RSV nanoparticle vaccine and preparation method therefor

Abstract

The present invention relates to the technical field of biomedicine. Specifically provided are a respiratory syncytial virus (RSV) Pre-F recombinant protein nanoparticle vaccine and a preparation method therefor. By performing amino acid mutation on an RSV Pre-F protein, and performing fusion expression on the mutated Pre-F protein and ferritin particles in eukaryotic cells, PreF-Ferritin fusion protein nanoparticles, in which eight Pre-F protein trimers are densely displayed on the surface of each ferritin particle, are obtained. By stabilizing and exposing antigen epitopes required to be displayed and disrupting or masking unrequired antigen epitopes, the PreF-Ferritin fusion protein nanoparticles effectively improve the immunogenicity and the production stability of the antigen. Experiments show that: when the PreF-Ferritin fusion protein is injected into mice, high-titer protective sera can be acquired, and the mice sera can yield relatively high neutralizing titers against RSV.

Description

A RSV nanoparticle vaccine and preparation method thereof Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a respiratory syncytial virus (RSV) pre-F recombinant protein nanoparticle vaccine and a preparation method thereof. Background Art

[0002] Human respiratory syncytial virus (HRSV) is a major viral pathogen that causes lower respiratory tract infections in infants, young adults with compromised immune systems, and the elderly. It is highly contagious and primarily transmitted through respiratory droplets and direct contact. In infants, HRSV infection can easily cause inflammation of the small airways in the lungs. In the elderly, infection can exacerbate existing lung and heart conditions, leading to death. Statistics show that HRSV has high infection rates, severe morbidity, and mortality rates in children and the elderly, making it a serious public health concern. The World Health Organization has prioritized the development of an HRSV vaccine.

[0003] RSV is an enveloped, nonsegmented, single-stranded, negative-sense RNA virus belonging to the order Monovirales, family Pneumoviridae, and genus Orthopneumovirus. RSV particles can be spherical or filamentous, with protrusions on the outer membrane and a size ranging from 150 to 300 nm. The RSV genome is approximately 15.2 kb in length and encodes 11 proteins, including three nonstructural proteins (NS1, NS2, and M2-2) and eight structural proteins. Three of these eight structural proteins are located on the viral surface membrane: small hydrophobic protein (SH), attachment glycoprotein G (G), and fusion glycoprotein (F); and five are located internally: nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 expressed from the M2 gene, and RNA-dependent RNA polymerase L (L) protein. Among them, F protein and G protein are crucial to the infectivity and pathogenesis of the virus, and are antigenic determinants that cause the host to produce neutralizing antibodies.

[0004] Research on HRSV vaccines began in the 1960s. The first-generation HRSV vaccine employed a whole-virus inactivated strategy, termed FI-RSV. This vaccine failed to induce a protective response in infants and young children. Instead, it caused enhanced respiratory disease (ERD) in subsequent primary infections and resulted in the deaths of two infants. This stalled HRSV vaccine development, and for over 30 years, HRSV vaccine development proceeded cautiously and slowly. Subsequent studies revealed that the RSV surface protein F, a class I membrane protein, undergoes a dramatic conformational transition during viral membrane fusion, from the Pre-F to the Post-F conformation, completing the early stages of viral infection. Due to the structural instability of the Pre-F conformation, researchers have long used the Post-F conformation as the primary conformation of the F protein in vaccine research, ultimately resulting in insufficient vaccine effectiveness. In 2013, Xiamen University collaborated with the National Institutes of Health (NIH) to identify a highly neutralizing monoclonal antibody that stabilizes Pre-F. This led to the identification of the molecular structure of Pre-F and its application in vaccine immunogen design. The resulting F protein, DS-Cav1, stabilizes the Pre-F conformation and induces unprecedented serum neutralizing responses in mice and cynomolgus macaques. This marked a shift in RSV vaccine research from empiricism to rational design, ushering in the era of structure-guided vaccine design. Pre-F has become a preferred target antigen for RSV vaccines.

[0005] With the continuous advancement and maturity of technology, in-depth research on the structure, function, and stabilization strategies of PreF, and the establishment of various vaccine platforms, RSV vaccines targeting PreF are being developed across various platforms. As of early 2023, six vaccine candidates are in Phase III clinical trials, five of which utilize a stabilized Pre-F format. Phase III clinical trial data released by Pfizer, GSK, and Moderna have demonstrated superior protective efficacy to previous studies. GSK's vaccine, which utilizes a T cell response-enhancing adjuvant to enhance immunity to RSV A2 Pre-F protein, has demonstrated 82% protection against RSV-associated lower respiratory tract illness in people over 60 years of age. Pfizer's unadjuvanted bivalent Pre-F vaccine strategy has demonstrated promising results in clinical trials targeting adults over 60 years of age and pregnant women. Maternally transmitted antibodies in pregnant women protect their newborns against severe HRSV-associated lower respiratory tract illness in 81% of cases within 90 days of vaccination, and 69% within 6 months of vaccination. Moderna used the same mRNA vaccine technology as its COVID-19 vaccine, and achieved an 84% lower respiratory tract protection rate in a Phase III clinical trial in people over 60 years old.

[0006] As can be seen from the numerous clinical trials currently underway worldwide, RSV vaccine development faces numerous challenges, including weak protection, complex target populations, inconsistent standards for evaluating immune-related protection, unclear clinical endpoints, and a lack of ideal animal models. RSV vaccine development remains a long and arduous journey. Therefore, continued research and innovation are crucial to developing a safe and highly immunogenic RSV vaccine, thereby reducing the disease and economic burden caused by RSV.

