A Respiratory Syncytial Virus F Protein Mutant and Its Application

Abstract

This invention belongs to the field of biomedicine, and particularly relates to respiratory syncytial virus (RSV) F protein mutants and their applications. The RSV F protein mutants include at least one pair of amino acid residues in the F1 and / or F2 subunits of the F protein polypeptide being replaced by cysteine, and / or the F protein signal peptide being replaced by a human immunoglobulin light chain signal peptide, and / or the polybasic sequence having alanine A at position 107 mutated to arginine R and lysine K at position 134 mutated to arginine R. This allows for the exposure of more neutralizing antibody epitopes, ensuring effective neutralizing antibody responses and antibody binding responses against RSV subtypes A and B while maintaining a stable pre-fusion conformation. It is suitable for various vaccine formulations using RSV F protein as an antigen.

Description

Technical Field

[0001] This invention is a divisional application of Chinese patent application filed on December 26, 2024, with application number CN202411939046.X and invention title "A Respiratory Syncytial Virus F Protein Mutant and Its Application".

[0002] This invention relates to the field of biomedicine, and in particular to a respiratory syncytial virus (RSV) F protein mutant and its application in the preparation of RSV vaccines. Background Technology

[0003] Respiratory syncytial virus (RSV) is one of the most important pathogens causing serious lower respiratory tract infections in infants and young children worldwide. Statistics show that as many as 34 million children under the age of 5 are infected with RSV globally. Because RSV infection does not provide lasting immunity, reinfection is common; almost 100% of infants and young children are infected more than once before the age of 2, resulting in 66,000-199,000 deaths. In single-pathogen infection factor analysis of all-cause mortality in children aged 1 month to 1 year, RSV infection caused a mortality rate as high as 6.7% in children under 1 year old, second only to malaria, posing a serious disease burden globally. Besides infants and young children, immunocompromised adults and the elderly are also high-risk groups for RSV infection. Each year, 3% to 10% of adult colds are caused by RSV, and the disease burden caused by RSV infection in the elderly is comparable to that caused by colds during the off-season. RSV infection leads to increased mucus secretion and inflammation, causing various serious complications such as heart failure and secondary bacterial pneumonia. RSV is also a significant iatrogenic pathogen, posing a great threat, particularly to premature infants, infants with congenital heart disease, bronchopulmonary dysplasia, and all immunosuppressed patients, with a substantial impact on human health, global healthcare, and the economy. Therefore, the WHO has listed RSV vaccines as one of the priority vaccines for global development in the 21st century, and the development of safe and effective RSV vaccines is an urgent need.

[0004] RSV belongs to the genus *Pneumovirus* of the family Paramyxoviridae and is a single-stranded negative-sense RNA virus. Its genome is 15.2 kb in length, transcribed into ten genomes, encoding ten major proteins, including three transmembrane proteins (G, F, and SH), two matrix proteins (M and M2), three nucleocapsid proteins (N, P, and L), and two non-structural proteins (NS1 and NS2). Among these, adhesion protein G and fusion protein F are the two most important viral proteins for inducing protective antibodies in the body. G protein mediates viral binding and determines the diversity of RSV antigens, classifying RSV into two subtypes, A and B, based on antigenic differences. F protein is primarily responsible for viral-host cell membrane fusion; its protein sequence is highly conserved across different subtypes and plays a crucial role in inducing immune protection and high levels of neutralizing antibodies in serum. Neutralizing antibodies induced by F protein can simultaneously inhibit RSV infection of both subtypes A and B and can induce a CD8+ cytotoxic T-cell response to clear the pathogen from the body. Therefore, F protein is an important antigenic target for RSV vaccine development.

[0005] The RSV F protein, a type I integrated membrane protein, is a major target protein for neutralizing antibodies. Its precursor protein, F0, consists of 574 amino acids, with three F0 fragments forming a trimer through hydrophobic interactions. During viral invasion-mediated membrane fusion, the precursor F0 is cleaved by the furin protease, releasing a short peptide P27 (109-127 amino acids). The remaining two fragments, F2 and F1, are linked by two disulfide bonds to form the mature F protein, which is then displayed on the cell membrane or virion surface via budding. At this stage, the F protein is in a high-energy metastable state and is highly unstable, known as pre-F. After cell infection, it spontaneously transforms into the low-energy post-fusion conformation. Extensive preclinical and clinical data indicate that highly neutralizing sites are primarily located in the pre-fusion conformation. Vaccines using the RSV pre-fusion conformation as an antigen can stimulate the body to produce higher levels of virus-neutralizing antibodies. Although RSV vaccines based on pre-F have shown promising application prospects, maintaining the pre-fusion conformation of the F protein remains a key challenge in vaccine development. To date, only two recombinant protein vaccines targeting the RSV F protein, developed by GSK and Pfizer, have been approved for marketing globally. Both products utilize the pre-fusion conformation of the F protein and are intended for individuals aged 60 and older in the United States. However, no related preventative vaccine products have been launched in China.

