Respiratory syncytial virus F protein mutant and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a respiratory syncytial virus F protein mutant and application thereof. The respiratory syncytial virus F protein mutant is characterized in that at least one pair of amino acid residues in an F1 subunit and / or an F2 subunit of F protein polypeptide are substituted by cysteine, and / or an F protein signal peptide part is substituted by a human immunoglobulin light chain signal peptide, and / or alanine A at the 107 site of a multi-base sequence is mutated into arginine R, lysine K at the 134 site is mutated into arginine R, and the amino acid residues in the F1 subunit and / or the F2 subunit of the F protein polypeptide are mutated into arginine. According to the invention, more neutralizing antibody epitopes can be exposed, the stable pre-fusion conformation is maintained, effective neutralizing antibody reaction and antibody binding reaction to the RSV subtypes A and B can be ensured, and the vaccine is suitable for various vaccine forms taking the RSV F protein as the antigen.

Description

Technical Field

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

[0002] This invention relates to the field of biomedicine, and particularly to a respiratory syncytial virus F protein mutant and its application in the preparation of a respiratory syncytial virus vaccine. Background Art

[0003] Respiratory Syncytial Virus is one of the important pathogens that cause severe lower respiratory tract infections in infants and young children worldwide. According to statistics, the number of children under 5 years old infected with RSV globally is as high as 34 million. Since there is no lasting immunity after RSV infection, repeated infections can occur. Almost 100% of infants and young children are infected more than once within 2 years, resulting in 66,000 - 199,000 deaths. In the analysis of single-pathogen infection factors for all-cause deaths in children aged 1 month to 1 year, RSV infection causes a fatality rate of up to 6.7% in children under 1 year old, second only to malaria, causing a serious disease burden globally. In addition to infants and young children, adults and the elderly with weakened immune functions are also high-risk groups for RSV infection. 3% - 10% of adult colds are caused by RSV every year, and the disease burden caused by RSV infection in the elderly population is comparable to that caused by colds in the non-epidemic season. After infection with RSV, it will lead to increased mucus secretion and inflammation, causing various serious complications such as heart failure and secondary bacterial pneumonia. RSV is also an important nosocomial infection pathogen, especially highly harmful to premature infants, infants with congenital heart disease, bronchopulmonary dysplasia, and all patients with immunosuppression, having a huge impact on human health, global healthcare, and the economy. Therefore, the WHO lists RSV vaccines as one of the vaccines that should be prioritized for development globally in the 21st century, and developing safe and effective RSV vaccines is an urgent current need.

[0004] RSV belongs to the genus Pneumovirus of the Paramyxoviridae family and is a single-stranded negative-sense RNA virus. Its genome is 15.2 kb in length and transcribes ten genes, encoding a total of 10 major proteins, including 3 transmembrane proteins (G, F, and SH), 2 matrix proteins (M and M2), 3 nucleocapsid proteins (N, P, and L), and 2 non-structural proteins (NS1 and NS2). Among them, the attachment protein G and the fusion protein F are the two most important viral proteins that stimulate the body to produce protective antibodies against RSV. The G protein mediates virus binding and determines the antigenic diversity of RSV. According to its antigenic differences, RSV is divided into two subtypes, A and B. The F protein is mainly responsible for virus-host cell membrane fusion. Its protein sequence is highly conserved among different subtypes and plays an important role in inducing immune protection and high-level serum neutralizing antibodies. The neutralizing antibodies induced by the F protein can simultaneously inhibit the infection of RSV viruses of both subtypes A and B and can induce a cellular immune CD8+ cytotoxic T cell response to clear pathogens in the body. Therefore, the F protein is an important antigen target for the development of RSV vaccines.

[0005] The F protein of RSV is the main target protein of neutralizing antibodies and belongs to type I integral membrane proteins. Its precursor protein F0 consists of 574 amino acids, and three F0s form trimers through hydrophobic interactions. During the process of virus invasion-mediated membrane fusion, the precursor F0 is cleaved by Furin protease and releases a short peptide P27 (109 - 127aa) containing 27 amino acids. The remaining two segments, F2 and F1, are connected by two disulfide bonds to form the mature F protein, which is displayed on the cell membrane or virion surface by budding. At this time, the F protein is in a high-energy metastable state and is very unstable, known as pre-F. After infecting cells, it will spontaneously change to the post-fusion conformation, a low-energy state. A large amount of preclinical and clinical data show that the sites with high neutralizing activity mainly exist in the pre-fusion conformation. Vaccines based on the pre-fusion conformation of RSV F can stimulate the body to produce higher levels of virus-neutralizing antibodies. Although RSV vaccines based on pre-F show good application prospects, maintaining the pre-fusion conformation of the F protein has become a key and difficult point in vaccine research and development. So far, only two recombinant protein vaccines targeting the RSV F protein, GSK and Pfizer, have been approved for marketing globally. Both products use the pre-fusion conformation of the F protein and are used for the population aged 60 and above in the United States, but there are no related prophylactic vaccine products on the market in China.