[0007] Nanoparticle vaccines are currently a key area of ​​research and development for novel vaccines. These vaccines primarily utilize strategies such as chemical conjugation and fusion expression to efficiently link antigens to biosynthetic protein backbones. Studies have shown that RSV nanoparticle proteins exhibit greater thermal stability and immunogenicity than non-nanoparticle RSV proteins, but further research is needed to determine their specific effectiveness.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a respiratory syncytial virus (RSV) pre-F recombinant protein nanoparticle vaccine and a preparation method thereof.

[0010] The present invention provides an RSV recombinant protein nanoparticle vaccine. By connecting the RSV Pre-F protein modified by amino acid mutation to a self-assembled ferritin nanoparticle carrier, the physical stability of the PreF antigen is further increased. At the same time, the PreF can be repeatedly presented on the surface of the nanoparticle, increasing the density of antigens and neutralizing epitopes, and more effectively stimulating the body to produce an immune response.

[0011] The present invention utilizes RSV protein nanoparticle vaccines to induce effective neutralizing antibody responses, and further develops a new, safe and effective RSV PreF nanoparticle vaccine (nanoparticles vaccine). This is of great significance for increasing the research and development of domestic products and accelerating the development of clinical trials of vaccines and drugs in my country, so as to protect children and the elderly from RSV infection-related diseases to the greatest extent, and fill the gap in intervention measures for RSV infection in high-risk groups.

[0012] The RSV nanoparticle vaccine provided by the present invention can rapidly activate antigen-presenting cells without the need for additional adjuvants or with reduced adjuvant addition, and promotes antigen uptake and cross-presentation, thereby enhancing subsequent immune responses. Compared to RSV recombinant protein vaccines, the RSV nanoparticle vaccine has greater structural stability and antigen clustering capabilities, and has a significant advantage in inducing neutralizing antibodies. This invention is of great significance for addressing diseases caused by RSV infection and is expected to provide a safe, effective, and widely applicable vaccine for clinical use.

[0013] The technical solutions to the technical problems of the present invention are as follows:

[0014] In a first aspect of the present invention, an RSV protein is provided. The RSV protein is an RSV Pre-F recombinant protein modified by amino acid mutations. The RSV Pre-F recombinant protein is obtained by any one of the following two methods:

[0015] (1) amino acid point mutations were performed on the full-length sequence of the wild-type pre-F protein as shown in SEQ ID NO. 1, such that T at position 324 was mutated to C and N at position 437 was mutated to C;

[0016] (2) First, the transmembrane region / intracellular region of the wild-type pre-F protein full-length sequence as shown in SEQ ID NO.1 was deleted, and the fibritin / Throm / 6his / Stretaq sequence was connected to its C-terminus, and then the T at position 324 was mutated to C, and the N at position 437 was mutated to C.

[0017] Furthermore, the amino acid sequence of the RSV Pre-F recombinant protein is shown in SEQ ID NO.4.

[0018] In a second aspect of the present invention, a fusion protein is provided. The fusion protein is an RSV PreF-Ferritin nanoparticle fusion protein modified by amino acid mutation. The fusion protein is obtained by any one of the following two methods:

[0019] (1) Based on the full-length wild-type pre-F protein sequence as shown in SEQ ID NO.1, amino acid point mutations were performed, and the ferritin sequence as shown in SEQ ID NO.5 was connected to its C-terminus; the mutation method was as follows: T at position 324 of the wild-type pre-fusion F protein amino acid sequence as shown in SEQ ID NO.1 was mutated to C, and N at position 437 was mutated to C;

[0020] (2) The transmembrane region / intracellular region in the full-length sequence of the wild-type pre-F protein as shown in SEQ ID NO.1 is deleted, and a ferritin sequence is connected to its C-terminus to obtain a mutant as shown in SEQ ID NO.2. Amino acid point mutations are further performed on the basis of SEQ ID NO.2; the mutation method is as follows: T at position 324 of the pre-fusion F protein amino acid sequence as shown in SEQ ID NO.2 is mutated to C, and N at position 437 is mutated to C.

[0021] Furthermore, the amino acid sequence of the RSV PreF-Ferritin nanoparticle fusion protein is shown in SEQ ID N0.3.

[0022] In a third aspect of the present invention, a biomaterial is provided, which is at least one of the following (1)-(4):

[0023] (1) a nucleic acid molecule encoding the RSV protein as described in the first aspect or the fusion protein as described in the second aspect;

[0024] (2) a recombinant expression vector containing the nucleic acid molecule described in (1);

[0025] (3) a recombinant microorganism containing the nucleic acid molecule described in (1) or a recombinant microorganism containing the recombinant expression vector described in (2);

[0026] (4) A recombinant cell line containing the nucleic acid molecule described in (1) or a recombinant cell line containing the recombinant expression vector described in (2).

[0027] In the fourth aspect of the present invention, a method for preparing the RSV protein as described in the first aspect or the fusion protein as described in the second aspect is provided, comprising the following steps: expressing a nucleic acid molecule encoding the RSV protein as described in the first aspect or the fusion protein as described in the second aspect in an organism or biological cell to obtain the RSV protein or fusion protein.

[0028] Furthermore, the preparation method comprises the following steps: introducing a nucleic acid molecule encoding the RSV protein as described in the first aspect or the fusion protein as described in the second aspect into CHO K1Q cells to obtain recombinant cells; and culturing the recombinant cells to obtain the RSV protein or fusion protein.

