Severe fever with thrombocytopenia syndrome virus-like particle, vaccine and preparation method of virus-like particle

Abstract

The invention discloses a fever with thrombocytopenia syndrome virus-like particle, a vaccine and a preparation method of the fever with thrombocytopenia syndrome virus-like particle. Based on SFTSV genome analysis, glycoproteins Gn and Gc and capsid protein NP are selected as core antigens, potential T cell epitopes of the Gn protein are predicted by utilizing bioinformatics, and the chimeric antigen Mosaic-Gn protein is designed. The optimized antigen gene is cloned to a baculovirus transfer vector, and three recombinant baculoviruses are constructed through a homologous recombination technology. The three recombinant viruses co-infect suspended insect cells, and the SFTSV virus-like particles self-assembled in the cells can be harvested after 72 hours. And mixing and emulsifying with an adjuvant to prepare the vaccine after ultra-filtration and ultra-separation concentration purification. The prepared virus-like particle vaccine can induce a body to generate a specific antibody when immunizing a BALB / c mouse, and has the advantages of strong immunogenicity, good safety, high hereditary stability and the like. And the vaccine is verified to have broad spectrum.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a virus-like particle for fever with thrombocytopenia syndrome, a vaccine, and a method for preparing the same. Technical Background

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic disease caused by a novel Bunyavirus (SFTSV). SFTSV is primarily transmitted through the bite of ticks (Haemaphysalis longhorn), but recent studies have confirmed that domestic animals (such as dogs, cats, and sheep) are the main vectors for transmission to humans. They are not only tick hosts and viral reservoirs, but can also transmit the virus to humans through direct contact (such as blood and secretions) or aerosol routes. Current control measures mainly focus on tick extermination and case management, which are insufficient to block zoonotic transmission. Domestic animals act as "amplifiers" of virus transmission, and their immunization is a crucial link in breaking the transmission chain.

[0003] Traditional inactivated or attenuated vaccines suffer from high production costs, insufficient immunogenicity, or biosafety risks. Furthermore, SFTSV exhibits significant genotypic diversity across different regions, meaning traditional single-antigen vaccines may offer limited protection, and the need for broad-spectrum protection against diverse genotypes remains unmet.

[0004] Therefore, it is necessary to develop a safe and broad-spectrum vaccine using new technologies to meet the needs of SFTS prevention and control. Summary of the Invention

[0005] Virus-like particles (VLPs) are non-infectious particles that self-assemble from structural proteins carrying viral antigens. They mimic the natural structure of viral particles, activate cellular and humoral immune responses, and induce a more comprehensive immune protection mechanism. VLP vaccines can be mass-produced in insect suspension cells using a baculovirus expression system. Mosaic sequences, based on viral gene diversity, utilize a specific genetic algorithm to synthesize a chimeric protein covering the most potential T-cell epitopes from a large number of natural sequences. This application employs a broad-spectrum design, enhancing the vaccine's cross-protective ability against heterologous strains by designing Gn-Mosaic chimeric sequences, providing a forward-looking solution to address viral evolution. Therefore, the production of SFTSV virus-like particle vaccines using a baculovirus expression system has significant application potential.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A virus-like particle for fever with thrombocytopenia syndrome comprises: a chimeric antigen Gn-Mosaic protein, a glycoprotein Gc of the 20-China_Henan-1 strain, and a nucleocapsid protein NP; wherein the chimeric antigen Gn-Mosaic protein is encoded by the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the Gc protein is encoded by the sequence shown in SEQ ID NO: 2 or its degenerate sequence; and the NP protein is encoded by the sequence shown in SEQ ID NO: 3 or its degenerate sequence.

[0008] On the other hand, a method for preparing the aforementioned virus-like particles for fever with thrombocytopenia syndrome is provided, comprising:

[0009] The Gn-Mosaic gene of SEQ ID NO: 1, the GC gene of SEQ ID NO: 2, and the gene of SEQ ID NO: 3 were obtained and cloned into the baculovirus transfer vector pVL1393, respectively, to obtain recombinant shuttle plasmids pVL-Gn-Mosaic, pVL-Gc, and pVL-NP.

