Recombinant oncolytic virus rVSV M51R -S Application in Hepatitis B-related Hepatocellular Carcinoma

Abstract

The present invention discloses the application of a recombinant oncolytic virus rVSV<supgt;M51R< / supgt>‑S in hepatitis B‑associated liver cancer, belonging to the field of biomedicine. The recombinant oncolytic virus of the present invention is a recombinant attenuated vesicular stomatitis virus carrying the HBV HBsAg gene and having an M‑>R mutation at the 51st amino acid of the M gene. The recombinant oncolytic virus has been proven to be effective in tumor models and can be used to prepare therapeutic vaccines for HBV‑positive liver cancer, diffuse large B‑cell lymphoma positive for HBV, and other HBV‑positive tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a recombinant oncolytic virus composed of an attenuated vesicular stomatitis virus and hepatitis B virus surface antigen (HBsAg) and its application. Background Art

[0002] Liver cancer is one of the major problems threatening human life and health. Hepatocellular carcinoma is the most common form of liver cancer, and its occurrence and development are related to chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body to attack tumor cells (highly specific cell lysis). However, in clinical practice, immunotherapy only brings durable survival benefits to a small number of patients. Most patients still face the problem of immunotherapy resistance, and once the first-line treatment fails, there is usually a lack of effective treatment means. Therefore, there is an urgent need to develop a suitable treatment strategy for HBV-positive liver cancer patients.

[0003] Oncolytic virus therapy is a novel tumor treatment method with a unique mechanism of action. Its antitumor effect mainly includes two aspects. On the one hand, it directly infects tumor cells and causes them to lyse and die. On the other hand, it indirectly activates the host's innate and adaptive antitumor immune responses. Vesicular stomatitis virus (VSV) is a member of the genus Vesiculovirus and belongs to the family Rhabdoviridae. The virus is a bullet-shaped enveloped virus with an 11 kb negative-strand RNA genome. It has a broad spectrum of tumor-killing ability, replicates in the cytoplasm, and its genome does not integrate into the host cell DNA, with good safety. Due to the defect of type I interferon signaling in tumor cells, the virus can preferentially replicate in many tumor cells.

[0004] Oncolytic viruses can not only directly kill tumors, but also are expected to stimulate the body's immune response during replication through the expression of specific antigens, induce immunogenic cell death, and at the same time stimulate the host's anti-tumor immunity to further enhance the anti-tumor effect. Oncolytic viruses lyse tumors locally in the tumor to release tumor-associated antigens (TAAs), which is similar to tumor vaccines, but may not be sufficient to induce tumor-specific T cell responses. By integrating the TAAs gene into the viral genome, more TAAs can be expressed to enhance specific T cell immune responses. Many studies have shown that tumor-specific antigens have high immunogenicity, and spontaneous neoantigen-specific T cells have been detected in cancer patients. Therefore, in the design of tumor therapeutic vaccines, tumor-specific antigens are preferentially selected as targets, which have higher immunogenicity than self-antigens and reduce the risk of autoimmunity. Chronic viral infections are prone to induce cancer by causing repeated tissue damage, inflammation, and the integration of viral genes into the host genome. The integrated viral genes can be expressed through host mechanisms to produce viral-derived protein fragments, which are then degraded into viral peptides and assembled with MHC class I to be presented on the cell surface. Therefore, these cancer cells are specifically labeled with viral peptides as tumor antigens. At the same time, these oncoproteins are also unique to tumors and can cause T cell recognition and killing of antigens.

[0005] HBV belongs to the Hepadnaviridae family, with a full-length genome of 3.2 kb, which is a partially single-stranded double-stranded circular DNA. The HBV genome has 4 open reading frames, encoding envelope protein (S), core protein (C), polymerase (P), and X protein (HBX), respectively. More than 90% of HBV-related HCC genomes contain the integration of HBV-DNA, that is, most cells of HBV-related HCC contain HBV-DNA fragments that do not encode complete HBV antigens. Studies have shown that HCC cells that do not express complete HBV antigens contain short HBV mRNAs, which encode epitopes recognized and activated by HBV-specific T cells. Some patients with secondary HCC can express complete HBV surface antigen epitopes. In these specific patients, recombinant oncolytic virus therapy provides us with feasibility guidance. Moreover, the "hot" tumor microenvironment generated by inflammatory cells recruited by oncolytic viruses can also ensure that tumor-specific cytotoxic T cells maintain optimal anti-tumor activity, which is lacking in current therapeutic HBV tumor vaccine methods. In summary, there is an urgent need in this field to develop effective therapeutic vaccines against HBV-positive liver cancer. Currently, there is no oncolytic virus drug based on vesicular stomatitis virus carrying HBV-related genes against HBV-positive liver cancer. Summary of the Invention