[0006] Therefore, developing a safe, highly expressed, and stable pre-fusion conformation F protein antigen has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention develops a pre-fusion conformation mutant of respiratory syncytial virus (RSV) F protein. This mutant protein possesses a stable pre-fusion conformation and high expression levels, and can form a stable trimer, thus making it suitable for the treatment of RSV infection. In 2013, McLellan's team et al. first developed the first stable pre-fusion conformation F protein, "DS-Cav1," through point mutation: intramolecular disulfide bonds were obtained by point mutations at S155C and S290C within the F1 fragment, while S190F and V207L ​​point mutations were added to enhance the binding activity of key neutralizing epitopes (DOI:10.1126 / science.1243283). This protein has been shown to elicit significantly higher levels of neutralizing antibody titers. Currently, stable pre-fusion conformation F protein vaccines developed based on structural biology have also entered the clinical stage. Therefore, the recombinant RSVF protein described in this invention introduces mutations into the amino acid sequence of DS_Cav1 (referred to as wild-type), including amino acid substitutions, deletions, and insertions.

[0008] A first aspect of the present invention provides a respiratory syncytial virus (RSV) F protein, said F protein comprising at least one mutation selected from the group consisting of:

[0009] (a) At least one pair of amino acid residues is replaced by cysteine;

[0010] (b) The F protein signal peptide was partially replaced by the human immunoglobulin light chain signal peptide;

[0011] (c) At least two sites of the multi-base sequence are replaced.

[0012] In some embodiments, the F protein comprises at least one pair of amino acid residues in the F1 subunit and / or F2 subunit of the F protein polypeptide being replaced by cysteine.

[0013] In some embodiments, the cysteine ​​substitution causes a non-natural disulfide bond to form between the F1 and F2 subunits of the F protein, the non-natural disulfide bond including disulfide bonds other than Cys69-Cys212 and Cys37-Cys439 formed between the F1 and F2 subunits.

[0014] In the pre-fusion conformation of RSV F protein, the β2 and β4 chains are close to each other, promoting the spatial folding of the two discontinuous epitopes at the 0 epitope. The addition of non-natural disulfide bonds can relatively stabilize the spatial position between the β2 and β4 chains, thereby enhancing the structural stability between the pre-fusion F1 and F2 subunits, preventing conformational changes in the heptapeptide repeat regions HRA and HRB, effectively stabilizing the pre-F protein structure, and maintaining or enhancing the binding activity of important sites such as the 0 and V epitopes, thus improving the pre-fusion F protein-specific immunogenicity of mutant antigens.

[0015] In some embodiments, the F protein includes at least one pair of cysteine ​​mutations from 102C+147C, 102C+149C, 102C+150C, 101C+147C, 101C+146C, and 100C+145C, with the mutation sites referencing the amino acid sequence of the wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:01.

[0016] In some embodiments, the F protein signal peptide portion replaced by the human immunoglobulin light chain signal peptide includes at least amino acid residues 1 to 25 of the sequence shown in SEQ ID NO:1.

[0017] In some embodiments, the human immunoglobulin light chain signal peptide is as shown in SEQ ID NO:2.

[0018] Wild-type F protein first forms F0 precursor protein, which is cleaved by furin protease to release a polypeptide pep27 composed of 27 amino acids. The remaining two segments, F2 and F1, are linked by two disulfide bonds to form mature F protein-pre-F, which is then displayed on the cell membrane or virion surface by budding.

[0019] In some implementations, two multibase sites on the F protein are replaced to improve the removal efficiency of the p27 peptide inside the F protein.

[0020] In some embodiments, the p27 polypeptide within the F protein includes at least amino acid residues 110-136 of the wild-type respiratory syncytial virus F protein.

[0021] In some embodiments, compared to the amino acid sequence of wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the polybasic sequences RARR and KKRKRR of the F protein have alanine A at position 107 mutated to arginine R and lysine K at position 134 mutated to arginine R.

[0022] In some embodiments, the F protein includes a cysteine ​​mutation 102C+147C, the F protein signal peptide portion is replaced by a human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the amino acid sequence of wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the polybasic sequences RARR and KKRKRR of the F protein have a mutation of alanine A at position 107 to arginine R and a mutation of lysine K at position 134 to arginine R.