[0006] Therefore, developing a pre-fusion conformation F protein antigen with good safety, high expression level, and stability has become a technical problem that needs to be urgently solved by those skilled in the art. Summary of the Invention

[0007] To solve the technical problems existing in the prior art, the present invention has developed a pre-fusion F protein mutant of respiratory syncytial virus. This mutant protein can have a stable pre-fusion conformation and a high expression level, and can form a stable trimer, thus being useful for the treatment of RSV infection. In 2013, the McLellan team et al. first developed the first F protein with a stable pre-fusion conformation "DS-Cav1" through point mutations: an intramolecular disulfide bond was obtained by point mutations of S155C and S290C within the F1 fragment, and at the same time, point mutations of S190F and V207L were added to enhance the binding activity of key neutralizing epitopes (DOI: 10.1126 / science.1243283). This protein was confirmed to be able to elicit significantly higher levels of neutralizing antibody titers. Currently, a stable pre-fusion conformation F protein vaccine developed based on structural biology has also entered the clinical stage. Therefore, the recombinant RSV F protein described in the present invention introduces mutations on the basis of the amino acid sequence of DS_Cav1 (referred to as wild type), and the mutations include amino acid substitutions, deletions, and insertions.

[0008] In a first aspect of the present invention, there is provided a respiratory syncytial virus F protein, the F protein comprising at least one mutation selected from the group consisting of: (a) at least one pair of amino acid residues is replaced by cysteine; (b) the signal peptide portion of the F protein is replaced by the signal peptide of human immunoglobulin light chain; (c) the sites of at least two polybasic sequences are replaced.

[0009] In some embodiments, at least one pair of amino acid residues in the F1 subunit and / or F2 subunit of the F protein polypeptide is replaced by cysteine.

[0010] In some embodiments, the cysteine substitution results in the formation of a non-natural disulfide bond connection between the F1 subunit and the F2 subunit of the F protein, and the non-natural disulfide bond includes disulfide bonds other than Cys69-Cys212 and Cys37-Cys439 formed between the F1 subunit and the F2 subunit.

[0011] In the pre-fusion conformation of the RSV F protein, β2 and β4 strands are close to each other, promoting the spatial folding of two discontinuous epitopes of the 0 epitope; by adding non-natural disulfide bonds, the spatial position between β2 and β4 strands can be relatively stabilized, so as to enhance the structural stability between the pre-fusion F1 subunit and F2 subunit, prevent conformational changes in the heptapeptide repeat regions HRA and HRB, well stabilize the Pre-F protein structure, and at the same time maintain or enhance the binding activity of important sites such as the 0 epitope and V epitope, and improve the pre-fusion F protein specific immunogenicity of the mutant antigen.

[0012] In some embodiments, the F protein comprises at least one pair of cysteine mutations among 102C+147C, 102C+149C, 102C+150C, 101C+147C, 101C+146C, 100C+145C, and the mutation sites refer to the sites of the wild-type respiratory syncytial virus F protein amino acid sequence shown in SEQ ID NO:01.

[0013] In some embodiments, the F protein signal peptide portion replaced by the human immunoglobulin light chain signal peptide comprises at least the 1st to 25th amino acid residues of the sequence shown in SEQ ID NO:1.

[0014] In some embodiments, the human immunoglobulin light chain signal peptide is as shown in SEQ ID NO:2.

[0015] The wild-type F protein first forms the F0 precursor protein, and after cleavage by furin protease, a 27-amino acid polypeptide pep27 is released, and the remaining two segments F2 and F1 are connected by two disulfide bonds to form the mature F protein - pre-F, which is displayed on the cell membrane or virion surface by budding.

[0016] In some embodiments, in order to improve the removal efficiency of the p27 polypeptide inside the F protein, two polybasic sites on the F protein are replaced.

[0017] In some embodiments, the p27 polypeptide inside the F protein comprises at least the 110th to 136th amino acid residues of the wild-type respiratory syncytial virus F protein.

[0018] In some embodiments, compared with the wild-type respiratory syncytial virus F protein amino acid sequence shown in SEQ ID NO:1, the alanine A at position 107 in the polybasic sequences RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0019] In some embodiments, the F protein comprises the cysteine mutation 102C+147C, the F protein signal peptide portion is replaced by the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence shown in SEQ ID NO:1, the alanine A at position 107 in the polybasic sequences RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0020] In some embodiments, the F protein comprises cysteine mutations 102C+149C, the signal peptide portion of the F protein is replaced with the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, alanine A at position 107 in the polybasic sequences RARR and KKRKRR of the F protein is mutated to arginine R, and lysine K at position 134 is mutated to arginine R.