[0006] To address the deficiencies of the existing technology, the objective of the present invention is to provide a recombinant oncolytic virus composed of an attenuated vesicular stomatitis virus and hepatitis B virus surface antigen, as well as its applications.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A recombinant oncolytic virus, which is a recombinant attenuated vesicular stomatitis virus carrying at least one HBV antigen gene. The HBV antigen gene includes the HBsAg gene.

[0009] In some embodiments, the recombinant oncolytic virus is a recombinant attenuated vesicular stomatitis virus carrying the HBV HBsAg gene.

[0010] In some embodiments, the attenuated vesicular stomatitis virus is a vesicular stomatitis virus with an M->R mutation at the 51st amino acid of the M gene.

[0011] In some embodiments, the HBsAg gene is derived from HBV genotype A, B, C, or D.

[0012] In some embodiments, the HBsAg gene is derived from D-type HBV, and its nucleotide sequence is as shown in SEQ ID NO: 1.

[0013] In some embodiments, the HBsAg gene is located between the G gene and the L gene of the vesicular stomatitis virus.

[0014] In some embodiments, the recombinant attenuated vesicular stomatitis virus further comprises one or more of the following genes:

[0015] (1) Reporter genes, such as sodium iodide symporter (NIS), green fluorescent protein (GFP), luciferase;

[0016] (2) Genes to be delivered to target cells or tissues, such as genes to be delivered to tumor cells and tumors;

[0017] (3) Immune checkpoint inhibitory antibodies, such as PD-1, PD-L1, CTLA-4, LAG-3, TIGIT;

[0018] (4) Immune stimulatory genes, such as IL-2, IL-12, IL-15, IFN-γ, GM-CSF;

[0019] (5) Tumor-associated antigen TAA for tumor vaccination.

[0020] Use of the above recombinant oncolytic virus in the preparation of a tumor therapeutic vaccine, wherein the tumors include HBV-positive liver cancer, HBV-positive diffuse large B-cell lymphoma, and other HBV-positive tumors.

[0021] A tumor therapeutic vaccine, wherein the active ingredient is the above recombinant oncolytic virus.

[0022] In the present invention, the gene encoding hepatitis B virus surface antigen is inserted into an attenuated oncolytic virus vector, and a large amount of hepatitis B virus surface antigen is expressed by infecting HBV-positive liver cancer cells, strengthening antigen delivery to DCs, promoting MHC I processing and presentation, enhancing tumor-specific cellular immune responses, and improving the clearance of tumor cells by cytotoxic T cells.

[0023] Advantages and beneficial effects of the present invention: The recombinant oncolytic virus rVSV M51R -S can significantly inhibit the tumor growth of mice in the HBV-positive liver cancer model. After treatment with rVSV M51R -S, the survival rate of HBV-positive liver cancer mice reached 44%, which was much higher than that of the control group; in the HBV-positive liver cancer ascites tumor model, the survival rate of liver cancer mice reached 100%, also much higher than that of the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of the modification of the recombinant oncolytic virus rVSV M51R -S.

[0025] Figure 2 is the construction of the virus packaging plasmid pXN2-M51R-HBsAg. Among them, Figure 1 shows the plasmid map of pXN2-M51R-HBsAg (A) and its restriction enzyme verification results (B).

[0026] Figure 3 is the construction and identification of the recombinant oncolytic virus rVSV M51R -S. Figure 3 (A) shows that the recombinant oncolytic virus rVSV M51R -S infects the mouse liver cancer cell line Hepa1-6, and Western Blot verifies the expression of the hepatitis B virus surface protein S-HBsAg; Figure 3 (B) shows that the recombinant oncolytic virus rVSV M51R -S infects the mouse liver cancer cell line Hepa1-6, and ELISA is used to verify the expression of the hepatitis B virus surface protein in the supernatant and cell lysate.