[0023] In some embodiments, the F protein includes a cysteine ​​mutation 102C+149C, the F protein signal peptide portion is replaced by a human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the amino acid sequence of wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the polybasic sequences RARR and KKRKRR of the F protein have a mutation of alanine A at position 107 to arginine R and a mutation of lysine K at position 134 to arginine R.

[0024] In some embodiments, the F protein includes a cysteine ​​mutation 102C+150C, the F protein signal peptide portion is replaced by a human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the amino acid sequence of wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the polybasic sequences RARR and KKRKRR of the F protein have a mutation of alanine A at position 107 to arginine R and a mutation of lysine K at position 134 to arginine R.

[0025] In some embodiments, the F protein includes a cysteine ​​mutation 101C+147C, the F protein signal peptide portion is replaced by a human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the amino acid sequence of wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the polybasic sequences RARR and KKRKRR of the F protein have a mutation of alanine A at position 107 to arginine R and a mutation of lysine K at position 134 to arginine R.

[0026] In some embodiments, the F protein includes a cysteine ​​mutation 101C+146C, the F protein signal peptide portion is replaced by a human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the amino acid sequence of wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the polybasic sequences RARR and KKRKRR of the F protein have a mutation of alanine A at position 107 to arginine R and a mutation of lysine K at position 134 to arginine R.

[0027] In some embodiments, the F protein includes a cysteine ​​mutation 100C+145C, the F protein signal peptide portion is replaced by a human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the amino acid sequence of wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the polybasic sequences RARR and KKRKRR of the F protein have a mutation of alanine A at position 107 to arginine R and a mutation of lysine K at position 134 to arginine R.

[0028] In some embodiments, the F protein comprises an amino acid sequence selected from any of SEQ ID NO:3-8.

[0029] In some embodiments, the F protein includes a protease cleavage site sequence, such as the HRV 3C cleavage site (LEVLFQGP), or a protein tag, such as a 6×His-tag (HHHHHH) and a Strep tag-II tag (WSHPQFEK), which is not essential for the function of the RSV F protein (e.g., inducing an immune response).

[0030] Another aspect of the present invention provides a recombinant nucleic acid comprising a nucleotide sequence encoding the respiratory syncytial virus F protein as described above.

[0031] In some implementations, the recombinant nucleic acid is codon-optimized for expression in selected prokaryotic or eukaryotic host cells.

[0032] Another aspect of the present invention provides an expression vector comprising the recombinant nucleic acid as described above.

[0033] Another aspect of the present invention provides a host cell comprising the recombinant nucleic acid or the expression vector described above.

[0034] In some embodiments, the host cell is selected from Escherichia coli, yeast cells, insect cells or mammalian cells, preferably, the mammalian cell is selected from ExpiCHO, VERO and Expi293, etc.

[0035] Another aspect of the present invention provides a method for preparing the respiratory syncytial virus F protein as described above, the method comprising:

[0036] S1: Culture the host cells as described above under conditions suitable for the expression of the respiratory syncytial virus F protein;

[0037] S2: Collect the expression product and purify the expression product to obtain the respiratory syncytial virus F protein.

[0038] Another aspect of the present invention provides the use of the respiratory syncytial virus F protein in the preparation of a vaccine to prevent respiratory syncytial virus infection.

[0039] The present invention also provides a vaccine for preventing respiratory syncytial virus infection, comprising the respiratory syncytial virus F protein as described above, or the respiratory syncytial virus F protein prepared by the preparation method described above, and a pharmaceutically acceptable carrier or excipient.

[0040] In some embodiments, the single-dose human vaccine contains 60-120 μg of the recombinant RSV F protein. In some embodiments, the single-dose human vaccine preferably contains 60 μg of the recombinant RSV F protein. In some embodiments, the single-dose human vaccine preferably contains 120 μg of the recombinant RSV F protein.

[0041] In some embodiments, the carrier or excipient comprises a buffer. Pharmaceutically acceptable carriers and excipients are well known in the art and can be selected by those skilled in the art. These include, but are not limited to: pH adjusters (such as phosphate buffers), surfactants (such as cationic, anionic, or nonionic surfactants), adjuvants, solubilizers, stabilizers, media for containing or administering therapeutic agents, and any combinations thereof. Those skilled in the art can select suitable excipients and carriers to produce formulations suitable for administration to a subject via a selected route of use. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including oils derived from petroleum, animals, plants, or synthetic sources. Suitable excipients include, but are not limited to, glycerol, polyethylene glycol, and salts of calcium, magnesium, zinc, and other divalent cations.

