EBV vaccine based on vesicular stomatitis virus vector and application thereof

Abstract

The invention discloses a VSV vector, prepared recombinant VSV virus particles can express EBV gp42 protein in host cells and can cause remarkable specific antibody reaction aiming at EBV surface glycoprotein gp42, a monovalent gp42 vaccine can initiate strong and lasting humoral immunity, and it is indicated that the VSV is an appropriate vaccine vector of gp42. The specific antibody generated by the VSV-based gp42 vaccine can inhibit EBV infection epithelial cells and B lymphocytes, which indicates that the VSV-based EBV vaccine has the potential for preventing and controlling EBV infection and related neoplastic and non-neoplastic diseases of people.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an EBV vaccine based on a vesicular stomatitis virus vector and application thereof. Background Art

[0002] Epstein-Barr virus (EBV) is the first human tumor-associated virus discovered and is closely related to the occurrence and development of a variety of human diseases. Although the development of vaccines against EBV has a history of decades, there is still no clinically available EBV vaccine.

[0003] Based on the molecular mechanism of EBV infection of host cells, vaccines targeting EBV gB, gHgL, and gp350 have been designed, but there are few reports on monovalent vaccines designed for EBV gp42. One of the important reasons is that it is not clear whether monovalent gp42 can induce neutralizing protective antibodies. The previously reported neutralizing antibody titer induced by gp42 displayed by hepatitis B virus core antigen virus-like particles is low and does not have clinical translation potential (Viruses, 2021). This shows that vaccines based on EBV gp42 need to creatively select suitable carriers.

[0004] Vesicular Stomatitis Virus (VSV) is a single-stranded negative-sense RNA virus and a new type of human vaccine development vector. VSV mainly infects rodents, cattle, pigs, and horses, and has a very low prevalence in the human population. There are currently reports of individual animal keepers and laboratory personnel being infected with VSV, but most of the symptoms after infection are mild or asymptomatic. The VSV genome is simple (containing only 5 genes) and can be inserted into larger exogenous genes; and its RNA genome cannot be integrated into the human genome to cause gene mutations. Due to the above advantages of the VSV vector, it has been reported that the glycoprotein G of the Ebola virus and the glycoprotein S of the new coronavirus are displayed on the surface of VSV for vaccine development. The former is produced by the Merck Group and has been approved by the United States and the European Union for clinical prevention of Ebola virus infection. The above evidence shows that displaying exogenous pathogen proteins on the surface of VSV and using them for vaccine development is a safe vaccine development program with clinical translation value. Summary of the invention

[0005] The purpose of the present invention is to provide an EBV vaccine based on a vesicular stomatitis virus vector and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a VSV vector, comprising (a) at least a portion of a VSV genome, wherein the at least a portion of the VSV genome comprises a VSV N gene, a VSV P gene, a VSV M gene and a VSV L gene; and (b) a polynucleotide sequence encoding an EBV gp42 protein or an immunogenic variant thereof.

[0008] In one or more embodiments, the VSV vector comprises (a) a VSV genome comprising a VSV N gene, a VSV P gene, a VSV M gene, a VSV G gene and a VSV L gene; and (b) a polynucleotide sequence encoding an EBV gp42 protein or an immunogenic variant thereof.

[0009] In one or more embodiments, the polynucleotide sequence encoding the EBV gp42 protein or an immunogenic variant thereof is located between the VSV N gene and the VSV P gene, or between the VSV M gene and the VSV G gene.

[0010] In one or more embodiments,

[0011] The VSV L gene is shown in SEQ ID NO.1;

[0012] The VSV N gene is shown in SEQ ID NO.2;

[0013] The VSV P gene is shown in SEQ ID NO. 3;

[0014] The VSV M gene is shown in SEQ ID NO.4;

[0015] VSV G gene is shown in SEQ ID NO.5;

[0016] The polynucleotide sequence encoding EBV gp42 protein or its immunogenic variant is shown in SEQ ID NO.6.

[0017] In one or more embodiments, the VSV vector is a pBS plasmid vector that integrates (a) a VSV genome comprising a VSV N gene, a VSV P gene, a VSV M gene, a VSV G gene, and a VSV L gene; and (b) a polynucleotide sequence encoding an EBV gp42 protein or an immunogenic variant thereof.

[0018] In one or more embodiments, the VSV vector sequence is shown as SEQ ID NO.7.

[0019] The second aspect of the present invention provides a recombinant VSV virus particle, which contains at least VSV N protein, VSV P protein, VSV M protein, VSV L protein and EBV gp42 protein or an immunogenic variant thereof.

[0020] In one or more embodiments, the recombinant VSV virus particle comprises VSV N protein, VSV P protein, VSVM protein, VSV G protein, VSV L protein and EBV gp42 protein or immunogenic variants thereof.

[0021] In one or more embodiments, the amino acid sequence of the VSV L protein is shown in SEQ ID NO.8;

[0022] The amino acid sequence of VSV N protein is shown in SEQ ID NO.9;

[0023] The amino acid sequence of VSV P protein is shown in SEQ ID NO.10;

[0024] The amino acid sequence of VSV M protein is shown in SEQ ID NO.11;

[0025] The amino acid sequence of VSV G protein is shown in SEQ ID NO.12;

[0026] The amino acid sequence of EBV gp42 protein or its immunogenic variant is shown in SEQ ID NO.13.

[0027] In one or more embodiments, the recombinant VSV virus particle is prepared by packaging the VSV vector of the first aspect via packaging cells.

[0028] The third aspect of the present invention provides a cell, which produces a recombinant VSV virus particle of the second aspect.

[0029] In one or more embodiments, the cell is a mammalian cell.

[0030] In one or more embodiments, the mammalian cell is a multipotent adult progenitor cell (MAPC), a neural stem cell (NSC), a mesenchymal stem cell (MSC), a HeLa cell, any HEK293 cell, a Vero cell, a bone marrow-derived tumor-infiltrating cell (BM-TIC), or a golden hamster kidney cell (BSR).

[0031] A fourth aspect of the present invention provides a method for preparing VSV virus particles, comprising:

[0032] Co-transfecting host cells with the recombinant VSV vector of the first aspect and the helper plasmid, collecting the supernatant, and passage to obtain recombinant VSV virus particles;

[0033] The auxiliary plasmids include: auxiliary plasmids VSV-N, VSV-P, VSV-L and VSV-G which contain N gene, P gene, L gene and G gene of VSV virus respectively.