[0027] Figure 4 shows that HBV does not affect the replication and oncolytic ability of rVSV M51R -GFP. Figure 4(A) Treat H22-DE-HBV cells with 1 μg / mL doxycycline (DOX), and detect the related indicators of HBV replication (HBsAg, HBeAg, HBV DNA) 10 days later. Figure 4 (B) Three groups of cells (including H22 group, H22-DE-HBV group, H22-DE-HBV+DOX group) were simultaneously infected with the virus rVSV M51R -GFP, and the replication and oncolytic ability of the virus rVSV M51R -GFP in different hepatocellular carcinoma cells were detected.

[0028] Figure 5 It is the evaluation of the therapeutic effect of the tumor therapeutic vaccine rVSV M51R -S on HBV-positive hepatocellular carcinoma. Figure 5 (A) shows the tumor volumes of different treatment groups in the subcutaneous tumor-bearing model of HBV-positive hepatocellular carcinoma; Figure 5 (B) shows the survival rates of different treatment groups in the subcutaneous tumor-bearing model of HBV-positive hepatocellular carcinoma.

[0029] Figure 6 It is the evaluation of the therapeutic effect of the tumor therapeutic vaccine rVSV M51R -S on HBV-positive hepatocellular carcinoma. Figure 6 (A) The changes in body weights of mice in different treatment groups in the ascites tumor model of HBV-positive hepatocellular carcinoma; Figure 6 (B) shows the survival rates of different treatment groups in the ascites tumor model of HBV-positive hepatocellular carcinoma. Detailed implementation manners

[0030] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the technical solutions of the present invention will be further described in detail below in combination with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] According to the information included in the present invention, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only used to illustrate specific aspects of the present invention schematically. In fact, all the various changes that those skilled in the art or related fields can obviously make to the embodiments of the present invention are covered by the scope of the appended claims.

[0032] For a better understanding of the present invention and not as a limitation on its scope, all numbers representing amounts, percentages, and other numerical values used in the present invention should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary depending on the desired properties to be obtained. Each numerical parameter should be considered at least as obtained by the reported significant digits and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0033] Unless otherwise specified, the following examples of the present invention are carried out under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in the four seasons without additional cooling or heating treatment. Generally, the normal temperature is controlled at 10-30°C, preferably 15-25°C.

[0034] Example 1

[0035] This example further describes the recombinant oncolytic virus rVSV using the vesicular stomatitis virus system as an example M51R -S (see the schematic diagram of the modification in Figure 1The construction process of ( ) specifically includes the following steps: By means of molecular biology experiments, an M->R mutation was made at the 51st amino acid of the M gene of VSV, and the mutant M gene was cloned into the plasmid pXN2-GFP containing the full-length genome of VSV (the plasmid pXN2-GFP was kindly provided by Professor John K Rose of Yale University. The construction of this plasmid is described in the literature 1. Dalton KP, Rose JK. Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles. Virology. 2001 Jan 20;279(2):414-21.) through the XbaI and MIuI restriction enzyme sites to obtain the plasmid pXN2-M51R-GFP (the construction of this plasmid is described in the literature 2. Gaddy DF, Lyles DS. Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways. J Virol. 2005 Apr;79(7):4170-9. doi: 10.1128 / JVI.79.7.4170-4179.2005.). Then we obtained the HBsAg sequence using the cloning plasmid pAAV-HBV1.3 (the construction of the plasmid pAAV-HBV1.3 is described in the literature 3. Xu Z, Zhao L, Zhong Y, Zhu C, Zhao K, Teng Y, Cheng X, Chen Q, Xia Y. A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA. Cell Mol Gastroenterol Hepatol. 2022;13(4):1001-1017.) as a template. According to the pXN2-M51R-GFP and HBsAg sequences, upstream and downstream primers for homologous recombination were designed, with the restriction enzyme sites Xho I and Nhe I added upstream and downstream respectively, and a Flag tag sequence added upstream. The primers are shown in the following table.

