Mutant of RSV pre-fusion conformational f protein and use thereof

Abstract

Provided is a mutant of a respiratory syncytial virus (RSV) pre-fusion conformational F protein. The mutant comprises an amino acid mutation relative to the amino acid sequence of a wild-type RSV F protein, the amino acid mutation being amino acids at a position 392 and a position 493 or a position near the position 493 being substituted by C, and a disulfide bond being formed between a position 392C and a position 493C or a position near the position 493C after the mutation. Also provided are a related DNA coding sequence, recombinant plasmid and mRNA sequence and a corresponding RSV vaccine. By introducing a disulfide bond at a suitable position, and combining same with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of an F protein having pre- and post-fusion conformations and a trimerized conformation are significantly improved, and the conformation of the F protein is significantly inhibited from being converted into the post-fusion conformation. The mutant is also applied to preparation of an RSV vaccine and RSV infection detection.

Description

Mutants of RSV prefusion conformation F protein and their applications

[0001] The present invention claims priority to a Chinese patent application filed with the Patent Office of China on August 7, 2023, with application number 2023109859128, entitled “Mutants of RSV prefusion conformation F protein and their applications”. The present invention also claims priority to a Chinese patent application filed with the Patent Office of China on July 16, 2024, with application number 2024109530454, entitled “Mutants of RSV prefusion conformation F protein and their applications”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and in particular relates to a mutant of the pre-fusion conformation of the respiratory syncytial virus F protein and its application in the development of respiratory syncytial virus vaccines and the diagnosis of respiratory syncytial virus infection. Background Art

[0003] Respiratory Syncytial Virus (RSV) infection causes substantial morbidity and mortality in infants, young children, and the elderly, with an estimated 64 million infections and 160,000 deaths worldwide each year. RSV-induced pneumonia is the leading cause of clinical mortality. To date, passive prophylaxis with monoclonal antibodies has been limited to high-risk infants, in part due to its modest efficacy. Currently, there are no approved safe and effective vaccines against RSV infection in my country, highlighting the urgent need for RSV vaccines that provide a protective immune response.

[0004] The RSV genome is approximately 15 kilobases (kb) long and contains 10 genes encoding 11 proteins, including eight structural proteins (F, G, M2-1, M2-2, SH, N, P, and L) and three nonstructural proteins (NS1, NS2, and NS3). Fusion glycoprotein (F) and attachment glycoprotein (G) are the two major envelope glycoproteins. The F protein, a type I viral fusion protein, mediates membrane fusion between the virus and host cells, allowing the viral envelope to fuse with the host cell membrane to form a polysyncytium. Neutralizing antibodies against the F protein can effectively inhibit RSV infection. Neutralizing antibodies induced by natural RSV infection in humans are primarily directed against the F protein. The amino acid sequence of the F protein is over 93% identical across RSV subtypes, making it much more genetically stable than the G protein. Furthermore, the F protein is an important target for inducing the production of CD8+ T cells. Therefore, the RSV F protein is a key target for RSV vaccine development.

[0005] The F protein of RSV has a trimer structure and mediates the fusion of the viral envelope and the host cell through conformational changes. Before the fusion is initiated, the F protein adopts a pre-fusion conformation, which is unstable and has a low energy barrier. During the process of RSV virus infection of host cells, the F protein conformation changes from the pre-fusion conformation to the post-fusion conformation, mediating the fusion of the virus and the host cell membrane. The F protein in the post-fusion conformation is very stable. The F protein in the pre-fusion conformation is very different from the F protein in the post-fusion conformation and has different antigenic epitopes. The pre-fusion conformation of the F protein has key neutralizing epitopes for RSV infection. When the F protein changes to the post-fusion conformation, most of the key neutralizing epitopes are lost. Therefore, it is crucial to maintain the stability of the pre-fusion trimer conformation of the F protein.

[0006] In order to solve the problem of unstable structure of pre-fusion F protein (pre-F), the protein structure was stabilized by introducing mutations at specific amino acid sites in the protein. While increasing the stability, the neutralization-sensitive epitope of the pre-F conformation of the F protein was also retained.