[0034] In one or more embodiments, the host cell is a host cell expressing T7 RNA polymerase.

[0035] In one or more embodiments, the host cell is a mammalian cell.

[0036] In one or more embodiments, the mammalian cell is a multipotent adult progenitor cell (MAPC), a neural stem cell (NSC), a mesenchymal stem cell (MSC), a HeLa cell, any HEK293 cell, a Vero cell bone marrow-derived tumor-infiltrating cell (BM-TIC), or a BSR cell.

[0037] In one or more embodiments, the mammalian cell is a BSR cell expressing T7 RNA polymerase.

[0038] The fifth aspect of the present invention provides an immunogenic composition, which comprises (a) VSV virus particles prepared by the VSV vector of the first aspect, or the recombinant VSV virus particles of the second aspect; and (b) a pharmaceutically acceptable carrier.

[0039] In one or more embodiments, the immunogenic composition is a vaccine.

[0040] In one or more embodiments, the vaccine further comprises an adjuvant.

[0041] Use of the VSV virus particle prepared by the VSV vector of the first aspect, the recombinant VSV virus particle of the second aspect, the VSV virus particle prepared by the method of the fourth aspect, or the immunogenic composition of the fifth aspect in at least one of the following:

[0042] (1) Preparation of drugs for treating and / or preventing EBV infection;

[0043] (2) Preparation of drugs for treating and / or preventing EBV infection-related neoplastic diseases or non-neoplastic diseases;

[0044] (3) Preparation of antibodies that inhibit EBV infection.

[0045] In one or more embodiments, the neoplastic disease comprises at least one of Burkitt's lymphoma, NK / T cell lymphoma, diffuse large B cell lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric cancer.

[0046] In one or more embodiments, the non-neoplastic disease comprises at least one of infectious mononucleosis, oral leukoplakia, X-linked lymphoproliferative syndrome, and virus-associated erythrophagocytosis.

[0047] The beneficial effects of the present invention are:

[0048] The present invention inserts EBV gp42 protein into the vesicular stomatitis virus genome by modifying the VSV genome, thereby obtaining VSV virus particles that can express EBV glycoprotein gp42, and uses the recombinant VSV virus particles for animal immunization, showing that the recombinant VSV virus particles can induce significant specific antibody responses against EBV surface glycoprotein gp42, and the monovalent gp42 vaccine can induce strong and lasting humoral immunity, indicating that VSV is a suitable vaccine vector for gp42. The specific antibodies produced by the VSV-based gp42 vaccine in the present invention can inhibit EBV infection of epithelial cells and B lymphocytes, indicating that the VSV-based EBV vaccine has the potential to be used for preventing and controlling EBV infection and related neoplastic and non-neoplastic diseases in the population. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 For packaging and amplification of VSV recombinant virus, Figure 1 A in the middle is the composition of the viral genome. Figure 1 B in the middle is the composition of the modified viral genome. Figure 1 C in the middle is the virus packaging process.

[0050] Figure 2 The results are from Coomassie Brilliant Blue staining.

[0051] Figure 3 The results of negative staining electron microscopy testing of virus sample quality.

[0052] Figure 4 The results of surface plasmon resonance detection of gp42 content in the supernatant after VSV-gp42 infected cells.

[0053] Figure 5 Serum gp42-specific antibody titer results.

[0054] Figure 6 Serum neutralizing antibody titer. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below by specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial sources or by prior art methods unless otherwise specified. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0056] Example 1 Preparation and Identification of VSV Recombinant Virus VSV-gp42-GP2

[0057] Packaging and amplification of VSV recombinant virus Figure 1 As shown, Figure 1 As shown in A, the whole genome of VSV is integrated into the pBS plasmid vector (pBS-VSV) (the pBS-VSV vector was donated by Professor Chen Mingzhou's research group at Wuhan University), and RNA synthesis is driven by the T7 promoter. After the pBS-VSV plasmid is transfected into the cell, it is transcribed and synthesized into an RNA chain under the action of T7 RNA polymerase, and the complementary chain of the VSV genome is obtained after processing by δ-ribozyme at the cleavage site at the end of the RNA chain. Using this as a template, the VSV RNA polymerase synthesizes the VSV genomic RNA and mRNA, and expresses the VSV proteins N, P, M, G, and L, and finally completes the assembly of VSV virus particles.

[0058] like Figure 1 As shown in B, on the basis of pBS-VSV plasmid, the coding gene of EBV gp42 is inserted into different positions of VSV genome to obtain different VSV genome backbone plasmids pBS-VSV-gp42 containing EBV gp42, so that viral genomes and viral particles containing EBV glycoprotein gp42 can be produced in cells after plasmid transfection. When EBV gp42 is inserted between VSV-N and VSV-P genes, it is named VSV-gp42-GP2, and the resulting plasmid is pBS-VSV-gp42-GP2, where GP is the abbreviation of Glycoprotein, i.e. glycoprotein. Similarly, EBV gp42 was inserted before VSV N, between VSV P and VSV M, between VSV M and VSV G, between VSV G and VSV L, and after VSV L and named VSV-gp42-GP1, VSV-gp42-GP3, VSV-gp42-GP4, VSV-gp42-GP5, and VSV-gp42-GP6, respectively. The packaged VSV particles can express EBV gp42 protein in host cells.

[0059] like Figure 1 As shown in Figure C, the whole process of packaging VSV recombinant virus is shown, which mainly includes plasmid transfection, primary transfection, and virus passage amplification.

[0060] Primary transfection: BSR-T7 cells are infected with poxvirus VTF3-7 to provide T7 RNA polymerase. Plasmids pBS-VSV-N, pBS-VSV-P, pBS-VSV-L, pCAGGS-VSV-G containing genes encoding VSV viral proteins N, P, L, and G and a VSV genome backbone plasmid containing EBV gp42 are then co-transfected into BSR-T7 cells infected with VTF3-7. The T7 RNA polymerase expressed in the cells will transcribe the complementary chain of the VSV genome, and the N, P, and L proteins will assemble into VSV RNA polymerase to synthesize VSV genomic RNA, transcribe VSV viral protein mRNA, synthesize VSV viral proteins, and ultimately produce the VSV recombinant virus VSV-gp42. Collect cells and supernatant into centrifuge tubes, freeze and thaw repeatedly three times to break the cells and release the virus particles in the cells, centrifuge to remove cell debris, filter the supernatant with a 0.22μm filter membrane to remove poxvirus, and store the first-generation virus supernatant at 4°C for subsequent amplification.