[0042] In some embodiments, the vaccine further includes an adjuvant.

[0043] In some embodiments, the adjuvant includes at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, CpG, and QS-21.

[0044] Generally, adjuvants should be selected to enhance Th1-biased immune responses in subjects or subject populations receiving the vaccine, and should be safe and effective in subjects or subject populations.

[0045] Optionally, the vaccine may also contain at least one other antigen from a pathogenic organism different from RSV, such as a virus different from RSV, such as varicella-zoster virus, human papillomavirus, hepatitis B virus, coronavirus, or influenza virus. Alternatively, the pathogenic organism may be bacteria, such as diphtheria, tetanus, or pneumococcus.

[0046] The present invention also provides a method for preparing the above-mentioned vaccine for preventing RSV infection, specifically comprising: packaging the purified F protein and adjuvant separately or mixing them thoroughly in a certain proportion.

[0047] In some embodiments, the method further includes additional steps such as gene synthesis, expression vector construction, and protein purification lyophilization. The expressed recombinant RSV F protein can be recovered and purified from recombinant cell cultures using any one or more of a variety of methods known in the art, including ammonium sulfate precipitation, filtration, ultrafiltration, affinity chromatography, anion / cation exchange chromatography, hydrophobic interaction chromatography, etc.

[0048] Compared with the prior art, the advantages and positive effects of the present invention are:

[0049] This invention, through the substitution and deletion of various amino acids in wild-type RSV F protein, obtained a pre-fusion conformation of RSV F protein capable of exposing more neutralizing antibody epitopes. While maintaining a stable pre-fusion conformation, it ensures that it can elicit effective neutralizing antibody responses and antibody binding responses against RSV subtypes A and B. Furthermore, this invention uses recombinant RSV F protein as an immunogen combined with an adjuvant, achieving stronger immunoinduction efficacy, not only enhancing humoral immune responses but also strongly stimulating Th1-type immunity, significantly improving the immunogenicity of the RSV antigen. The mutation methods disclosed in this invention are applicable to other human RSV strains; and applicable to various vaccine forms using RSV F protein as an antigen, such as recombinant protein vaccines, nucleic acid vaccines, virus-like particle vaccines, and vector vaccines. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0051] Figure 1 This is a map of the pCAGGS plasmid.

[0052] Figure 2 The results of Western blot analysis were used to detect the expression of the F protein mutant.

[0053] Figure 3 The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified F protein mutant HN108-05 and control protein Ds_Cav1 under reducing conditions.

[0054] Figure 4 The results of Western blot analysis for detecting the co-expression of F protein and Furin protease are presented. Detailed Implementation

[0055] The present invention will be further described below with reference to specific embodiments:

[0056] To facilitate a better understanding of the present invention, but not to limit the invention, the experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments, unless otherwise specified, were all purchased from conventional biochemical reagent stores.

[0057] Example 1: Design and gene synthesis of mutants that stabilize the pre-fusion conformation of F protein by introducing disulfide bonds through cysteine ​​substitution:

[0058] By observing the spatial structure of the pre-fusion F protein of respiratory syncytial virus (RSV), amino acid mutations that stabilize the pre-fusion conformation were designed based on structural biology principles. Using the amino acid sequence (SEQ ID No. 01) of the wild-type RSV pre-fusion F protein (Ds_Cav1) as a template, disulfide bonds were introduced through cysteine ​​substitution to enhance the structural stability between the pre-fusion F1 and F2 subunits, thereby preventing conformational changes in the heptapeptide repeat regions HRA and HRB. Simultaneously, the binding activity of important sites such as the Φ and V epitopes was maintained or enhanced, improving the pre-fusion F protein-specific immunogenicity of the mutant antigen. To promote the secretory expression of the F protein, the F protein signal peptide (1–25 aa) was replaced with the human immunoglobulin light chain signal peptide “MRVPAQLLGLLLLWLRGARC (SEQ ID No. 02)”. To improve the removal efficiency of the p27 polypeptide (110–136 aa) within the F protein, two multibase sites (RARR109 and KKRKRR134) on the F protein were replaced with (RRRR107 and KKRRRR134). Based on the original sequence DS_Cav1, six protein variants were designed, with amino acid sequences shown in SEQ ID NO:3,4,5,6,7 and8, respectively.

[0059] The nucleotide sequences of the F protein variants designed according to the table below were submitted to Beijing Qingke Biotechnology Co., Ltd. Codon optimization was performed on the host Expi293F cells to determine the nucleic acid sequences, which were then inserted into the eukaryotic expression pCAGGS (plasmid map shown). Figure 1 The genome was purchased from Beijing Qingke Biotechnology Co., Ltd. (the sequence is shown in SEQ ID NO.9) and the whole genome was synthesized.