[0010] Based on the principle of homologous recombination, recombinant shuttle plasmids pVL-Gn-Mosaic, pVL-Gc, and pVL-NP were co-transfected with linearized baculovirus genomic DNA into insect cells to rescue recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP. The successfully rescued recombinant baculoviruses were purified and transferred into suspended insect cells for large-scale culture to obtain recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP, respectively.

[0011] Recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP were co-infected with suspended insect cells at an MOI ratio of 1:1:1. After culturing, the cell supernatant was collected, and fever-associated thrombocytopenia syndrome virus-like particles (SFTSV-VLPs) containing Gn-Mosaic, Gc, and NP proteins were harvested.

[0012] In some embodiments, the insect cells are Sf9 cells.

[0013] In some embodiments, the baculovirus is the alfalfa silver-striped moth multinucleate polyhedrovirus (AcMNPV).

[0014] On the other hand, a virus-like particle for fever with thrombocytopenia syndrome is provided, which is prepared by the aforementioned preparation method.

[0015] On the other hand, a vaccine is provided, comprising a pharmaceutically acceptable carrier and an immunizing dose of the aforementioned fever with thrombocytopenia syndrome virus-like particles.

[0016] Furthermore, the pharmaceutically acceptable carrier includes an adjuvant, wherein the adjuvant is a SEPPIC adjuvant.

[0017] Furthermore, the viral-like particles of fever with thrombocytopenia syndrome are emulsified by mixing the immunizing dose with the adjuvant at a volume ratio of 1:1.

[0018] On the other hand, the use of the aforementioned fever with thrombocytopenia syndrome virus-like particles in the preparation of medicaments for the prevention and / or treatment of diseases caused by fever with thrombocytopenia syndrome is provided.

[0019] On the other hand, the use of the vaccine is provided in the preparation of a medicament for the prevention and / or treatment of diseases caused by fever with thrombocytopenia syndrome.

[0020] The vaccine prepared in this application was used to immunize BALB / c mice. Blood samples were collected two weeks after the first immunization and one, two, and three weeks after the second immunization to detect IgG antibodies.

[0021] The broad-spectrum nature of the virus-like vaccine was verified by reacting immunized mouse serum with different genotypes of SFTSV-Gn protein.

[0022] This invention leverages the unique advantages of the insect cell-baculovirus expression system, including its high safety, high protein expression levels, and ease of large-scale production. Utilizing the Ph promoter and multiple cloning site provided by the baculovirus transfer vector pVL1393, and selecting broad-spectrum antigens Gn-Mosaic, Gc, and NP proteins as target proteins for vaccine design, recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP were constructed. The biological characteristics of the viruses, including growth, genetic stability, and exogenous protein expression, were identified. The conditions for forming virus-like particles were optimized, and a virus-like particle vaccine was prepared. Its immunogenicity in BALB / c mice was evaluated. The above findings provide a new approach for the development of SFTSV vaccines and demonstrate the promising application prospects of this vaccine in the prevention and control of SFTSV.

[0023] The beneficial effects of this invention are as follows: (1) Currently, there are no approved vaccines against SFTS reported globally. Existing prevention and control measures mainly rely on tick control and case management, but these are difficult to block animal-borne transmission. Domestic animals act as "amplifiers" for virus transmission, and their immunization is the core link in cutting off the transmission chain. SFTS exhibits genotypic diversity in different regions (e.g., differences in prevalent strains in China, South Korea, and Japan), and traditional single-antigen vaccines may have limited protection. This application adopts a broad-spectrum design, which enhances the cross-protection ability of the vaccine against heterologous strains by designing Gn-Mosaic chimeric sequences, providing a forward-looking solution to address viral evolution. (2) The prepared SFTSV-VLP vaccine, the three recombinant baculovirus (rBac-Gn-Mosaic, rBac-Gc, rBac-NP) subunit vaccine (prepared by mixing the three recombinant baculoviruses with BEI inactivation and adjuvant), and the inactivated SFTSV were injected intramuscularly into four-week-old BALB / c mice at a dose of 15 μg. All vaccines could induce IgG antibodies, and the level of IgG antibodies induced by the VLP vaccine was significantly higher than that induced by the subunit vaccine and the inactivated vaccine. The prepared SFTSV-VLP vaccine, recombinant baculovirus subunit vaccine rBac-Gn-Mosaic, and recombinant baculovirus subunit vaccine rBac-Gn-E (expressing type E SFTSV-Gn protein) were administered intramuscularly to four-week-old BALB / c mice at a dose of 15 μg. The immunized mouse serum reacted with different genotypes of SFTSV-Gn protein, verifying the broad-spectrum nature of the virus-like vaccines. All vaccines induced IgG antibodies, and the IgG antibody level induced by the VLP vaccine was significantly higher than that of the two subunit vaccines. The recombinant baculovirus subunit vaccine rBac-Gn-Mosaic induced IgG antibodies superiorly to the recombinant baculovirus subunit vaccine rBac-Gn-E (expressing type E SFTSV-Gn protein). Attached Figure Description