[0021] In some embodiments, the F protein comprises cysteine mutations 102C+150C, the signal peptide portion of the F protein is replaced with the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, alanine A at position 107 in the polybasic sequences RARR and KKRKRR of the F protein is mutated to arginine R, and lysine K at position 134 is mutated to arginine R.

[0022] In some embodiments, the F protein comprises cysteine mutations 101C+147C, the signal peptide portion of the F protein is replaced with the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, alanine A at position 107 in the polybasic sequences RARR and KKRKRR of the F protein is mutated to arginine R, and lysine K at position 134 is mutated to arginine R.

[0023] In some embodiments, the F protein comprises cysteine mutations 101C+146C, the signal peptide portion of the F protein is replaced with the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, alanine A at position 107 in the polybasic sequences RARR and KKRKRR of the F protein is mutated to arginine R, and lysine K at position 134 is mutated to arginine R.

[0024] In some embodiments, the F protein comprises cysteine mutations 100C+145C, the signal peptide portion of the F protein is replaced with the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, alanine A at position 107 in the polybasic sequences RARR and KKRKRR of the F protein is mutated to arginine R, and lysine K at position 134 is mutated to arginine R.

[0025] In some embodiments, the F protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 3-8.

[0026] In some embodiments, the F protein comprises a protease cleavage site sequence, such as an HRV 3C protease cleavage site (LEVLFQGP) or a protein tag, such as a 6×His-tag (HHHHHH) and a Strep tag-II tag (WSHPQFEK), and these sequences are not essential for the function of the RSV F protein (e.g., inducing an immune response).

[0027] 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.

[0028] In some embodiments, the recombinant nucleic acid is codon-optimized for expression in a selected prokaryotic or eukaryotic host cell.

[0029] Another aspect of the present invention provides an expression vector comprising the recombinant nucleic acid as described above.

[0030] Another aspect of the present invention provides a host cell comprising the recombinant nucleic acid as described above or the expression vector as described above.

[0031] In some embodiments, the host cell is selected from Escherichia coli, yeast cells, insect cells or mammalian cells. Preferably, the mammalian cells are selected from ExpiCHO, VERO, Expi293, etc.

[0032] Another aspect of the present invention provides a method for preparing the respiratory syncytial virus F protein as described above, the method comprising: S1: Culturing the host cell as described above under conditions suitable for the expression of the respiratory syncytial virus F protein; S2: Collecting the expression product and purifying the expression product to obtain the respiratory syncytial virus F protein.

[0033] Another aspect of the present invention also provides the use of the respiratory syncytial virus F protein as described above in the preparation of a vaccine for preventing respiratory syncytial virus infection.

[0034] 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 as described above, and a pharmaceutically acceptable carrier or excipient.

[0035] 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.

[0036] In some embodiments, the carrier or excipient comprises a buffering agent. 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 regulators (such as phosphate buffer), surfactants (such as cationic, anionic or non-ionic surfactants), adjuvants, solubilizers, stabilizers, media for accommodating or administering therapeutic agents, and any combination thereof. Those skilled in the art can select suitable excipients and carriers to produce a formulation suitable for administration to a subject via a selected route of use. A pharmaceutically acceptable carrier can be a sterile liquid, such as water and oils, including oils derived from petroleum, animals, plants or synthesis. Suitable excipients include, but are not limited to, glycerol, polyethylene glycol, and salts related to calcium ions, magnesium ions, zinc ions and other divalent cations, etc.

[0037] In some embodiments, the vaccine further comprises an adjuvant.

[0038] In some embodiments, the adjuvant comprises at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, CpG, and QS-21.

[0039] Generally, when selecting an adjuvant, it should be able to enhance the Th1-biased immune response in the subject or group of subjects receiving the vaccine administration, and be safe and effective in the subject or group of subjects.

[0040] Optionally, the vaccine may further comprise at least one other antigen of a pathogenic organism different from RSV. For example, the pathogenic organism is a virus different from RSV, such as varicella zoster virus, human papillomavirus, hepatitis B virus, coronavirus or influenza virus. Or the pathogenic organism can be a bacterium, such as diphtheria, tetanus or pneumococcus, etc.

[0041] The present invention also provides a method for preparing the above-mentioned vaccine for preventing RSV infection, specifically including: separately packaging or fully mixing the purified F protein and the adjuvant in proportion.

