSSG between amino acids 108 and 136.

[0017] Replace the amino acid sequence from position 104 to 144 with GS or E between amino acid positions 103 and 145.

[0018] The amino acid sequence from position 109 to position 135 was replaced with GGSSG between amino acids 108 and 136, and amino acid 106 was replaced with Q.

[0019] In some embodiments, a proline mutation is also introduced, the proline mutation being selected from at least one of the following: E161P, N175P, Q210P, S211P, I214P, S215P.

[0020] In some embodiments, the mutant is a combination of the various mutations or substitutions described above, with preferred combinations including any of the following: substitution of the lysine residue at position 176 and the disulfide bond mutation.

[0021] The combination of lysine substitution at position 176 and the linker mutation,

[0022] The combination of lysine substitution at position 176 and the cavity-filling mutation, or the combination of lysine substitution at position 176, the disulfide bond mutation, and the cavity-filling mutation.

[0023] A combination of lysine substitution at position 176, disulfide bond mutation, and electrostatic mutation.

[0024] In some embodiments, the mutant is a combination of the various mutations or substitutions described above, with preferred combinations including any of the following: substitution of serine at position 186 and the disulfide bond mutation.

[0025] The combination of serine substitution at position 186 and the linker mutation,

[0026] The combination of serine substitution at position 186 and the cavity-filling mutation,

[0027] The combination of serine substitution at position 186, the disulfide bond mutation, and the cavity-filling mutation,

[0028] A combination of the substitution of serine at position 186, the disulfide bond mutation, and the electrostatic mutation.

[0029] In some embodiments, the cavity-filling mutation is preferably S190F and / or V207L; and / or the disulfide bond mutation is selected from S155C-S290C, or S466C-S443C.

[0030] In some embodiments, the cavity-filling mutation is preferably K87M or T54H; and / or the disulfide bond mutation is selected from T103C-I148C or D392C-S493C.

[0031] In some implementations, the mutation includes D392C-S493C and / or K176P.

[0032] In some implementations, the mutation includes D392C-S493C and / or K176S.

[0033] In some implementations, the mutation includes D392C-S493C and / or K176T.

[0034] In some implementations, the mutation includes D392C-S493C and / or K176Q.

[0035] In some implementations, the mutation includes D392C-S493C and / or K176V.

[0036] In some implementations, the mutation includes D392C-S493C and / or K176I.

[0037] In some implementations, the mutation includes D392C-S493C and / or K176L.

[0038] In some implementations, the mutation includes D392C-S493C and / or K176M.

[0039] In some implementations, the mutation includes D392C-S493C and / or K186P.

[0040] In some implementations, the mutation includes D392C-S493C and / or K186H.

[0041] In some implementations, the mutation includes D392C-S493C and / or K186K.

[0042] In some implementations, the mutations include D392C-S493C and / or K186D.

[0043] In some implementations, the mutations include D392C-S493C and / or K186E.

[0044] In some implementations, the mutation includes D392C-S493C and / or K186T.

[0045] In some implementations, the mutation includes D392C-S493C and / or K186N.

[0046] In some implementations, the mutations include D392C-S493C and / or K186Q.

[0047] In some implementations, the mutation includes D392C-S493C and / or K186I.

[0048] In some implementations, the mutation includes D392C-S493C and / or K186L.

[0049] In some implementations, the mutation includes D392C-S493C and / or K186M.

[0050] In some implementations, the mutations include S155C-S290C, S190F-V207L, and / or K176P.

[0051] In some embodiments, the mutations include S155C-S290C, S190F-V207L, and / or S186P.

[0052] In some implementations, the mutations include S466C-S443C, S190F-V207L, and / or K176P.

[0053] In some embodiments, the mutations include S466C-S443C, S190F-V207L, and / or S186P.

[0054] In some embodiments, the mutations include T103C-I148C, S190I, D486S, and / or K176P.

[0055] In some embodiments, the mutations include T103C-I148C, S190I, D486S, and / or S186P.

[0056] In some embodiments, the mutations include D392C-S491C, S190F-V207L, and / or K176P.

[0057] In some embodiments, the mutations include D392C-S491C, S190F-V207L, and / or S186P.

[0058] In some implementations, the mutations include D392C-S493C, K87M, and / or K176P.

[0059] In some implementations, the mutations include D392C-S493C, K87M, and / or S186P.

[0060] In some embodiments, the RSV wild-type F protein is derived from an RSV strain, which is either an A subtype strain or a B subtype strain.

[0061] In some embodiments, the subtype A strains include, but are not limited to, RSV A2, RSV Long, RSV2, RSV ON1 and / or RSV 19; and the subtype B strains include, but are not limited to, RSV 18537, RSV B1, RSV BA9 and / or RSV 9320.

[0062] A second aspect of the present invention is to provide a nucleic acid sequence comprising a mutant of the RSV F protein described in any of the preceding claims; the nucleic acid sequence is a DNA sequence or an RNA sequence; further, the RNA is mRNA.

[0063] A third aspect of the present invention is to provide a recombinant plasmid or vector having inserted a coding sequence encoding any of the mutants described above.