[0024] Figure 1 This is an electrophoretic identification diagram of the recombinant shuttle plasmid DNA. Line A 1 shows the pVL1393 vector and the target gene NP fragment, while line 2 shows the pVL1393 vector and the target gene Gc fragment; line B shows the pVL1393 vector and the target gene Gn-Mosaic fragment. M represents a 1kb DNA ladder maker.

[0025] Figure 2 The diagram shows the results of identifying the target protein using an indirect immunofluorescence assay; A is recombinant baculovirus rBac-Gn-Mosaic; B is recombinant baculovirus rBac-Gc; C is recombinant baculovirus rBac-NP; and D is the Sf9 cell control.

[0026] Figure 3The results of Western blot identification of the target gene after infection of Sf9 cells with the prepared recombinant baculovirus seed virus are shown. A is recombinant baculovirus rBac-Gn-Mosaic; B is recombinant baculovirus rBac-Gc; C is recombinant baculovirus rBac-NP.

[0027] Figure 4 The images show the PCR identification of recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP; line 1 represents the Gn-Mosaic gene, line 2 represents the Gc gene, and line 3 represents the NP gene. M represents a 1kb DNA ladder maker.

[0028] Figure 5 Images for SFTSV-VLP identification. A is a transmission electron microscope (TEM) image of SFTSV-VLP; B is a Western blotting (WB) identification result of SFTSV-VLP.

[0029] Figure 6 , Figure 7 , Figure 8 , Figure 9 The results of Gn antibody testing were obtained on days 14, 21, 28, and 35 after vaccination.

[0030] Figure 10 , Figure 11 , Figure 12 , Figure 13 The results of Gc antibody testing were obtained on days 14, 21, 28, and 35 after vaccination.

[0031] Figure 14 , Figure 15 , Figure 16 , Figure 17 The results of NP antibody testing were obtained on days 14, 21, 28, and 35 after vaccination.

[0032] Figure 18 , Figure 19 The results of the reaction between serum IgG antibodies and type A SFTSV-Gn protein were measured on days 14 and 28 after vaccination, respectively.

[0033] Figure 20 , Figure 21 The results of the reaction between serum IgG antibodies and type B SFTSV-Gn protein were measured on days 14 and 28 after vaccination, respectively.

[0034] Figure 22 , Figure 23 The results of the reaction between serum IgG antibodies and type C SFTSV-Gn protein were measured on days 14 and 28 after vaccination, respectively.

[0035] Figure 24 , Figure 25 The results of the reaction between serum IgG antibodies and type D SFTSV-Gn protein were measured on days 14 and 28 after vaccination, respectively.

[0036] Figure 26 , Figure 27 The results of the reaction between serum IgG antibodies and type E SFTSV-Gn protein were measured on days 14 and 28 after vaccination, respectively. Detailed Implementation

[0037] The embodiments of the present invention are illustrated by the following examples. These examples are for illustrative purposes only, and the embodiments of the present invention are not limited to the specific details of the examples. The scope of the present invention is defined by the appended claims.

[0038] Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available. Unless otherwise specified, the experimental methods described are conventional experimental methods.

[0039] Example 1: Construction and identification of recombinant baculovirus plasmid.