[0060] Table 1 shows the design of mutants that introduce disulfide bonds and multiple base site substitutions through cysteine ​​substitution. Detailed sequences can be found in SEQ ID No. 03 to No. 08.

[0061] Table 1. Mutant design for cysteine ​​substitution to introduce disulfide bonds and multi-base site substitution.

[0062]

[0063] Example 2: Recombinant F protein expression and preliminary screening:

[0064] 1. Cell transfection and growth curve monitoring:

[0065] Expression vectors synthesized using genetic engineering techniques were cloned, transformed, and plasmidized before being used to transfect Expi293 cells for recombinant protein expression. Expi293 cells in logarithmic growth phase were passaged at high density one day before transfection, with a viable cell density of 2-3 × 10⁶ / mL. After overnight cell growth, the cell density was diluted to 3 × 10⁶ / mL. 2.5 mL of cell suspension was used per well for transfection. 2 μL of DNA was used per well, diluted with Opti-MEM medium and gently mixed. Immediately afterward, 8 μL of PEI40000 was gently added dropwise to the diluted DNA, and the cells were incubated at room temperature for 10-20 min to prepare the DNA complex. After incubation, the DNA complex was added dropwise to the cell suspension to be transfected, and the cells were transferred to a shaker at 37°C with 8% CO₂ and cultured at 225 rpm. 500 μL of feed was added 18-22 h after transfection, and the cells were cultured further. Table 2 shows the changes in cell viability and density during transfection of different protein variants.

[0066] Table 2. Changes in cell viability and density during transfection with different protein variants

[0067]

[0068] Identification of expression of different F protein mutants:

[0069] 2.1 Western Blot analysis of expression of different protein variants:

[0070] (1) On the 3rd day of culture, the cell culture supernatant was collected by centrifugation at 4700rpm for 40min at 4℃ and then filtered with a 0.22um filter membrane for sterilization.

[0071] (2) Add β-mercaptoethanol and loading buffer to the sample, heat denature at 100℃ for 10 min, load 25 μl / well of cell supernatant electrophoresis sample, load 5 μg of purified DS_Cav1 protein from the control group, electrophoresis at 120V for 60 min.

[0072] (3) After electrophoresis, the protein was transferred to the PVDF membrane by dry transfer and blocked with TBS solution containing 5% skim milk powder for 2 hours.

[0073] (4) His mouse monoclonal antibody was diluted 1:2000 with TBS buffer, and the PDVF membrane was transferred to the diluted antibody and incubated overnight at 4°C.

[0074] (5) Clean with TBST three times every other day, 5 minutes each time.

[0075] (6) Dilute HRP-Goat anti Mouse secondary antibody with TBS containing 5% skim milk powder, transfer PVDF membrane into the diluted secondary antibody, and incubate on a shaker at room temperature for 1 hour.

[0076] (7) Wash with TBST three times, 10 min each time, add substrate developing solution, develop in the dark for 10 min and take pictures.

[0077] (8) Figure 2 As shown, Western blotting was used to analyze the expression level of the F protein mutant in Expi293 cells. The detection antibody was His tag. In the figure, the first lane is the marker, the second lane is the control pre-fusion F protein Ds_Cav1, and the third to eighth lanes are the F protein mutants HN108-01 to HN108-06.

[0078] 2.2 Detection of expression levels of different protein variants by double-sandwich ELISA:

[0079] Based on the results of Western blotting, D25 (which specifically recognizes the F protein in the pre-fusion conformation of respiratory syncytial virus) was used as the coating antibody, and MPE8 was used as the marker antibody for detection pairing to further examine the expression levels of each mutant. The specific steps are as follows:

[0080] Antibody coating: Dilute D25 antibody to 100 ng / ml with coating buffer, add 100 μl / well to the well of the ELISA plate, and coat overnight at 4°C.

[0081] Blocking: Wash plate with 300 μl / well, wash twice and pat dry, add blocking solution 300 μl / well, and incubate at 37°C for 2 hours.

[0082] Sample addition: Wash the plate twice, add serially diluted standard and test sample, 100 μl / well, and incubate at room temperature for 1.5 h.

[0083] Primary antibody incubation: After washing the plate 5 times, add 100 μl of detection antibody MPE8 at a final concentration of 100 ng / mL to each well and incubate at room temperature for 1.5 h.

[0084] Secondary antibody incubation: Wash the plate 5 times, add 100 μl of HRP-labeled anti-IgG antibody per well, and incubate at room temperature for 1 h.

