Bacteria-like particle-based tick-borne encephalitis virus vaccine and methods of making and using the same

Abstract

The application belongs to the technical field of biological medicine, and provides a tick-borne encephalitis virus vaccine based on bacterial-like particles, a preparation method and application thereof, wherein the tick-borne encephalitis virus vaccine is EDc2&MAB2560-GEM co-display bacterial-like particles, which are prepared by co-displaying recombinant antigen protein TBEV E-DIII-Dcpep2-PA3 and recombinant adjuvant protein MAB2560-PA3 on the surface of GEM particles; the prepared tick-borne encephalitis virus vaccine has good immunogenicity and can induce animals to produce efficient and persistent specific immune responses.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a tick-borne encephalitis virus vaccine based on bacterial-like particles, its preparation method and application. Background Technology

[0002] Tick-borne encephalitis (TBE), also known as tick-borne encephalitis, is a zoonotic disease primarily affecting the nervous system, caused by the tick-borne encephalitis virus (TBEV). It can cause fatal encephalitis in humans and animals, accompanied by long-term sequelae, posing a serious threat to human health. With global climate change, the number and activity range of ticks are constantly expanding, and the incidence of TBE is currently showing an upward trend year by year, posing a dual threat to public health and livestock production. As one of the important tick-borne pathogens, vaccine development against it has never stopped. However, existing inactivated vaccines have problems such as poor safety and insufficient duration of immunity. How to solve these problems is a hot topic and direction of research in this field.

[0003] Tick-borne encephalitis virus (TEV) is a single-stranded positive-sense RNA virus belonging to the genus Flaviviridae in the family Flaviviridae. It is approximately 11 kb long and contains one open reading frame (ORF). This single ORF encodes three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Flavivir are enveloped viruses with an average diameter of 40-60 nm. The surface of mature viral particles consists of dimers of 90 E and M proteins. A lipid bilayer surrounds the nucleocapsid (composed of the C protein), within which lies the single-stranded positive-sense RNA genome. The flavivirus C protein can form the nucleocapsid together with the viral genome and can also interact with liposomes, participating in cell membrane remodeling, but it does not induce the production of neutralizing antibodies. The transmembrane domain of the C protein can serve as an additional anchor and signaling sequence for the prM protein. The prM protein is cleaved by host furin protease, producing the pr fragment and the M protein. The M protein is anchored to the lipid envelope via two transmembrane domains and interacts with the transmembrane domains of the E protein in mature granules. The E protein is an envelope protein containing neutralizing antibody epitopes and plays an important role in immunoprophylaxis.

[0004] Bacterial-like particles (BLPs) are a novel, broad-spectrum biomimetic nanoparticle vaccine platform composed of Gram-positive bacterial enhancer matrix (GEM) particles and ptein anchor (PA) proteins. They are widely used as antigen delivery vectors in subunit vaccine development and hold great potential in vaccine research. BLPs offer several advantages over traditional vaccines: their main component, peptidoglycan, is a natural TLR agonist that stimulates innate immunity without additional adjuvants and can be effectively recognized by immune system-related cells and molecules; derived from edible microorganisms such as Lactococcus lactis, they exhibit excellent biocompatibility and contain no nucleic acid, making them safer for immunocompromised vaccine recipients; they can display antigens in multiple forms and simultaneously display both antigens and adjuvant molecules. Currently, numerous studies have used BLPs as vectors to develop novel vaccines, such as those for porcine epidemic diarrhea virus and Newcastle disease virus, significantly enhancing systemic and mucosal immune responses in animal models. Various bacterial-like particle vaccines produced using different expression systems are in the preclinical development stage. Summary of the Invention

[0005] The purpose of this invention is to provide a tick-borne encephalitis virus vaccine based on bacterial-like particles, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A tick-borne encephalitis virus vaccine based on bacterial-like particles, comprising EDc2 & MAB2560-GEM co-displaying bacterial-like particles, is prepared by co-displaying recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3 and recombinant adjuvant protein MAB2560-PA3 on the surface of GEM particles.

[0008] Furthermore, the amino acid sequence of the recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3 is shown in the sequence listing SEQ ID NO:8.

[0009] Furthermore, the amino acid sequence of the recombinant adjuvant protein MAB2560-PA3 is shown in the sequence listing SEQ ID NO:10.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned tick-borne encephalitis virus vaccine based on bacterial-like particles, which includes the following steps:

[0011] Construction and expression of recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3: Using a plasmid containing the target gene TBEV E-DⅢ-Dcpep1-PA3 as a template, the TBEV E-DⅢ-Dcpep2-PA3 gene was amplified using primers TBEV E-DⅢ-Dcpep2-PA3-F and TBEV E-DⅢ-Dcpep2-PA3-R, cloned into a eukaryotic expression vector, and then transfected into host cells for expression. Cell culture supernatant containing the recombinant antigen protein was collected. The nucleotide sequence of TBEV E-DⅢ-Dcpep1-PA3 is shown in SEQ ID NO:5; the nucleotide sequence of the TBEV E-DⅢ-Dcpep2-PA3 gene is shown in SEQ ID NO:7.