[0029] In a fifth aspect of the present invention, there is provided the use of the RSV protein as described in the first aspect, or the fusion protein as described in the second aspect, or the biomaterial as described in the third aspect, or the protein or fusion protein prepared according to the method described in the fourth aspect in any one of the following (1) to (4):

[0030] (1) As an immunogen;

[0031] (2) preparing anti-RSV products;

[0032] (3) preparing products for preventing and / or treating RSV infection;

[0033] (4) Preparation of products for preventing and / or treating diseases caused by RSV.

[0034] In the sixth aspect of the present invention, a vaccine is provided, the active ingredient of which is the RSV protein as described in the first aspect, or the fusion protein as described in the second aspect, or the biological material as described in the third aspect, or the protein or fusion protein prepared according to the method described in the fourth aspect.

[0035] Furthermore, the vaccine is in the form of an aqueous solution or a lyophilized preparation.

[0036] Furthermore, the vaccine contains an adjuvant.

[0037] More preferably, the adjuvant is any one of CpG, QS21, aluminum phosphate, a mixture of CpG and aluminum phosphate, or a mixture of QS21 and aluminum phosphate.

[0038] In a seventh aspect of the present invention, there is provided a use of the vaccine according to the sixth aspect in any one of the following (1)-(3):

[0039] (1) preparing anti-RSV products;

[0040] (2) preparing products for preventing and / or treating RSV infection;

[0041] (3) Preparation of products for preventing and / or treating diseases caused by RSV.

[0042] The present invention has the following technical effects:

[0043] (1) The effective immunogenicity and stability of the Pre-F protein in the RSV vaccine were enhanced through specific antigenic mutation design, and the immunogenicity was further enhanced by exposing the required epitopes on the surface of nanoparticles. Experiments have shown that the RSV nanoparticle vaccine prepared by the present invention can achieve a good immune effect at a low dose, among which the neutralization titer of the RSV PreF-Ferritin group can reach 8560.

[0044] (2) The present invention solves the problem of poor stability of wild antigens. The PreF-Ferritin fusion protein prepared by the present invention can induce RSV antibodies with neutralizing activity after entering the body, thereby giving the body corresponding immune protection.

[0045] (3) The present invention designs amino acid mutations in RSV Pre-F protein and fuses the Pre-F mutant protein with ferritin particles in eukaryotic cells to obtain PreF-Ferritin fusion protein nanoparticles in which eight Pre-F protein trimers are concentratedly displayed on the surface of ferritin particles. The PreF-Ferritin fusion protein nanoparticles effectively improve the immunogenicity and production stability of the antigen by stabilizing and exposing the antigenic epitopes that need to be displayed and destroying or hiding the unnecessary antigenic epitopes. Experiments have shown that when the PreF-Ferritin fusion protein prepared by the present invention is injected into mice, serum with high protective titer can be obtained, and the mouse serum can produce a high neutralization titer against RSV virus.

[0046] (4) The RSV preF-Ferritin nanoparticle vaccine prepared by the present invention can reduce the use of adjuvants or eliminate the use of adjuvants while still achieving a good immune effect; thereby, the side effects caused by the use of adjuvants can be reduced, the safety is higher, and the production cost is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is an SDS-PAGE gel image of RSV PreF-Ferritin protein purity detection.

[0048] Figure 2 is a transmission electron micrograph of RSV PreF-Ferritin protein.

[0049] FIG3 shows the antibody titer test results of mice immunized with RSV protein vaccine before and after modification (without adjuvant).

[0050] FIG4 shows the antibody titer test results of mice immunized with RSV protein vaccine before and after modification (with adjuvant).

[0051] FIG5 shows the results of the protein antibody titer detection in the immune sera of mice treated with preparation a0 (preF) and preparation b0 (preF-Ferritin).

[0052] FIG6 shows the results of the protein antibody titer detection in the immune sera of mice of preparation a1 (preF+Alum) and preparation b1 (preF-Ferritin+Alum).

[0053] FIG7 shows the results of the protein antibody titer detection in the immune sera of mice of preparation a2 (preF+CpG+Alum) and preparation b2 (preF-Ferritin+CpG+Alum).

[0054] FIG8 shows the results of the protein antibody titer detection in the immune sera of mice of preparation a2 (preF+CpG+Alum) and preparation b1 (preF-Ferritin+Alum). DETAILED DESCRIPTION

[0055] In order to more concisely and clearly demonstrate the technical solutions, objectives and advantages of the present invention, the technical solutions of the present invention are described in detail below with reference to specific embodiments and accompanying drawings.

[0056] Example 1: Preparation of RSV recombinant protein

[0057] (1) Protein construction

[0058] The term "wild type" as used herein refers to a protein that exists in nature and has not been artificially modified or processed. It is understood by those skilled in the art that wild RSV F proteins can be a variety of sequences, which may have slight differences but have essentially the same biological activity.

[0059] The wild-type full-length F protein mentioned in the present invention refers to the sequence provided by GenBank, and the specific sequence is shown in SEQ ID NO. 1 (Fusion glycoprotein F0 OS=Human respiratory syncytial virus A (strain A2) OX=11259GN=F PE=1SV=1).

[0060] (1) Amino acid mutation modification

[0061] The RSV Pre-F nanoparticle protein of the present invention can be obtained by the following two methods:

[0062] Method 1: Based on the full-length wild-type pre-F protein sequence as shown in SEQ ID NO.1, amino acid point mutations are performed, and the ferritin sequence as shown in SEQ ID NO.5 is connected to its C-terminus; the specific mutation method is as follows: the T at position 324 of the wild-type pre-fusion F protein amino acid sequence as shown in SEQ ID NO.1 is mutated to C, and the N at position 437 is mutated to C.