[0061] Virus passage amplification: Since the titer of the virus particles obtained by the first transfection packaging is extremely low and may contain a large number of structurally incomplete virus particles, it is generally necessary to amplify through several passages to obtain high-titer, high-quality virus products. During the amplification process, Vero cells are used as host cells, and the cells are inoculated into culture dishes. After the cells are fully grown, the virus supernatant obtained from the above primary transfection is added to the well plate and incubated with the cells for 6-12 hours. After the incubation is completed, the supernatant is discarded, the cells are washed with PBS, and fresh high-glucose medium containing 10% volume ratio of fetal bovine serum is added. Continue to culture for 48 hours to 72 hours, and collect cells and supernatants. The recovery ratio of the virus in the virus supernatant is maximized by freezing, thawing and centrifugation. 4-5 rounds of the above passage operation are performed to obtain high-quality virus supernatant. After that, Vero cells are still used as host cells, and a large amount of virus supernatant is obtained through large-volume passage.

[0062] Among them, the above-designed VSV-gp42-GP1, VSV-gp42-GP2, VSV-gp42-GP3, VSV-gp42-GP4, VSV-gp42-GP5, and VSV-gp42-GP6 successfully packaged and produced VSV-gp42-GP2 and VSV-gp42-GP4 viruses, while VSV-gp42-GP1, VSV-gp42-GP3, VSV-gp42-GP5, and VSV-gp42-GP6 were not successfully packaged.

[0063] The specific steps for packaging VSV-gp42-GP2 are as follows:

[0064] Packaging of recombinant virus VSV-gp42-GP2

[0065] (1) BSR-T7 cells (frozen in the inventor's laboratory) were inoculated into a 10 cm culture dish and cultured in DMEM medium (Gibco TM , Catalog No.: C11995500BT), cultured at 37°C, cell confluence reached 80%-90%. The culture medium was discarded, and poxvirus VTF7-3 (frozen in the inventor's laboratory) was diluted with DMEM medium without fetal bovine serum, and BSR-T7 cells were infected with the virus at a multiplicity of infection of 5 for 1 hour, and the culture dish was gently shaken once every 15 minutes. After the poxvirus VTF7-3 infected BSR-T7 cells, T7 RNA polymerase was expressed in the cells.

[0066] (2) During the poxvirus infection, prepare the transfection reagent. Add 3 μg of pBS-VSV-N plasmid, 5 μg of pBS-VSV-P plasmid, 1 μg of pBS-VSV-L plasmid, 8 μg of pCAGGS-VSV-G plasmid, and 5 μg of pBS-VSV-gp42-GP2 plasmid (sequence shown in SEQ ID NO.7) to 500 μL of opti-MEM medium (Gibco TM , Catalog No.: 51985034), mix well, add 66 μL lipo2000 transfection reagent to another 500 μL opti-MEM, mix well by pipetting, and let stand at room temperature for 5 minutes. Mix the prepared 500 μL plasmid-opti-MEM and 500 μL lipo2000-opti-MEM, mix well by pipetting, and let stand at room temperature for 20 minutes.

[0067] (3) After 1 hour of poxvirus infection, discard the supernatant, wash the culture dish 3-5 times with PBS, discard the supernatant, add 5 mL of opti-MEM, evenly add the prepared transfection reagent to the culture dish, and gently shake it. Incubate at 37°C, 5% CO2 for 4-6 hours, discard the supernatant, replace with DMEM medium containing 10% fetal bovine serum, and culture at 37°C, 5% CO2 for 48 hours. During this period, observe the cell pathological changes.

[0068] (4) 48 hours after transfection, collect cells and supernatant, freeze and thaw three times repeatedly to fully release the virus. Centrifuge at 500g for 10 minutes to discard cell debris, filter the supernatant with a 0.22μm filter 2-3 times to remove poxvirus, and freeze the virus solution in a -80℃ refrigerator for later use.

[0069] Amplification of primary recombinant virus VSV-gp42-GP2

[0070] (1) Inoculate Vero cells into a 10 cm culture dish, add 10% fetal bovine serum DMEM medium, and culture at 37° C., 5% CO 2 until the cell confluence reaches 80%-90%.

[0071] (2) Discard the culture medium, add 5 mL of the virus solution collected in the above-mentioned "Packaged Recombinant Virus VSV-gp42-GP2", and infect for 2 hours, shaking the culture dish 2-3 times during the period. Discard the supernatant virus solution, replace it with DMEM medium containing 5% fetal bovine serum, and culture at 37°C, 5% CO2 for 48 hours, and observe the pathological changes of the cells.

[0072] (3) When most cells showed cytopathic effects, the cells and supernatant were collected and centrifuged at 4000 g for 10 min to remove cell debris. The supernatant was filtered through a 0.22 μm filter membrane and stored in a -80°C refrigerator until use.

[0073] Amplification of recombinant virus VSV-gp42-GP2

[0074] (1) Vero cells were inoculated into a 15 cm culture dish and cultured in DMEM medium containing 5% fetal bovine serum at 37° C. and 5% CO 2 until the cell confluence reached 80%-90%.

[0075] (2) Take the amplified virus supernatant obtained in the above "Amplification of primary recombinant virus VSV-gp42-GP2" and add 2 mL to each culture dish to infect Vero cells for 2 hours. During this period, shake the culture dish 2-3 times. Discard the supernatant and replace it with DMEM medium containing 5% fetal bovine serum. Incubate at 37°C, 5% CO2 for 48 hours, and observe the pathological changes of the cells during this period.

[0076] (3) When most cells show cytopathic effects, collect the supernatant virus solution, centrifuge at 5000 g for 10 min to remove cell debris, filter the supernatant with a 0.22 μm filter membrane, and store in a refrigerator at 4°C for later use.

[0077] Purification and identification of VSV-gp42-GP2

[0078] Virus purification by density gradient centrifugation

[0079] The collected virus supernatant was transferred to a high-speed centrifuge tube, centrifuged and discarded the supernatant, and the virus precipitate was resuspended in sterile PBS (100-200 times volume concentration). The obtained virus concentrated product was centrifuged for 1 hour with a 20%-50% sucrose density gradient, and the sample was separated layer by layer and identified by polyacrylamide gel electrophoresis and Coomassie brilliant blue staining, and the sample layer containing the virus VSV-gp42-GP2 was collected, and the sample was diluted with PBS, and the sucrose contained in the sample was separated by centrifugation. After the centrifugation, the supernatant was discarded, and the precipitate was resuspended with an appropriate amount of PBS and frozen in a -80°C refrigerator for later use.