[0040] This invention, based on viral gene diversity, synthesizes a Gn-Mosaic sequence covering the most potential T-cell epitopes from a large number of natural sequences, aiming to induce broader protection in the body. All SFTSV-Gn protein amino acid sequences were downloaded from GenBank, and incomplete sequences that were too short were removed, leaving 560 sequences. These 560 sequences were exported as FASTA format using MEGA. The Gn.fasta file was uploaded to the Mosaic Vaccine Designer website, and the parameters were set as follows: "Generate mosaic cocktail" function; Cocktail Size was set to "1" to obtain a Gn-Mosaic sequence for the next step of plasmid construction. Nucleotide sequences of Gc protein (OM452767.1) and NP protein (OM452068) were obtained from publicly available sequences in GenBank. The Gn-Mosaic gene (SEQ ID NO: 1), the GC gene (SEQ ID NO: 2), and the gene (SEQ ID NO: 3) were obtained through gene synthesis and cloned into the pVL-1393 shuttle vector. Primers were designed using SnapGene 3.1.1 software based on the gene sequence Gn-Mosaic, Gc, and NP to identify the target gene. Primers and gene sequences were synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0041] Figure 1 Electrophoretic identification diagram of recombinant shuttle plasmid DNA. Figure 1 In section A: line 1 is the pVL1393 vector and the target gene NP fragment, while line 2 is the pVL1393 vector and the target gene Gc fragment; Figure 1 In the diagram, B represents the pVL1393 vector and the Gn-Mosaic fragment of the target gene. M represents a 1kb DNA ladder maker.

[0042] Example 2: Rescue and identification of recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP.

[0043] (1) Obtaining recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP.

[0044] The recombinant shuttle plasmids pVL-Gn-Mosaic, pVL-Gc, and pVL-NP, along with linearized baculovirus genomic DNA from the alfalfa silver-striped moth multinucleate polyhedrovirus AcMNPV, were co-transfected into Sf9 cells in logarithmic growth phase. A simplified procedure is as follows:

[0045] Add Sf9 cells, 1×10⁶, to a six-well cell plate. 6 / well, place the cell plate horizontally in a 27°C incubator and incubate for 1 hour, while preparing transfection samples;

[0046] Prepare the DNA transfection complex: Dilute the recombinant shuttle plasmid to a concentration of 100 ng / μL for later use; preheat the transfection reagent to room temperature and mix gently; prepare two sterile EP tubes and add 95 μL of serum-free culture medium to each; add 5 µL of transfection reagent to one tube and gently tap the EP tube to mix well; add 5 µL (40 ng) of Bacmid DNA to the other tube, and then add 3 µL (300 ng) of transfer vector plasmid DNA to the centrifuge tube and mix thoroughly.

[0047] Will Adding the solution Mix thoroughly and let stand at room temperature for 20-30 minutes.

[0048] After culturing cells for 1 hour, remove the culture medium, slowly add 1 mL of serum-free culture medium along the well wall, and gently wash the cell monolayer twice.

[0049] Add the cells to a monolayer and incubate at 27°C for 4-6 days;

[0050] Collect the cell culture supernatant and centrifuge at 3000 rpm for 10 min to harvest the P0 generation recombinant baculovirus. Aliquot the harvested cell supernatant and store it protected from light. For short-term storage, keep at 4°C; for long-term storage, keep at -70°C.

[0051] (2) Identification of the expression of the target proteins Gn-Mosaic, Gc, and NP.

[0052] ① Indirect immunofluorescence detection of target proteins Gn-Mosaic, Gc, and NP. Figure 2 This is a diagram showing the results of identifying the target protein using an indirect immunofluorescence assay. A represents recombinant baculovirus rBac-Gn-Mosaic; B represents recombinant baculovirus rBac-Gc; C represents recombinant baculovirus rBac-NP; and D represents Sf9 cell control.

[0053] ②Western-blot of the target protein. Figure 3 The images show the Western blot (WB) results of the target gene after infecting Sf9 cells with the prepared recombinant baculovirus seed virus. A represents recombinant baculovirus rBac-Gn-Mosaic; B represents recombinant baculovirus rBac-Gc; and C represents recombinant baculovirus rBac-NP.

[0054] ③ DNA was extracted from the recombinant baculovirus and identified by PCR. Figure 4 PCR identification diagrams for recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP; line 1 is the Gn-Mosaic gene, line 2 is the Gc gene, and line 3 is the NP gene. M represents a 1kb DNA ladder maker.

[0055] Example 3: Identification of the genetic stability of recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP.

[0056] (1) Sf9 cell suspension culture.

[0057] ① After Sf9 cells are revived, they are cultured in T25 square flasks for more than 3 generations until the cells are in good condition. Then they can be transferred to Erlenmeyer flasks for shaking suspension culture.