[0063] Method 2: The transmembrane / intracellular region in the full-length sequence of the wild-type pre-F protein as shown in SEQ ID NO.1 is deleted, and a linker ferritin sequence is connected to its C-terminus to obtain a mutant with a sequence as shown in SEQ ID NO.2. Amino acid point mutations are then performed on the basis of SEQ ID NO.2. The specific mutation method is as follows: T at position 324 of the wild-type pre-fusion F protein amino acid sequence as shown in SEQ ID NO.2 is mutated to C, and N at position 437 is mutated to C to obtain a full-length F protein mutant, whose amino acid sequence is shown in SEQ ID NO.3.

[0064] In this example, the second method was used to obtain the full-length mutant of the Pre-F nanoparticle protein.

[0065] This embodiment also relates to RSV Pre-F recombinant protein, which can be obtained by the following two methods:

[0066] (1) amino acid point mutations were performed on the full-length sequence of the wild-type pre-F protein as shown in SEQ ID NO. 1, such that T at position 324 was mutated to C and N at position 437 was mutated to C;

[0067] (2) First, the transmembrane region / intracellular region in the wild-type pre-F protein full-length sequence as shown in SEQ ID NO.1 was deleted, and the fibritin / Throm / 6his / Stretaq sequence was connected to its C-terminus. Then, the T at position 324 was mutated to C, and the N at position 437 was mutated to C to obtain the full-length mutant of the Pre-F protein as shown in SEQ ID NO.4.

[0068] In this example, the second method was used to obtain RSV Pre-F recombinant protein.

[0069] The sequences of SEQ ID NO.1 to SEQ ID NO.5 are shown below:

[0070] SEQ ID NO.1:

[0071] >sp|P03420|FUS_HRSVA Fusion glycoprotein F0 OS=Human respiratory syncytial virus A(strain A2)OX=11259GN=F PE=1SV=1

[0072] SEQ ID NO.2:

[0073] >RSV F-Ferritin

[0074] SEQ ID NO.3:

[0075] >RSV F-Ferritin T324C N437C

[0076] SEQ ID NO.4:

[0077] >RSV F T324C N437C

[0078] SEQ ID NO.5:

[0079] >Ferritin

[0080] (2) Synthesis of target gene

[0081] The RSV PreF-Ferritin protein target gene and the RSV PreF protein target gene were synthesized by the following methods:

[0082] (1) Synthesis of RSV PreF-Ferritin protein target gene

[0083] The complete wild-type pre-F was truncated to remove the transmembrane and intracellular regions, and a linker ferritin sequence was attached to its C-terminus to obtain a mutant with the sequence shown in SEQ ID NO. 2. Further amino acid point mutations were performed on SEQ ID NO. 2, mutating T at position 324 to C and N at position 437 to C, to obtain a full-length F protein mutant with the sequence shown in SEQ ID NO. 3. Based on the amino acid sequence of the RSV PreF-Ferritin protein, SEQ ID NO. 3, and the codon preference of the host cell, the corresponding coding sequence was determined. The restriction endonuclease EcoRI sequence was added to the C-terminus of the gene in this segment, and the restriction endonuclease XbaI sequence was added to the N-terminus. The designed nucleotide sequence was then chemically synthesized.

[0084] (2) Synthesis of RSV PreF protein target gene

[0085] The complete wild-type pre-F protein was truncated to remove the transmembrane and intracellular regions. The fibritin / Throm / 6his / Stretaq sequence was then ligated to its C-terminus. The T at position 324 was mutated to a C, and the N at position 437 was mutated to a C, resulting in a full-length mutant of the Pre-F protein as shown in SEQ ID NO. 4. Based on the amino acid sequence of the RSV Pre-F protein, SEQ ID NO. 4, and the codon preference of the host cell, the corresponding coding sequence was determined. The restriction endonuclease sequence EcoRI was added to the C-terminus of this segment, and the restriction endonuclease sequence XbaI was added to the N-terminus. The designed nucleotide sequence was then chemically synthesized.

[0086] (3) Plasmid amplification and target gene extraction

[0087] The pUC19 plasmid vector was double-digested with EcoRI and XbaI restriction enzymes, ligated with the synthesized gene, and introduced into the amplification host DH5α. Single clones were screened using LB (Amp+) agar solid medium. Single clones containing the target gene were inoculated into LB (Amp+) liquid medium and cultured and amplified at 37°C, 200 rpm. The plasmid pUC19-preF was extracted using the Sigma-Aldrich GenEluteTMHP Plasmid MidiPrep Kit. The extracted plasmid was double-digested with EcoRI and XbaI restriction enzymes, and the target gene fragment was recovered using the TaKaRa MiniBestAgarose Gel Extraction Kit.

[0088] (4) Construction of eukaryotic expression vector

[0089] The mammalian cell expression plasmid pGN-M, containing the CMV promoter and the dihydrofolate reductase (DHFR) gene, was double-digested with EcoRI and XbaI restriction enzymes, and the vector DNA fragment was recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0. The vector DNA fragment and the target gene fragment were ligated via sticky-ends and introduced into the DH5α amplification host, and a single clone containing the eukaryotic expression plasmid pGN-M_preF was screened. The culture was inoculated in LB (Amp+) for amplification, and the amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit.