[0080] The specific steps are as follows:

[0081] (1) Centrifuge the amplified virus solution at 100,000 g at 4°C for 2 h, and resuspend the precipitate in 1 mL of PBS.

[0082] (2) A 20%-50% sucrose density gradient was prepared using a density gradient preparation apparatus. The concentrated VSV-gp42-GP2 was ultracentrifuged at 4°C, 100,000 g for 1 hour. A clear white band was seen in the centrifuge tube, which was the virus sample layer.

[0083] (3) Use a density gradient separator to separate the samples after ultracentrifugation into layers. Take 40 μL of each sample and prepare gel electrophoresis samples. Perform polyacrylamide gel electrophoresis. Stain the gel with Coomassie Brilliant Blue dye and analyze the characteristic protein bands of each layer of the sample. Based on the characteristic bands of the VSV virus N, P, M, G, and L proteins, determine which layer may be the complete virus sample.

[0084] (4) Collect samples containing recombinant viruses, dilute with PBS, centrifuge at 4°C, 100,000 g for 2 hours, desugar, and resuspend the precipitate with PBS to obtain a virus liquid sample.

[0085] Identification of virus samples

[0086] The isolated and purified virus samples were identified using Coomassie brilliant blue staining, negative staining electron microscopy, surface plasmon resonance and other experiments.

[0087] Coomassie Brilliant Blue Staining After density gradient centrifugation, the virus sample obtained is separated by polyacrylamide gel electrophoresis, and the gel is stained with Coomassie Brilliant Blue dye to detect the position of characteristic protein bands, such as the position of VSV genome encoded proteins N, P, M, and L and the size of the corresponding molecular weight of the band. Based on this, the integrity of the virus particles in the corresponding sample is judged to determine whether the virus sample separation is successful.

[0088] The results are as follows Figure 2 As shown, the results showed that high-purity VSV-gp42 virus was obtained after density gradient centrifugation.

[0089] Negative stain electron microscopy After preparing the virus samples obtained above, the completeness of the virus particles was observed under a negative staining electron microscope to see whether they had the classic VSV rhabdoviral particle structure.

[0090] Negative staining electron microscopy test results of virus sample quality are as follows Figure 3 As shown, the results showed that the obtained virus particles exhibited a classic rhabdovirus structure under negative staining electron microscopy.

[0091] Surface plasmon resonance experiment Using the Biacore 8K instrument, the monoclonal antibody of gp42 was fixed to the protein A chip, and then the supernatant of Vero cells infected with the virus samples obtained above was passed through the corresponding channels on the chip to detect whether the cell supernatant samples with different dilution multiples would show concentration-dependent binding reactions (the response value of the reaction curve decreases with the increase of the dilution multiple).

[0092] The specific steps are as follows:

[0093] (1) Using a Biacore 8K instrument, fit the Protein A chip into the chip compartment.

[0094] (2) The gp42 monoclonal antibody 2C1 (for details about 2C1, see WO2024187675A1) was diluted to a concentration of 10 μM using a buffer containing 150 mM NaCl, 10 mM HEPES (pH 7.5), 3 mM EDTA, and 0.05% Tween-20, and the supernatant of the above-mentioned virus-infected Vero cells was diluted 2-fold in a continuous gradient.

[0095] (3) The antibody obtained in (2) and the supernatant dilution were sequentially passed through the protein A chip. The reaction temperature was 25°C, the detection frequency was 1 Hz, the flow rate was 30 μL / min, the binding time was 60 seconds, the dissociation time was 120 seconds, and the chip was regenerated using a solution containing 10 mM glycine (pH 1.5).

[0096] (4) Observe whether the response value of the virus sample to the antibody binding reaction changes with the dilution of the supernatant.

[0097] Surface plasmon resonance detection of gp42 expression in the supernatant of cells infected with VSV-gp42-GP2 Figure 4 As shown, the results showed that with the increase in the dilution multiple of the cell supernatant after infection, the response value of 2C1 to the supernatant binding showed a gradient decrease, confirming that EBV gp42 was indeed expressed in the cell supernatant after VSV-gp42-GP2 infection.

[0098] VSV-gp42-GP2 virus titer titration

[0099] Since the surface of the virus particles carries G protein, the virus titer can be determined by the plaque method. The virus solution obtained by the above separation and purification is diluted in a continuous 10-fold gradient, and the dilution is added to a 6-well plate that has been overgrown (100% confluence) with Vero cells at 100 μL per well. Incubate for 2 hours, discard the supernatant, and gently wash the cells with sterile PBS buffer. Cover the wells with DMEM culture medium containing methylcellulose. Incubate for 72-96 hours, add a paraformaldehyde solution containing crystal violet to each well, fix the cells and stain. Rinse the well plate gently with running water, discard the fluid in the well, dry it, count the number of plaques in the lowest concentration virus wells where plaques appear on the well plate, and calculate the virus titer according to the formula.

[0100] The specific steps are as follows:

[0101] (1) Since the virus particles carry G protein on their surface, the virus titer can be determined by plaque assay. The virus solution obtained by separation and purification is diluted 10 times in a continuous gradient using DMEM medium containing 10% by volume fetal bovine serum to obtain 10 0 times up to 10 12 Serial virus dilutions.

[0102] (2) 100 μL of the dilution solution was added to each well of a 6-well plate containing confluent (100% confluence) Vero cells.

[0103] (3) Incubate at 37°C, 5% CO2 for 2 hours, gently shaking the plate every 30 minutes.

[0104] (4) After 2 hours, discard the cell supernatant and gently wash the cells 3-5 times with sterile PBS buffer, 300 μL per well.

[0105] (5) The supernatant was discarded and the wells were covered with 2 mL of a buffer solution containing 0.83% (mass volume ratio) methylcellulose, 16.7% (volume ratio) PBS, 1.7% (volume ratio) fetal bovine serum, 81.7% (volume ratio) DMEM medium and 0.5% (double antibody).

[0106] (6) Place the plate at 37°C and 5% CO2 for 72-96 hours.

[0107] (7) Add 500 μL of 4% (mass volume ratio) paraformaldehyde solution containing 0.25% (mass volume ratio) crystal violet to each well to fix the cells and stain them, and incubate them at room temperature for 12 hours.

[0108] (8) Gently rinse the plate with running water, discard the fluid in the wells, dry them, count the number of plaques in the wells with the lowest virus concentration where plaques appear, and calculate the virus titer according to the following formula.