[0058] ② With an initial density of 1×10 6 The inoculum size was gradually increased to 250 mL Erlenmeyer flasks, and cells were cultured in Sf900 III SFM serum-free medium with 80 mL of cell culture solution added. The culture temperature was 27°C and the rotation speed was 120 rpm. Cell counts were performed daily during the cell culture period.

[0059] ③ When the cell growth density reaches 5×10 6 ~6×106 Passage was performed at a cell density of 100 cells / mL, and the high-density cell solution was diluted with fresh culture medium to a density of 1×10⁻⁶. 6 The cells were collected at a concentration of 80 cells / mL and then transferred to a new sterile Erlenmeyer flask, maintaining the total cell volume at 80 mL.

[0060] Sf9 cells, after being passaged for more than 3 generations in shake flask suspension culture and exhibiting no cell aggregation and in good condition, can be used for subsequent amplification of recombinant baculovirus.

[0061] (2) Amplification of recombinant viruses.

[0062] ①The initial density of Sf9 cells was 1×10⁻⁶ 6 Inoculate at a rate of cells / mL into a 250 mL shake flask, and culture in a volume of 80 mL.

[0063] ② Dilute rBac-Gn-Mosaic-P0, rBac-Gc-P0, and rBac-NP-P0 by 10-fold.

[0064] ③ Take 500 μL of virus dilution and add it to a 250 mL shake flask. Place the flask in a shaker and suspend it by shaking. The incubation temperature is 27℃ and the shaker speed is 120 rpm.

[0065] ④ After culturing for 4 consecutive days, harvest the cell supernatant, which is the P1 generation recombinant baculovirus rBac-Gn-Mosaic, rBac-Gc, and rBac-NP.

[0066] (3) Virus titer assay (TCID) 50 )

[0067] ①Lay up 96-well cell culture plates with a cell quantity of 1×10⁶ cells / wells. 5 cells / well, let stand at 27°C for 1 h.

[0068] ② The recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP were serially diluted 10-fold using Sf900 III SFM medium.

[0069] ③ Discard the culture medium from the 96-well cell plate and take 10g of virus. -1 ~10 -6 The serial dilution buffer was added sequentially to the corresponding wells of the 96-well cell plate. The virus inoculation volume was 100 μL per well, and each dilution was repeated to inoculate 8 wells.

[0070] ④ Cover the lid and allow it to absorb at 27°C for 1 hour.

[0071] ⑤ Discard the virus solution and replace it with Excell-420 containing 5% serum, then incubate at 27°C.

[0072] ⑥ After continuous culture for 4 days, IFA was performed to determine and calculate the viral infection based on the expression of gp64 protein.

[0073] (4) Recombinant baculovirus was used to infect shake-flask Sf9 cells with an MOI of 0.01 and the virus was passaged from P1 to P4.

[0074] Example 4: TCID of passaged recombinant baculovirus 50 Identification

[0075] (1) Determination of recombinant baculovirus titer in passaged strains (TCID) 50 The results are shown in Table 1.

[0076] Table 1: Results of viral titer (TCID50) determination for recombinant baculoviruses from generation 1 to generation 4

[0077]

[0078] Example 5: Harvesting and Identification of VLPs.

[0079] (1) Assembly of VLP.

[0080] SFTSV-VLP was assembled by co-infecting Sf9 cells with recombinant baculoviruses. Three baculovirus strains, rBac-Gn-Mosaic, rBac-Gc, and rBac-NP, were used to infect Sf9 cells at an MOI of 1:1:1, with a cell density of 10⁶ cells / year. 6 The cell culture supernatant was collected at 100 mL per 100 mL cell culture depth. After 72 h of infection, the cell culture supernatant was harvested, centrifuged at 3000 rpm for 10 min to remove cell debris, and then briefly stored at 4 °C.

[0081] (2) The benefits of VLP.

[0082] The supernatant harvested in step (1) was centrifuged using an Amicon Ultra-15 centrifuge equipped with an Ultracel-50 filter membrane.

[0083] The solution was concentrated using a filter. The conditions were 5000g for 30-60 minutes, and the concentrate was collected. The obtained concentrate was then purified by ultracentrifugation at 100000g for 2 hours to obtain purified SFTSV-VLP.

[0084] (3) Identification of VLP.