[0007] Summary of the Invention

[0008] Based on this, the purpose of the present invention is to provide a mutant of RSV prefusion conformation F protein and its application, wherein the mutant of prefusion conformation F protein can improve the expression level and stability of prefusion trimerization conformation F protein.

[0009] To achieve the above objectives, the following technical solutions may be included.

[0010] The first aspect of the present invention is to provide a mutant of the RSV prefusion conformation F protein, which comprises an amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, wherein the amino acids at positions 392 and 493 or the amino acids near position 493 are replaced by cysteine, and a disulfide bond is formed between the mutated 392C and 493C, or after the mutation, the positions near 392C and 493 are mutated to cysteine.

[0011] In some embodiments, the position near position 493 includes position 491, which is mutated to 491C.

[0012] In some embodiments, the amino acid sequence of the wild-type RSV F protein is shown in SEQ ID NO: 1.

[0013] In some embodiments, the mutant further introduces a cavity-filling mutation, and the cavity-filling mutation is selected from at least one of the following: E60I, E60Y, S62V, K80M, K87M, K87Y, V90I, S150L, ​​S150V, S150F, S150W, V154L, K168Y, K168L, K191V, K191L, K191M, K427Y, D448V, S19 0F and V207L, E60I and K191V, S150L and K191V, K191V and D448V, D448Y, V154L and K191V, K168Y and K191V, K191V and K427Y, S150L and V154L, K427Y and D448V, E60I and S62V, S190F and V207L, or the above mutation sites are replaced by any hydrophobic amino acid.

[0014] Further preferably, the cavity-filling mutation is selected from at least one of the following: K80M, K87M, K87Y, V90I, V154L, K427Y, D448Y, 190F and V207L.

[0015] In some embodiments, the mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity-filling mutation, wherein the cavity-filling mutation includes at least one selected from K80M, K87M, K87Y, V154L, D448Y, S190F and V207L.

[0016] In some embodiments, the mutant includes a disulfide bond formed between the mutated D392C and S491C, and a cavity-filling mutation, wherein the cavity-filling mutation includes S190F and V207L ​​mutations.

[0017] In some embodiments, a proline mutation is also introduced, and the proline mutation is selected from at least one of the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P.

[0018] In some embodiments, the mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity-filling mutation of K87M, or S190F and V207L, and further includes at least one proline mutation selected from the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P.

[0019] In some embodiments, the mutants include the formation of a disulfide bond between the mutated D392C and S491C, as well as S190F, V207L ​​cavity-filling mutations, and I214P mutations.

[0020] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO: 3.

[0021] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO:5.

[0022] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO:7.

[0023] In some embodiments, the amino acid sequence of the mutant is shown in any one of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39.

[0024] The second aspect of the present invention is to provide a DNA sequence encoding a mutant of the RSV pre-fusion conformation F protein.

[0025] In some embodiments, the coding sequence is as shown in any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40.

[0026] The third aspect of the present invention is to provide a recombinant plasmid into which is inserted the coding sequence of any one of the mutants encoding the RSV pre-fusion conformation F protein.

[0027] The fourth aspect of the present invention is to provide an mRNA sequence encoding a mutant of the RSV pre-fusion conformation F protein, which can be obtained by enzyme digestion and transcription of a recombinant plasmid inserted with a coding sequence encoding any of the mutants.

[0028] The fifth aspect of the present invention is to provide the use of the above-mentioned mRNA sequence or the mutant in the preparation of RSV vaccine.

[0029] The sixth aspect of the present invention is to provide the use of the mutant in the detection of respiratory syncytial virus infection.

[0030] The sixth aspect of the present invention is to provide a respiratory syncytial virus (RSV) vaccine, characterized in that it comprises at least one ribonucleic acid polynucleotide having an open reading frame encoding any of the above-mentioned RSV prefusion conformation F protein mutants or immunogenic fragments thereof, and a pharmaceutically acceptable carrier.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. By introducing amino acids at appropriate positions (using amino acids at positions 392 and 493 or 491) that can limit the positional movement of the (HRB) region, disulfide bonds can stabilize the pre- and post-fusion conformations and trimerization conformations of the F protein and significantly inhibit the transition of the F protein to the post-fusion conformation.