[0064] A fourth aspect of the invention is to provide an mRNA sequence encoding the mutant. This sequence can be obtained by enzyme digestion and transcription of a recombinant plasmid containing a coding sequence encoding any of the mutants described above.

[0065] A fifth aspect of the present invention is the use of the above-mentioned nucleic acid sequence, the above-mentioned DNA coding sequence, the above-mentioned mRNA sequence, or the above-mentioned recombinant plasmid, or the above-mentioned vector, or any of the above-mentioned RSV F protein mutants in the preparation of RSV vaccines, or in improving the expression and / or stability of RSV pre-fusion (Pre-F) conformational protein or RSV pre-fusion trimer conformational protein.

[0066] A sixth aspect of the present invention is to provide a respiratory fusion virus (RSV) vaccine comprising at least one nucleic acid having an open reading frame encoding a mutant or immunogenic fragment of any of the aforementioned RSV F proteins, and a pharmaceutically acceptable vector.

[0067] A seventh aspect of the present invention is to provide the use of any of the above-mentioned RSV F protein mutants in the detection of respiratory syncytial virus infection.

[0068] An eighth aspect of the present invention provides a method for stabilizing the pre-fusion conformation of an RSV F protein by introducing a coding sequence encoding any of the mutants described above into the RSV wild-type F protein.

[0069] A ninth aspect of the present invention provides a lipid nanoparticle comprising any of the aforementioned RSV F protein mutants, nucleic acids, plasmids or vectors, and a nanolipid carrier.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] 1. By introducing one or more amino acid substitutions at specific positions (including substitution of amino acid at position 176 with proline, arginine, histidine, serine, threonine, or asparagine), glutamine, valine, isoleucine, leucine, or methionine, and / or substitution of amino acid at position 186 with proline, arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan), the expression levels and stability of the pre-fusion conformation and trimer conformation of F protein can be significantly improved; furthermore, while significantly improving the expression levels and stability of the pre-fusion conformation and trimer conformation of F protein, the conversion of F protein to the post-fusion conformation can also be significantly inhibited.

[0072] 2. Based on the amino acid substitution at the specific positions mentioned above, this invention further combines with disulfide bonds, cavity filling, and / or electrostatic mutations to significantly improve the expression level and stability of the pre-fusion conformation and pre-fusion trimer conformation of F protein; furthermore, it can significantly improve the expression level and stability of the pre-fusion conformation and pre-fusion trimer conformation of F protein while significantly inhibiting the transformation of F protein to the post-fusion conformation. Attached Figure Description

[0073] Figure 1 pNeoCura-Bvac plasmid map.

[0074] Figure 2 In vitro expression of mRNA encoding RSV wild-type F protein and single-site mutant RSV F protein.

[0075] Figure 3 : In vitro expression of RSV F protein mutant mRNA containing a combination of mutations at position 176 or 186 and disulfide bond mutations.

[0076] Figure 4 : In vitro expression of RSV F protein mutant mRNA containing 176 or 186 site mutations combined with the linker.

[0077] Figure 5 In vitro expression of RSV F protein mutant mRNA encoding a combination of mutations at K176P or S186P sites and a stable Pre-F conformation. Detailed Implementation

[0078] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0079] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0080] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0081] To facilitate understanding of this technology, some terms and phrases are defined below.

[0082] RSV stands for Respiratory Syncytial Virus. RSV F protein refers to the fusion protein of RSV.

[0083] Pre-F (pre-fusion) is the pre-fusion conformation of the F protein. Post-F (post-fusion) is the post-fusion conformation of the F protein.

[0084] As used herein, the term "wild-type" refers to any naturally occurring RSV strain, including those isolated from natural sources such as mammalian subjects. Exemplary wild-type RSV strain subtypes include, but are not limited to, RSV subtypes A and B. Exemplary subtype A strains include, but are not limited to, RSV A2, RSV Long, RSV S2, RSV ON1, and / or RSV19; subtype B strains include, but are not limited to, RSV 18537, RSV B1, RSV BA9, and / or RSV 9320.

[0085] A disulfide bond is a chemical bond that connects the thiol groups of two different cysteine ​​residues in different peptide chains or in the same peptide chain.

[0086] (1) Disulfide bond mutation: As shown in Table 1, D392C and S493C undergo mutation, causing them to form a disulfide bond.

[0087] (2) Cavity filling: The conformational grooves were modified (as shown in Table 1, S190F, V207L) to fill them, as shown in RF-KW5~RF-KW34.

[0088] (3) Electrostatic mutation: altering the charge distribution on the surface of antibody molecules to optimize their interaction with antigens or other molecules. Such mutations typically involve replacing neutral amino acids with charged amino acids (such as lysine, glutamic acid, etc.) to enhance or adjust electrostatic interactions. For example, in this invention, aspartic acid (D) is replaced with serine (S).

[0089] In this article, specific binding refers to an antibody binding to a target molecule with a higher affinity than it binds to other substances being tested. For example, an antibody that specifically binds to RSV F protein in its pre-fusion conformation binds to RSV F protein in its pre-fusion conformation with a higher affinity than it binds to RSV F protein in its post-fusion conformation.