[0042] In some embodiments, the method further comprises additional steps such as gene synthesis, expression vector construction, freeze-drying of purified protein, etc. The expressed recombinant RSV F protein can be recovered and purified from recombinant cell cultures by any one or several of many methods well known in the art, including ammonium sulfate precipitation, filtration, ultrafiltration, affinity chromatography, anion / cation exchange chromatography, hydrophobic interaction chromatography, etc.

[0043] Compared with the prior art, the advantages and positive effects of the present invention are: By substituting and deleting various amino acids in the wild-type RSV F protein, the present invention obtains a pre-fusion conformation of the RSV F protein that can expose more neutralizing antibody epitopes, ensuring an effective neutralizing antibody response and antibody binding response to RSV subtypes A and B while maintaining a stable pre-fusion conformation. Moreover, the present invention uses the recombinant RSV F protein as an immunogen in combination with an adjuvant, obtaining a stronger immune induction effect, not only improving the humoral immune response but also strongly stimulating Th1-type immunity, greatly enhancing the immunogenicity of the RSV antigen. The mutation methods disclosed in the present invention are applicable to other virus strains of human RSV; applicable to various vaccine forms using the RSV F protein as an antigen, such as recombinant protein vaccines, nucleic acid vaccines, virus-like particle vaccines, and vector vaccines, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for the description of the embodiments.

[0045] Figure 1 It is the plasmid map of pCAGGS.

[0046] Figure 2 It is the result of detecting the expression of the F protein mutant by immunoblotting (Western blot).

[0047] Figure 3 It is the analysis result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified proteins of the F protein mutant HN108-05 and the control protein Ds_Cav1 under reducing conditions.

[0048] Figure 4 It is the result of detecting the co-expression of the F protein and Furin protease by immunoblotting (Western blot). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] The following further elaborates on the present invention in combination with the specific embodiments: It is convenient to better understand the present invention, but does not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from regular biochemical reagent stores unless otherwise specified.

[0050] Example 1: Design and gene synthesis of mutants that introduce disulfide bonds to stabilize the pre-fusion conformation of the F protein by cysteine substitution: By observing the spatial structure of the pre-fusion F protein of respiratory syncytial virus, amino acid mutations that can stabilize the pre-fusion conformation are designed according to the principles of structural biology. Using the amino acid sequence (SEQ ID No. 01) of the wild-type respiratory syncytial virus pre-fusion F protein (Ds_Cav1) as a template, disulfide bonds are introduced by cysteine substitution to enhance the structural stability between the pre-fusion F1 subunit and F2 subunit and prevent conformational changes in the heptapeptide repeat regions HRA and HRB. At the same time, the binding activity of important sites such as the Φ epitope and V epitope is maintained or enhanced to improve 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 (1-25aa) is replaced with the human immunoglobulin light chain signal peptide "MRVPAQLLGLLLLWLRGARC (SEQ ID No. 02)". To improve the removal efficiency of the internal p27 polypeptide (110-136aa) of the F protein, two polybasic sites (RARR109 and KKRKRR134) on the F protein are replaced with (RRRR107 and KKRRRR134). Based on the original sequence DS_Cav1, a total of 6 protein variants are designed, and the amino acid sequences are shown in SEQ ID NO:3, 4, 5, 6, 7, and 8, respectively.

[0051] Submit the nucleotide sequences of the F protein variants designed according to the following table to Beijing Tsingke Biotechnology Co., Ltd. Codon optimization is performed according to the host Expi293F cells to determine the nucleic acid sequences, and they are inserted into the eukaryotic expression pCAGGS (plasmid map see Figure 1 , purchased from Beijing Tsingke Biotechnology Co., Ltd., and the sequence is shown in SEQID NO.9) sequence for full gene synthesis.

[0052] Table 1 Design of mutants with disulfide bond introduction by cysteine substitution and polybasic site replacement. The detailed sequences are shown in SEQ ID No.03-No.08.

[0053] Table 1 Design of mutants with disulfide bond introduction by cysteine substitution and polybasic site replacement

[0054] Example 2: Recombinant F protein expression and preliminary screening: 1. Cell transfection and growth curve monitoring: The expression vector synthesized by genetic engineering technology is subjected to clone transformation and large-scale plasmid extraction, and then used to transfect Expi293 cells for recombinant protein expression. One day before transfection, Expi293 cells in the logarithmic growth phase are passaged at high density, and the viable cell density is 2 - 3×106 / mL. After the cells grow overnight, the cell density is diluted to 3×106 / mL. 2.5 mL of cell suspension is taken from each well for transfection. 2 μL of DNA is used per well, diluted with Opti-MEM medium and gently mixed, and then 8 μL of PEI40000 is gently added dropwise to the diluted DNA, and left standing at room temperature for 10 - 20 min to prepare the DNA complex. After the incubation, the DNA complex is added dropwise to the cell solution to be transfected, and transferred to a shaker at 37°C with 8% CO2 and cultured at a rotation speed of 225 rpm. 500 μL of feed is added 18 - 22 h after transfection and the culture is continued. Table 2 shows the changes in cell viability and density during transfection of different protein variants.