[0012] Construction and expression of recombinant adjuvant protein MAB2560-PA3: Using a plasmid containing the target gene MAB2560-PA3 as a template, the MAB2560-PA3 gene was amplified using primers MAB2560-PA3-F and MAB2560-PA3-R, cloned into a eukaryotic expression vector, and then transfected into host cells for expression. Cell culture supernatant containing the recombinant adjuvant protein was collected; the nucleotide sequence of MAB2560-PA3 is shown in SEQ ID NO:9.

[0013] Preparation of EDc2-GEM BLPs: Cell culture supernatant containing recombinant antigen protein was mixed with GEM particles and incubated to obtain EDc2-GEM BLPs;

[0014] Preparation of EDc2 & MAB2560-GEM co-display bacterial-like particles: Cell culture supernatant containing recombinant antigen protein was mixed with EDc2-GEM BLPs and incubated to obtain the tick-borne encephalitis virus vaccine.

[0015] Furthermore, the eukaryotic expression vector is pCDNA3.4, and the host cell is HEK293F cell.

[0016] Furthermore, the nucleotide sequences of the primers TBEV E-DⅢ-Dcpep2-PA3-F and TBEV E-DⅢ-Dcpep2-PA3-R are shown in the sequence listing SEQ ID NO:11-12, respectively.

[0017] Furthermore, the nucleotide sequences of the primers MAB2560-PA3-F and MAB2560-PA3-R are shown in the sequence listing SEQ ID NO:15-16, respectively.

[0018] Another object of the present invention is to provide the application of the above-mentioned tick-borne encephalitis virus vaccine based on bacterial-like particles in the preparation of a drug for preventing tick-borne encephalitis virus.

[0019] This invention provides a tick-borne encephalitis virus vaccine based on bacterial-like particles, which is EDc2 & MAB2560-GEM co-displaying bacterial-like particles. The expression is mainly performed using the 293F eukaryotic expression system. It is prepared by co-displaying the recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3 and the recombinant adjuvant protein MAB2560-PA3 obtained from the 293F eukaryotic expression system on the surface of GEM particles. The tick-borne encephalitis virus vaccine prepared by this invention has good immunogenicity and can induce a highly efficient and durable specific immune response in animals. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the recombinant plasmid pCDNA3.4-TBEV E-DⅢ-Dcpep2-PA3 in Example 1.

[0021] Figure 2 The results are PCR amplification results of the TBEV E-DⅢ-Dcpep2-PA3 gene in Example 1; where M: DL5K DNA Marker; 1: PCR amplification results of the TBEV E-DⅢ-Dcpep1-PA3 gene; 2: PCR amplification results of the TBEV E-DⅢ-Dcpep2-PA3 gene.

[0022] Figure 3 The identification results of the recombinant plasmid pCDNA3.4-TBEV E-DⅢ-Dcpep2-PA3 in Example 1; where M: DL5K DNA Marker; 1: TBEV E-DⅢ-Dcpep1-PA3 bacterial culture PCR identification results; 2: TBEV E-DⅢ-Dcpep2-PA3 bacterial culture PCR identification results.

[0023] Figure 4 The Western Blot results for the recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3 in Example 1 are shown below; where M: protein marker; 1: cell control; 2: cell culture supernatant of 293F suspension cells expressing TBEV E-DⅢ-Dcpep1-PA3; 3: cell culture supernatant of 293F suspension cells expressing TBEV E-DⅢ-Dcpep2-PA3.

[0024] Figure 5 The identification results of EDc2-GEM BLPs in Example 2 are shown below; where A: Western Blot identification results; B: Indirect immunofluorescence identification results (Ba: empty GEM particles; Bb: EDc2-GEM).

[0025] Figure 6 This is a schematic diagram of the recombinant plasmid pCDNA3.4-MAB2560-PA3 in Example 3.

[0026] Figure 7 The result is the PCR amplification of the MAB2560-PA3 gene in Example 3; where M: DL5K DNA Marker; 1: PCR amplification product of the target fragment MAB2560-PA3.

[0027] Figure 8 The results are for the identification of the recombinant plasmid pCDNA3.4-MAB2560-PA3 in Example 3; where M: DL5K DNA Marker; 1-2: are the results of bacterial PCR identification of pCDNA3.4-MAB2560-PA3.

[0028] Figure 9 The Western Blot identification results of the recombinant adjuvant protein MAB2560-PA3 in Example 3 are shown; where M: protein marker; 1: cell culture supernatant sample of 293F suspension cells expressing MAB2560-PA3.

[0029] Figure 10 The identification results of MAB2560 BLPs in Example 4 are shown below; where A: Western Blot identification results (M: Protein Marker 1: Blank GEM particle sample 2: MAB2560-GEM sample); B: Indirect immunofluorescence identification (Ba: Empty GEM particle; Bb: MAB2560-GEM BLPs).

[0030] Figure 11 The identification results of EDc2 & MAB2560 BLPs in Example 5 are shown below; where A: Western Blot identification results (M: Protein Marker 1: EDc2 & MAB2560-GEM sample); B: Indirect immunofluorescence identification results.

[0031] Figure 12 The results of the detection of TBEV IgG antibodies in mouse serum in Example 6 are shown.

[0032] Figure 13 The results of flow cytometry analysis of DC cell activation in inguinal lymph nodes after the first immunization in Example 6; where A: CD11c + CD80 + Percentage of double-positive cells; B: CD11c + CD86 + Percentage of double-positive cells; C:CD11c + MHCⅠ +Percentage of double-positive cells; D:CD11c + MHCⅡ + The proportion of double-positive cells.