[0090] In this document, "vaccine" refers to a pharmaceutical composition containing an immunogen capable of evoking a preventive or therapeutic immune response in an individual. Typically, vaccines evoke a specific immune response against pathogens, such as viral pathogens like the respiratory syncytial virus antigen of this invention, for the prevention and treatment of diseases caused by respiratory syncytial virus.

[0091] Coding sequence: The DNA sequence in a gene that encodes a protein. It contains the genetic information that guides protein synthesis and is usually composed of three nucleotides per codon, with each codon corresponding to one amino acid. The coding sequences in this article are the DNA sequences encoding the mutant and wild-type F proteins of the corresponding RSV fusion preconformation F protein.

[0092] In accordance with conventional representation in the art, the amino acids involved in the mutant are represented in different ways as follows.

[0093]

[0094] This invention significantly enhances the expression of both the pre-fusion and pre-fusion trimer conformations of the F protein by introducing point mutations at specific amino acid sites into the F protein sequence of respiratory syncytial virus (RSV). This pre-fusion F protein can form a stable trimer and possesses a stable trimer conformation. Using the mutant F protein as an immunogen or vaccine can significantly increase the level of neutralizing antibodies induced against RSV.

[0095] RSV virus has two antigenic subtypes, A and B, mainly differing in their G glycoproteins, while the F protein sequence is more conserved between the two subtypes. Furthermore, the mature processed F proteins of the two subtypes share approximately 93% amino acid sequence identity. In almost all identified natural RSV F0 precursor proteins, the furin cleavage site falls at the same amino acid position. Therefore, in this embodiment of the invention, the F protein sequence of RSV A2 strain is used as the base sequence for mutant mutations. However, the base sequence for mutant mutations in this invention is not limited to RSV A2 strain, but also includes other RSV types, such as other type A strains and clinically isolated strains, and other type B strains and clinically isolated strains. Those skilled in the art will readily understand that the concept of this invention can be applied to other RSV types, and it is expected that these mutations can significantly improve the expression level and stability of the pre-fusion conformation and pre-fusion trimer conformation of the F protein.

[0096] As used herein, amino acid positions refer to the sequence (SEQ ID NO:1) of the RSV F protein (F protein or a mutant of the F protein) from the A2 virus strain. As used herein, the phrase "amino acid at position "x" of the RSV F protein thus refers to the amino acid corresponding to position "x" in the RSV F protein of the RSV A2 virus strain of SEQ ID NO:1. Note that in the sequence numbering system used herein, 1 refers to the N-terminal amino acid of an immature F protein (corresponding to the first amino acid of SEQ ID NO:1). When an RSV virus strain other than the A2 virus strain is used, the amino acid positions of the F protein will be numbered by inserting gaps where necessary to align the sequences of other RSV virus strains with the F protein of SEQ ID NO:1, referring to the numbering of the F protein of the A2 virus strain of SEQ ID NO:1. Those skilled in the art can readily compare amino acid positions between different native RSV F sequences to identify corresponding RSV F amino acid positions between different RSV strains and subtypes. Sequence alignment can be performed using methods well known in the art, such as CLUSTALW, Bioedit, or CLC workbenches.

[0097] The following are examples of the amino acid and nucleic acid sequences of some of the mutated F proteins. For other amino acid and nucleic acid sequences not given, you can obtain the corresponding amino acid and nucleic acid sequences by referring to the mutation positions given in Table 1, the wild-type sequences, and the sequences below, using conventional techniques.

[0098] The present invention will be further described in detail below with reference to specific embodiments.

[0099] Example 1: Preparation of plasmid templates encoding RSV wild-type F protein and single-site F protein mutant.

[0100] Based on the wild-type F protein sequence of RSV A2 strain (WT, as shown in SEQ ID NO:1, and the wild-type F protein in the following examples is also the same), the codon-optimized DNA sequence encoding the wild-type F protein sequence (as shown in SEQ ID NO:2) was recombined into the Bam H Ⅰ and Sac Ⅰ restriction sites of the pNeoCura-Bvac plasmid vector through gene synthesis to obtain the recombinant plasmid pNeoCura-Bvac-WT, which serves as a plasmid template for preparing the mRNA encoding the wild-type F protein (mWT, as shown in SEQ ID NO:3).

[0101] SEQ ID NO: 3

[0102]

[0103] Based on the amino acid sequence corresponding sites of the mutants shown in Table 1, point mutations were performed on the recombinant plasmid pNeoCura-Bvac-WT to obtain the corresponding recombinant plasmid.

[0104] Table 1:

[0105]

[0106]

[0107]

[0108] Example 2: Preparation and expression detection of mRNA encoding RSV wild-type F protein and single-site F protein mutant.

[0109] 2.1 Linearization Template Preparation

[0110] The recombinant plasmids described in Example 1 were digested with restriction endonuclease BspQ I (NEB, catalog number: R0712L). The linearized plasmids after digestion were purified using VAHTS DNA Clean Beads (Novizan, N411-01) to obtain purified linearized plasmids for in vitro transcription.