[0109]

[0110] The results of the VSV-gp42-GP2 titer determination showed that the virus stock solution with a virus titer of 1E8 was finally obtained, which was packaged into 200 μL per tube and frozen at -80°C for later use.

[0111] The specific steps of packaging VSV-gp42-GP4 are the same as the steps of packaging VSV-gp42-GP2 mentioned above, the only difference is that the transfected plasmid is pBS-VSV-gp42-GP4, that is, based on the pBS-VSV plasmid, the coding gene of EBV gp42 is inserted between VSV M and VSV G in the VSV genome to obtain a plasmid.

[0112] Example 2 Packaging and Preparation of VSV Recombinant Virus VSV-gp42-GP2-ΔG

[0113] Wherein, ΔG indicates that the VSV G gene in the VSV genome has been knocked out. Different from Example 1, since the VSV-G gene in the VSV genome has been knocked out, and EBV gp42 cannot independently mediate viral infection of host cells, it is necessary to transiently transfer the plasmid to supplement VSV G during the preparation of VSV-gp42-GP2-ΔG. The prepared VSV-gp42-GP2-ΔG can only infect host cells once, and the virus particles released from the host cells do not have the ability to infect other host cells.

[0114] The VSV-ΔG genome is integrated into the pBS plasmid vector (pBS-VSV-ΔG), and RNA synthesis is driven by the T7 promoter. After the pBS-VSV-ΔG plasmid is transfected into the cell, it is transcribed and synthesized into an RNA chain under the action of T7 RNA polymerase, and the complementary chain of the VSV genome is obtained after processing by the δ-ribozyme at the cleavage site at the end of the RNA chain. Using this as a template, the VSV RNA polymerase synthesizes the VSV genomic RNA and mRNA, and expresses the VSV proteins N, P, M, G, and L, and finally completes the assembly of VSV virus particles.

[0115] On the basis of pBS-VSV plasmid, the coding gene of EBV gp42 was inserted into different positions of VSV genome. When EBVgp42 was inserted between VSV-N and VSV-P genes, it was named VSV-gp42-GP2-ΔG, and the obtained plasmid was pBS-VSV-gp42-GP2-ΔG. During the preparation process, pCAGGS-VSV-G plasmid was added, so that the packaged VSV particles (VSV-gp42-GP2-ΔG) can express EBV gp42 protein in host cells.

[0116] The whole process of packaging VSV recombinant virus mainly includes plasmid transfection, initial transfection, and virus passage and amplification.

[0117] Primary transfection: BSR-T7 cells are infected with poxvirus VTF3-7 to provide T7 RNA polymerase. Plasmids pBS-VSV-N, pBS-VSV-P, pBS-VSV-L, pCAGGS-VSV-G containing genes encoding VSV viral proteins N, P, L, and G, and VSV-ΔG genome backbone plasmid containing EBV gp42 are co-transfected into BSR-T7 cells infected with VTF3-7. The T7 RNA polymerase expressed in the cells will transcribe the complementary chain of the VSV genome, and the N, P, and L proteins will assemble into VSV RNA polymerase to synthesize VSV genomic RNA, transcribe VSV viral protein mRNA, synthesize VSV viral protein, and finally produce VSV recombinant virus VSV-gp42. Collect cells and supernatant into centrifuge tubes, freeze and thaw repeatedly three times to break the cells and release the virus particles in the cells, centrifuge to remove cell debris, filter the supernatant with a 0.22μm filter membrane to remove poxvirus, and store the first-generation virus supernatant at 4°C for subsequent amplification.

[0118] Virus passage amplification: Since the titer of the virus particles obtained by the first transfection packaging is extremely low and may contain a large number of structurally incomplete virus particles, it is generally necessary to amplify through several passages to obtain high-titer, high-quality virus products. During the amplification process, Vero cells are used as host cells, and the cells are inoculated into culture dishes. After the cells grow to a density of 60%, the pCAGGS-VSV-G plasmid is transfected. After the cells are full, the virus supernatant obtained in the above primary transfection is added to the well plate and incubated with the cells for 6-12 hours. After the incubation is completed, the supernatant is discarded, the cells are washed with PBS, and fresh high-glucose medium containing 10% volume ratio of fetal bovine serum is added. Continue to culture for 48 hours to 72 hours, and collect cells and supernatants. The recovery ratio of the virus in the virus supernatant is maximized by freeze-thaw and centrifugation. 4-5 rounds of the above passage operation are performed to obtain high-quality virus supernatant. After that, Vero cells are still used as host cells, and a large amount of virus supernatant is obtained through large-volume passage.

[0119] The specific steps are as follows:

[0120] Packaging of recombinant virus VSV-gp42-GP2-ΔG

[0121] (1) Inoculate BSR-T7 cells (frozen in the inventor's laboratory) into a 10 cm culture dish and culture at 37°C with DMEM medium containing 10% (volume ratio) fetal bovine serum until the cell confluence reaches 80%-90%. Discard the culture medium, dilute the poxvirus VTF7-3 (frozen in the inventor's laboratory) with DMEM medium without fetal bovine serum, and infect BSR-T7 cells for 1 hour at a virus dosage of 5, gently shaking the culture dish once every 15 minutes. After the poxvirus VTF7-3 infects BSR-T7 cells, it will express T7 RNA polymerase in the cells.

[0122] (2) During the poxvirus infection, prepare the transfection reagent. Add 3 μg of pBS-VSV-N plasmid, 5 μg of pBS-VSV-P plasmid, 1 μg of pBS-VSV-L plasmid, 8 μg of pCAGGS-VSV-G plasmid, and 5 μg of pBS-VSV-gp42-GP2-ΔG plasmid to 500 μL of opti-MEM medium and mix well. Add 66 μL of lipo2000 to another 500 μL of opti-MEM, pipette to mix well, and let stand at room temperature for 5 minutes. Mix the prepared 500 μL plasmid-opti-MEM and 500 μL of lipo2000-opti-MEM, pipette to mix well, and let stand at room temperature for 20 minutes.

[0123] (3) After 1 hour of poxvirus infection, discard the supernatant, wash the culture dish 3-5 times with PBS, discard the supernatant, add 5 mL of opti-MEM, evenly add the prepared transfection reagent to the culture dish, and gently shake it. Incubate at 37°C, 5% CO2 for 4-6 hours, discard the supernatant, replace with DMEM medium containing 10% fetal bovine serum, and culture at 37°C, 5% CO2 for 48 hours. During this period, observe the cell pathological changes.