[0055] Table 2 Changes in cell viability and density during transfection of different protein variants

[0056] Expression identification of different F protein mutants: 2.1 Detection of the expression of different protein variants by Western Blot: (1) On the 3rd day of culture, the cell culture supernatant is collected by centrifugation at 4700 rpm for 40 min at 4°C and filtered through a 0.22-μm filter membrane for sterilization.

[0057] (2) β-mercaptoethanol and Loading buffer are added to the sample, and heat-denatured at 100°C for 10 min. The loading amount of the cell supernatant electrophoresis sample is 25 μL / well, and 5 μg of the purified DS_Cav1 protein in the control group is loaded. Electrophoresis is carried out at a voltage of 120 V for 60 minutes.

[0058] (3) After electrophoresis, the protein is transferred to a PVDF membrane by dry transfer method and blocked with TBS solution containing 5% skim milk powder for 2 hours.

[0059] (4) The His mouse monoclonal antibody is diluted with TBS buffer at 1:2000, and the PDVF membrane is transferred to the diluted antibody and incubated overnight at 4°C.

[0060] (5) The next day, wash three times with TBST, 5 min each time.

[0061] (6) Dilute the HRP-Goat anti Mouse secondary antibody with TBS containing 5% skim milk powder, transfer the PVDF membrane to the diluted secondary antibody, and incubate on a shaker at room temperature for 1 h.

[0062] (7) Wash three times with TBST for 10 min each time. Add the substrate chromogenic solution, develop color for 10 min in the dark and take pictures.

[0063] (8) As Figure 2 shown, Western Blot was used to analyze the expression levels of F protein mutants in Expi293 cells. The detection antibody was His tag. The first lane in the figure was Marker, the second lane was the pre-fusion F protein Ds_Cav1 as a control, and the third to eighth lanes were F protein mutants HN108-01 to HN108-06.

[0064] 2.2 Detection of the expression levels of different protein variants by double sandwich ELISA: Based on the results of Western Blot, D25 (the D25 antibody can specifically recognize the pre-fusion conformation of the F protein of respiratory syncytial virus) was used as the coating antibody, and MPE8 was used as the labeled antibody for pairing to further detect the expression levels of each mutant. The specific steps are as follows: Antibody coating: Dilute the D25 antibody to 100 ng / ml with the coating solution, add 100 μl / well to the wells of the enzyme-linked immunosorbent assay (ELISA) plate, and coat overnight at 4°C.

[0065] Blocking: Wash the plate with 300 μl / well, wash 2 times and pat dry, add 300 μl / well of the blocking solution, and incubate at 37°C for 2 hours.

[0066] Sample addition: Wash the plate 2 times, add the serially diluted standards and samples to be tested, 100 μl / well, and incubate at room temperature for 1.5 h.

[0067] Primary antibody incubation: After washing the plate 5 times, add the detection antibody MPE8 with a final concentration of 100 ng / mL, 100 μl / well, and incubate at room temperature for 1.5 h.

[0068] Secondary antibody incubation: Wash the plate 5 times, add 100 μl / well of the HRP-labeled anti-IgG antibody, and incubate at room temperature for 1 h.

[0069] Color development: Add 100 μl / well of the chromogenic solution, and react at room temperature in the dark for 15 minutes.

[0070] Reading: Turn on the microplate reader in advance. After complete color development, add 50 μl / well of the stop solution to the reaction wells of the ELISA plate, and read the values on the microplate reader (450 nm). The results are expressed as OD values. The results are shown in Table 3: Table 3 Results of ELISA detection of the expression levels of different mutants

[0071] After comprehensively considering the expression level, the cysteine-substituted mutants pHN108-03, pHN108-04, pHN108-05, and pHN108-06 showed better performance and had an expression level comparable to that of the wild-type DS_Cav1 protein. Moreover, under the condition that the rest of the mutation designs were the same, pHN108-05 with the 101C-146C mutation could improve the protein expression level to a certain extent and could be used for the next round of screening and evaluation.