[0033] Figure 14 The results of ELISpot detection of IFN-γ and IL-4 in spleen cells in Example 6 are shown; where A: ELISpot detection result of IFN-γ in spleen cells; B: ELISpot detection result of IL-4 in spleen cells.

[0034] Figure 15 The results are from the spleen cell proliferation experiment in Example 6.

[0035] Figures 16-18 The results are from flow cytometry analysis of mouse spleen cells after three immunizations in Example 6; whereby, Figure 16 Recruiting and activating mouse spleen B cells (Aa:CD19) + CD40 + Percentage of double-positive cells; Ab: CD19 + CD69 + (proportion of double-positive cells); Figure 17 Recruitment and activation of mouse spleen T cells (Ba:CD4) + CD69 + Percentage of double-positive cells; Bb:CD8 + CD69 + (proportion of double-positive cells); Figure 18 Production of memory T cells in the mouse spleen (Ca:CD4) + / CD44 + CD62L + Percentage of double-positive cells; Cb:CD8 + / CD44 + CD62L + (Percentage of double-positive cells). Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, the materials and reagents involved are all commercially available products; unless otherwise specified, the experimental methods used are all conventional methods.

[0038] Example 1: This example provides a method for constructing and identifying the recombinant antigen protein TBEV E-DⅢ (Domain III)-Dcpep2-PA3, as detailed below:

[0039] 1. Primer Design and Synthesis: Specific amplification primers were designed (as shown in Table 1). Primers were designed using the previously constructed eukaryotic expression plasmid pCDNA3.4-TBEV E-DⅢ-Dcpep1-PA3 as a template. A pair of amplification primers (TBEV E-DⅢ-Dcpep2-PA3-F and TBEV E-DⅢ-Dcpep2-PA3-R) and a pair of identification primers (JD-F and JD-R) were designed. It should be noted that the construction process of pCDNA3.4-TBEV E-DⅢ-Dcpep1-PA3 in the laboratory was as follows: Based on the TBEV gene sequence published in GenBank, the Far Eastern subtype strain WH2012 (GenBank: KJ755186) was selected, and the E protein (prM-E) gene sequence was synthesized (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2). The Domain III gene sequence of the E protein was predicted. Using the peptide Dcpep (nucleotide sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:4), upstream and downstream amplification primers were designed to amplify the TBEV E-DⅢ-Dcpep1 gene fragment. Simultaneously, the sequence containing the synthesized anchor protein PA3 was amplified. After amplification, TBEV E-DⅢ-Dcpep1, PA3, and the previously stored pCDNA3.4 linearized vector were seamlessly ligated together to construct pCDNA3.4-TBEV E-DⅢ-Dcpep1-PA3. Sequencing confirmed the successful construction of the plasmid. The nucleotide sequence of TBEV E-DⅢ-Dcpep1-PA3 is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6.

[0040] Table 1 Primer Information

[0041]

[0042] 2. For example Figure 1 As shown, the specific methods for constructing and identifying the recombinant plasmid pCDNA3.4-TBEV E-DⅢ-Dcpep2(Linker)-PA3 are as follows:

[0043] (1) Amplification of the target gene TBEV E-DⅢ-Dcpep2-PA3: Using the plasmid pCDNA3.4-TBEV E-DⅢ-Dcpep1-PA3 containing the target gene TBEV E-DⅢ-Dcpep1-PA3 as a template, the TBEV E-DⅢ-Dcpep2-PA3 gene (nucleotide sequence as shown in SEQ ID NO:7) was amplified using primers TBEV E-DⅢ-Dcpep2-PA3-F and TBEV E-DⅢ-Dcpep2-PA3-R (Table 1). The reaction conditions are shown in Table 2. The target fragment was separated and recovered by 1% agarose gel electrophoresis. The amplification results showed that the product was the same size as the target fragment ( Figure 2 The TBEV E-DⅢ-Dcpep2-PA3 gene was successfully amplified. Then, it was seamlessly cloned and ligated into the previously stored pCDNA3.4 vector in the laboratory at a certain ratio, and the reaction was carried out at 50℃ for 15 min. The ligation system is shown in Table 3.

[0044] Table 2 PCR reaction conditions

[0045]

[0046] Table 3 Seamless Connection System

[0047]

[0048] (2) Identification of recombinant plasmid pCDNA3.4-TBEV E-DⅢ-Dcpep2-PA3: The complete pCDNA3.4-TBEV E-DⅢ-Dcpep2-PA3 gene amplified by PCR was transformed into DH5α competent cells, plated on ampicillin-resistant solid LB plates, and incubated upside down at 37℃ for 12 h. After that, single clones of the strain were picked and cultured in ampicillin-resistant liquid LB plates with shaking for 12 h. The bacterial culture was collected and identified by PCR using 2×Taq PCR StarMix (Dye). Figure 3 The correct bacterial culture sample was identified, and the plasmid was extracted for sequence analysis. The results showed that its sequence was 100% homologous to the TBEV E-DⅢ-Dcpep2-PA3 gene sequence. The recombinant plasmid containing the TBEVE-DⅢ-Dcpep2-PA3 gene was successfully constructed and named pCDNA3.4-TBEV E-DⅢ-Dcpep2-PA3.