[0124] (4) 48 hours after transfection, collect cells and supernatant, freeze and thaw three times repeatedly to fully release the virus. Centrifuge at 500g for 10 minutes to discard cell debris, filter the supernatant with a 0.22μm filter 2-3 times to remove poxvirus, and freeze the virus solution in a -80℃ refrigerator for later use.

[0125] Amplification of the primary recombinant virus VSV-gp42-GP2-ΔG

[0126] (1) Inoculate Vero cells into a 10 cm culture dish, add 10% fetal bovine serum DMEM medium, and culture at 37°C, 5% CO2. When the cell confluence reaches 60%, add 10 μg of pCAGGS-VSV-G into 1 mL of opti-MEM medium, pipette and mix evenly, then add 30 μg of PEI200, pipette and mix evenly, and let stand at room temperature for 10 minutes.

[0127] (2) When the cell confluence reaches 80-90%, discard the culture medium, add 5 mL of the virus solution collected in the above-mentioned "Packaged Recombinant Virus VSV-gp42-GP2-ΔG", and infect for 2 hours, shaking the culture dish 2-3 times during the period. Discard the supernatant virus solution, replace with DMEM medium containing 5% fetal bovine serum, culture at 37°C, 5% CO2 for 48 hours, and observe the pathological changes of the cells.

[0128] (3) When most cells showed cytopathic effects, the cells and supernatant were collected and centrifuged at 4000 g for 10 min to remove cell debris. The supernatant was filtered through a 0.22 μm filter membrane and stored in a -80°C refrigerator until use.

[0129] Amplification of recombinant virus VSV-gp42-GP2-ΔG

[0130] (1) Inoculate Vero cells into a 10 cm culture dish, add 10% fetal bovine serum DMEM medium, and culture at 37°C, 5% CO2. When the cell confluence reaches 60%, add 10 μg of pCAGGS-VSV-G into 1 mL of opti-MEM medium, pipette and mix evenly, then add 30 μg of PEI200, pipette and mix evenly, and let stand at room temperature for 10 minutes.

[0131] (2) When the cell confluence reaches 80-90%, take the amplified virus supernatant obtained in the above "Amplification of primary recombinant virus VSV-gp42-GP2-ΔG", add 2 mL to each culture dish, and infect Vero cells for 2 hours, shaking the culture dish 2-3 times during the period. Discard the supernatant and replace it with DMEM medium containing 5% fetal bovine serum. Incubate at 37°C, 5% CO2 for 48 hours, and observe the pathological changes of the cells during the period.

[0132] (3) When most cells show cytopathic effects, collect the supernatant virus solution, centrifuge at 5000 g for 10 min to remove cell debris, filter the supernatant with a 0.22 μm filter membrane, and store in a refrigerator at 4°C for later use.

[0133] Purification and identification of VSV-gp42-GP2-ΔG

[0134] Virus purification by density gradient centrifugation

[0135] The collected virus supernatant was transferred to a high-speed centrifuge tube, centrifuged and discarded the supernatant, and the virus precipitate was resuspended in sterile PBS (100-200 times volume concentration). The obtained virus concentrated product was centrifuged for 1 hour with a 20%-50% sucrose density gradient, and the sample was separated layer by layer and identified by polyacrylamide gel electrophoresis and Coomassie brilliant blue staining, and the sample layer containing the virus VSV-gp42-GP2-ΔG was collected, and the sample was diluted with PBS, and the sucrose contained in the sample was separated by centrifugation. After the centrifugation, the supernatant was discarded, and the precipitate was resuspended with an appropriate amount of PBS and frozen in a -80°C refrigerator for later use.

[0136] The specific steps are as follows:

[0137] (1) Centrifuge the amplified virus solution at 100,000 g at 4°C for 2 h, and resuspend the precipitate in 1 mL of PBS.

[0138] (2) A 20%-50% sucrose density gradient was prepared using a density gradient preparation apparatus. The concentrated VSV-gp42-GP2-ΔG was ultracentrifuged at 4°C, 100,000 g for 1 hour. A clear white band was seen in the centrifuge tube, which was the virus sample layer.

[0139] (3) Use a density gradient separator to separate the samples after ultracentrifugation into layers. Take 40 μL of each sample and prepare gel electrophoresis samples. Perform polyacrylamide gel electrophoresis. Stain the gel with Coomassie Brilliant Blue dye and analyze the characteristic protein bands of each layer of the sample. Based on the characteristic bands of the VSV virus N, P, M, and L proteins, determine which layer may be the complete virus sample.

[0140] (4) Collect samples containing recombinant viruses, dilute with PBS, centrifuge at 4°C, 100,000 g for 2 hours, desugar, and resuspend the precipitate with PBS to obtain a virus liquid sample.

[0141] Identification of virus samples

[0142] The isolated and purified virus samples were identified using Coomassie brilliant blue staining, negative staining electron microscopy, surface plasmon resonance and other experiments.

[0143] Coomassie Brilliant Blue Staining After density gradient centrifugation, the virus sample obtained is separated by polyacrylamide gel electrophoresis, and the gel is stained with Coomassie Brilliant Blue dye to detect the position of characteristic protein bands, such as the position of VSV genome encoded proteins N, P, M, and L and the size of the corresponding molecular weight of the band. Based on this, the integrity of the virus particles in the corresponding sample is judged to determine whether the virus sample separation is successful.

[0144] Negative stain electron microscopyAfter preparing the virus samples obtained above, the completeness of the virus particles was observed under a negative staining electron microscope to see whether they had the classic VSV rhabdoviral particle structure.

[0145] Surface plasmon resonance experiment Using the Biacore 8K instrument, the monoclonal antibody of gp42 was fixed to the protein A chip, and then the supernatant of Vero cells infected with the virus samples obtained above was passed through the corresponding channels on the chip to detect whether the cell supernatant samples with different dilution multiples would show concentration-dependent binding reactions (the response value of the reaction curve decreases with the increase of the dilution multiple).

[0146] The specific steps are as follows:

[0147] (1) Using a Biacore 8K instrument, fit the Protein A chip into the chip compartment.

[0148] (2) The gp42 monoclonal antibody 2C1 (for details about 2C1, see WO2024187675A1) was diluted to a concentration of 10 μM using a buffer containing 150 mM NaCl, 10 mM HEPES (pH 7.5), 3 mM EDTA, and 0.05% Tween-20, and the supernatant of the above-mentioned virus-infected Vero cells was diluted 2-fold in a continuous gradient.

