Recombinant oncolytic virus rVSV M51R Use of -C in hepatitis b liver cancer

Abstract

The application discloses a recombinant oncolytic virus rVSV M51R The application of C in hepatitis B liver cancer belongs to the field of biological medicine. The recombinant oncolytic virus is a recombinant attenuated vesicular stomatitis virus carrying an HBV HBcAg gene and having an M-R mutation at the 51st amino acid of the M gene. The recombinant oncolytic virus is proved to have a curative effect in a tumor model, and can be used for preparing a therapeutic vaccine for HBV-positive liver cancer, HBV-positive diffuse large B-cell lymphoma and other HBV-positive tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a recombinant oncolytic virus composed of attenuated vesicular stomatitis virus and hepatitis B virus core antigen (HBcAg) and its applications. Background Technology

[0002] Liver cancer is one of the major challenges threatening human life and health, with hepatocellular carcinoma being the most common form. Its development is associated with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. The theoretical basis of tumor immunotherapy is the immune system's ability to recognize tumor-associated antigens and regulate the body's attack on tumor cells (highly specific cell lysis). However, in clinical practice, immunotherapy only provides lasting survival benefits to a small number of patients; most patients still face immunotherapy resistance, and once first-line treatment fails, there is usually a lack of effective treatment options. Therefore, there is an urgent need to develop suitable treatment strategies for HBV-positive liver cancer patients.

[0003] Oncolytic virus therapy is a novel cancer treatment method with a unique mechanism of action. Its anti-tumor effects mainly involve two aspects: firstly, directly infecting tumor cells and causing them to lyse and die; secondly, indirectly activating the host's innate and adaptive anti-tumor immune responses. Vesicular stomatitis virus (VSV) is a member of the genus *Vesicularis*, belonging to the family *Rhabdoviridae*. This virus is a bullet-shaped enveloped virus with an 11kb negative-sense RNA genome. It possesses broad-spectrum tumor-killing capabilities, replicates in the cytoplasm, and its genome does not integrate into the host cell's DNA, exhibiting good safety. Due to defects in type 1 interferon signaling in tumor cells, this virus can preferentially replicate in many tumor cells.

[0004] Oncolytic viruses can not only directly kill tumors, but also stimulate the body's immune response during replication by expressing specific antigens, inducing immunogenic cell death and stimulating the host's anti-tumor immunity, thus further enhancing the anti-tumor effect. Oncolytic viruses lyse tumors locally, releasing tumor-associated antigens (TAAs), similar to tumor vaccines, but this may not be sufficient to induce a tumor-specific T-cell response. Integrating TAA genes into the viral genome can enhance the expression of more TAAs and thus improve the specific T-cell immune response. Many studies have shown that tumor-specific antigens are highly immunogenic, and spontaneous neoantigen-specific T cells have been detected in cancer patients. Therefore, in the design of therapeutic cancer vaccines, tumor-specific antigens are preferentially selected as targets, as they have higher immunogenicity and reduce the risk of autoimmunity compared to autoantigens. Chronic viral infection, by causing repeated tissue damage, inflammation, and integration of viral genes into the host genome, can easily induce cancer. Integrated viral genes can be expressed through host mechanisms to produce virus-derived protein fragments, which are then degraded into viral peptides and assembled with MHC class I antigens for presentation on the cell surface. Therefore, these cancer cells serve as viral peptide-specific markers of tumor antigens. These cancer proteins are also tumor-specific and can induce T cells to recognize and kill antigens.

[0005] HBV is a hepatotropic DNA virus with a full-length genome of 3.2 kb, consisting of partially single-stranded double-stranded circular DNA. The HBV genome has four open reading frames, encoding the envelope protein (S), core protein (C), polymerase (P), and X protein (HBX). More than 90% of HBV-associated hepatocellular carcinoma (HCC) genomes contain integrated HBV-DNA, meaning that most HBV-associated HCC cells 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 encoding epitopes that are recognized and activated by HBV-specific T cells. Some secondary HCC patients can express complete HBV surface antigen epitopes. These findings provide a feasible guide for recombinant oncolytic virus therapy in these specific patients. Furthermore, the "hot" tumor microenvironment generated by inflammatory cells recruited by oncolytic viruses can 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 are no oncolytic virus drugs based on vesicular stomatitis virus carrying HBV-related genes to combat HBV-positive liver cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a recombinant oncolytic virus composed of attenuated vesicular stomatitis virus and hepatitis B virus core antigen, and its application.