[0085] Color development: Add 100 μl of color development solution per well and react at room temperature in the dark for 15 minutes.

[0086] Reading: Turn on the microplate reader in advance. After complete color development, add 50 μl of stop solution to each well of the microplate and read the value using the microplate reader (450 nm). The results are expressed as OD values. The results are shown in Table 3.

[0087] Table 3. Results of ELISA detection of expression levels of different mutants

[0088]

[0089] Based on a comprehensive consideration of expression levels, the cysteine-substituted mutants pHN108-03, pHN108-04, pHN108-05, and pHN108-06 showed superior performance, exhibiting expression levels comparable to wild-type DS_Cav1 protein. Furthermore, with all other mutation designs remaining the same, the introduction of the 101C-146C mutation in pHN108-05 can significantly increase protein expression levels, making it suitable for further screening and evaluation.

[0090] Example 3: Preparation and stability assay of recombinant preF protein:

[0091] High protein expression:

[0092] Recombinant proteins DS_Cav1 and pHN108-05 were expressed by transient transfection of Expi293F cells using PEI40000. Expi293F cells in logarithmic growth phase were passaged at high density (2.5 × 10⁶ cells / year) one day prior to transfection. 6 Cells were seeded at a density of / ml in 1L shake flasks. On the day of transfection, the cell density was diluted to 3×10⁹ / ml. 6 Dilute the expression plasmid DNA to Opti-MEM at a concentration of / ml, then immediately add PEI 40000 transfection reagent to the mixed DNA and mix well. Incubate at room temperature for 15 min. Add the DNA-PEI complex to the cells and incubate in a CO2 shaker at 37°C, 8% CO2, and 125 rpm. On day 4 post-transfection, centrifuge at 4700 rpm for 40 min and collect the cell culture supernatant. After sterilization by 0.22 μm filtration, purify the protein using StrepII affinity chromatography.

[0093] Protein purification:

[0094] Prepare a 30 mL gravity column. Filter the cell supernatant to remove impurities and prevent column clogging. Purify the highly expressed recombinant protein using a one-step affinity chromatography method.

[0095] Column packing: Take an empty column, press the lower gasket against the bottom of the column and compact it. Rinse the gasket with deionized water, and close the lower outlet immediately after the water flows out. Suspend the resin, and add an appropriate amount of slurry to the column using a nozzle (the ratio of preservation solution to packing material is 1:1). Open the lower outlet to let the preservation solution drain. Add an appropriate amount of deionized water to rinse the column material, and close the lower outlet after the column material has drained. Install the upper gasket, ensuring that there are no gaps between the gasket and the column material. Be careful not to force the gasket to prevent damage to the column material.

[0096] Equilibration: Equilibrate with 5 column volumes of equilibration buffer to ensure the packing material is in the same buffer system as the target protein.

[0097] Combine: Take an appropriate amount of sample, suspend the well-equilibrated packing material, and transfer the sample-packing mixture into the whole sample. Combine overnight on a shaker at 4°C.

[0098] Sample loading: Add the sample to the equilibrated column. Multiple loadings can be performed to increase binding efficiency and collect the flow-through liquid.

[0099] Equilibration / washing: Wash with 10 column volumes of washing buffer to remove non-specifically bound proteins and collect the washing buffer.

[0100] Elution: Elute the target protein with 5 column volumes of elution buffer and collect the eluent in separate tubes.

[0101] Concentration and media exchange: The collected protein samples were concentrated and the media exchanged using a 30kDa ultrafiltration tube.

[0102] Quantitative analysis: After mixing the concentrated protein, quantification was performed using UV280nm. A small amount of protein was then taken for electrophoresis detection. The results are as follows: Figure 3 As shown, the remaining portions are then placed in... Store frozen at ℃.

[0103] Stability of purified protein expression levels as detected by double-antibody sandwich ELISA:

[0104] The purified protein was aliquoted into 50 μL tubes, with two tubes per group. These tubes were incubated at 4°C for 0, 7, 14, and 28 days in the dark. A double-antibody sandwich ELISA method was then used, with D25 as the coating antibody and MPE8 as the labeling antibody for detection pairing. The expression levels of pDs-Cav1 and the mutant pHN108-05 purified proteins after incubation at 4°C for different times were detected. The expression level was expressed as the concentration of the epitope-specific neutralizing antibody. The results are shown in Table 4.