[0111] 2.2 In vitro transcription to prepare mRNA

[0112] Following the instructions of the T7 High Yield RNA Transcription Kit (N1-Me-PseudoUTP) (Novizan, DD4202), mRNA was obtained by in vitro transcription using the linearized plasmid prepared in section 2.1 as a template. The mRNA was then purified using VAHTS RNA Clean Beads (Vazyme, N412-01) according to the manufacturer's instructions.

[0113] 2.3 Capping and Purification

[0114] The purified mRNA was capped using the mRNA Cap 2'-O-Methyltransferase (Novizan, DD4110) capping kit. The capped mRNA was then purified using VAHTS RNA Clean Beads (Vazyme, catalog number: N412-01) to obtain the mRNA with the corresponding sequence.

[0115] 2.4 In vitro expression detection of RSV wild-type F protein and single-site F protein mutant mRNA

[0116] 2.4.1 A549 cell plating

[0117] A549 cells were seeded in 24-well plates (5 × 10⁻⁶ cells per well). 5 / well), and cultured in DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C and 5% CO2 for 18 hours until the cell density reached 80% confluence before transfection.

[0118] 2.4.2 mRNA transfection of A549 cells

[0119] The cell culture medium was replaced with serum-free Opti-MEM medium (Thermo Fisher Scientific), and mRNA (0.3 μg, 1 μg RNA / well) was mixed with Lipofectamine™ Messenger MAX™ transfection reagent (Thermo Fisher Scientific) before transfection. Six hours later, the cell culture medium was replaced with DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% penicillin and streptomycin, and the cells were cultured for another 24 hours.

[0120] 2.4.3 Flow cytometry detection of F protein expression on the surface of HEK293T cells

[0121] After culturing transfected cells for 24 hours, the cells were centrifuged at 1000g for 10 minutes, collected, and incubated with fluorescently labeled monoclonal antibodies. Flow cytometry (BD, FACS Celesta) was used to detect the mean fluorescence intensity (MFI) and cell percentage of F protein on the cell surface bound to monoclonal antibodies D25, AM14, or 4D7.

[0122] The monoclonal antibody D25 in this invention (D25 was prepared using the method disclosed in McLellan, JS, Chen, M., Leung, S., Graepel, KW, Du, X., Yang, Y., Zhou, T., Baxa, U., Yasuda, E., Beaumont, T., Kumar, A., Modjarrad, K., Zheng, Z., Zhao, M., Xia, N., Kwong, PD, & Graham, BS (2013). Structure of RSV fusion glycoprotein trimer bound to aprefusion-specific neutralizing antibody. Science (New York, NY), 340(6136), 1113–1117. https: / / doi.org / 10.1126 / science.1234914) represents the expression level of F protein at the prefusion conformational epitope, characterized by the mean fluorescence intensity (MFI) of D25 binding to cell surface F protein.

[0123] The monoclonal antibody AM14 in this invention: (AM14 was prepared using the method disclosed in Gilman, MS, Moin, SM, Mas, V., Chen, M., Patel, NK, Kramer, K., Zhu, Q., Kabeche, SC, Kumar, A., Palomo, C., Beaumont, T., Baxa, U., Ulbrandt, ND, Melero, JA, Graham, BS, & McLellan, JS (2015). Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSVFusion Glycoprotein. PLoS pathogens, 11(7), e1005035. https: / / doi.org / 10.1371 / journal.ppat.1005035). The mean fluorescence intensity (MFI) of AM14 binding to cell surface F protein characterizes the expression level of F protein at the pre-fusion trimeric conformation epitope.

[0124] The monoclonal antibody 4D7 in this invention was prepared using the method disclosed in Zhang, L., Durr, E., Galli, JD, Cosmi, S., Cejas, PJ, Luo, B., Touch, S., Parmet, P., Fridman, A., Espeseth, AS, & Bett, AJ (2018). Design and characterization of a fusion glycoprotein vaccine for Respiratory Syncytial Virus with improved stability. Vaccine, 36(52), 8119–8130. https: / / doi.org / 10.1016 / j.vaccine.2018.10.032. The mean fluorescence intensity (MFI) of 4D7 binding to the cell surface F protein characterizes the expression level of the F protein at the post-fusion conformational epitope.

[0125] 2.5 Test Results

[0126] like Figure 2 As shown in Figure A, compared with wild-type F protein (WT), mutations at position 176 of amino acid K (lysine) to P (proline), S (serine), T (threonine), N (asparagine), Q (glutamine), V (valine), I (isoleucine), L (leucine), or M (methionine) all increased the mean fluorescence intensity (MFI) of cells binding to D25, indicating that the above-mentioned amino acid point mutations at position 176 can effectively increase the expression level of the pre-fusion (Pre-F) conformation of F protein.

[0127] like Figure 2 As shown in B: Compared with wild-type F protein (WT), mutations at position 176 of amino acid K (lysine) to P (proline), R (arginine), or H (histidine), or S (serine), or T (threonine), or N (asparagine), or Q (glutamine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine) all increased the mean fluorescence intensity (MFI) of cells binding to AM14. Figure 2 A and Figure 2 (D) indicates that the above-mentioned amino acid point mutation at position 176 can effectively increase the expression level of the pre-F trimer conformation of the F protein.