[0033] 2. Based on the disulfide bond D392C-S491C, introducing a single-point cavity-filling mutation, or based on D392C-S493C, introducing specific single-point cavity-filling mutations and double-point cavity-filling mutations, will help stabilize the conformation of the F protein and increase protein expression.

[0034] 3. The combination of disulfide bonds and cavity filling, electrostatic mutation, and introduction of proline mutations at specific positions described in the present invention can further significantly improve the expression and stability of F proteins with pre- and post-fusion conformations and trimerization conformations, and significantly inhibit the transition of F proteins to the post-fusion conformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1: Plasmid map of pNeoCura-Bvac.

[0036] Figure 2: Analysis of monoclonal antibody binding in RF-NeoC-K1 mRNA-transfected cells, including: A. Average fluorescence intensity of binding to monoclonal antibody D25, B. Average fluorescence intensity of binding to monoclonal antibody AM14, C. Average fluorescence intensity of binding to monoclonal antibody 4D7, D. Ratio of binding to monoclonal antibody D25, E. Average fluorescence intensity of binding to monoclonal antibody AM14, F. Ratio of binding to monoclonal antibody 4D7.

[0037] Figure 3: Binding analysis of cells transfected with RF-NeoC-K24 and RF-NeoC-K35 mRNA and monoclonal antibodies, including A. D25 monoclonal antibody, B. AM14 monoclonal antibody, and C. 4D7 monoclonal antibody.

[0038] Figure 4: Binding analysis of cells transfected with mRF-NeoC-K133 mRNA and monoclonal antibodies, including A. D25 monoclonal antibody, B. AM14 monoclonal antibody, and C. 4D7 monoclonal antibody.

[0039] Figure 5: Protein expression levels of mutant mRNA containing disulfide bond mutations relative to mRNA encoding RSV wild-type F protein, where: A. protein bound to the D25 monoclonal antibody indicates the expression level of the pre-fusion conformation F protein; B. protein bound to the AM14 monoclonal antibody indicates the expression level of the pre-fusion trimeric conformation F protein; C. protein bound to the 4D7 monoclonal antibody indicates the expression level of the post-fusion conformation F protein.

[0040] Figure 6: Protein expression levels of mutant mRNAs containing disulfide bond mutations and single cavity-filling mutations relative to the mRNA encoding RSV wild-type F protein, where: A. protein bound to the D25 monoclonal antibody indicates the expression level of the pre-fusion conformation F protein; B. protein bound to the AM14 monoclonal antibody indicates the expression level of the pre-fusion trimeric conformation F protein; C. protein bound to the 4D7 monoclonal antibody indicates the expression level of the post-fusion conformation F protein.

[0041] Figure 7: Protein expression levels of mutant mRNAs containing disulfide bond mutations and two cavity-filling mutations relative to the mRNA encoding RSV wild-type F protein, where: A. protein bound to the D25 monoclonal antibody indicates the expression level of the pre-fusion conformation F protein; B. protein bound to the AM14 monoclonal antibody indicates the expression level of the pre-fusion trimeric conformation F protein; C. protein bound to the 4D7 monoclonal antibody indicates the expression level of the post-fusion conformation F protein.

[0042] Figure 8: Protein expression levels of mutant mRNAs containing disulfide bond mutations, single cavity-filling mutations, and proline mutations at the hinge region linking α-helices to α-helices or β-sheets relative to the mRNA encoding RSV wild-type F protein, where: A. protein bound to the D25 monoclonal antibody represents the expression level of the pre-fusion conformation F protein; B. protein bound to the AM14 monoclonal antibody represents the expression level of the pre-fusion trimeric conformation F protein; C. protein bound to the 4D7 monoclonal antibody represents the expression level of the post-fusion conformation F protein. DETAILED DESCRIPTION

[0043] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0044] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0046] definition

[0047] To facilitate understanding of this technology, certain terms and phrases are defined below.

[0048] A disulfide bond is a chemical bond connecting the sulfhydryl groups of two different cysteine ​​residues in different peptide chains or in the same peptide chain.

[0049] pre fusion pre-fusion conformation.