[0036]

[0037] Amplification of HBsAg: Using pAAV-HBV1.3 as a template, PCR amplification was carried out under the following conditions: The PCR reaction system included pAAV-HBV1.3: 1 μL; pXN2-HBsAg-F: 1 μL; pXN2-HBsAg-R: 1 μL; 2× Primer Star Mix: 25 μL; ddH2O: 22 μL. The PCR reaction conditions were: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 35 PCR cycles, sufficient extension at 72°C for 10 min, and storage at 4°C. After 1% agarose gel electrophoresis of the PCR products, they were photographed using a gel imaging system, the target bands were cut, and DNA gel recovery was performed according to the instructions of the Axygen gel recovery kit.

[0038] Digestion of pXN2-M51R-GFP vector: The pXN2-M51R-GFP vector was digested with Xho I and Nhe I restriction endonucleases. The reaction system was as follows: pXN2-M51R-GFP: 2 μg; Xho I: 1 μL; Nhe I: 1 μL; 10×M Buffer: 5 μL. Digestion was carried out at 37°C for 2 h, and the digestion products were detected by 1% DNA agarose gel electrophoresis and the gel was cut for recovery.

[0039] Ligation of the target gene and the vector. The ligation system was as follows: HBsAg target gene fragment: 3.5 μL; pXN2-M51R-GFP digestion product: 2.5 μL; 2×Basic assembly mix: 6 μL, reaction at 50°C for 30 min.

[0040] Transformation of the ligation system: 50 μL of stable competent cells were added to the 12 μL ligation system, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then incubated on ice for 2 min. 1 mL of LB medium without antibiotics was added, and the cells were cultured with shaking at 37°C and 250 rpm for 1 h. Centrifuged at 12000 rpm for 1 min, the supernatant was discarded, and the bacteria were resuspended in 100 μL of LB medium and evenly spread on an LB solid plate containing Kana antibiotic. After incubation at 37°C in an inverted position for 8 - 12 h, single colonies were picked and amplified in liquid LB for 8 - 12 h. Referring to the "AXYGEX Plasmid Mini Extraction Kit Instruction Manual", the pXN2-M51R-HBsAg plasmid was extracted in small amounts.

[0041] Verification of plasmid digestion and sequencing. Digestion was carried out at 37°C for 2 h, the digestion products were detected by 1% DNA agarose gel electrophoresis, and the plasmids with correct digestion results were selected and sent to the company for sequencing.

[0042] In this embodiment, we found the DNA sequence of the surface antigen of hepatitis B virus of genotype D and designed primers using plasmid AAV-HBV1.3 as a template. The S-HBs gene fragment was obtained by PCR. The target gene was cloned between the G and L genes of the pXN2-M51R vector with a mutation at position 51 of the M gene through homologous recombination, resulting in the recombinant plasmid pXN2-M51R-HBsAg. The recombinant plasmid map is as shown in Figure 2 A. Through double digestion with Xho Ⅰ and Nhe Ⅰ and sequencing verification, the digestion results are as shown in Figure 2 B. Lanes 1, 3, and 4 are all plasmids with correct digestion. The excised vector fragment is greater than 10 kb, and the inserted target gene is 681 bp.

[0043] Prepare 4 plasmids required for the packaging of recombinant vesicular stomatitis virus: pXN2-M51R-GFP / pXN2-M51R-HBsAg, pP, pN, pL (the plasmids pP, pN, pL were kindly provided by Professor John K Rose of Yale University, and their construction is described in the literature 1. Dalton KP, Rose JK. Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles. Virology. 2001 Jan 20;279(2):414-21.). When the growth density of BSR cells (a cloned cell line of the commercial cell line BHK-21 cells, with the T7 promoter inserted into its genome) reaches 80% in a 10 cm cell culture dish, add 10 μL of vaccinia virus containing T7 RNA polymerase (its construction is described in the literature 4. Yang F, Tan J, Fang Y, Chen G, Zhang Y, Hu Q, Han W, Liu Y, Fu B, Jing Z, Li W. The Multiplicity of Infection of Recombinant Vaccinia Virus Expressing the T7 RNA Polymerase Determines the Rescue Efficiency of Vesicular Stomatitis Virus. Front Microbiol. 2022 Apr 4;13:846426.) to 10 mL of DMEM basal medium and infect BSR cells for 2 h. After infecting BSR cells, use liposome transfection method (Lipo3000 transfection reagent) to transfect the vesicular stomatitis virus packaging plasmids pXN2-M51R-GFP / pXN2-M51R-HBsAg (10 μg), pP (5 μg), pN (4 μg), pL (1 μg) into them. After 6 h, change the cell medium. After 48 h, collect the supernatant and filter it through a 0.22 μm filter membrane. Infect Vero cells again with the collected virus solution. After 24 h, observe the cytopathic effect. If the cells become round, it indicates that the VSV virus is successfully packaged in vitro. By means of reverse genetics, the mutant attenuated strain rVSV M51R -GFP, rVSV M51R -S is rescued. Then utilize rVSV M51R-S infects Hepa1-6 (mouse liver cancer cells), and the S-HBsAg protein of HBV is verified to be highly expressed in the lysate and supernatant of infected tumor cells through Western blot and ELISA experiments. The specific results are shown in Figure 3 A and B.