[0128] like Figure 2 As shown in C: Compared with wild-type F protein (WT), the mutation of amino acid K (lysine) at position 176 to any amino acid also reduced the mean fluorescence intensity (MFI) of cells binding to 4D7, indicating that point mutation of any amino acid at position 176 effectively reduced the expression level of the post-F trimeric conformation of F protein.

[0129] Figure 2 The AC results indicate that mutants with mutations at position 176, particularly RSVF-K1, K2, K3, K6, K7, K8, K9, K13, K14, K15, and K16, effectively increase the expression level of the pre-F conformation or trimer conformation of the F protein, and effectively decrease the expression level of the post-F trimer conformation of the F protein.

[0130] like Figure 2As shown in D: Compared with wild-type F protein (WT), mutations at position 186, such as S (serine) to P (proline), H (histidine), K (lysine), D (aspartic acid), E (glutamic acid), T (threonine), N (asparagine), Q (glutamine), C (cysteine), A (alanine), V (valine), I (isoleucine), L (leucine), M (methionine), F (phenylalanine), Y (tyrosine), or W (tryptophan), all increased the mean fluorescence intensity (MFI) of cells binding to antibody D25. This indicates that point mutations at position 186 can effectively increase the expression level of the pre-fusion (Pre-F) conformation of F protein.

[0131] like Figure 2 As shown in E, compared with wild-type F protein (WT), mutations at position 186 of the amino acid S (serine) to P (proline), R (arginine), H (histidine), K (lysine), D (aspartic acid), E (glutamic acid), T (threonine), N (asparagine), Q (glutamine), C (cysteine), A (alanine), V (valine), I (isoleucine), L (leucine), M (methionine), F (phenylalanine), Y (tyrosine), or W (tryptophan) all increased the mean fluorescence intensity (MFI) of cells binding to antibody AM14. This indicates that the above-mentioned point mutations at position 186 can effectively increase the expression level of the pre-fusion (Pre-F) trimer conformation of F protein.

[0132] like Figure 2 As shown in F: Compared with wild-type F protein (WT), the mutation of amino acid S (serine) at position 186 to any native amino acid did not significantly increase the mean fluorescence intensity (MFI) of cells binding to 4D7.

[0133] Figure 2 Overall, compared to wild-type F protein (WT), the DF mutation at position 186, except for the (S186G)S11 mutant, effectively increased the expression levels of the pre-fusion (Pre-F) conformation or trimer conformation of F protein, but did not significantly increase the expression level of the post-fusion (Post-F) trimer conformation. Although the mutation of the amino acid at position 186 from S (serine) to P (proline) slightly increased the mean fluorescence intensity (MFI) of cell binding to antibody 4D7 by approximately 22%, it also increased the mean fluorescence intensity (MFI) of cell binding to antibody D25 by approximately 64% and the mean fluorescence intensity (MFI) of cell binding to antibody AM14 by approximately 91%, indicating that the mutation of the amino acid at position 186 from S (serine) to P (proline) more significantly increased the expression levels of both the pre-fusion (Pre-F) and pre-fusion trimer conformations.

[0134] Figure 2 The DF indicates that mutants with mutations at position 186, except for the S11 mutant, can effectively increase the expression levels of the pre-F conformation or trimer conformation of the F protein.

[0135] Example 3: Preparation of plasmid templates encoding F protein mutants containing a combination of mutations at 176 or 186 sites and disulfide bond mutations.

[0136] The pre-fusion conformation of the F protein was stabilized by introducing disulfide bonds at specific sites. F protein mutants were constructed by combining natural amino acid substitutions that enhance the Pre-F and Pre-F trimers with disulfide bond mutations (D392C-S493C) using single-point mutations at positions 176 or 186, as shown in Table 2.

[0137] Based on the amino acid sequence corresponding sites of the mutants shown in Table 2, point mutations and sequence substitutions were performed on the recombinant plasmid pNeoCura-Bvac-WT to obtain the corresponding recombinant plasmids.

[0138] Table 2

[0139]

[0140]

[0141] Example 4: Preparation and in vitro expression detection of F protein mutant mRNA encoding a combination of mutations at or above the 176 or 186 site and disulfide bond mutations.

[0142] 4.1 Test Methods

[0143] Same as in Example 2, transfect the mRNA corresponding to the protein mutants shown in Table 2.

[0144] 4.2 Test Results

[0145] like Figure 3 As shown in Figure A: Compared with wild-type F protein (WT) and F protein mutants containing only disulfide bond mutations (D392C-S493C), mutations of the amino acid at position 176 from K (lysine) to P (proline), S (serine), T (threonine), Q (glutamine), V (valine), I (isoleucine), L (leucine), or M (methionine) all increased the mean fluorescence intensity (MFI) of cells binding to D25. This indicates that the above-mentioned amino acid point mutations at position 176 can effectively increase the expression level of the pre-fusion (Pre-F) conformation of F protein.