[0072] Example 3: Preparation of recombinant preF protein and detection of protein stability: Large-scale protein expression: The recombinant proteins DS_Cav1 and pHN108-05 were expressed by transient transfection using PEI40000 in Expi293F cells. One day before transfection, the exponentially growing Expi293F cells were subcultured at a high density and inoculated into a 1L shake flask at a cell density of 2.5×10 6 / ml. On the day of transfection, the cell density was diluted to 3×10 6 / ml. The expression plasmid DNA was diluted with Opti-MEM, and then the transfection reagent PEI40000 was immediately added to the mixed DNA and mixed well. The mixture was allowed to stand at room temperature for 15 min. The DNA-PEI complex was added to the cells, and the cells were cultured in a carbon dioxide shaker at 37°C, 8% CO2, and 125 rpm. On the 4th day after transfection, the cells were centrifuged at 4700 rpm for 40 min, and the cell culture supernatant was collected. After filtering and sterilizing with a 0.22-μm filter, the protein was purified by StrepII affinity chromatography.

[0073] Protein purification: Prepare a 30-mL gravity column, and filter the cell supernatant to remove impurities to prevent column clogging. The recombinant protein expressed in large quantities was purified using one-step affinity chromatography.

[0074] Column packing: Take an empty column, press the lower gasket at the bottom of the column and compact it. Rinse the gasket with deionized water. Immediately close the lower outlet after the water flows out from the lower outlet; suspend the resin, and use a pipette to take an appropriate amount of slurry and add it to the column (the ratio of the storage solution to the packing is 1:1). Open the lower outlet to drain the protective liquid; add an appropriate amount of deionized water to wash the column packing, and close the lower outlet after the column packing has drained; install the upper gasket, ensuring that there is no gap between the gasket and the column packing. Note that do not apply force to the gasket to prevent damage to the column packing.

[0075] Equilibration: Equilibrate with 5 column volumes of equilibration buffer to bring the packing into the same buffer system as the target protein.

[0076] Binding: Take an appropriate amount of the sample and suspend the equilibrated packing, and transfer the sample-packing mixture to all the samples. Incubate overnight with shaking at 4°C.

[0077] Loading: Add the sample to the equilibrated column. Multiple loadings can be performed during this process to increase the binding efficiency and collect the flow-through fraction.

[0078] Equilibration / Washing: Wash with 10 column volumes of washing buffer to remove non-specifically bound proteins and collect the washing fraction.

[0079] Elution: Elute the target protein with 5 column volumes of elution buffer and collect in separate tubes.

[0080] Concentration and Buffer Exchange: Concentrate and exchange the buffer of the collected protein sample using a 30 kDa ultrafiltration tube.

[0081] Quantification: Mix the concentrated protein and quantify it at UV280 nm. Take a small amount of the protein for SDS-PAGE analysis. The results are shown as Figure 3 follows. Aliquot the remaining protein and store it at °C.

[0082] Detection of the stability of the purified protein expression level by double antibody sandwich ELISA: Aliquot the purified protein into 50 μl / tube. Take 2 tubes for each group and place them in the dark at 4°C for 0 d, 7 d, 14 d, and 28 d. Then, use the double antibody sandwich ELISA method with D25 as the coating antibody and MPE8 as the labeled antibody for detection. Detect the changes in the expression levels of the purified proteins pDs-Cav1 and the mutant pHN108-05 after incubation at 4°C for different times. Among them, the expression level is represented by the activity concentration detected by the epitope-specific neutralizing antibody. The results are shown in Table 4: Table 4 Comparison of the expression levels between the mutant and the wild type

[0083] Detection of the structural stability of the purified protein by SEC-HPLC: To analyze the stability of the designed protein mutant structure, the present invention uses size exclusion chromatography (SEC-HPLC) to separate and detect the purified samples, and calculates the relative proportion of the main peak according to the peak area of the chromatographic peak. The SEC purity of the purified samples of the control sample pDs_Cav1 and the mutant pHN108-05 was measured after being placed at 4°C for different times. The specific implementation method is as follows: The chromatographic column is TSKgel G3000SWXL (7.8*300mm 5μm), the detection wavelength is 280nm, the mobile phase is phosphate buffer solution, the flow rate is 0.6ml / min, the injection volume is 12μg, and isocratic elution is carried out for 15min. The preparation of the test sample solution is as follows: Blank solution: Take 0.5ml of Buffer into the injection vial for standby; Take 200μl of the sample and add it to the inner liner tube in the injection vial, and remove the bubbles for standby. It is confirmed by detection that the mutant described in the present invention is in a trimer conformation, and the antigen chromatographic detection after purification shows a single protein peak, meeting the purity requirements. The results of detecting the relative proportion of the trimer are shown in Table 5.