[0085] ① Electron microscopy observation of the SFTSV-VLP structure.

[0086] ②SFTSV-VLP Western blot identification.

[0087] Figure 5 Images for SFTSV-VLP identification. A is a transmission electron microscope (TEM) image of SFTSV-VLP; B is a Western blotting (WB) identification result of SFTSV-VLP.

[0088] Example 6: Vaccine preparation.

[0089] The concentrations of antigen proteins were determined using a BCA protein quantification kit (Beyotime). Harvested VLPs and inactivated recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP were mixed at a 1:1 ratio of antigen to adjuvant. The mixture was homogenized by intermittent vigorous shaking at 6000 rpm for 30 minutes to ensure thorough emulsification. Emulsification continued until the mixture formed a uniform, milky-white emulsion. A dosage form test was performed by adding a small amount of sample to tap water; the emulsion appeared as a non-dispersible water-in-oil emulsion.

[0090] Example 7: Vaccine Immunization Experiment.

[0091] Thirty-six four-week-old BALB / c mice were randomly divided into five groups: SFTSV-VLPs immunization group (n=6), rBac-Gn-Mosaic immunization group (n=6), rBac-Gc immunization group (n=6), rBac-NP immunization group (n=6), inactivated SFTSV immunization group (n=6), and PBS group (n=6). Vaccinations were administered via intramuscular injection in the leg. The immunization dose for each group is shown in Table 2. The unimmunized group received 500 μL of PBS as a control.

[0092] Table 2: Immunoassay Groups in BALB / c Mice

[0093]

[0094] Blood was collected from the mandibular sinus at 14, 21, 28, and 35 days post-immunization to separate serum. Gn, Gc, and NP proteins were used as detection antigens, and the antibody levels of immunized mice were determined using an indirect ELISA method.

[0095] Figures 6 to 17 The results showed serum antibody levels on days 14, 21, 28, and 35 post-vaccination. Figures 6 to 9 This refers to the results of Gn antibody testing after immunization; Figures 10 to 13 This refers to the results of Gc antibody testing after immunization; Figures 14 to 17 This is the result of NP antibody detection after immunization.

[0096] The results showed that, after immunization, the antibody responses against specific proteins induced by the SFTSV-VLPs immunization group, the rBac-Gn-Mosaic immunization group, the rBac-Gc immunization group, the rBac-NP immunization group, and the inactivated SFTSV immunization group were all significantly higher than those induced by the PBS group. Furthermore, the antibody responses against specific proteins induced by the SFTSV-VLPs immunization group, the rBac-Gn-Mosaic immunization group, the rBac-Gc immunization group, and the rBac-NP immunization group were all significantly higher than those induced by the inactivated SFTSV vaccine group. Notably, the levels of Gn and Gc antibodies produced by the SFTSV-VLPs immunization group were significantly higher than those produced by the respective subunit vaccines immunization groups.

[0097] Example 8: Vaccine broad-spectrum validation experiment

[0098] SFTSV viral sequences expressing Gn protein (genotypes A, B, C, D, and E) were downloaded from GenBank. These sequences were synthesized and constructed on the eukaryotic expression plasmid Pet-32a, and the five genotypes of Gn protein were expressed in BL-21 supercompetent prokaryotic cells. The Gn protein of the five genotypes AE was purified using Beyotime's His-tag purification kit. Twenty-four four-week-old BALB / c mice were randomly divided into four groups: SFTSV-VLPs immunization group (n=6), rBac-Gn-Mosaic immunization group (n=6), rBac-Gn-E immunization group (n=6), and PBS group (n=6). Vaccinations were administered via intramuscular injection in the leg. The immunization dose for each group is shown in Table 3. The unimmunized group received 500 μL of PBS as a control.

[0099] Table 3: Immunoassay Groups in BALB / c Mice

[0100]

[0101] Blood was collected from the mandibular sinus at 14 and 28 days post-immunization to separate serum. Five different genotypes of Gn protein expressed in prokaryotes were used as detection antigens, and the antibody levels of immunized mice were determined by indirect ELISA.

[0102] Figures 18-27 The results of the broad-spectrum validation experiment were obtained by detecting the reactivity of serum IgG antibodies with SFTSV-Gn proteins of five different genotypes (A, B, C, D, and E) on days 14 and 28 after vaccine immunization.