[0049] 3. Expression of recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3: HEK293F cells were cultured in OPM-293 CD05 Medium in an incubator at 37℃, 85% humidity, and 5% CO2 with shaking at 120 rpm. When the cell density reached 1.0 × 10⁻⁶ cells / year... 6Transfection was performed at a concentration of 10 cells / mL (pCDNA3.4-TBEV E-DⅢ-Dcpep2-PA3 plasmid to PEI reagent ratio of 1:4). 24 h after transfection, OPM-293 PFeed was added at a 1:20 volume ratio, and 48 h later, dextrorotatory glucose was added to a final concentration of 3 g / L. Five days after transfection, cells were removed by centrifugation at 1500 rpm for 10 min at room temperature, and the cell culture supernatant was collected.

[0050] 4. Identification of recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3: Samples were prepared from the supernatant of HEK293F cells 5 days after transfection. Western blotting was performed using 1:300 diluted anti-TBEV EDⅢ mouse serum as the primary antibody and 1:10000 diluted HRP-labeled goat anti-mouse IgG as the secondary antibody. Results showed a specific target protein band at 72 kDa. Figure 4 According to the Editseq software, the TBEV E-DⅢ-Dcpep2-PA3 protein is approximately 72 kDa. In summary, this indicates that the recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3 was successfully expressed in the eukaryotic expression system HEK293F cells, and its amino acid sequence is shown in SEQ ID NO:8.

[0051] Example 2: This example provides a method for the preparation and identification of EDc2-GEM BLPs, as detailed below:

[0052] 1. Preparation of EDc2-GEM BLPs: HEK293F cells were transiently transfected with pCDNA3.4-TBEV E-DⅢ-Dcpep2-PA3 plasmid. 24 h after transfection, OPM-293 PFeed was added at a 1:20 volume ratio. 48 h later, dextrorotatory glucose was added to a final concentration of 3 g / L. Five days after transfection, cells were removed by centrifugation at 1500 rpm for 10 min at room temperature. The cell culture supernatant was collected, and GEM particles were added at a 1:10 volume ratio. The mixture was shaken at 37°C for 1 h to ensure complete binding. BLPs were then separated by centrifugation at 8000 rpm for 10 min at 4°C and resuspended in PBS, followed by five centrifugation and washing. Finally, the cells were resuspended in PBS (1 U GEM resuspended in 200 μL PBS). The prepared BLPs were named EDc2-GEM and stored at -80°C for later use.

[0053] 2. Western Blot Identification: Protein samples were prepared from the EDc2-GEM BLPs. A 1:300 dilution of anti-TBEV ED III mouse serum was used as the primary antibody, and a 1:10000 dilution of HRP-labeled goat anti-mouse IgG was used as the secondary antibody for Western Blot identification. Results showed a specific protein band appearing at 70-100 kDa. Figure 5 The A result indicates that EDc2-GEM BLPs were successfully prepared.

[0054] 3. Indirect Immunofluorescence Identification: 200 μL of the prepared EDc2-GEM BLPs was added to a 1.5 mL centrifuge tube. The BLPs were separated by centrifugation at 8000 rpm for 10 minutes at 4°C, resuspended in PBS solution with 3% BSA, and blocked by inversion for 30 minutes at room temperature. The sample was then separated by centrifugation at 8000 rpm for 10 minutes at 4°C, and resuspended in PBST buffer for five consecutive centrifugations and washes. Anti-TBEV-E-DⅢ serum (1% BSA dilution) diluted 1:200 was used as the primary antibody and incubated at 37°C for 1 hour. FITC-labeled goat anti-mouse IgG diluted 1:200 was used as the secondary antibody and incubated at 37°C for 1 hour in the dark. After each step, the sample was resuspended and washed five times in PBST buffer, and finally resuspended in PBS buffer. A small amount of sample was added to a glass slide and observed under a fluorescence microscope. The results showed that, compared with the blank GEM control, EDc2-GEM BLPs exhibited obvious green fluorescence under a fluorescence microscope. Figure 5 The presence of Ba and Bb indicates that EDc2-GEM BLPs were successfully prepared.

[0055] Example 3: This example provides a method for constructing and identifying the recombinant adjuvant protein MAB2560-PA3, as detailed below:

[0056] 1. Primer design and synthesis: Design specific amplification primers (Table 4). A total of one pair of amplification primers (MAB2560-PA3-F+MAB2560-PA3-R) and one pair of identification primers (JDM-F+JDM-R) were designed.

[0057] Table 4 Primer Information

[0058]

[0059] 2. For example Figure 6 As shown, the specific methods for constructing and identifying the recombinant plasmid pCDNA3.4-MAB2560-PA3 are as follows:

[0060] (1) Amplification of the target gene MAB2560-PA3: Using the plasmid pFBD-MAB2560-PA3, which was previously constructed in the laboratory and contains the target gene MAB2560-PA3, as a template, the MAB2560-PA3 gene (nucleotide sequence as shown in SEQ ID NO:9) was amplified using MAB2560-PA3-F and MAB2560-PA3-R primers (Table 4). The reaction conditions are shown in Table 2. The target fragment was separated and recovered by 1% agarose gel electrophoresis. The amplification results showed that the product was the same size as the target fragment ( Figure 7 The MAB2560-PA3 gene was successfully amplified, and then it was seamlessly cloned and ligated into the previously stored pCDNA3.4 vector in the laboratory at a certain ratio. The reaction was carried out at 50℃ for 15 min. The system is shown in Table 3.