[0105] Table 4. Comparison of expression levels between mutant and wild type

[0106]

[0107] SEC-HPLC detection of purified protein structural stability:

[0108] To analyze the stability of the designed protein mutant structure, this invention employs size exclusion chromatography (SEC-HPLC) to separate and detect the purified sample, calculating the relative proportion of the main peak based on the peak area. The SEC purity of the control sample pDs_Cav1 and the mutant pHN108-05 was determined after being stored at 4℃ for different times. The specific implementation method was as follows: the chromatographic column was a TSKgel G3000SWXL (7.8*300mm 5μm), the detection wavelength was 280nm, the mobile phase was phosphate buffer, the flow rate was 0.6ml / min, the injection volume was 12μg, and isocratic elution was performed for 15min. The test solution was prepared as follows: blank solution: 0.5ml of buffer was added to the injection vial for later use; 200μl of sample was added to the inner liner of the injection vial, and air bubbles were removed for later use. Tests confirmed that the mutant described in this invention has a trimer conformation, and the purified antigen chromatographic detection showed a single protein peak, meeting the purity requirements. The results of the relative proportion of trimers are shown in Table 5.

[0109] Table 5. Trimer detection results between mutant and wild type

[0110]

[0111] After comprehensively considering expression levels and structural stability, the cysteine-substituted mutant pHN108-05 showed superior performance. Compared with wild-type DS-Cav1, after storage at 4°C for 28 days, the D25 activity concentration showed no significant decrease or no decrease, and the pre-fusion conformation retention was comparable to or showed higher stability than DS-Cav1.

[0112] Example 4: Detection of mouse immune and neutralizing antibodies:

[0113] Animal immunization:

[0114] Female BALB / c mice aged 6-8 weeks were immunized and randomly divided into four groups of 6 mice each: a negative control group, an HN108-05+HA201 group, an HN108-05+HA208 group, a Ds-Cav1+HA201 group, and a Ds-Cav1+HA208 group. Specific groupings and numbering are shown in Table 6. The negative control group received PBS buffer; the test groups received either 10 μg of the target protein mixed with 25 μL of HA201 or HA208 adjuvant. Immunization was performed via intramuscular injection in the hind limb, with an injection volume of 100 μL per mouse. Each mouse was immunized twice, with an interval of 3 weeks. Two weeks after the second immunization, blood was collected from each group of mice, and the collected serum was stored at -20°C for subsequent neutralizing antibody detection.

[0115] Table 6. Grouping and numbering of immunized mice

[0116]

[0117] Neutralizing antibody testing:

[0118] The specific implementation steps are as follows: (1) After immunization, the serum is incubated in a water bath at 56℃ for 30 min; (2) The inactivated serum sample is started at an appropriate multiple and diluted 3 times in a 96-well plate with DMEM medium containing 2% FBS, for a total of 6 gradients, with 4 replicates for each sample; (3) 50-100 PFU of RSV A2 and RSV B strains are added to the wells respectively, mixed and incubated at 37℃ and 5% CO2 for 1 h; (4) The virus and serum mixture is transferred to a 96-well plate pre-inoculated with Hep-2 cells and cultured at 37℃ and 5% CO2 for 5-10 days; (5) Observe the cytopathic effect and calculate the neutralizing antibody titer using Reed-Muench. The neutralizing antibody titer is defined as the highest serum dilution with more than 50% intact Hep-2 cells.

[0119] As shown in Tables 7 and 8, compared with the negative control PBS group, the serum of mice in each experimental group induced higher levels of neutralizing antibodies two weeks after the second immunization, and the serum of mice in each experimental group three weeks after the second immunization efficiently neutralized the replication of type A and type B wild-type live viruses in in vitro cells. The geometric mean titer of neutralizing antibodies induced by the candidate antigen HN108-05 + adjuvant HA201 was comparable to that induced by HN108-05 + adjuvant HA208. HN108-05 + HA208 was slightly higher than HN108-05 + HA201, but the difference was not statistically significant. However, the geometric mean titer of neutralizing antibodies induced by both was significantly higher than that induced by DS-Cav1 + HA201 and DS-Cav1 + HA208.

[0120] Table 7. Serum neutralizing antibody titer test results (Type A)

[0121]

[0122] Table 8. Serum neutralizing antibody titer detection results (type B)

[0123]

[0124] In summary, the RSV F protein mutant HN108-05 provided by this invention can induce a higher level of neutralizing antibody titer compared to DS-Cav1, and when used as a vaccine product, it can induce cross-protection against both strains A and B.