[0146] like Figure 3As shown in B, compared with wild-type F protein (WT) and F protein mutants containing only disulfide bond mutations (D392C-S493C), mutations of amino acid at position 176 from K (lysine) to P (proline), S (serine), T (threonine), Q (glutamine), V (valine), I (isoleucine), L (leucine), or M (methionine) significantly increased the mean fluorescence intensity (MFI) of cells binding to AM14. This indicates that the above-mentioned amino acid point mutations at position 176 can effectively increase the expression level of the pre-fusion (Pre-F) trimer conformation of F protein.

[0147] like Figure 3 As shown in C: Compared with wild-type F protein (WT), the disulfide bond mutation (D392C-S493C) and the mutation of the amino acid at position 176 from K (lysine) to P (proline), or S (serine), or T (threonine), or Q (glutamine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine) all significantly reduced the mean fluorescence intensity (MFI) of cells binding to 4D7, indicating that the above-mentioned amino acid point mutations at position 176 significantly reduced the expression level of the post-F trimer conformation of F protein.

[0148] like Figure 3 As shown in D: Compared with wild-type F protein, mutations at position 186, such as S (serine) to P (proline), H (histidine), K (lysine), D (aspartic acid), E (glutamic acid), T (threonine), N (asparagine), Q (glutamine), I (isoleucine), L (leucine), or M (methionine), significantly increased the mean fluorescence intensity (MFI) of cells binding to antibody D25. This indicates that point mutations at position 186 can effectively increase the expression level of the pre-fusion (Pre-F) conformation of F protein.

[0149] like Figure 3 As shown in E, compared with wild-type F protein, mutations at position 186, such as S (serine) to P (proline), H (histidine), K (lysine), D (aspartic acid), E (glutamic acid), T (threonine), N (asparagine), Q (glutamine), I (isoleucine), or M (methionine), significantly increased the mean fluorescence intensity (MFI) of cell binding to antibody D25. This indicates that the above-mentioned point mutations at position 186 effectively improve the expression level of the pre-fusion (Pre-F) trimer conformation of F protein.

[0150] like Figure 3As shown in Figure F: Compared with the wild-type F protein, mutations at position 186 of the amino acid S (serine) to P (proline), H (histidine), K (lysine), D (aspartic acid), E (glutamic acid), T (threonine), N (asparagine), Q (glutamine), I (isoleucine), L (leucine), or M (methionine) significantly reduced the mean fluorescence intensity (MFI) of the cell binding to 4D7. This indicates that the above-mentioned point mutations at position 186 significantly and effectively reduced the expression level of the post-fusion (Post-F) trimer conformation of the F protein.

[0151] Example 5: Preparation of plasmid templates encoding F protein mutants containing 176 or 186 site mutations combined with Linker.

[0152] One known strategy for improving the pre-fusion conformation of the F protein is to add a linker between the F1-F2 domains to replace or stabilize the fusion peptide at its stationary position, preventing its release from the head region. Therefore, we introduced different linker sequences into different regions of F1-F2, as shown in Table 3.

[0153] In Example 1, it was found that substitution of P (proline) at position 176 had the best effect on stabilizing the expression of Pre-F and Pre-F trimer conformation proteins. Substitution of P (proline) at position 186 improved the expression of Pre-F and Pre-F trimer conformation proteins and also improved the expression of Post-F conformation proteins. Therefore, substitution of P (proline) at position 176 or P (proline) at position 186 was used in combination with the linker to construct F protein mutants, as shown in Table 3.

[0154] Based on the amino acid sequence corresponding sites of the mutants shown in Table 3, point mutations and sequence substitutions were performed on the recombinant plasmid pNeoCura-Bvac-WT to obtain the corresponding recombinant plasmid.

[0155] Δ109R-135R, GGSSG, R106Q: The amino acid sequence GGSSG replaces amino acid positions 109 to 135 of the wild-type F protein sequence (WT, as shown in SEQ ID NO:1) of the RSV A2 strain, and amino acid position 106 is replaced with Q.

[0156] Δ104N-144V, GS: The amino acid sequence GS replaces positions 104 to 144 of the wild-type F protein sequence (WT, as shown in SEQ ID NO:1) of the RSV A2 strain.

[0157] Δ108R-144V, E: Replace amino acid E with the amino acid sequence from position 104 to position 144 of the wild-type F protein sequence (WT, as shown in SEQ ID NO:1) of the RSV A2 strain.

[0158] Table 3

[0159]

[0160] Example 6: Preparation and in vitro expression detection of F protein mutant mRNA encoding a combination of mutations at site 176 or 186 and a linker.

[0161] 6.1 Test Methods

[0162] Same as in Example 2, transfect the mRNA corresponding to the protein mutants shown in Table 3.

[0163] 6.2 Test Results

[0164] like Figure 4 As shown in Figure A: Compared to the wild-type F protein (WT), cells transfected with F protein mutant mRNA containing Linker1 (Lin1) or Linker2 (Lin2) combined with K176P or S186P, respectively, showed a decrease in mean fluorescence intensity (MFI) binding to D25, indicating that these mutations reduced the expression level of the pre-F conformational protein before fusion. Conversely, cells transfected with F protein mutant mRNA containing Linker3 (Lin3) combined with K176P or S186P, respectively, showed an increase in mean fluorescence intensity (MFI) binding to D25, indicating that these mutations promoted an increase in the expression level of the pre-F conformational protein before fusion. Compared to mutants containing only Linker2 or Linker3, the K176P mutation promoted an increase in the expression level of the pre-F conformational protein before fusion.