[0149] (3) The antibody and virus dilutions obtained in (2) above were passed through the protein A chip in sequence. The reaction temperature was 25°C, the detection frequency was 1 Hz, the flow rate was 30 μL / min, the binding time was 60 seconds, and the dissociation time was 120 seconds. The chip was regenerated using a solution containing 10 mM glycine (pH 1.5).

[0150] (4) Observe whether the response value of the virus sample to the antibody binding reaction changes with the dilution of the virus.

[0151] VSV-gp42-GP2-ΔG virus titration

[0152] Although the virus particles carry G protein on their surface, there is no VSV-G gene in the virus particle genome. When the virus infects host cells, the virus produced again will not be able to infect other host cells, so the virus titer cannot be determined by plaque assay. The virus surface protein will be quantified by Western Blot and ELISA, and the plaque-qPCR curve will be established by VSV-gp42-GP2 virus to titrate the VSV-gp42-GP2-ΔG titer.

[0153] The specific steps are as follows:

[0154] (1) Take a small amount of virus liquid sample and perform western blot and ELISA experiments to detect the amount of gp42 displayed by the recombinant virus.

[0155] (2) Take a small amount of virus liquid sample, extract the viral genomic RNA, and measure its genomic nucleic acid content by RT-q-PCR. By comparing it with the wild-type virus of known titer, the relative titer of the recombinant virus is calculated.

[0156] Example 3 Detection of the immune effect of VSV recombinant virus

[0157] 1. Immunization of mice

[0158] VSV-gp42-GP2 and VSV-gp42-GP4 prepared in Example 1 were used for immune testing. Balb / c mice (Vitamin Liva, catalog number: A11) were used for immune testing. Six-week-old mice were selected and inoculated with VSV-gp42 by intramuscular injection. They were inoculated sequentially at week 0, week 2, and week 4, and blood was collected from the posterior canthal venous plexus at week -1, week 2, week 4, week 6, and every 4 weeks thereafter. The serum was separated and stored at -80°C for later use. The state and weight of the mice were observed during the inoculation process to determine whether adverse reactions occurred.

[0159] The specific steps are as follows:

[0160] (1) Balb / c mice, 6 weeks old, were inoculated with VSV-gp42-GP2 and VSV-gp42-GP4 (10 mg / kg / day, respectively) by intramuscular injection on the right hind limb. 1 pfu, 10 3 pfu, 10 5 pfu), 200 μL per mouse.

[0161] (2) Vaccination was performed at week 0, week 2, and week 4, and blood was collected from the retrocanthal venous plexus at week -1, week 2, week 4, week 6, and every 4 weeks thereafter.

[0162] (3) Collect blood into non-anticoagulant tubes and let stand at room temperature for 2-4 hours to allow the whole blood to coagulate.

[0163] (4) Centrifuge at 15,000 g for 30 min at 4°C, collect the supernatant as serum, and incubate at 56°C for 30 min to inactivate complement.

[0164] (5) Store at -80°C for later use.

[0165] There was no obvious abnormal change in the body weight of mice after immunization, and no obvious adverse reactions were observed.

[0166] 2. Serum antibody titer detection

[0167] In order to determine whether the vaccine can induce a high level of immune response, the total IgG antibody titer in the serum of mice after immunization was detected by enzyme-linked immunosorbent assay. In order to determine the characteristics of the immune response after immunization, the titer of IgG subtypes such as IgG1, IgG2a, IgG2b, IgG2c, and IgG3 in the serum of mice was detected by enzyme-linked immunosorbent assay.

[0168] The specific steps are as follows:

[0169] (1) Coat 100 ng of gp42 protein per well into an ELISA plate.

[0170] (2) Incubate at 4°C for 12 hours, discard excess liquid in the wells, and wash the plate 3-5 times with PBS (PBST) solution containing 0.1% (volume ratio) Tween 20.

[0171] (3) Use PBS containing 5% (mass-volume ratio) bovine serum albumin and 0.1% (volume ratio) Tween 20 (BSA-PBST) to graded dilute the immunized mouse serum into a well plate coated with the target antigen (100-fold, 300-fold, 900-fold, 2700-fold, 8100-fold, 24300-fold, 72900-fold, 218700-fold), with a final volume of 100 μL.

[0172] (4) Incubate at 37°C for 1 hour, discard the liquid in the plate, and wash the plate 3-5 times with PBST.

[0173] (5) Dilute HRP-labeled goat anti-mouse IgG or IgG subtype antibody using BSA-PBST solution and add 100 μL to each well.

[0174] (6) Incubate at 37°C for 1 hour and wash the plate 3-5 times with PBST.

[0175] (7) Add 100 μL of TMB solution to each well and incubate at room temperature for 15 minutes.

[0176] (8) Retain the liquid in the wells and add 100 μL of diluted hydrochloric acid solution to each well to terminate the color development reaction.

[0177] (9) Use an ELISA reader to read the absorbance values ​​at 450 nm and 630 nm for calculation of relative titer of serum antibodies.

[0178] Serum gp42-specific antibody titer results Figure 5As shown, the experimental results showed that VSV-gp42-GP2 and VSV-gp42-GP4 could induce high levels of serum antibodies against EBV gp42, and the serum IgG subtypes were mainly IgG2a and IgG1. Moreover, the level of serum antibodies against EBV gp42 induced by VSV-gp42-GP2 was significantly higher than that induced by VSV-gp42-GP4.

[0179] 3. In vitro micro-neutralization test protection effect detection

[0180] In order to detect the level of antibodies that can block EBV-infected cells in the serum of immunized mice, an in vitro microneutralization experiment was performed. Since EBV can infect B lymphocytes and epithelial cells, Akata cells (frozen in the inventor's laboratory) and HEK293 (frozen in the inventor's laboratory) cells were used as representatives of B cells and epithelial cells, respectively. In order to facilitate detection, an EBV strain with green fluorescent protein (GFP) - CNE2-EBV-GFP was used to infect host cells.

[0181] The specific steps are as follows:

[0182] CNE2-EBV-GFP preparation

[0183] (1) CNE2 cells carrying the virus strain were cultured in RPMI1640 (Gibco TM , catalog number: C11875500BT) culture medium, and G418 (Yisheng Bio, catalog number: 60220ES08) with a final concentration of 0.7 mg / mL was used for positive clone screening.

[0184] (2) When the cells grow to 90% density, TPA (Biyuntian, catalog number: S1819-1mg) and NaB (sodium butyrate) (Biyuntian, catalog number: S1539-10g) are added for induction, with concentrations of 20 ng / ml and 2.5 mM, respectively.