[0061] (2) Identification of recombinant plasmid pCDNA3.4-MAB2560-PA3: The complete pCDNA3.4-MAB2560-PA3 gene amplified by PCR was transformed into DH5α competent cells, plated on ampicillin-resistant solid LB plates, and incubated upside down at 37℃ for 12 h. After that, single clones of the strain were picked and cultured in ampicillin-resistant liquid LB plates with shaking for 12 h. The bacterial culture was collected and identified by PCR using 2×Taq PCRStarMix (Dye). Figure 8 The correct bacterial culture sample was identified, and the plasmid was extracted for sequence analysis. The results showed that its sequence was 100% homologous to the MAB2560-PA3 gene sequence. The recombinant plasmid containing the MAB2560-PA3 gene was successfully constructed and named pCDNA3.4-MAB2560-PA3.

[0062] 3. Expression of recombinant adjuvant protein MAB2560-PA3: HEK293F cells were expressed in OPM-293 CD05 Medium at 37°C, 85% humidity, and 5% CO2. 2 The cells were cultured in an incubator with shaking at 120 rpm. When the cell density reached 1.0 × 10⁻⁶ cells / year... 6 Transfection was performed at a concentration of 10 cells / mL (pCDNA3.4-MAB2560-PA3 plasmid to PEI reagent ratio of 1:4). 24 h after transfection, OPM-293 PFeed was added at a 1:20 volume ratio, and 48 h later, dextrorotatory glucose was added to a final concentration of 3 g / L. Five days after transfection, cells were removed by centrifugation at 1500 rpm for 10 min at room temperature, and the cell culture supernatant was collected.

[0063] 4. Identification of recombinant adjuvant protein MAB2560-PA3: Samples were prepared from the supernatant of HEK293F cells 5 days after transfection. Western blotting was performed using 1:300 diluted anti-MAB2560 rabbit serum as the primary antibody and 1:10000 diluted HRP-labeled goat anti-rabbit IgG as the secondary antibody. Results showed a specific target protein band at 50 kDa. Figure 9 According to the Editseq software, the MAB2560-PA3 protein is approximately 50 kDa. In summary, this indicates that the recombinant adjuvant protein MAB2560-PA3 was successfully expressed in the eukaryotic expression system HEK293F cells, and its amino acid sequence is shown in SEQ ID NO:10.

[0064] Example 4: This example provides a method for the preparation and identification of MAB2560-GEM BLPs, as detailed below:

[0065] 1. Preparation of MAB2560-GEM BLPs: HEK293F cells were transiently transfected with pCDNA3.4-MAB2560-PA3 plasmid. 24 h after transfection, OPM-293 PFeed was added at a 1:20 volume ratio. 48 h later, dextro-glucose was added to a final concentration of 3 g / L. Five days after transfection, cells were removed by centrifugation at 1500 rpm for 10 min at room temperature. The cell culture supernatant was collected, and GEM particles were added at a 1:10 volume ratio. The mixture was shaken at 37°C for 1 h to ensure complete binding. BLPs were then separated by centrifugation at 8000 rpm for 10 min at 4°C and resuspended in PBS, followed by five centrifugation and washing. Finally, the cells were resuspended in PBS (1 μg GEM resuspended in 200 μL PBS). The prepared BLPs were named MAB2560-GEM and stored at -80°C for later use.

[0066] 2. Western Blot Identification: Protein samples were prepared from the MAB2560-GEM BLPs. A 1:300 dilution of anti-MAB2560 rabbit serum was used as the primary antibody, and a 1:10000 dilution of HRP-labeled goat anti-rabbit IgG was used as the secondary antibody for Western Blot identification. Results showed a specific protein band at 50-70 kDa. Figure 10 The result of A indicates that MAB2560-GEM BLPs were successfully prepared.

[0067] 3. Indirect Immunofluorescence Identification: 200 μL of the prepared MAB2560-GEM BLPs was added to a 1.5 mL centrifuge tube. The BLPs were separated by centrifugation at 8000 rpm for 10 minutes at 4°C, resuspended in PBS solution with 3% BSA, and blocked by inversion for 30 minutes at room temperature. The sample was then separated by centrifugation at 8000 rpm for 10 minutes at 4°C, and resuspended in PBST buffer for five consecutive centrifugations and washes. Anti-MAB2560 serum (1% BSA dilution) diluted 1:200 was used as the primary antibody and incubated at 37°C for 1 hour. FITC-labeled goat anti-rabbit IgG diluted 1:200 was used as the secondary antibody and incubated at 37°C for 1 hour in the dark. After each step, the sample was resuspended and washed five times in PBST buffer, and finally resuspended in PBS buffer. A small amount of sample was added to a glass slide and observed under a fluorescence microscope. The results showed that, compared with the blank GEM control, MAB2560-GEM BLPs exhibited obvious green fluorescence under a fluorescence microscope. Figure 10 The presence of Ba and Bb indicates that MAB2560-GEM BLPs were successfully prepared.