[0165] like Figure 4 As shown in Figure B: Compared to the wild-type F protein (WT), cells transfected with F protein mutant mRNA containing Linker1 or Linker2 combined with K176P or S186P showed a decrease in mean fluorescence intensity (MFI) binding to AM14, indicating that these mutations reduced the expression level of the pre-fusion Pre-F trimer conformation protein. Conversely, cells transfected with F protein mutant mRNA containing Linker3 combined with K176P or S186P showed an increase in mean fluorescence intensity (MFI) binding to AM14, indicating that it promoted an increase in the expression level of the pre-fusion Pre-F trimer conformation protein. Compared to mutants containing only Linker2 or Linker3, the K176P mutation promoted an increase in the expression level of the pre-fusion Pre-F trimer conformation protein.

[0166] like Figure 4As shown in Figure C: Compared to the wild-type F protein (WT), cells transfected with F protein mutant mRNA containing Linker1 combined with K176P or S186P showed a decrease in mean fluorescence intensity (MFI) binding to 4D7, indicating that these mutations reduced the expression level of the Post-F trimer conformation protein after fusion. Conversely, cells transfected with F protein mutant mRNA containing Linker2 or Linker3 combined with K176P or S186P showed an increase in mean fluorescence intensity (MFI) binding to 4D7, indicating that these mutations promoted an increase in the expression level of the Post-F trimer conformation protein after fusion. Compared to mutants containing only Linker2 or Linker3, the K176P or S186P mutations promoted an increase in the expression level of the Post-F trimer conformation protein after fusion.

[0167] Example 7: Preparation of plasmid templates for F protein mutants encoding combinations of mutations at K176P or S186P sites with other stable Pre-F conformations.

[0168] In addition to adding a linker between the F1-F2 domains to improve the pre-fusion conformation of the F protein, we can also introduce cavity-filling mutations, electrostatic mutations, disulfide bonds, or combinations of the above mutation strategies at specific sites. Therefore, we added K176P or S186P site mutations to stabilize the pre-fusion conformation of the F protein, as shown in Table 4.

[0169] Based on the amino acid sequence corresponding sites of the mutants shown in Table 4, point mutations and sequence substitutions were performed on the recombinant plasmid pNeoCura-Bvac-WT to obtain the corresponding recombinant plasmid.

[0170] Table 4

[0171]

[0172]

[0173] 7.1 Test Methods

[0174] Same as in Example 2, transfect the mRNA corresponding to the protein mutants shown in Table 4.

[0175] 7.2 Test Results

[0176] like Figure 5As shown in Figure A: Compared with wild-type F protein (WT), cells transfected with F protein mutant mRNA containing K176P or S186P and a stable Pre-F conformation showed increased mean fluorescence intensity (MFI) binding to D25, indicating increased expression of the pre-F conformation protein before fusion. Compared with no addition of K176P or S186P mutation, the addition of K176P or S186P further promoted the increase in expression of the pre-F conformation protein before fusion.

[0177] like Figure 5 As shown in B: Compared with wild-type F protein (WT), cells transfected with F protein mutant mRNA containing K176P or S186P in combination with a stable Pre-F conformation showed increased mean fluorescence intensity (MFI) binding to AM14, indicating increased expression of K176P or S186P Pre-F trimer protein. Compared with no addition of K176P or S186P mutation, the addition of K176P or S186P further promoted the increase in expression of the Pre-F trimer conformation protein before fusion.

[0178] like Figure 5 As shown in C: Compared with wild-type F protein (WT), the mean fluorescence intensity (MFI) of F protein mutant mRNA transfected with K176P or S186P combined with a stable Pre-F conformation was reduced, indicating that the expression level of Post-F trimer conformation protein increased after fusion. Compared with not adding K176P or S186P mutation, the addition of K176P or S186P further reduced the expression level of Post-F trimer conformation protein after fusion.

[0179] In summary, the amino acid mutations designed in this invention at positions 176 and 186 include: at position 176, the amino acid is mutated from K (lysine) to P (proline), or R (arginine), or H (histidine), or S (serine), or T (threonine), or N (asparagine), or Q (glutamine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine); at position 186, the amino acid is mutated from S (serine) to P (proline) or R (arginine). The following ingredients, or H (histidine), or K (lysine), or D (aspartic acid), or E (glutamic acid), or T (threonine), or N (asparagine), or Q (glutamine), or C (cysteine), or A (alanine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine), or F (phenylalanine), or Y (tyrosine), or W (tryptophan), can significantly improve the expression level and stability of the pre-fusion conformation and pre-fusion trimer conformation of protein F.