[0084] Table 5 Detection results of trimers of mutants and wild types

[0085] After comprehensively considering the expression level and structural stability, the cysteine-substituted mutant pHN108-05 shows better performance. Compared with the wild type DS-Cav1, the detected active concentration of D25 does not decrease significantly or does not decrease after being stored at 4°C for 28 days, and the pre-fusion conformation retention rate is equivalent to or has higher stability than that of DS-Cav1.

[0086] Example 4: Mouse immunization and neutralizing antibody detection: Animal immunization: Female BALB / C mice aged 6-8 weeks were selected for immunization, randomly grouped, 6 mice / group, which were negative control group, HN108-05+HA201 group, HN108-05+HA208 group, Ds-Cav1+HA201 group, Ds-Cav1+HA208 group respectively. The specific grouping and numbering are shown in Table 6. The animals in the negative control group were given PBS buffer solution; the test sample groups were respectively given 10μg of the target protein formulated with 25μL of HA201 or HA208 adjuvant. The immunization method was intramuscular injection in the hind limbs, and the injection volume was 100ul / dose / animal. Each mouse was immunized 2 times, and the immunization interval was 3 weeks. Blood was collected from each group of mice two weeks after the second immunization, and the collected serum was stored at -20°C for subsequent neutralizing antibody detection.

[0087] Table 6 Grouping and numbering of immunized mice

[0088] Neutralizing antibody detection: The specific implementation steps are as follows: (1) Incubate the post-immunization serum in a water bath at 56 °C for 30 min; (2) Starting from the inactivated serum sample at an appropriate multiple, dilute it in a 96-well plate with DMEM medium containing 2% FBS in a 3-fold gradient for a total of 6 gradients, with 4 replicates for each sample; (3) Add 50 - 100 PFU of RSV A2 and RSV B strains to each well respectively, mix well, and incubate at 37 °C under 5% CO₂ for 1 h; (4) Transfer the virus-serum mixture to a 96-well plate pre-inoculated with Hep-2 cells, and culture it at 37 °C and 5% CO₂ for 5 - 10 days; (5) Observe the cytopathic effect, and use Reed-Muench to calculate the neutralizing antibody titer. The neutralizing antibody titer is defined as the highest serum dilution with more than 50% intact Hep-2 cells.

[0089] As shown in the results of Table 7 and Table 8, compared with the negative control PBS group, the sera of each experimental group of mice two weeks after the second immunization could induce a relatively high level of neutralizing antibodies, and the sera of each experimental group of mice three weeks after the second immunization could efficiently neutralize the replication of wild-type live viruses of type A and type B in vitro cells. Among them, the geometric mean titer of neutralizing antibodies induced by the candidate antigen HN108-05 + adjuvant HA201 of the present invention was comparable to that of HN108-05 + adjuvant HA208. HN108-05 + HA208 was slightly higher than HN108-05 + HA201, but there was no significant difference. However, the geometric mean titers of neutralizing antibodies induced by both were significantly higher than those of DS-Cav1 + HA201 and DS-Cav1 + HA208.

[0090] Table 7 Detection results of serum neutralizing antibody titers (type A)

[0091] Table 8 Detection results of serum neutralizing antibody titers (type B)

[0092] In summary, compared with DS-Cav1, the RSV F protein mutant HN108-05 provided by the present invention can induce a higher level of neutralizing antibody titer, and when used as a vaccine product, it can induce cross-protection against both A / B strains.

[0093] Example 5: Co-expression of furin protein promotes F protein expression: Cell transfection and growth curve monitoring: Furin protease is an accelerator for the maturation of pre-F. Integrating the Furin expression cassette into the vector during cell development can further increase the yield of F protein. The research found that it might be due to the use of different promoters. If Furin protease and F protein are co-expressed, a higher level of expression can be achieved. Based on this research and the screening results of the above mutants, Furin protease was co-transfected with the control protein Ds_Cav1 and the mutant pHN108-05 into Expi293 cells respectively. The specific implementation plan is shown in Table 9: Table 9 Implementation plan for co-expression of furin

[0094] 2. Effect of co-expression of furin protease on the expression level of recombinant F protein: 2.1 Detection of the expression of F protein in the co-expression supernatant samples by Western Blot: (1) Cultivate until the 3rd - 4th day, centrifuge the cell culture supernatant at 4700 rpm for 40 min at 4°C, and filter and sterilize it with a 0.22 μm filter membrane.

[0095] (2) Add β-mercaptoethanol and Loading buffer to the sample, heat-denature it at 100°C for 10 min. The loading volume of the cell supernatant electrophoresis sample is 10 μl / well, and 3 μg of the purified DS_Cav1 protein in the control group is loaded. Electrophoresis is carried out at a voltage of 120 V for 60 minutes.