[0044] Discard the original medium of the cultured Vero cells, add 10 mL of DMEM medium containing 2% serum to each dish of cells, and infect the Vero cells with the recombinant VSV virus (rVSV M51R -GFP, rVSV M51R -S) at a dose of MOI = 0.01. Since the VSV virus can be transmitted through the aerosol route, the cells infected with VSV need to be cultured separately to prevent cross-contamination. Observe the cell morphology 24 h after infection. After the recombinant VSV virus successfully infects the Vero cells, the cells become round. When the vast majority of the cells become round and more than 50% of the cells float, collect the amplified VSV virus, centrifuge at 3000 rpm for 20 min, and discard the cell debris.

[0045] Prepare solutions of 40% PEG6000 / 2.5 M NaCl, 25%, 40%, 55% sucrose solutions, and 1× TNF buffer, all ensuring sterility. For every 40 mL of the VSV virus stock solution, add 10 mL of 40% PEG6000 / 2.5 M NaCl solution to form a mixed solution with a final concentration of 8% PEG6000 / 0.5 M NaCl. Mix well and let it stand overnight at 4°C. Centrifuge the virus solution that has stood overnight at 12000 rpm for 1 h. Precipitated virus will appear on the centrifuge tube wall. Resuspend the virus with 2 mL of 1× TNE buffer.

[0046] Next, a sucrose density gradient needs to be laid to perform isopycnic ultracentrifugation on the virus. Sequentially add 3 mL of 55% sucrose solution, 3 mL of 40% sucrose solution, 3 mL of 25% sucrose solution, 2 mL of the virus suspension, and 1 mL of 1× TNF buffer into the horizontal centrifuge tube. Slowly add the above solutions to ensure obvious stratification between layers. After balancing the horizontal centrifuge tube, perform isopycnic ultracentrifugation on it with an ultracentrifuge. Adjust the acceleration and deceleration of the centrifuge to the lowest level and perform isopycnic ultracentrifugation for 2 h. After the horizontal centrifugation is completed, aspirate the purified virus in the middle (at the interface of 25% and 40% sucrose) with a 1 mL syringe and place it into a 1.5 mL EP tube. Transfer the virus solution to a 25 mL ultracentrifuge tube, fill it with 1× TNF buffer, and perform vertical ultracentrifugation for 3 h. After the centrifugation is completed, resuspend it with 300 μL of 1× TNF buffer, aliquot it into 6 tubes, and store it at -80°C.

[0047] Example 2

[0048] This example specifically describes the treatment of murine HBV-positive liver cancer with oncolytic virus rVSV M51R -S as an example.

[0049] This example is an evaluation of the therapeutic effect of rVSV M51R -S on HBV-positive liver cancer, using a viral vector system to express hepatitis B virus antigens and activate specific anti-tumor immune responses. The specific implementation cases involve the treatment implementation with virus antigens expressed by a specific purified recombinant non-segmented, negative-strand RNA viral vector.