[0050] (1) Disulfide bond: As shown in Table 1, D392C and S493C are mutated to form a disulfide bond between them.

[0051] (2) Cavity filling: The two grooves of the conformation were modified (such as S190F and V207L ​​in Table 1) to fill them, as shown in RF-KW5 to RF-KW34.

[0052] The present invention introduces point mutations at amino acid sites in the F protein sequence of respiratory syncytial virus (RSV) to form disulfide bonds, and / or cavity filling, and / or a combination of disulfide bonds and cavity filling, thereby significantly improving the expression of the pre-fusion F protein; this pre-fusion F protein can form a stable trimer and has a stable trimerized conformation. Using the mutant F protein as an immunogen or vaccine can significantly increase the level of neutralizing antibodies induced against RSV.

[0053] The present invention uses the F protein sequence of RSV A2 strain as the basic sequence for the mutant mutation, but the basic sequence for the mutant mutation of the present invention is not limited to RSV A2 strain, and also includes other RSV types such as RSV type B and clinical strains.

[0054] As is well known, RSV has two antigenic subtypes, A and B. The amino acid sequences of the mature processed F proteins of the two subtypes are approximately 93% identical. As used in the present invention, amino acid positions are given with reference to the sequence (SEQ ID NO: 1) of the RSV F protein (F protein or a mutant of the F protein) from the A2 strain. As used herein, the expression "amino acid at position "x" of the RSV F protein" thus refers to the amino acid corresponding to the amino acid at position "x" in the RSV F protein of the RSV A2 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 strain other than the A2 strain is used, the amino acid positions of the F protein will be numbered with reference to the numbering of the F protein of the A2 strain of SEQ ID NO: 1 by aligning the sequence of the other RSV strain with the F protein of SEQ ID NO: 1 by inserting gaps where necessary. Sequence alignment can be performed using methods well known in the art, for example, by CLUSTALW, Bioedit or CLC Workbench.

[0055] The following lists the amino acid sequences and nucleic acid sequences of some of the mutant F proteins. For other amino acid and nucleic acid sequences not shown, the corresponding amino acid and nucleic acid sequences can be obtained according to the mutation positions given in Table 1, with reference to the given wild-type sequence and the following sequence, using conventional techniques.

[0056] SEQ ID NO.1

[0057] SEQ ID NO.2

[0058] SEQ ID NO.3 (RF-NeoC-K1)

[0059] SEQ ID NO.4 (dRF-NeoC-K1)

[0060] SEQ ID NO.5 (RF-NeoC-K24)

[0061] SEQ ID NO.6 (d RF-NeoC-K24)

[0062] SEQ ID NO:7 (RF-NeoC-K35)

[0063] SEQ ID NO:8(d RF-NeoC-K35)

[0064] SEQ ID NO:9 (RF-NeoC-K131)

[0065] SEQ ID NO:10(d RF-NeoC-K131)

[0066] SEQ ID NO:11 (RF-NeoC-K132)

[0067] SEQ ID NO:12(d RF-NeoC-K132)

[0068] SEQ ID NO:13 (RF-NeoC-K133) <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0069] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:14(d RF-NeoC-K133)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0070] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15(RF-NeoC-K5)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0071] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:16(d RF-NeoC-K5)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0072] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:17(RF-NeoC-K6)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0073] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:18(d RF-NeoC-K6)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0074] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:19(RF-NeoC-K7)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0075] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:20(d RF-NeoC-K7)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0076] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:21(RF-NeoC-K21)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0077] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:22(d RF-NeoC-K21)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0078] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:23(RF-NeoC-K82)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:24(d RF-NeoC-K82)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0080] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:25(RF-NeoC-K83)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0081] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:26(d RF-NeoC-K83)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0082] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:27(RF-NeoC-K84)<h2 style=";text-align:left;direction:ltr">

[0083] SEQ ID NO:28(d RF-NeoC-K84)

[0084] SEQ ID NO:29 (RF-NeoC-K85)

[0085] SEQ ID NO:30(d RF-NeoC-K85)

[0086] SEQ ID NO:31 (RF-NeoC-K89)