[0096] (3) After electrophoresis, transfer the protein to the PVDF membrane by dry transfer method, and block it with TBS solution containing 5% skim milk powder for 2 hours.

[0097] (4) Dilute the anti-RSV F protein monoclonal antibody with TBS buffer at a ratio of 1:2000, transfer the PDVF membrane to the diluted antibody, and incubate it overnight at 4°C.

[0098] (5) Wash it three times with TBST the next day, 5 min each time.

[0099] (6) Dilute the HRP-Goat anti Rabbit secondary antibody with TBS containing 5% skim milk powder, transfer the PVDF membrane to the diluted secondary antibody, and incubate it on a shaker at room temperature for 1 h.

[0100] (7) Wash it three times with TBST, 10 min each time, add the substrate chromogenic solution, develop the color in the dark for 10 min and take pictures.

[0101] (8) As Figure 4As shown, Western Blot was used to analyze the expression level of the F protein mutant in Expi293 cells. The detection antibody was the anti-RSV F protein. The first lane in the figure was the 235KDa protein Marker, the second lane was the negative control empty pCAGGS, the third lane was the purified protein Ds_Cav1 of the control sample, and the fourth to seventh lanes were the expression of HN108-05 and Ds_Cav1 with and without Furin protease.

[0102] 3. Detection of the expression of the F protein in the co-expression supernatant sample by double antibody sandwich ELISA: Based on the results of Western Blot, using D25 (the D25 antibody can specifically recognize the pre-fusion conformation of the respiratory syncytial virus F protein) as the coating antibody and MPE8 as the labeled antibody for detection pairing, the changes in the expression levels of the pre-F protein after co-expression of the control sample DS_Cav1 and the mutant pHN108-05 with Furin were further detected. The results are shown in Table 10.

[0103] Table 10 Expression levels of mutants and wild types after co-expression with Furin:

[0104] The above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

[0105] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration and not for limiting the scope of the present invention. Those skilled in the art should also understand that various modifications can be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A respiratory syncytial virus F protein, characterized in that, The F protein includes at least one pair of cysteine mutations of 101C+147C and 101C+146C, and the mutation sites refer to the sites of the wild-type respiratory syncytial virus F protein amino acid sequence shown in SEQ ID NO:

01.

2. The respiratory syncytial virus F protein according to claim 1, characterized in that, The signal peptide of the human immunoglobulin light chain replaces the signal peptide of the F protein, and the signal peptide of the F protein includes at least the amino acid residues at positions 1 to 25 of the sequence shown in SEQ ID NO:

1.

3. The respiratory syncytial virus F protein according to claim 1, characterized in that, The signal peptide of the human immunoglobulin light chain is as shown in SEQ ID NO:

2.

4. The respiratory syncytial virus F protein according to any one of claims 1-3, characterized in that, Compared with the wild-type respiratory syncytial virus F protein amino acid sequence shown in SEQ IDNO:1, in the polybasic sequences RARR and KKRKRR of the F protein, alanine A at position 107 is mutated to arginine R, and lysine K at position 134 is mutated to arginine R. Optionally, the F protein includes 6×His and Strep tag-II tags.

5. The respiratory syncytial virus F protein according to any one of claims 1-4, characterized in that, The F protein includes an amino acid sequence selected from any one of SEQ ID NO:06-07.

6. A recombinant nucleic acid comprising a nucleotide sequence encoding the respiratory syncytial virus F protein according to any one of claims 1-5.

7. An expression vector comprising the recombinant nucleic acid according to claim 6.

8. A host cell comprising the recombinant nucleic acid according to claim 6 or the expression vector according to claim 7.

9. The host cell according to claim 8, characterized in that, The host cell is selected from Escherichia coli, yeast cells, insect cells or mammalian cells.

10. A method for preparing a respiratory syncytial virus F protein as described in any one of claims 1-5, characterized in that, The method comprises: S1: Culturing the host cell according to claim 8 or 9 under conditions suitable for the expression of the respiratory syncytial virus F protein; S2: Collecting the expression product and purifying the expression product to obtain the respiratory syncytial virus F protein.

11. Use of the respiratory syncytial virus F protein according to any one of claims 1-5, or the respiratory syncytial virus F protein prepared by the preparation method according to claim 10 in the preparation of a vaccine for preventing respiratory syncytial virus infection.

12. A vaccine for preventing respiratory syncytial virus infection, comprising the respiratory syncytial virus F protein according to any one of claims 1-5, or the respiratory syncytial virus F protein prepared by the preparation method according to claim 10, and a pharmaceutically acceptable carrier or excipient.

13. The vaccine according to claim 12, wherein The vaccine further comprises an adjuvant, and the adjuvant includes at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, CpG and QS-21.

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