[0090] (II) Protein expression and cloning screening

[0091] CHO K1 cells purchased from ATCC were used as host cells. After cell recovery, they were cultured in DMEM medium (Sigma-Aldrich) supplemented with 10% newborn calf serum and passaged every 3 days. After passage 2, the cells were observed to be growing well, and then CHO K1 cells were plated at 0.75×10 6 Three 9.6cm cells / well 2 Wells were filled with Iscove's optimized DMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (IMEM+FBS) (Gibco). Cells were incubated in a humidified incubator with 5% CO₂ and 37°C. 4 μg of pcDNARSV vector was added to each well. DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two of the wells. Lipofectamine 2000 alone was added to the third well as a negative control. After 48 hours, the medium was removed, the cells were centrifuged at 200×g for 5 minutes, and the supernatant was stored at -20°C. IMDM+FBS medium and 10 μg / mL blasticidin-HCl (Invitrogen) were added to one well of transfected cells. The other well was washed with PBS, and the cells were lysed with 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v / v) Triton X-100 containing complete, EDA-free protease inhibitor cocktail (Roche Diagnostics). The cells were centrifuged at 16,000 × g for 10 minutes at 4°C, and the lysate was stored at -20°C. The supernatant and lysate were assayed for the presence of recombinant protein by Western blot. After 5 days of culture in selective medium, the cells were eluted with trypsin (Invitrogen) and seeded onto 9 cm Petri dishes. Serial dilutions were performed to isolate single clones. Over the next 7–11 days, 42 single clones were selected and transferred to wells of a 96-well plate. The culture supernatant was analyzed by Western blot to screen for highly expressed proteins. The clones secreting the highest amount of RSV protein were selected for the next round of screening, and the cells were finally expanded and 30 new clones were selected and preserved.

[0092] The selected clones were expanded into three T175 flasks (NETS). Trypsin was added for digestion, washed with PBS, and resuspended in 100 mL of ProCHO4 (Lonza) in a 250 mL spinner flask, supplemented with 1 × ProHT, 4 mM L-glutamine, and 2% FBS (Lonza). Culture was carried out in a humidified incubator at 37 ° C, 5% CO2, with a stirring speed of 90 rpm and a lid slightly opened to ensure air diffusion. Samples were taken daily, stained with trypan blue (Sigma-Aldrich), cells were counted, and passaged every 3-5 days. When the viable cell concentration was higher than 0.3 × 10 6 cells / mL, after the plateau phase when the number of viable cells exceeds 90%. When the cells adapt and grow well, BFS is gradually removed, and the cells are considered to be fully suitable for serum-free suspension growth.

[0093] (III) Protein production in bioreactors

[0094] A 1.5-liter perfusion culture was set up in a 3-liter bioreactor equipped with a 10 μm spin filter. Culture parameters were as follows: temperature maintained at 37°C using a heating blanket, pH adjusted to 6.9 with CO₂ or 0.3 M sodium hydroxide, agitation at 200-300 rpm, and dissolved oxygen (dO₂) maintained at 40% of saturated air using a mixture of N₂ and O₂ at a maximum flow rate of 200 mL / min. The perfusion rate was 0.3 to 0.8 dilutions / day, and culture fluid was sampled daily for cell counts. Supernatants were stained with trypan blue, and glucose and lactate concentrations were measured offline.

[0095] A total of 12.5 liters of cell-free culture fluid was collected and centrifuged at 8,000 × g for 30 minutes at 4°C. The fluid was filtered through a 0.45 μm membrane and then concentrated by ultrafiltration using a 10 kDa membrane pack. Ultrafiltration was performed with buffer and the sample solution was concentrated to 0.5 liters. After adding 0.5 liters of PBS, the sample solution was concentrated to 0.5 liters. This procedure was repeated five times.

[0096] (IV) Protein purification

[0097] The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column and washed with 20mM Tris-HCl pH 7.5. The column was then washed with 20mM Tris-HCl pH 7.5 to further remove adsorbed protein impurities. The pre-F protein was eluted with a solution in which the sodium chloride concentration was increased to 300mM. Ammonium sulfate was added to the combined solution to a concentration of 800mM, loaded onto a Butyl-Sepharose (GE Bioscience) column, and washed with phosphate buffered saline (PBS, 6mM Na2HPO4, 1.5mM KH2PO4, 0.15M sodium chloride pH 6.8) with 800mM ammonium sulfate added to the column. The column was then washed with a PBS solution containing 400mM ammonium sulfate, and the RSV protein was finally eluted with purified water. Finally, Sephacryl S-400HR (GE Bioscience) was loaded, the column was washed with PBS, the protein peak was collected, a cosolvent was added, the protein was lyophilized in a vacuum freeze dryer, and stored at -70°C until use.

[0098] RSV modified preF, RSV modified preF-Ferritin and RSV modified preF protein were prepared respectively using the above method.

[0099] As can be seen from Figure 1, the molecular weight of the PreF-Ferritin protein is 69 kDa. The electron microscopy image in Figure 2 shows that the molecule forms uniform nanoparticles (24-mers). One Pre-F ferritin nanoparticle presents 8 preF protein trimers on its surface, making the PreF-Ferritin nanoparticle protein structure more stable and exposing more antigenic epitopes required for presentation, which can effectively improve the immunogenicity and stability of the RSV preF protein antigen.

[0100] Example 2: Preparation of immune preparation

[0101] Preparation a0 / Preparation b0 / Preparation c0:

[0102] 200 ug / mL of RSV modified preF / preF-Ferritin / pre-modified preF protein obtained by the method of Example 1 was added to phosphate buffer pH 5.8 buffer, sterilized and filtered with a 0.22 μm membrane, stirred at 4°C for 1 hour, aseptically divided into 0.7 mL / bottle, and stored at 4°C for immunization.