[0180] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A mutant of the RSV F protein, characterized in that, The mutant contains amino acid mutations relative to the RSV wild-type F protein, including: the amino acid at position 176 being replaced by P (proline), or R (arginine), or H (histidine), or S (serine), or T (threonine), or N (asparagine), or Q (glutamine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine); and / or The amino acid at position 186 is replaced by P (proline), or R (arginine), or H (histidine), or K (lysine), or D (aspartic acid), or E (glutamic acid), or T (threonine), or N (asparagine), or Q (glutamine), or C (cysteine), or A (alanine), or V (valine), or I (isoleucine), or L (leucine), or M (methionine), or F (phenylalanine), or Y (tyrosine), or W (tryptophan).

2. The mutant according to claim 1, characterized in that, The RSV wild-type F protein is derived from RSV type A or type B strains.

3. The mutant according to claim 1, characterized in that, The amino acid at position 176 is replaced by proline; and / or the amino acid at position 186 is replaced by proline.

4. The mutant according to any one of claims 1-3, characterized in that, The mutant also introduces at least one type of mutation from the following A, B, C, and D: A. Disulfide bond mutation: The disulfide bond mutation is selected from at least one of the following: S155C-S290C, S466C-S443C, T103C-I148C, S55C-L188C, D392C-S491C, D392C-S493C, or the disulfide bond mutation is that an amino acid near the above mutation site is replaced by a cysteine. B. Cavity filling mutation: The cavity filling mutation is selected from at least one of the following: T54H, N67I, K80M, K87Y, K87M, S190F and V207L; C. The mutant also introduces an electrostatic mutation: the electrostatic mutation is D486S; D. Proline mutation: The proline mutation is selected from at least one of the following: E161P, N175P, Q210P, S211P, I214P, S215P.

5. The mutant according to any one of claims 1-4, characterized in that, The mutant also introduces a linker mutation between F1 and F2, the linker mutation being derived from at least one of the following: Replace the amino acid sequence from position 109 to 135 with GGSSG between amino acids 108 and 136. Replace the amino acid sequence from position 104 to 144 with GS or E between amino acid positions 103 and 145. The amino acid sequence from position 109 to position 135 was replaced with GGSSG between amino acids 108 and 136, and amino acid 106 was replaced with Q.

6. The mutant according to claim 1, characterized in that, The mutant has any of the following combinations of mutations: The combination of the substitution of the lysine at position 176 as described in any one of claims 1-3 and the disulfide bond mutation as described in claim 4; The combination of the substitution of the lysine at position 176 as described in any one of claims 1-3 and the linker mutation as described in claim 5; The combination of the substitution of lysine at position 176 as described in any one of claims 1-3 and the cavity-filling mutation as described in claim 4; The substitution of the lysine at position 176 as described in any one of claims 1-3, and the combination of the disulfide bond mutation and the cavity-filling mutation as described in claim 4; The substitution of the lysine at position 176 as described in any one of claims 1-3, the disulfide bond mutation as described in claim 4, and a combination of the electrostatic mutations described therein; The combination of the 186-position serine substitution as described in any one of claims 1-2, 4 and the disulfide bond mutation as described in claim 5; The combination of the 186-position serine substitution as described in any one of claims 1-3 and the linker mutation as described in claim 5; The combination of the 186-position serine substitution as described in any one of claims 1-3 and the cavity-filling mutation as described in claim 4; The 186-position serine substitution as described in any one of claims 1-3, and the combination of disulfide bond mutation and cavity-filling mutation as described in claim 4; The combination of the 186-position serine substitution as described in any one of claims 1-3, the disulfide bond mutation as described in claim 4, and the electrostatic mutation.

7. The mutant according to claim 6, characterized in that, The cavity filling mutation is S190F and / or V207L; and / or the disulfide bond mutation is selected from S155C-S290C, or S466C-S443C.

8. The mutant according to claim 6, characterized in that, The cavity-filling mutation is K87M or T54H; and / or the disulfide bond mutation is selected from T103C-I148C or D392C-S493C.

9. A DNA sequence encoding a mutant of the RSV F protein according to any one of claims 1-8.

10. A recombinant plasmid or vector, characterized in that, Its insertion contains a DNA sequence encoding a mutant of the RSV F protein according to any one of claims 1-8.

11. An mRNA sequence encoding a mutant of the RSV F protein according to any one of claims 1-8.

12. The use of the mutant of RSV F protein according to any one of claims 1-8, the DNA coding sequence according to claim 9, the recombinant plasmid or vector according to claim 10, or the mRNA sequence according to claim 11 in the preparation of RSV vaccines, or in improving the expression and / or stability of RSV pre-fusion (Pre-F) conformational protein or RSV pre-fusion trimer conformational protein.

13. A respiratory syncytial virus (RSV) vaccine, characterized in that, It comprises at least one nucleic acid having an open reading frame encoding a mutant of the RSV F protein of any one of claims 1-8 or an immunogenic fragment thereof, and a pharmaceutically acceptable vector.

14. A lipid nanoparticle, characterized in that, It comprises an active substance and a nanoliposome carrier, wherein the active substance is selected from any one of the following: a mutant of the RSV F protein according to any one of claims 1-8, the DNA coding sequence according to claim 9, the recombinant plasmid or vector according to claim 10, or the mRNA sequence according to claim 11.

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