[0125] Example 5: Co-expression of furinase protein to promote F protein expression:

[0126] Cell transfection and growth curve monitoring:

[0127] Furin protease is an accelerator of pre-F maturation. Integrating the Furin expression cassette into a vector during cell development can further increase the yield of F protein. Studies have found that, possibly due to the use of different promoters, co-expression of Furin protease and F protein can achieve higher expression levels. Based on this study and the results of mutant screening, Furin protease was co-transfected with the control protein Ds_Cav1 and the mutant pHN108-05 into Expi293 cells. Specific implementation schemes are shown in Table 9.

[0128] Table 9. Co-expression furinase implementation plan

[0129]

[0130] 2. Effect of co-expression of furin protease on the expression level of recombinant F protein:

[0131] 2.1 Western Blot analysis of F protein expression in co-expression supernatant samples:

[0132] (1) After culturing for 3-4 days, the cell culture supernatant was collected by centrifugation at 4700rpm for 40min at 4℃ and then filtered through a 0.22um filter membrane for sterilization.

[0133] (2) Add β-mercaptoethanol and loading buffer to the sample, heat denature at 100℃ for 10 min, load 10 μl of cell supernatant electrophoresis sample per well, load 3 μg of purified DS_Cav1 protein from the control group, electrophoresis at 120V for 60 min.

[0134] (3) After electrophoresis, the protein was transferred to the PVDF membrane by dry transfer and blocked with TBS solution containing 5% skim milk powder for 2 hours.

[0135] (4) The anti-RSV F protein monoclonal antibody was diluted with TBS buffer at a ratio of 1:2000. The PDVF membrane was transferred to the diluted antibody and incubated overnight at 4°C.

[0136] (5) Clean with TBST three times every other day, 5 minutes each time.

[0137] (6) Dilute HRP-Goat anti Rabbit secondary antibody with TBS containing 5% skim milk powder, transfer PVDF membrane into the diluted secondary antibody, and incubate on a shaker at room temperature for 1 hour.

[0138] (7) Wash with TBST three times, 10 min each time, add substrate developing solution, develop in the dark for 10 min and take pictures.

[0139] (8) Figure 4As shown, Western blotting was used to analyze the expression level of the F protein mutant in Expi293 cells. The antibody used for detection was anti-RSV F protein. In the figure, the first lane represents the 235 kDa protein marker, the second lane represents the negative control empty vector pCAGGS, the third lane represents the purified control protein Ds_Cav1, and the fourth to seventh lanes represent the expression of HN108-05 and Ds_Cav1 with and without Furin protease.

[0140] 3. Detection of F protein expression in co-expression supernatant samples using double-antibody sandwich ELISA:

[0141] Based on the results of Western Blot, D25 (the D25 antibody can specifically recognize the F protein in the pre-fusion conformation of respiratory syncytial virus) was used as the coating antibody and MPE8 was used as the labeling antibody to detect pairing. The changes in the expression level of pre-F protein after co-expression of control sample DS_Cav1 and mutant pHN108-05 with Furin were further detected. The results are shown in Table 10.

[0142] Table 10 Expression levels of mutants and wild-type after co-transfection with Furin:

[0143] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

[0144] While specific embodiments of the invention have been described in detail by way of example, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of the invention. It should also be understood that various modifications can be made to the embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims

1. A respiratory syncytial virus (RSV) F protein, characterized in that, The amino acid sequence of the F protein is shown in SEQ ID NO:

6.

2. A recombinant nucleic acid encoding the respiratory syncytial virus F protein as described in claim 1.

3. An expression vector comprising the recombinant nucleic acid of claim 2.

4. A host cell comprising the recombinant nucleic acid as described in claim 2 or the expression vector as described in claim 3.

5. The host cell as described in claim 4, characterized in that, The host cell is selected from Escherichia coli, yeast cells, insect cells, or mammalian cells.

6. A method for preparing the respiratory syncytial virus F protein as described in claim 1, characterized in that, The method includes: S1: Culture the host cells as described in claim 4 or 5 under conditions suitable for the expression of the respiratory syncytial virus F protein; S2: Collect the expression product and purify the expression product to obtain the respiratory syncytial virus F protein.

7. The use of the respiratory syncytial virus F protein as described in claim 1, or the respiratory syncytial virus F protein prepared by the preparation method as described in claim 6, in the preparation of a vaccine to prevent respiratory syncytial virus infection.

8. A vaccine for preventing respiratory syncytial virus infection, comprising the respiratory syncytial virus F protein as described in claim 1, or the respiratory syncytial virus F protein prepared by the method described in claim 6, and a pharmaceutically acceptable carrier or excipient.

9. The vaccine as described in claim 8, characterized in that, The vaccine further includes an adjuvant, which includes at least one of aluminum adjuvant, squalene, tocopherol, MPL, CpG, and QS-21.

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