[0103] Preparation a1 / Preparation b1 / Preparation c1:

[0104] 200 ug / mL of RSV modified preF / preF-Ferritin / pre-modified preF protein obtained by the method of Example 1 was added to phosphate buffer pH 5.8 buffer, sterilized and filtered with a 0.22 μm membrane, and sterile aluminum phosphate gel (Benetag) was added. The mixture was stirred at 4°C for 1 hour, and aseptically divided into 0.7 mL / bottles and stored at 4°C for immunization.

[0105] Preparation a2 / Preparation b2:

[0106] 200 ug / mL of RSV modified preF / preF-Ferritin protein obtained by the method of Example 1 was added with CpG (Genscript), followed by phosphate buffer pH 5.8 buffer, sterilized and filtered with a 0.22 μm membrane, and then sterile aluminum phosphate gel (Benetag) was added. The mixture was stirred at 4°C for 1 hour, aseptically divided into 0.7 mL / bottle, and stored at 4°C for immunization.

[0107] Example 3: Immunization of mice with RSV protein vaccines before and after modification and blood collection

[0108] Female BALB / c mice aged 4-6 weeks were randomly divided into 4 groups of 10 mice each. Each group was given the following immune preparations: preparation a0, preparation a1, preparation c0, and preparation c1, prepared using Example 2. The mice were subcutaneously immunized once every two weeks with 0.1 mL each time, for a total of two immunizations. Blood was collected 35 days after immunization, and the blood was then allowed to stand at room temperature for 4 hours and centrifuged at 10,000 RPM at room temperature. The supernatant serum was aspirated and stored at -70°C for testing.

[0109] Example 4: ELISA detection of pre-F protein antibody titers in the immune serum of mice immunized with RSV recombinant protein vaccine before and after modification

[0110] Prepare a 1 mg / mL stock solution of purified pre-F protein in 1× PBS and store in a 4°C refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer and coat the ELISA plate with 100 μL of coating solution per well. Incubate overnight at room temperature. Wash four times with plate wash buffer, add 150 μL of blocking buffer, incubate at 37°C for 2 hours, and wash three times with 300 μL per well of plate wash buffer. Store at 4°C for one week.

[0111] Dilute the corresponding test serum obtained from the mice vaccinated in Example 3 to prepare working sample serum. Dilute the serum appropriately and add 100 μl per well to the first row of wells on an ELISA plate. Perform a 2-fold serial dilution from the first row downwards and incubate at 37°C for 2 hours. Wash each well three times with 300 μl of plate wash buffer. Add 100 μl / well of AP-labeled goat anti-mouse secondary antibody at a 1:2000 dilution and incubate at 37°C for 1 hour. Wash the plate three times using a plate washer according to the protocol. Add 100 μl / well of pNPP substrate solution and read the plate on a microplate reader set to a wavelength of 405 nm.

[0112] The test results are shown in Figures 3-4. The results in Figure 3 show that: when the immune preparations do not contain adjuvants, the antibody titer of mice immunized with the RSV preF recombinant protein vaccine after amino acid modification (preparation a0) is significantly higher than that of the RSV preF protein vaccine before modification (preparation c0); and after the serum of the immunized mice is diluted at different multiples, at each concentration, the antibody titer of the RSV preF recombinant protein vaccine after amino acid modification (preparation a0) is still significantly higher than that of the RSV preF protein vaccine before modification (preparation c0), and as the serum dilution multiple gradually increases, the antibody titer of both RSV preF proteins before and after modification shows a downward trend.

[0113] The results in Figure 4 show that when both immune preparations contain adjuvants, the antibody titer of mice immunized with the amino acid-modified RSV preF recombinant protein vaccine (preparation a1) is significantly higher than that of the unmodified RSV preF protein vaccine (preparation c1); and when the serum of the immunized mice is diluted at different multiples, the antibody titer of the amino acid-modified RSV preF recombinant protein vaccine (preparation a1) is still higher than that of the unmodified RSV preF protein vaccine (preparation c1) at each concentration, and as the serum dilution multiple gradually increases, the antibody titers of both the unmodified and modified RSV preF proteins show a downward trend.

[0114] It can be seen that compared with the RSV preF protein before modification, the RSV PreF recombinant protein vaccine after amino acid modification can obtain serum with higher protective titer, indicating that the RSV PreF recombinant protein vaccine prepared by the present invention after amino acid modification has better immune effect.

[0115] Example 5: Immunization of mice with RSV recombinant protein vaccine and RSV recombinant protein nanoparticle vaccine and blood collection

[0116] Female BALB / c mice aged 4-6 weeks were randomly divided into 6 groups of 9 mice each. The immune preparations in each group were prepared using Example 2: Preparation a0, Preparation a1, Preparation a2, Preparation b0, Preparation b1, and Preparation b2. 0.1 mL of the vaccine was administered subcutaneously every two weeks for a total of two immunizations. Blood was collected after the second immunization and then allowed to stand at room temperature for 4 hours. The blood was centrifuged at 10,000 RPM at room temperature, and the supernatant serum was collected and stored at -70°C for testing.

[0117] Example 6: ELISA for detecting pre-F protein antibody titers in sera of mice immune to RSV recombinant protein vaccine and RSV recombinant protein nanoparticle vaccine

[0118] Prepare a 1 mg / mL stock solution of purified pre-F protein in 1× PBS and store in a 4°C refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer and coat the ELISA plate with 100 μL of coating solution per well. Incubate overnight at room temperature. Wash four times with plate wash buffer, add 150 μL of blocking buffer, incubate at 37°C for 1 hour, and wash three times with 300 μL per well of plate wash buffer. Store at 4°C for one week.