[0068] Example 5: This example provides a method for preparing and identifying tick-borne encephalitis virus vaccine EDc2&MAB2560-GEM BLPs, as detailed below:

[0069] 1. Preparation of EDc2&MAB2560-GEM BLPs: HEK293F cells were transiently transfected with pCDNA3.4-MAB2560-PA3 plasmid. 24 h after transfection, OPM-293 PFeed was added at a 1:20 volume ratio. 48 h later, dextro-glucose was added to a final concentration of 3 g / L. Five days after transfection, cells were removed by centrifugation at 1500 rpm for 10 min at room temperature. The cell culture supernatant was collected, and EDc2-GEM BLPs were added at a 1:10 volume ratio. The mixture was incubated at 37°C with shaking for 1 h to ensure complete binding. The BLPs were then separated by centrifugation at 8000 rpm for 10 min at 4°C and resuspended in PBS, followed by five centrifugation and washing. Finally, the BLPs were resuspended in PBS (1 U GEM resuspended in 200 μL PBS). The prepared BLPs were named EDc2&MAB2560-GEM and stored at -80°C for later use.

[0070] 2. Western Blot Identification: Protein samples were prepared using the EDc2 & MAB2560-GEM BLPs. A 1:300 dilution of anti-MAB2560 rabbit serum was used as the primary antibody, and a 1:10000 dilution of HRP-labeled goat anti-rabbit IgG was used as the secondary antibody for Western Blot identification. Results showed a specific protein band at 50-70 kDa. Figure 11The A result indicates that EDc2-GEM BLPs were successfully prepared.

[0071] 3. Indirect Immunofluorescence Identification: 200 μL of the prepared MAB2560-GEM BLPs was added to a 1.5 mL centrifuge tube. The BLPs were separated by centrifugation at 8000 rpm for 10 minutes at 4°C, resuspended in PBS solution with 3% BSA, and blocked by inversion for 30 minutes at room temperature. The sample was then separated by centrifugation at 8000 rpm for 10 minutes at 4°C, and resuspended in PBST buffer for five consecutive centrifugations and washes. Anti-MAB2560 serum (1% BSA dilution) diluted 1:200 was used as the primary antibody and incubated at 37°C for 1 hour. FITC-labeled goat anti-rabbit IgG diluted 1:200 was used as the secondary antibody and incubated at 37°C for 1 hour in the dark. After each step, the sample was resuspended and washed five times in PBST buffer, and finally resuspended in PBS buffer. A small amount of sample was added to a glass slide and observed under a fluorescence microscope. The results showed that, compared with the blank GEM control, MAB2560-GEM BLPs exhibited obvious green fluorescence under a fluorescence microscope. Figure 11 The presence of B indicates that MAB2560-GEM BLPs were successfully prepared.

[0072] Example 6: This example provides a method for evaluating the immunization efficacy of the tick-borne encephalitis virus vaccine EDc2&MAB2560-GEM BLPs, as detailed below:

[0073] 1. Mouse Immunization: To evaluate the immunization effect of EDc2 & MAB2560-GEM BLPs, 50 healthy female BALB / c mice weighing 16-18g were randomly divided into 5 groups (Table 5). Mice in the experimental groups were injected intramuscularly with 100μL of immunogen from either EDc2 & MAB2560-GEM BLPs or EDc2-GEM + MAB2560-GEM BLPs. The grouping scheme is as follows:

[0074] Table 5 Immunization and Grouping Protocols for BALB / c Mice

[0075]

[0076] 2. Detection of TBEV IgG antibodies in mouse serum: Using PBS-treated mouse serum as a negative control, the TBEV antibody indirect ELISA method was used to detect the IgG antibody levels in mouse serum at weeks 1, 3, 5, 7, 9, 11, 13, 17, and 21 after immunization with EDc2&MAB2560-GEM BLPs, EDc2-GEM+MAB2560-GEM BLPs, and other control groups. The procedure was as follows: TBEV E-DⅢ, a recombinant antigen protein purified from prokaryotic expression at a final concentration of 1 μg / mL, was used as the coating antigen and coated overnight at 4°C. 5% skim milk powder was used as the blocking buffer; mouse serum was serially diluted 2-fold (starting at 1:2500) as the primary antibody; and HRP-labeled goat anti-mouse IgG antibody diluted 1:40000 was used as the secondary antibody. After 30 min of TMB staining, the staining was stopped with 0.5 M H2SO4. The OD values ​​of each well were read at a wavelength of 450 nm using an ELISA reader. The highest serum dilution with an OD450 ratio of test serum to negative serum ≥ 2.1 was taken as the ELISA antibody titer. The results showed ( Figure 12 ): TBEV-specific IgG antibodies were detectable in the serum of mice 1 week after the first immunization with EDc2 & MAB2560-GEM BLPs; the level of IgG antibodies in the serum of mice was significantly increased after the third immunization, reaching a maximum of 1:10. 5.8 The ratio remained at 1:10 four months after the three exemptions. 4 .

[0077] 3. Flow cytometry analysis of immune cells in mouse inguinal lymph nodes: One week after the first immunization, three mice were randomly selected from each of the EDc2&MAB2560-GEM, EDc2-GEM+MAB2560-GEM, EDc2-GEM+alum adjuvant, EDc2-GEM and PBS groups. Lymphocyte suspensions were prepared from the inguinal lymph nodes of the mice, the cells were stained, and the fluorescence signal was detected by flow cytometry.