[0185] (3) Replace the culture medium with fresh one before induction and replace the medium 12 hours after induction.

[0186] (4) 48-72 hours after the medium change, the culture supernatant was collected to separate and purify the virus. The supernatant was directly aspirated and centrifuged at 200g for 5 minutes at room temperature.

[0187] (5) The supernatant after centrifugation was filtered through a 0.45 filter.

[0188] (6) The filtered supernatant was used as the virus stock solution and stored at 4°C for later use.

[0189] Serum neutralization protection effect detection

[0190] (1) Akata cells and HEK293 cells were cultured using RPMI1640 and DMEM culture media containing 5% (volume ratio) fetal bovine serum, respectively.

[0191] (2) Add 3.8 μL of the immunized mouse serum collected above into a 96-well cell culture plate according to the first well, and add 58.7 μL of the corresponding culture medium and mix well.

[0192] (3) Use the serum-containing culture medium of the corresponding cells to make 5-fold dilutions, with 6 gradients (50-fold, 250-fold, 1250-fold, 6250-fold, 31250-fold, 156250-fold, relative to the final volume).

[0193] (4) Add 50 μL of CNE2-EBV-GFP supernatant to each well and incubate at 37°C, 5% CO2 for 2 hours.

[0194] (5) Add 15,000 Akata or HEK293 cells in good condition to each well in a volume of 50 μL.

[0195] (6) Incubate at 37°C, 5% CO2 for 48 hours.

[0196] (7) For Akata cells, 32 μL of 4% paraformaldehyde (White Shark, Catalog No.: BL539A) solution was added to each well and fixed at room temperature for 20 minutes. The proportion of cells with FITC signal positive relative to the negative control in each well was detected using the CytoFLEX flow cytometer B525 channel.

[0197] (8) For HEK293 cells, discard the supernatant, add 50 μL of trypsin (Suzhou Xinsaimei, catalog number: C100C1) to each well, incubate at 37°C for 5 minutes, and add 100 μL of DMEM medium containing 5% FBS to each well to terminate the digestion. Perform the same operation as in (7) and add paraformaldehyde for fixation to detect the proportion of FITC-positive cells.

[0198] The experimental results are as follows Figure 6 The results showed that the mice were inoculated with a dose of 10 5 PFU of VSV-gp42-GP2 and VSV-gp42-GP4 can produce high levels of neutralizing antibodies in lymphocytes and epithelial cells, and the lymphocytes and epithelial cells neutralizing antibodies produced by mice vaccinated with VSV-gp42-GP2 are significantly higher than those produced by mice vaccinated with VSV-gp42-GP4. In other words, after booster immunization, VSV-gp42 can produce high levels of serum neutralizing antibodies that can protect target cells from viral infection.

[0199] The immunogenicity comparison of the EBV vaccine prepared in Example 1 of the present invention and the EBV vaccine reported in the prior art is shown in the following table:

[0200]

[0201] *The authors did not indicate whether the antibody titers tested were endpoint titers or EC50.

[0202] According to the data in the above table, although there have been reports on the use of other viral vectors to develop EBV vaccines, or the use of EBV glycoproteins gB and / or gHgL as vaccine design targets, or the use of VSV viruses to display other viral glycoproteins and use them as vaccines and other purposes, in this Example 1, EBV gp42 was inserted into the front end of the VSV genome through genome modification and screening to obtain a VSV vector vaccine that stably and efficiently expresses monovalent EBV gp42, and the immunized serum can achieve complete inhibition of EBV-infected target cells, and the immunized serum shows a high competitive activity with gp42 neutralizing antibodies, and compared with ordinary recombinant protein vaccines and granule protein vaccines, it has extremely strong immunogenicity. The above unique immune effects clearly indicate that the VSV-gp42 vaccine is an independent, novel, and transformative EBV vaccine.

[0203] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A VSV vector, comprising (a) at least a portion of a VSV genome, wherein the at least a portion of the VSV genome comprises a VSV N gene, a VSV P gene, a VSV M gene and a VSV L gene; and (b) a polynucleotide sequence encoding an EBV gp42 protein or an immunogenic variant thereof.

2. The VSV vector according to claim 1, characterized in that The VSV vector comprises (a) a VSV genome comprising a VSV N gene, a VSV P gene, a VSV M gene, a VSV G gene and a VSV L gene; and (b) a polynucleotide sequence encoding an EBV gp42 protein or an immunogenic variant thereof.

3. The VSV vector according to claim 2, characterized in that The polynucleotide sequence encoding the EBV gp42 protein or an immunogenic variant thereof is located between the VSV N gene and the VSV P gene, or between the VSV M gene and the VSV G gene.

4. A recombinant VSV virus particle, which comprises at least VSV N protein, VSV P protein, VSVM protein, VSV L protein and EBV gp42 protein or an immunogenic variant thereof.

5. The recombinant VSV virus particle according to claim 4, characterized in that The recombinant VSV virus particle comprises VSV N protein, VSV P protein, VSV M protein, VSV G protein, VSV L protein and EBV gp42 protein or immunogenic variants thereof.

6. A cell, which produces the recombinant VSV virus particle of claim 4 or 5.

7. A method for preparing VSV virus particles, comprising: Co-transfecting host cells with the recombinant VSV vector and the helper plasmid according to any one of claims 1 to 3, and passage them to obtain recombinant VSV virus particles; The auxiliary plasmids include: auxiliary plasmids VSV-N, VSV-P, VSV-L and VSV-G which contain N gene, P gene, L gene and G gene of VSV virus respectively.

8. An immunogenic composition, comprising (a) VSV virus particles prepared by the VSV vector described in any one of claims 1 to 3 or the recombinant VSV virus particles described in claim 4 or 5; and (b) a pharmaceutically acceptable carrier.

9. The immunogenic composition according to claim 8, characterized in that The immunogenic composition is a vaccine.

10. Use of a VSV virus particle prepared by the VSV vector according to any one of claims 1 to 3, a recombinant VSV virus particle according to claim 4 or 5, a VSV virus particle prepared by the method according to claim 7, or an immunogenic composition according to claim 8 or 9 in at least one of the following: (1) Preparation of drugs for treating and / or preventing EBV infection; (2) Preparation of drugs for treating and / or preventing EBV infection-related neoplastic diseases or non-neoplastic diseases; (3) Preparation of antibodies that inhibit EBV infection.

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