[0119] The corresponding test serum obtained from the mice vaccinated in Example 5 was diluted to form working sample serum. Appropriate dilutions were added to the first row of wells of an ELISA plate, 100 μl per well. Four-fold serial dilutions were performed starting from the first row downwards, and the plate was incubated at 37°C for 2 hours. Each well was washed three times with 300 μl of plate wash buffer, and 100 μl of HRP-labeled goat anti-mouse antibody (1:2000 dilution) was added. The plate was incubated at 37°C for 1 hour. Each well was washed three times with 300 μl of plate wash buffer, and 100 μl of TMB substrate solution was added to each well. The plate was read at 450 nm.

[0120] The test results are shown in Figures 5, 6, 7 and 8, where the results in Figure 5 show that when no adjuvant is added to the immune preparation, the antibody titer of mice immunized with the RSV preF-Ferritin recombinant protein vaccine (preparation b0) is higher than that of the RSV preF recombinant protein vaccine (preparation a0); and the serum of the immunized mice was diluted at different multiples, especially at dilution multiples of 1:400, 1:1600, 1:6400, and 1:25600, the antibody titer of the RSV preF-Ferritin recombinant protein vaccine (preparation b0) was significantly higher than that of the RSV preF recombinant protein vaccine (preparation a0), and as the serum dilution multiple gradually increased, the antibody titers of both preparations gradually decreased.

[0121] The results in Figure 6 show that when the same adjuvant was added to the immune preparations, the antibody titer of mice immunized with the RSV preF-Ferritin recombinant protein vaccine (preparation b1) was significantly higher than that of the RSV preF recombinant protein vaccine (preparation a1); and the serum of the immunized mice was diluted at different multiples, especially at dilution multiples of 1:100, 1:400, 1:1600, 1:6400, and 1:25600, the antibody titer of the RSV preF-Ferritin recombinant protein vaccine (preparation b1) was significantly higher than that of the RSV preF recombinant protein vaccine (preparation a1), and as the serum dilution multiple gradually increased, the antibody titers of both preparations gradually decreased.

[0122] The results in Figure 7 show that when the same two adjuvants were added to the immune preparations, the antibody titer of mice immunized with the RSV preF-Ferritin recombinant protein vaccine (preparation b2) was significantly higher than that of the RSV preF recombinant protein vaccine (preparation a2); and when the serum of the immunized mice was diluted at different multiples, the antibody titer of the RSV preF-Ferritin recombinant protein vaccine (preparation b2) was significantly higher than that of the RSV preF recombinant protein vaccine (preparation a2) at different concentrations, and as the serum dilution multiple gradually increased, the antibody titers of both preparations gradually decreased.

[0123] The results in Figure 8 show that when different adjuvants were added to the immune preparations, that is, preparation a2 added an additional CpG adjuvant compared to preparation b1, the antibody titer of mice immunized with the RSV preF-Ferritin recombinant protein vaccine (preparation b1) was higher than that of the RSV preF recombinant protein vaccine (preparation a2); and when the serum of the immunized mice was diluted at different multiples, especially at dilution multiples of 1:100, 1:400, and 1:1600, the antibody titer of the RSV preF-Ferritin recombinant protein vaccine (preparation b1) was significantly higher than that of the RSV preF recombinant protein vaccine (preparation a2), and as the serum dilution multiple gradually increased, the antibody titers of both preparations gradually decreased.

[0124] Thus, it can be seen that when adding or not adding an adjuvant to the preparation, as well as when adding different adjuvants, the RSV preF-Ferritin recombinant protein vaccine can obtain serum with higher protective titers compared to the RSV preF recombinant protein vaccine, indicating that the RSV preF-Ferritin recombinant protein vaccine prepared by the present invention has a better immune effect. Moreover, the serum protective titer obtained by the vaccine with the adjuvant added is higher than that without the adjuvant, indicating that the addition of the adjuvant has a certain enhancing auxiliary effect on the immune effect of the vaccine; and the results of Figure 8 show that although preparation a2 adds an additional CpG adjuvant compared to preparation b1, the antibody titer of mice immunized with the RSV preF-Ferritin recombinant protein vaccine (preparation b1) is higher than that of the RSV preF recombinant protein vaccine (preparation a2), indicating that the RSV preF-Ferritin recombinant protein vaccine prepared by the present invention can reduce the use of adjuvants while still achieving a good immune effect; on the other hand, it can also reduce the side effects caused by the use of adjuvants, have higher safety, and reduce production costs.

[0125] Example 7: RSV-Ferritin virus A2 neutralization test results

[0126] (1) Immunization of mice and blood collection

[0127] Female BALB / c mice aged 4-6 weeks were randomly divided into 3 groups of 9 mice each. Each group was given the immune preparations prepared in Example 2: Preparation a1, Preparation b1, and Preparation c1. 0.1 mL of the vaccine was administered subcutaneously once every two weeks for a total of two immunizations. Blood was collected after the second immunization and then allowed to stand at room temperature for 4 hours. The blood was centrifuged at 10,000 RPM at room temperature, and the supernatant serum was collected and stored at -70°C for testing.

[0128] (2) RSV A2 virus neutralization titer

[0129] RSV type A was cultured in Hep-2 cells in DMEM supplemented with 10% bovine serum. 20,000 Hep-2 cells were plated in a 96-well plate with 100 μL of complete medium per well. The cells were incubated at 37°C, 5% CO₂ for 24 hours until the cell density reached 60-80%. The medium was replaced by aspirating the medium from the 96-well plate and adding 100 μL of maintenance medium per well. The serum was heat-inactivated in a 56°C waterbath for 30 minutes. The serum was aliquoted into at least four tubes at a rate of 15 μL per tube and stored at -80°C. Antiserum was diluted in a 96-well V-shaped plate at the appropriate dilution factor, with 70 μL per well plated in duplicate for each dilution. The virus and antiserum at different dilutions were mixed, diluted to 100 TCID₅₀ in DMEM, and 70 μL per well plated into the 96-well V-shaped plate. Incubate at 37°C for 1 hour; aspirate 100 μL / well and transfer to the above-mentioned Hep2 cell plate; culture at 37°C, 5% CO2 for about 3-5 days; observe CPE every day, and stain the cells with 5% glutaraldehyde containing 0.25% crystal violet.