[0078] Mouse inguinal lymph node cells via CD80 + MHC I + MHC II + and CD11c + After staining with fluorescent antibody, CD11c was detected. + CD80 + CD11c + MHC Ⅰ + and CD11c + MHC II + The proportion of double-positive cells was observed. Results showed that CD11c in the EDc2 & MAB2560-GEM group... + CD86 + CD11c + CD80+ CD11c + MHCⅠ + CD11c + MHCⅡ + The proportion of double-positive cells was significantly higher in both the PBS control group and the EDc2-GEM group than in the PBS control group and the EDc2-GEM group. Figure 13 The results (AD) indicate that after immunization with EDc2&MAB2560-GEM, the antigen can be captured by the inguinal lymph nodes of mice, stimulating dendritic cell (DC) activation and antigen processing, followed by antigen presentation to lymphocytes. This suggests that EDc2&MAB2560-GEM can stimulate DC activation as early as one day after the initial immunization, creating conditions for antigen presentation and the initiation of the immune response. In conclusion, the initial immunization of mice with EDc2&MAB2560-GEM BLPs promotes DC activation.

[0079] 4. Spleen cell activation analysis: Mouse spleen cells were isolated, and the spleen cell concentration was adjusted to 2.5 × 10⁻⁶ cells according to experimental needs. 6 Quantity / mL, for later use.

[0080] (1) ELISpot detection: The detection was performed according to the instructions of the mouse IFN-γ and IL-4 ELISpot detection kit. The steps are as follows: Add 5×10 to each well. 5 10 μg / mL of TBEV ED III was added as a stimulant to each spleen cell. The ELISpot plate was wrapped in aluminum foil and cultured at 37°C in a 5% CO2 incubator for 38 h. A 1:1000 dilution of biotin-labeled detection antibody (BVD6-24G2-biotin) was used as the primary antibody, and a 1:1000 dilution of Streptavidin-HRP was used as the secondary antibody. After incubation at room temperature, TMB chromogenic solution was added, and the plates were developed in the dark until clear spots appeared. The ELISpot plate was then rinsed with tap water to stop the development. The ELISpot plates were air-dried in the dark. The spots were automatically acquired and counted using an AID enzyme-linked spot image analyzer, and the number of spot-forming cells (SFCs) and spot images for each well were saved. The number of cell spots in the immunized group and the control group in the presence of the stimulant was plotted. The results showed ( Figure 14Following stimulation with TBEV-specific antigen, the number of IFN-γ and IL-4 secreting cells in the spleen cells of mice immunized with EDc2 & MAB2560-GEM was significantly higher than that in the PBS control group and the EDc2-GEM group. Furthermore, compared with the EDc2-GEM + alum adjuvant group and the EDc2-GEM + MAB2560-GEM group, the number of IFN-γ and IL-4 secreting cells in the spleen of mice immunized with the EDc2 & MAB2560-GEM co-display particle vaccine group was significantly increased. IFN-γ and IL-4 are representative cytokines of Th1-type cellular immunity and Th2-type humoral immunity, respectively. These results indicate that co-display of antigen and adjuvant on the BLPs platform has a regulatory and ameliorative effect on both Th1 (Th1 cytokine IFN-γ) and Th2 (Th2 cytokine IL-4) immunity.

[0081] (2) Splenic lymphocyte proliferation test: Isolated splenic lymphocytes were subjected to a 2.5×10⁻⁶ test. 5 Spinal lymphocytes were seeded per well in 96-well cell culture plates and stimulated with recombinant TBEV E-DIII antigen protein (final concentration 10 μg / mL). Cells were incubated at 37℃ in a 5% CO2 cell culture incubator for 44 h. The in vitro proliferation capacity of spinal lymphocytes was detected using the CCK-8 assay. 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 4 h. The OD450 value was then measured using a microplate reader. The results showed (…). Figure 15 The immunization groups containing EDc2-GEM particles showed significantly higher levels of stimulation than the PBS group, but the stimulation index (SI) of the EDc2 & MAB2560-GEM co-exhibition particle vaccine group was significantly higher than that of other groups. In summary, after triple immunization with EDc2 & MAB2560-GEM BLPs, mouse splenic lymphocytes showed significant proliferation under the action of specific antigens.

[0082] (3) Detection of spleen lymphocytes: Collect spleen lymphocytes after centrifugation in the previous step, add fluorescent antibody premixed solution, and use flow cytometry to detect the fluorescence signal of lymphocytes.

[0083] I. Recruitment and activation of mouse spleen B cells: Mouse spleen cells are recruited and activated via CD19. + CD40 + and CD69 + Fluorescent antibody staining to detect CD19 + CD40 + and CD19 + CD69 + The proportion of double-positive cells. Results showed that CD19 was present in lymphocytes infected with the EDc2 & MAB2560-GEM vaccine. + CD69 + CD19 +CD40 + The proportion of double-positive cells was the highest in both groups, significantly higher than that in the PBS control group and the EDc2-GEM group, but not significantly different from that in the EDc2-GEM + alum adjuvant group and the EDc2-GEM + MAB2560-GEM group (P > 0.05). Figure 16 (Aa, Ab). In summary, after triple immunization with EDc2 & MAB2560-GEM BLPs, splenic B cells are recruited and / or activated under the stimulation of specific antigens.