[0087] SEQ ID NO:32(d RF-NeoC-K89)

[0088] SEQ ID NO:33 (RF-NeoC-K93)

[0089] SEQ ID NO:34 (d RF-NeoC-K93)

[0090] SEQ ID NO:35 (RF-NeoC-K94)

[0091] SEQ ID NO:36 (d RF-NeoC-K94)

[0092] SEQ ID NO:37 (RF-NeoC-K96)

[0093] SEQ ID NO:38 (d RF-NeoC-K96)

[0094] SEQ ID NO:39 (RF-NeoC-K103)

[0095] SEQ ID NO:40(d RF-NeoC-K103)

[0096] The present invention is further described in detail below with reference to specific embodiments.

[0097] Example 1 Preparation of plasmid templates encoding RSV wild-type F protein and mutants.

[0098] Based on the wild-type F protein sequence of the RSV A2 strain (shown in SEQ ID NO:1) and the optimized DNA sequence encoding the wild-type F protein sequence of the RSV A2 strain (shown in SEQ ID NO:2), the DNA sequence of the wild-type F protein sequence was recombined into the pNeoCura-Bvac plasmid vector between the restriction sites Bam HI and Sac I by gene synthesis. This generated the recombinant plasmid pNeoCura-Bvac-WT, which served as a plasmid template for preparing wild-type F protein mRNA (mWT). Figure 1 shows the plasmid map of pNeoCura-Bvac.

[0099] Point mutations were made in the recombinant plasmid pNeoCura-Bvac-WT according to the corresponding sites in the amino acid sequences of the mutants shown in Table 1, resulting in the corresponding recombinant plasmids pNeoCura-Bvac-RF-NeoC-K1 to pNeoCura-Bvac-RF-NeoC-K133 (DNA sequences: dRF-NeoC-K1 to dRF-NeoC-K133). We also constructed the recombinant plasmid pNeoCura-Bvac-Ds-Cav1, which encodes the same amino acid mutation sites as Ds-Cav1, but retains the original amino acid sequences of the transmembrane region (TM) and intracellular region (CT) of F1 at the C-terminus.

[0100] Table 1:

[0101] Example 2 Preparation of mRNA encoding RSV wild-type F protein and mutants

[0102] 2.1. Linearized template preparation

[0103] The recombinant plasmids were digested with restriction endonuclease BspQ I (NEB, catalog number: R0712L), and the linearized plasmids were purified using VAHTS DNA Clean Beads (Norway, N411-01).

[0104] 2.2 Preparation of mRNA by in vitro transcription

[0105] Following the instructions for the T7 High Yield RNA Transcription Kit (N1-Me-Pseudo UTP) (Novozymes, DD4202), in vitro transcription was performed using the linearized plasmid prepared in 2.1 as a template to generate mRNA. mRNA was purified using VAHTS RNAClean Beads (Vazyme, N412-01) according to the instructions.

[0106] 2.3 Capping and purification

[0107] The purified mRNA was capped using the mRNACap 2'-O-Methyltransferase capping kit (Novozyme, DD4110) and purified using VAHTS RNA Clean Beads (Vazyme, Cat. No. N412-01) to obtain mRNA of the corresponding sequence.

[0108] Example 3 mRNA transfected cells and detection of cell surface F protein expression

[0109] 3.1 HEK293T cell plating

[0110] HEK293T cells were seeded in 24-well plates (2.5-3×10 5 The cells were cultured in DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO2 for 18 hours until the cell density reached 80% confluency before transfection.

[0111] 3.2 mRNA transfection into HEK293T cells

[0112] The cell culture medium was replaced with serum-free Opti-MEM medium (Thermo Fisher Scientific), and mRNA (0.3 μg or 1 μg RNA / well) was added with Lipofectamine TM Messenger MAX TM The cells were transfected with the transfection reagent (Thermo Fisher Scientific). After 6 hours, the cell culture medium was replaced with DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the cells were cultured for another 24 hours.