[0050] (1) Culturing of H22-DE-HBV tumor cell line, establishment of subcutaneous liver cancer tumor-bearing model and immunotherapy

[0051] Obtain the murine hepatoma cell line H22-DE-HBV positive for HBV. The specific construction method is as follows: First, obtain two transfection plasmids, namely pWPI-tet-off and pCMV-DE-HBV (for the construction of pWPI-tet-off and pCMV-DE-HBV plasmids, see Reference 5. Guo H, Jiang D, Zhou T, Cuconati A, Block TM, Guo JT. Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation. J Virol. 2007 Nov;81(22):12472-84.). The construction strategy of the pCMV-DE-HBV plasmid is to perform a point mutation at the start codon of the e antigen on the basis of the pCMV-HBV plasmid, so that it cannot form the HBV e antigen, and the e antigen can only be formed after the transfected cells are circularized to form a complete cccDNA. First, linearize the plasmids in advance. Digest the two plasmids with ScaI restriction endonuclease respectively and perform gel extraction. After the pCMV-DE-HBV is linearized, there is only one band, and directly perform gel extraction. After the pWPI-tet-off plasmid is digested with a single enzyme, there are two bands, one with a size of about 7800 bp and the other with a size of about 4300 bp. Gel extract the band around 7800 bp. Transfect the two linearized plasmids into H22 cells at the same time. After 48 h of transfection, kill the H22 cells with 1640 medium added with puromycin resistance. After 10 days of stable killing, obtain the H22-DE-HBV polyclonal cell line. Then, further obtain the monoclonal cell line of H22-DE-HBV. Plate according to the amount of 0-1 cell / 96-well plate. After 14 days of cell growth, it can be clearly observed that the cell clusters grow aggregately. Select the wells with only one cell cluster in the well and make marks. Collect a small amount of supernatant and detect the S and e antigens of HBV. Among the cell lines with confirmed stable integration of linear HBV, continue to select the cell lines regulated by Dox. After 10 days of Dox treatment, detect the expression changes of the S and e antigens of HBV and HBV DNA. The specific results are shown in Figure 4 A. Finally, successfully obtained the H22-DE-HBV cell line regulated by doxycycline (Dox) and stably integrated with linear HBV. At the same time, use an equal dose of rVSV M51RThree groups of cells were simultaneously infected with -GFP, namely the H22 group, the H22-DE-HBV group, and the H22-DE-HBV+DOX regulation group. After 24 hours of infection, the GFP positive rate of the cells was detected. It was found that HBV did not affect the replication ability of the oncolytic virus rVSV M51R -GFP. Similarly, three types of cells were infected with different doses of the virus. After 48 hours, the cell viability was detected by the CCK8 method. The results showed that the integration of linear HBV did not affect the oncolytic ability of the oncolytic virus rVSV M51R -GFP. The specific results are shown in Figure 4 B.

[0052] H22-DE-HBV cells were cultured in complete 1640 medium. When the cell number reached 70-80% density, tumor cells were inoculated. Observe their status before inoculating tumor cells, and the cell growth rate should be maintained in the exponential growth phase. One day before cell inoculation, the right thigh lateral of the mouse was shaved with an electric hair clipper. The type of tumor-bearing mice selected was 6-8-week-old C57BL / 6 male mice. H22-DE-HBV tumor cells were resuspended in PBS, and the inoculated cell resuspension density was 5×10 6 / mL. 200 μL of tumor cells were subcutaneously inoculated at the inguinal area of the right thigh lateral of each mouse. Tumors generally formed in about 6 days. Measure the tumor size and group them after statistical results. The volume of the mouse tumor generally remained at 70 mm 3 . On the 6th day, the mice with appropriate tumor sizes in the HBV-positive liver cancer model were divided into 3 groups: the normal saline Saline group, the oncolytic virus rVSV M51R -GFP group, and the oncolytic virus rVSV M51R -S group. On the 6th day, 1 mL of insulin syringe was used to inject the virus intratumorally, and the dosing dose was 1×10 8 PFU (50 μL). On the 8th and 10th days, intratumoral administration was still carried out according to the previous treatment method. The tumor size was statistically counted every other day, and the tumor volume was calculated. The tumor volume calculation formula: V = 0.52×L×W 2 (L = length, W = width). When the tumor volume reached 1500 mm 3 , according to the experimental humane endpoint, the mice were sacrificed by cervical dislocation.