[0007] The objective of this invention is achieved through the following technical solution:

[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 HBcAg gene.

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

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

[0011] In some implementations, the HBcAg gene is derived from HBV genotypes A, B, C, or D.

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

[0013] In some implementations, the HBcAg gene is located between the G and L genes of 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-iodine transporter (NIS), green fluorescent protein (GFP), and luciferase (luciferase).

[0016] (2) Genes delivered to target cells or tissues, such as genes 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) Immunostimulatory genes, such as IL-2, IL-12, IL-15, IFN-γ, GM-CSF;

[0019] (5) Tumor-associated antigens (TAAs) used for tumor inoculation.

[0020] The above-mentioned recombinant oncolytic virus is used in the preparation of tumor therapeutic vaccines, 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, the active ingredient of which is the above-mentioned recombinant oncolytic virus.

[0022] This invention inserts the gene encoding the hepatitis B virus core antigen into an attenuated oncolytic virus vector. By infecting HBV-positive liver cancer cells, the hepatitis B virus core antigen is expressed in large quantities, which enhances antigen delivery to dendritic cells (DCs), promotes MHC I processing and presentation, enhances tumor-specific cellular immune responses, and improves the clearance of tumor cells by cytotoxic T cells.

[0023] Advantages and beneficial effects of the present invention: The recombinant oncolytic virus rVSV constructed in this invention M51R -C significantly inhibited tumor growth in HBV-positive liver cancer model mice. After rVSV... M51R Treatment with -C resulted in a 33% survival rate in HBV-positive liver cancer mice, significantly higher than the control group; in the HBV-positive liver cancer ascites tumor model, the survival rate in liver cancer mice reached 75%, also significantly higher than the control group. Attached Figure Description

[0024] Figure 1 It is recombinant oncolytic virus rVSV M51R -C Modification Diagram.

[0025] Figure 2 This involves the construction of the viral packaging plasmid pXN2-HBcAg. Figure 2 The image of plasmid pXN2-M51R-HBcAg (A) and its enzyme digestion verification results (B) are shown.

[0026] Figure 3 It is a recombinant oncolytic virus rVSV M51R Construction and identification of -C. Figure 3 (A) Shows recombinant oncolytic virus rVSV M51R -C infection of mouse hepatocellular carcinoma cells Hepa1-6, Western Blot verification of hepatitis B virus core protein expression; Figure 3 (B) showcases the recombinant oncolytic virus rVSV. M51R The expression of hepatitis B virus core protein was verified in mouse hepatocellular carcinoma cells Hepa1-6 by ELISA in cell pellet and supernatant.

[0027] Figure 4 It is a recombinant oncolytic virus rVSV M51R -C、rVSV M51R - Comparison of GFP replication capacity in different hepatocellular carcinoma cell lines. Figure 4(A)-(C) Recombinant oncolytic virus rVSV at an MOI of 0.1 M51R -C、rVSV M51R -GFP was used to infect different liver cancer cells, and the one-step growth curve of the virus was detected.

[0028] Figure 5 HBV does not affect rVSV M51R -GFP replication and oncolytic abilities. Figure 5 (A) H22-DE-HBV cells were treated with 1 μg / mL doxycycline (DOX), and HBV replication-related indicators (HBsAg, HBeAg, HBV DNA) were detected after 10 days. Figure 5 (B) Three groups of cells (including the H22 group, the H22-DE-HBV group, and the H22-DE-HBV+DOX group) were simultaneously infected with the rVSV virus. M51R -GFP, for detecting the rVSV virus M51R -GFP's replication and oncolytic ability in different liver cancer cells.

[0029] Figure 6 It is a cancer therapeutic vaccine rVSV M51R -C Evaluation of the therapeutic effect of HBV-positive liver cancer. Figure 6 (A) Shows the tumor volume of different treatment groups in a subcutaneous tumor-bearing HBV-positive hepatocellular carcinoma model; Figure 6 (B) shows the survival rates of different treatment groups in a subcutaneous tumor-bearing HBV-positive hepatocellular carcinoma model.