[0113] 3.3 Detection of F protein expression on the surface of HEK293T cells by flow cytometry

[0114] After 24 hours of incubation, transfected cells were centrifuged at 1000 g for 10 minutes, harvested, and incubated with fluorescently labeled monoclonal antibodies. Flow cytometry (BD, FACS Celesta) was used to measure the mean fluorescence intensity (MFI) and percentage of cells expressing F protein on the cell surface that bound to monoclonal antibody D25, monoclonal antibody AM14, or monoclonal antibody 4D7. The monoclonal antibody D25 of the present 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 a prefusion-specific neutralizing antibody. Science (New York, NY), 340(6136), 1113–1117. https: / / doi.org / 10.1126 / science.1234914). The mean fluorescence intensity (MFI) of D25 binding to cell surface F protein represents the expression level of F protein in the prefusion conformational epitope.

[0115] The monoclonal antibody AM14 in the present 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 RSV Fusion 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 in the prefusion trimerization conformation epitope;

[0116] The monoclonal antibody 4D7 of the present invention: (4D7 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 the binding of 4D7 to the cell surface F protein characterizes the expression level of the F protein in the post-fusion conformational epitope.

[0117] Experimental results:

[0118] (1) The second heptad repeat (HRB) region is one of the unstable regions, forming the trimeric helical stem region in the prefusion conformation of the F protein. During the transition from the prefusion conformation to the postfusion conformation, the second heptad repeat (HRB) region participates in the formation of three α helices in the six-helix bundle in the postfusion conformation. The amino acid at position 392 forms a disulfide bond with the amino acid at position 493 or 491, restricting the positional movement of the (HRB) region and thus stabilizing the prefusion conformation.

[0119] As shown in Figure 2, compared with WT mRNA encoding wild-type F protein, cells transfected with RF-NeoC-K1 mRNA showed significantly increased mean fluorescence intensity (MFI) and ratio of D25 binding (Figures 2A and 2D), indicating that the disulfide bond D392C-S493C mutation significantly increased the expression level and ratio of the prefusion conformation of F protein. Cells transfected with RF-NeoC-K1 mRNA showed significantly increased mean fluorescence intensity (MFI) and ratio of AM14 binding (Figures 2B and 2E), indicating that the disulfide bond D392C-S493C mutation also significantly increased the expression level and ratio of the prefusion conformation of F protein trimer. Furthermore, due to the unstable conformation of the wild-type F protein encoded by WT mRNA, the mean fluorescence intensity (MFI) and ratio of 4D7 binding in cells transfected with WT mRNA were high, whereas the MFI and ratio of 4D7 binding in cells transfected with mRF-NeoC-K1 mRNA were significantly reduced (Figures 2C and 2F), approaching those in the untransfected mRNA group (no mRNA). This suggests that the disulfide bond D392C-S493C mutation significantly reduces the expression level and ratio of the post-fusion conformation of the F protein trimer.

[0120] The combination of the disulfide bond D392C-S493C with cavity filling and proline mutations at specific positions, such as the mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K24 and RF-NeoC-K35 binding to D25 and AM14, was significantly higher than that of Ds-Cav1 (a stabilized prefusion F protein containing the RSV F transmembrane domain that has been disclosed in the prior art), indicating that the level of trimerized F proteins encoded by RF-NeoC-K24 and RF-NeoC-K35 in the prefusion conformation was significantly higher than that of Ds-Cav1. 1 (Figures 3A and 3B); the mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K24 and RF-NeoC-K35 and the antibody 4D7 that characterizes the post-fusion conformation was significantly lower than that of Ds-Cav1, indicating that the levels of trimerized F proteins in the post-fusion conformation encoded by RF-NeoC-K24 and RF-NeoC-K35 were significantly lower than those of Ds-Cav1, and they had a more stable pre-fusion conformation (Figure 3C).