[0103] The study found that, after immunization, the antibody responses against specific proteins induced by the SFTSV-VLPs immunization group, the rBac-Gn-Mosaic immunization group, and the rBac-Gn-E immunization group were significantly higher than those induced by the PBS group, and the antibody levels induced by the SFTSV-VLPs immunization group were significantly higher than those induced by the subunit vaccine immunization group.

[0104] In summary, the recombinant baculovirus-based fever with thrombocytopenia syndrome virus-like particle (SFSV-VLPs) vaccine induced a rapid antibody response in mice and exhibited strong immunogenicity. Furthermore, the immunization efficacy of the SFTSV-VLPs vaccine group was superior to that of the subunit vaccine group and the inactivated vaccine group. The SFTSV-VLPs vaccine, designed with chimeric antigens to cover different genotypes of viral strains, produced antibodies that reacted with proteins from five different genotypes of viruses, providing cross-protective efficacy against prevalent strains in East Asia.

[0105] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the implementation of the present invention. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the present invention without departing from the inventive concept are within the scope of protection of the present invention.

Claims

1. A virus-like particle for fever with thrombocytopenia syndrome, characterized in that, include: The chimeric antigen Gn-Mosaic protein, the glycoprotein Gc of the 20-China_Henan-1 strain, and the nucleocapsid protein NP; wherein, the chimeric antigen Gn-Mosaic protein is encoded by the sequence shown in SEQ ID NO: 1 or its degenerate sequence; the Gc protein is encoded by the sequence shown in SEQ ID NO: 2 or its degenerate sequence; and the NP protein is encoded by the sequence shown in SEQ ID NO: 3 or its degenerate sequence.

2. The method for preparing virus-like particles for fever with thrombocytopenia syndrome according to claim 1, characterized in that, include: The Gn-Mosaic gene of SEQ ID NO: 1, the GC gene of SEQ ID NO: 2, and the gene of SEQ ID NO: 3 were obtained and cloned into the baculovirus transfer vector pVL1393, respectively, to obtain recombinant shuttle plasmids pVL-Gn-Mosaic, pVL-Gc, and pVL-NP. Based on the principle of homologous recombination, recombinant shuttle plasmids pVL-Gn-Mosaic, pVL-Gc, and pVL-NP were co-transfected with linearized baculovirus genomic DNA into insect cells to rescue recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP. The successfully rescued recombinant baculoviruses were purified and transferred into suspended insect cells for large-scale culture to obtain recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP, respectively. Recombinant baculoviruses rBac-Gn-Mosaic, rBac-Gc, and rBac-NP were co-infected with suspended insect cells at an MOI ratio of 1:1:

1. After culturing, the cell supernatant was collected, and virus-like particles containing Gn-Mosaic, Gc, and NP proteins were harvested to induce fever with thrombocytopenia syndrome.

3. The method for preparing virus-like particles for fever with thrombocytopenia syndrome according to claim 2, characterized in that, The insect cells used were Sf9 cells.

4. The method for preparing virus-like particles for fever with thrombocytopenia syndrome according to claim 2, characterized in that, The baculovirus used is AcMNPV, a multinucleate polyhedrovirus of the alfalfa silver-striped moth.

5. A virus-like particle for fever with thrombocytopenia syndrome, characterized in that, It is prepared by the preparation method according to any one of claims 2-4.

6. A vaccine, characterized in that, Including pharmaceutically acceptable carriers and immunizing doses of the virus-like particles for fever with thrombocytopenia syndrome as described in claims 1 and 5.

7. The vaccine according to claim 6, characterized in that, The pharmaceutically acceptable carrier includes an adjuvant, wherein the adjuvant is a SEPPIC adjuvant.

8. The vaccine according to claim 7, characterized in that, The viral-like particles of the fever with thrombocytopenia syndrome were emulsified by mixing the immunizing dose with the adjuvant in a 1:1 volume ratio.

9. The use of the viral-like particles of fever with thrombocytopenia syndrome as described in claims 1 and 5 in the preparation of medicaments for the prevention and / or treatment of diseases caused by fever with thrombocytopenia syndrome.

10. The use of the vaccine according to any one of claims 6-8 in the preparation of a medicament for the prevention and / or treatment of diseases caused by fever with thrombocytopenia syndrome.

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