[0030] Figure 7 This study evaluates the therapeutic efficacy of the tumor therapeutic vaccine rVSVM51R-C against HBV-positive liver cancer. Figure 7 (A) Changes in body weight in mice in different treatment groups in an HBV-positive hepatocellular carcinoma ascites model; Figure 7 (B) shows the survival rates of different treatment groups in an HBV-positive hepatocellular carcinoma ascites model. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and experimental example are commercially available. The specific embodiments described herein are only for explaining this invention and are not intended to limit this invention.

[0032] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0033] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this invention should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "approximately" means within 10%, preferably within 5%, of a given value or range.

[0034] Unless otherwise specified, the following embodiments of the present invention are carried out under normal temperature conditions, which refers to the natural room temperature conditions in all four seasons, without additional cooling or heating treatment. Generally, the room temperature is controlled at 10 to 30°C, preferably 15 to 25°C.

[0035] Example 1

[0036] This implementation uses the vesicular stomatitis virus system as an example to further describe the recombinant oncolytic virus rVSV. M51R -C (See the diagram for the modification) Figure 1The construction process of the plasmid pXN2-M51R-GFP involves the following steps: Using molecular biology techniques, an M->R mutation was performed at amino acid position 51 of the VSV M gene. The mutated M gene was cloned into the plasmid pXN2-GFP containing the full-length VSV genome (plasmid pXN2-GFP was kindly provided by Professor John K. Rose of Yale University; its construction can be found in reference 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.) to obtain the plasmid pXN2-M51R-GFP (its construction can be found in reference 2. Gaddy DF, Lyles DS. Vesicularstomatitis 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.). Next, we obtained the HBcAg sequence and used the cloning plasmid pAAV-HBV1.3 (the construction of plasmid pAAV-HBV1.3 can be found in reference 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. Based on the pXN2-M51R-GFP vector and the HBcAg sequence, homologous recombination upstream and downstream primers were designed, and restriction enzyme sites Xho I and Nhe I were added upstream and downstream, respectively. A Flag tag sequence was added upstream. The primers are shown in the table below.

[0037]

[0038] HbcAg amplification: Using pAAV-HBV1.3 as a template, PCR amplification was performed under the following conditions: The PCR reaction system included pAAV-HBV1.3: 1 μL; pXN2-HBcAg-F: 1 μL; pXN2-HBcAg-R: 1 μL; 2× Primer Star Mix: 25 μL; ddH2O: 22 μL. PCR reaction conditions: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 PCR cycles, 72℃ final extension for 10 min, and storage at 4℃. After PCR products were subjected to 1% agarose gel electrophoresis, images were taken using a gel imaging system, the target band was cut, and DNA was recovered from the gel according to the instructions of the Axygen gel recovery kit.

[0039] Enzyme digestion of pXN2-M51R-GFP vector: The pXN2-M51R-GFP vector was double-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℃ for 2 h. The digestion products were detected by 1% DNA agarose gel electrophoresis and then recovered by gel extraction.

[0040] The target gene was ligated to the vector using the following ligation system: HbcAg target gene fragment: 3.5 μL; pXN2-M51R-GFP enzyme digestion product: 2.5 μL; 2×Basic assembly mix: 6 μL. The reaction was carried out at 50℃ for 30 min.

[0041] Ligation system transformation: Add 50 μL of stable competent cells to 12 μL of ligation system, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and incubate on ice for 2 min. Add 1 mL of antibiotic-free LB medium, and incubate at 37℃ with shaking at 250 rpm for 1 h. Centrifuge at 12000 rpm for 1 min, discard the supernatant, and resuspend the cells in 100 μL of LB medium. Spread evenly on LB agar plates containing Kana antibiotic, and incubate upside down at 37℃ for 8-12 h. Then, pick single colonies and amplify them in liquid LB for 8-12 h. Extract pXN2-M51R-HBcAg plasmid in small quantities according to the instructions of the AXYGEX plasmid mini-extraction kit.