[0121] We also found that when the amino acid near position 493 (such as position 491) mutated to cysteine, a disulfide bond was formed between 392C and 491C, which also helped to stabilize the pre-fusion conformation of the F protein, as shown in Figure 4. Compared with the wild-type F protein sequence (WT), the combination of the disulfide bond D392C-S491C and cavity filling and proline mutations at specific positions, such as the mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K133 and bound to D25 and AM14, was significantly increased, indicating that the level of trimerized F protein in the pre-fusion conformation encoded by RF-NeoC-K133 was significantly higher than that of mDs-Cav1 (Figure 4A, Figure 4B); the mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K131, RF-NeoC-K132, and RF-NeoC-K133 and the antibody 4D7 that characterizes the post-fusion conformation was significantly reduced, indicating that the level of post-fusion conformation of the F proteins encoded by RF-NeoC-K131, RF-NeoC-K132, and RF-NeoC-K133 was significantly reduced, with a more stable pre-fusion conformation (Figure 4C). Although only the 392C and 491C mutations did not significantly increase the mean fluorescence intensity (MFI) of cell surface F protein binding to D25 and AM14, this may be due to the lack of overall F protein levels after RF-NeoC-K131 transfection in cells; however, the 392C and 491C mutations significantly reduced the expression of the post-fusion F protein conformation, indicating that the 392C and 491C mutations can effectively stabilize the F protein in the pre-fusion trimerization conformation. In summary, mutations at amino acid positions near positions 392C and 493 (such as position 491) to cysteine ​​(C) can also effectively stabilize the pre-fusion conformation of the F protein and effectively inhibit the post-fusion conformational transition of the F protein.

[0122] To further evaluate the role of cysteine ​​substitutions at amino acids 392 and 493, or at positions near 493, in forming disulfide bonds between 392C and 493C, or between 392C and positions near 493, the cysteine ​​mutation D392C-S491C was introduced at amino acids 392 and 491.

[0123] The results showed that compared with WT mRNA encoding wild-type F protein, the mean fluorescence intensity (MFI) and ratio of binding to D25, AM14, and 4D7 in cells transfected with RF-NeoC-K131 mRNA were extremely low ( Figure 5 ), indicating that the D392C-S491C mutation significantly reduced the expression of the post-fusion conformation F protein.

[0124] Introducing hydrophobic amino acid mutations to fill the cavity helps stabilize the F protein's conformation and increase protein expression. Based on the disulfide bond D392C-S491C, single-point cavity-filling mutations were introduced. As shown in Figure 6 , the cavity-filling mutations K80M, K87M, K87Y, V154L, and K427Y further increased expression of the F protein in the prefusion (Figure 6A) and prefusion trimer (Figure 6B) conformations, while decreasing expression of the F protein in the postfusion conformation (Figure 6C).

[0125] Furthermore, based on the disulfide bond D392C-S491C, two cavity-filling mutations were introduced, as shown in Figure 7 : the combination of cavity-filling mutations S190F and V207 can further increase the expression of F protein before fusion ( Figure 7A ) and in the pre-fusion trimer conformation ( Figure 7B ), while reducing the expression of F protein in the post-fusion conformation ( Figure 7C ).

[0126] Based on the disulfide bond D392C-S491C, the introduction of a combination of two cavity-filling mutations: E60I, K191V; E60I, K191V; S150L, ​​K191V, K191V, D448V; V154L, K191V; K168Y, K191V; K191V, K427Y; S150L, ​​V154L; K427Y, D448V; E60I, S62V reduced the expression of F protein before protein fusion (Figure 7A), pre-fusion trimeric conformation (Figure 7B), and post-fusion conformation (Figure 7C).

[0127] Furthermore, the introduction of specific proline mutations at the hinge region linking α helices and α helices or β sheets helps to stabilize the conformation of the F protein and increase protein expression. Proline mutations were added on the basis of the disulfide bond D392C-S491C and cavity filling K87M mutations (RF-NeoC-K6). The results showed that compared with the design without proline mutation sites, RF-NeoC-K6, RF-NeoC-K82 (containing I214P), RF-NeoC-K83 (containing S215P), RF-NeoC-K93 (containing E161P), and RF-NeoC-K94 (containing Q210P) further increased the expression of the pre-fusion conformation F protein; RF-NeoC-K82 (containing I214P), RF-NeoC-K83 (containing S215P), RF -NeoC-K85 (containing I217P), RF-NeoC-K93 (containing E161P), RF-NeoC-K95 (containing S213P, I214P), RF-NeoC-K96 (containing I214P, S215P), and RF-NeoC-K97 (containing S21PP, N216P) all further increased the expression of the pre-fusion trimeric conformation F protein; at the same time, as shown in Figure 8, the addition of proline mutation sites did not increase the expression of the post-fusion conformation F protein.