[0130] Table 1. Results of RSV-Ferritin virus A2 neutralization experiments

[0131] RSV A2 virus neutralization titer tests showed that the mean neutralization titer of PreF-Ferritin mouse serum was 8560, approximately 1.76 times that of RSV Pre-F mouse serum and more than five times that of the serum of mice immunized with the modified pre-F protein vaccine. This indicates that the RSV PreF-Ferritin nanoparticle vaccine prepared by the present invention can be injected into mice to obtain serum with high protective titers, and the mouse serum can produce high neutralizing antibody titers against the prevalent RSV A strain.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be included within the scope of protection of the claims of the present invention to be approved.

Claims

1. A RSV protein, characterized in that The RSV protein is an RSV Pre-F recombinant protein modified by amino acid mutation, and the RSV Pre-F recombinant protein is obtained by any one of the following two methods: (1) amino acid point mutations were performed on the full-length sequence of the wild-type pre-F protein as shown in SEQ ID NO. 1, such that T at position 324 was mutated to C and N at position 437 was mutated to C; (2) First, the transmembrane region and the intracellular region of the wild-type pre-F protein full-length sequence as shown in SEQ ID NO.1 were deleted, and a 6his sequence was connected to its C-terminus. Then, the T at position 324 was mutated to C, and the N at position 437 was mutated to C; its amino acid sequence is shown in SEQ ID NO.

4.

2. A fusion protein, characterized in that The fusion protein is an RSV PreF-Ferritin nanoparticle fusion protein modified by amino acid mutation, and the fusion protein is obtained by any one of the following two methods: (1) Based on the full-length wild-type pre-F protein sequence as shown in SEQ ID NO.1, amino acid point mutations were performed, and the ferritin sequence as shown in SEQ ID NO.5 was connected to its C-terminus; the mutation method was as follows: T at position 324 of the wild-type pre-fusion F protein amino acid sequence as shown in SEQ ID NO.1 was mutated to C, and N at position 437 was mutated to C; (2) The transmembrane region and the intracellular region of the wild-type pre-F protein full-length sequence as shown in SEQ ID NO.1 were deleted, and the Ferritin sequence was connected to its C-terminus to obtain a mutant with a sequence as shown in SEQ ID NO.

2. Amino acid point mutations were further performed on the basis of SEQ ID NO.2; the mutation method was as follows: T at position 324 of the pre-fusion F protein amino acid sequence as shown in SEQ ID NO.2 was mutated to C, and N at position 437 was mutated to C.

3. The fusion protein according to claim 2, characterized in that The amino acid sequence of the RSV PreF-Ferritin nanoparticle fusion protein is shown in SEQ ID N0.

3.

4. A biomaterial, characterized in that At least one of the following (1)-(4): (1) a nucleic acid molecule encoding the RSV protein according to claim 1 or the fusion protein according to any one of claims 2 to 3; (2) a recombinant expression vector containing the nucleic acid molecule described in (1); (3) a recombinant microorganism containing the nucleic acid molecule described in (1) or a recombinant microorganism containing the recombinant expression vector described in (2); (4) A recombinant cell line containing the nucleic acid molecule described in (1) or a recombinant cell line containing the recombinant expression vector described in (2).

5. A method for preparing the RSV protein according to claim 1 or the fusion protein according to any one of claims 2-3, comprising the steps of expressing a nucleic acid molecule encoding the RSV protein according to claim 1 or the fusion protein according to any one of claims 2-3 in an organism or biological cell to obtain the RSV protein or fusion protein.

6. The preparation method according to claim 5, characterized in that: The method comprises the following steps: introducing a nucleic acid molecule encoding the RSV protein according to claim 1 or the fusion protein according to any one of claims 2 to 3 into CHO K1Q cells to obtain recombinant cells; and culturing the recombinant cells to obtain the RSV protein or fusion protein.

7. Use of the RSV protein according to claim 1, or the fusion protein according to any one of claims 2-3, or the biomaterial according to claim 4, or the protein or fusion protein prepared by the method according to any one of claims 5-6 in any of the following (1)-(3): (1) preparing anti-RSV products; (2) preparing products for preventing and / or treating RSV infection; (3) Preparation of products for preventing and / or treating diseases caused by RSV.

8. A vaccine, the active ingredient of which is the RSV protein according to claim 1, the fusion protein according to any one of claims 2-3, the biological material according to claim 4, or the protein or fusion protein prepared according to any one of claims 5-6.

9. The vaccine according to claim 8, characterized in that The vaccine is in the form of an aqueous solution or a lyophilized preparation.

10. The vaccine according to claim 8, characterized in that The vaccine contains an adjuvant.

11. The vaccine according to claim 10, characterized in that The adjuvant is any one of CpG, QS21, aluminum phosphate, a mixture of CpG and aluminum phosphate, or a mixture of QS21 and aluminum phosphate.

12. Use of the vaccine according to claim 8 in any one of the following (1)-(3): (1) preparing anti-RSV products; (2) preparing products for preventing and / or treating RSV infection; (3) Preparation of products for preventing and / or treating diseases caused by RSV.

Images