[0053] The pharmacological performance of the oncolytic virus rVSV M51R -S, as Figure 5 shown, after 3 consecutive alternate-day injection treatments, compared with rVSV M51R -S and the control virus rVSV M51R -GFP, it could significantly inhibit tumor growth and improve the survival period of tumor-bearing mice. Through analysis, it can be seen that on the 14th day after virus treatment, the average tumor volume of the normal saline treatment group was 1075 mm 3 , rVSVM51R -GFP treatment group, the average tumor volume was about 496 mm 3 or so, while the mice received rVSV M51R -S treatment, the average volume was about 270 mm 3 . And by comparing the survival rates, it was found that in the rVSV M51R -S treatment group, the complete remission rate of mice was 44%, which was significantly higher than that of the normal saline treatment group and the rVSV M51R -GFP treatment group.

[0054] (2) Establishment of hepatocellular carcinoma ascites tumor model and immunotherapy

[0055] To establish a mouse HBV-positive hepatocellular carcinoma ascites tumor model, the type of tumor-bearing mice selected was 6-8-week-old C57BL / 6 male mice. The H22-DE-HBV tumor cells were resuspended with PBS, and the inoculation cell resuspension densities were 5×10 6 cells / mL, and 200 μL of tumor cells were inoculated into the abdominal cavity of each mouse. On the 5th day, the mice with appropriate tumor sizes in the mouse HBV-positive liver cancer model were divided into 3 groups: normal saline treatment group, rVSV M51R -GFP group and rVSV M51R -S group. On the same day, a 1 mL syringe was used to inject the virus intraperitoneally, and the dosage of the drug was 1×10 8 PFU. On the 7th and 9th days, the treatment method on the 5th day was still followed for intraperitoneal administration. The body weights of the mice were weighed every day to observe their weight changes. When the body weight of the mice exceeded 30 g, it was considered that the mice died by default.

[0056] Comparing the body weights of the mice in different treatment groups, the average body weight of the normal saline treatment group was 26.6 g, and that of the rVSV M51R -GFP treatment group was 24.7 g, and that of the rVSV M51R -S treatment group was 22.4. After rVSV M51R -S treatment, it could significantly inhibit the formation of liver ascites. And for the statistical analysis of the 40-day survival rate of the mice, the rVSV M51R -S treatment group could achieve a survival rate of 100%, which was significantly higher than that of the rVSV M51R -GFP group and the normal saline treatment group. The above results suggest that the insertion of hepatitis B surface antigen into the attenuated vesicular stomatitis virus plays an important role in anti-mouse tumors. The specific results are shown in Figure 6 .

[0057] The present invention designs and develops an attenuated vector system (rVSV M51R-S) to fill the long-term unsolved problem of this demand at home and abroad. In the present invention, the nucleotide sequence encoding the hepatitis B antigen is chimerically incorporated into a modified viral expression vector by means of gene editing, and a stable attenuated virus system expressing the chimeric hepatitis B antigen is recombinantly obtained in specific eukaryotic cells, and rVSV M51R -S oncolytic virus that can highly express the hepatitis B antigen in tumor tissues is screened. At the same time, the efficacy of the oncolytic virus on mouse tumors is further evaluated in an HBV-positive liver cancer model, providing a new technical solution and option for the development of therapeutic products for solid tumors.

[0058] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant oncolytic virus, characterized in that: The recombinant oncolytic virus is a recombinant attenuated vesicular stomatitis virus carrying at least one HBV antigen gene; the HBV antigen gene is the HBsAg gene, and its nucleotide sequence is as shown in SEQ ID NO.1; the attenuated vesicular stomatitis virus is a vesicular stomatitis virus with an M->R mutation at the 51st amino acid of the M gene; the HBsAg gene is located between the G gene and the L gene of the vesicular stomatitis virus.

2. The recombinant oncolytic virus according to claim 1, characterized in that: The recombinant attenuated vesicular stomatitis virus further comprises one or more of the following genes: (1) A reporter gene; (2) A gene to be delivered to a target cell or tissue; (3) An immune checkpoint inhibitory antibody; (4) An immune stimulatory gene; (5) A tumor-associated antigen for tumor vaccination.

3. Use of the recombinant oncolytic virus according to claim 1 or 2 in the preparation of a tumor therapeutic vaccine, characterized in that: The tumor is HBV-positive liver cancer.

4. A tumor therapeutic vaccine, characterized in that: The active ingredient of the tumor therapeutic vaccine is the recombinant oncolytic virus described in claim 1 or 2; the tumor is HBV-positive liver cancer.

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