[0128] Based on the design of RF-NeoC-K132 containing disulfide bonds D392C-S491C and cavity-filling mutations S190F and V207, the proline mutation I214P was added to improve the expression of F protein in the pre-fusion (Figure 8A) and pre-fusion trimeric conformation (Figure 8B) of the F protein, while reducing the expression of F protein in the post-fusion conformation (Figure 8C).

[0129] In summary, the disulfide bonds designed in the present invention, and / or their combination with cavity filling and proline mutations at specific positions can significantly improve the expression level of F protein with a pre-fusion trimer conformation and maintain the stability of the pre-fusion trimer conformation of the F protein.

[0130] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A mutant of RSV prefusion conformation F protein, characterized in that: The mutant comprises an amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, wherein the amino acids at positions 392 and 493 are mutated to amino acids at positions 392 and 493, or the amino acids at positions near position 493 are replaced by cysteine, respectively, and a disulfide bond is formed after the positions between the mutated 392C and 493C, or the positions near the mutated 392C and 493 are mutated to cysteine.

2. The mutant according to claim 1, characterized in that: The positions near position 493 include position 491, which is mutated to 491C.

3. The mutant according to claim 1, characterized in that: The mutant also introduces a cavity filling mutation, and the cavity filling mutation is selected from at least one of the following: E60I, E60Y, S62V, K80M, K87M, K87Y, V90I, S150L, ​​S150V, S150F, S150W, V154L, K168Y, K168L, K191V, K191L, K191M, K427Y, D448V, D448Y, S190F and V207L, E60I and K191V, S150L and K191V, K1 91V and D448V, V154L and K191V, K168Y and K191V, K191V and K427Y, S150L and V154L, K427Y and D448V, E60I and S62V, S190F and V207L, or the above mutation sites are replaced by any hydrophobic amino acid; preferably, the cavity filling mutation is selected from at least one of the following: K80M, K87M, K87Y, V90I, V154L, K427Y, D448Y and 190F and V207L.

4. The mutant according to claim 1, characterized in that: The mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity filling mutation, wherein the cavity filling mutation includes at least one selected from K80M, K87M, K87Y, V154L, D448Y, S190F and V207L; or The mutants include a disulfide bond formed between the mutated D392C and S491C, and a cavity filling mutation, wherein the cavity filling mutation includes S190F and V207L ​​mutations.

5. The mutant according to any one of claims 1 to 4, characterized in that: The mutant also introduces a proline mutation, and the proline mutation is selected from at least one of the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P.

6. The mutant according to any one of claims 1 to 4, characterized in that: The mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity-filling mutation of K87M, or S190F and V207L, and also includes at least one proline mutation selected from the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P; or The mutants include a disulfide bond formed between the mutated D392C and S491C, as well as S190F and V207L ​​cavity filling mutations, and also include I214P mutation.

7. The mutant according to claim 1, characterized in that: The amino acid sequence of the mutant is shown in any one of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:

39.

8. A DNA coding sequence encoding a mutant of the RSV prefusion conformation F protein according to any one of claims 1 to 7.

9. The DNA coding sequence according to claim 8, characterized in that The coding sequence is shown in any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:

40.

10. A recombinant plasmid, into which the DNA coding sequence according to any one of claims 8 to 9 is inserted.

11. An mRNA sequence encoding a mutant of the RSV prefusion conformation F protein according to any one of claims 1 to 7.

12. A respiratory syncytial virus (RSV) vaccine, characterized in that It comprises at least one ribonucleic acid polynucleotide having an open reading frame encoding a mutant of the RSV F protein pre-fusion conformation or an immunogenic fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

13. Use of the mutant of RSV prefusion conformation F protein according to any one of claims 1 to 7 and the mRNA sequence according to claim 11 in the preparation of RSV vaccine.

14. Use of the mutant of RSV prefusion conformation F protein according to any one of claims 1 to 7 in detecting respiratory syncytial virus infection.

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