[0084] II. Recruiting and / or activating mouse spleen T cells: Mouse spleen cells via CD4 + CD8 + and CD69 + Fluorescent antibody staining to detect CD4 + CD69 + and CD8 + CD69 + The proportion of double-positive cells was observed. Results showed that CD4+ cells in the EDc2 & MAB2560-GEM group lymphocytes... + The percentage of T lymphocytes was significantly higher in the EDc2-GEM group, the EDc2-GEM + alum adjuvant group, and the EDc2-GEM + MAB2560-GEM group; meanwhile, the CD8+ cells in the lymphocytes of the EDc2 & MAB2560-GEM groups were also significantly higher. + The percentage of T lymphocytes was the highest and significantly higher than that of the PBS control group and the EDc2-GEM group, but there was no significant difference compared with the EDc2-GEM + alum adjuvant group and the EDc2-GEM + MAB2560-GEM vaccine group (P>0.05). Figure 17 (Ba, Bb). In summary, after triple immunization with EDc2 & MAB2560-GEM BLPs, spleen CD4 under specific antigen stimulation. + Enhanced recruitment and / or activation of T lymphocytes.

[0085] III. Generation of memory T cells in the mouse spleen: Mouse spleen cells via CD4 + CD8 + CD44 + and CD62L+ + Fluorescent antibody staining to detect CD4 + and CD8 + CD44 in positive cells + CD62L + The proportion of double-positive cells. Results showed that compared with the PBS group, EDc2-GEM group, and EDc2-GEM+MAB2560-GEM group, the proportion of CD4+ cells in mouse spleen cells in the EDc2&MAB2560-GEM group was significantly higher. + CD44 in cell subsets+ The proportion of CD62L⁺ cells was significantly different and higher than that of the EDc2-GEM + alum adjuvant group, but the difference was not significant (P>0.05). Figure 18 (Ca, Cb). In summary, CD4 was produced in the spleen of mice immunized with EDc2 & MAB2560-GEM BLPs. + and CD8 + Central memory T cells.

[0086] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A tick-borne encephalitis virus vaccine based on bacterial-like particles, characterized in that, The tick-borne encephalitis virus vaccine is an EDc2&MAB2560-GEM co-display bacterial-like particle, which is prepared by co-displaying recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3 and recombinant adjuvant protein MAB2560-PA3 on the surface of GEM particles.

2. The tick-borne encephalitis virus vaccine based on bacterial-like particles according to claim 1, characterized in that, The amino acid sequence of the recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3 is shown in the sequence listing SEQ ID NO:

8.

3. The tick-borne encephalitis virus vaccine based on bacterial-like particles according to claim 1, characterized in that, The amino acid sequence of the recombinant adjuvant protein MAB2560-PA3 is shown in the sequence listing SEQ ID NO:

10.

4. A method for preparing a tick-borne encephalitis virus vaccine based on bacterial-like particles as described in any one of claims 1-3, characterized in that, Includes the following steps: Construction and expression of recombinant antigen protein TBEV E-DⅢ-Dcpep2-PA3: Using a plasmid containing the target gene TBEV E-DⅢ-Dcpep1-PA3 as a template, the TBEV E-DⅢ-Dcpep2-PA3 gene was amplified using primers TBEV E-DⅢ-Dcpep2-PA3-F and TBEV E-DⅢ-Dcpep2-PA3-R, cloned into a eukaryotic expression vector, and then transfected into host cells for expression. Cell culture supernatant containing the recombinant antigen protein was collected. The nucleotide sequence of TBEV E-DⅢ-Dcpep1-PA3 is shown in SEQ ID NO:5; the nucleotide sequence of the TBEV E-DⅢ-Dcpep2-PA3 gene is shown in SEQ ID NO:

7. Construction and expression of recombinant adjuvant protein MAB2560-PA3: Using a plasmid containing the target gene MAB2560-PA3 as a template, the MAB2560-PA3 gene was amplified using primers MAB2560-PA3-F and MAB2560-PA3-R, cloned into a eukaryotic expression vector, and then transfected into host cells for expression. Cell culture supernatant containing the recombinant adjuvant protein was collected; the nucleotide sequence of MAB2560-PA3 is shown in SEQ ID NO:

9. Preparation of EDc2-GEM BLPs: Cell culture supernatant containing recombinant antigen protein was mixed with GEM particles and incubated to obtain EDc2-GEM BLPs; Preparation of EDc2 & MAB2560-GEM co-display bacterial-like particles: Cell culture supernatant containing recombinant antigen protein was mixed with EDc2-GEM BLPs and incubated to obtain the tick-borne encephalitis virus vaccine.

5. The method for preparing a tick-borne encephalitis virus vaccine based on bacterial-like particles according to claim 4, characterized in that, The eukaryotic expression vector is pCDNA3.4, and the host cell is HEK293F cell.

6. The method for preparing a tick-borne encephalitis virus vaccine based on bacterial-like particles according to claim 4, characterized in that, The nucleotide sequences of the primers TBEV E-DⅢ-Dcpep2-PA3-F and TBEV E-DⅢ-Dcpep2-PA3-R are shown in the sequence listing SEQ ID NO:11-12, respectively.

7. The method for preparing a tick-borne encephalitis virus vaccine based on bacterial-like particles according to claim 4, characterized in that, The nucleotide sequences of the primers MAB2560-PA3-F and MAB2560-PA3-R are shown in SEQ ID NO:15-16, respectively.

8. The use of a tick-borne encephalitis virus vaccine based on bacterial-like particles as described in any one of claims 1-3 in the preparation of a medicament for the prevention of tick-borne encephalitis virus.

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