[0042] Plasmid digestion verification and sequencing: The plasmids were digested at 37℃ for 2 hours. The digestion products were detected by 1% DNA agarose gel electrophoresis. Plasmids with correct digestion results were sent to the company for sequencing.

[0043] In this embodiment, we found the DNA sequence of the hepatitis B virus (HBV) core antigen (HBeAg) type D, and designed primers using plasmid AAV-HBV1.3 as a template. We obtained the HBV core gene fragment by PCR, and cloned the target gene into the G and L genes of the pXN2-M51R vector with a mutation at position 51 of the M gene via homologous recombination, obtaining the recombinant plasmid pXN2-M51R-HBcAg. The recombinant plasmid map is shown below. Figure 2 A, verified by double digestion with Xho I and Nhe I and sequencing, the digestion results are as follows: Figure 2 B.

[0044] Four plasmids were prepared for packaging recombinant vesicular stomatitis virus: pXN2-M51R-GFP / pXN2-M51R-HBcAg, pN, pL, and pP plasmids (pP, pN, and pL plasmids were kindly provided by Professor John K. Rose of Yale University, and their construction can be found in reference 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 BSR cells (a clone of the commercially available BHK-21 cell line with an inserted T7 promoter in its genome) reached a confluence density of 80% in 10 cm cell culture dishes, 10 μL of poxvirus containing T7 RNA polymerase (RNAP) was added to 10 mL of DMEM medium and infected for 2 h. (The construction of this virus can be found in reference 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.) After infecting BSR cells, the vesicular stomatitis virus (VSV) packaging plasmid pXN2-M51R-GFP / pXN2-M51R-HBcAg (10 μg), pP (5 μg), pN (4 μg), and pL (1 μg) were transfected into the cells using liposome transfection (Lipo3000 transfection reagent). The medium was changed after 6 hours, and the supernatant was collected after 48 hours and filtered through a 0.22 μm filter membrane. The collected virus solution was then used to infect Vero cells. Cytopathic effects were observed after 24 hours; rounded cells indicated successful in vitro packaging of the VSV virus. A mutant attenuated strain, rVSV, was rescued from BSR cells using reverse genetics. M51R -GFP, rVSV M51R -C. Then use rVSVM51R -C was used to infect mouse hepatocellular carcinoma cells Hepa1-6 (mouse hepatocellular carcinoma cells). Western blotting and EILSA experiments confirmed the efficient expression of HBV Core and S-HBs proteins in the lysate of infected tumor cells. Specific results can be found in [link to results]. Figure 3 Next, we validated rVSV expressing the hepatitis B virus core antigen. M51R -C replication capability, utilizing recombinant oncolytic virus rVSV M51R -C、rVSV M51R -GFP was used to infect different liver cancer cells, including Huh7, HepG2, H22, and Hepa1-6. One-step viral growth curves revealed that the recombinant oncolytic virus rVSV... M51R -C and rVSV M51R Compared to -GFP, the virus's replication ability remained unchanged; see the detailed results below. Figure 4 .

[0045] Discard the original culture medium from the cultured Vero cells, add 10 mL of DMEM medium containing 2% serum to each plate of cells, and then add the recombinant VSV virus (rVSV). M51R -GFP, rVSV M51R -C) Infect Vero cells with an MOI of 0.01. Since VSV can spread via aerosols, infected cells must be cultured separately to prevent cross-contamination. Cell morphology was observed 24 hours after infection. Successful VSV infection resulted in rounded cells. When the majority of cells were rounded and more than 50% of cells were floating, the amplified VSV was collected, centrifuged at 3000 rpm for 20 minutes, and cell debris was discarded.

[0046] Prepare sterile solutions of 40% PEG6000 / 2.5M NaCl, 25%, 40%, and 55% sucrose, and 1×TNF buffer. Add 10 mL of 40% PEG6000 / 2.5M NaCl solution to every 40 mL of VSV virus stock solution to form a final concentration of 8% PEG6000 / 0.5M NaCl. Mix thoroughly and incubate overnight at 4°C. Centrifuge the overnight virus solution at 12000 rpm for 1 hour. Virus precipitate will appear on the centrifuge tube wall; resuspend the virus in 2 mL of 1×TNF buffer.

[0047] Next, a sucrose density gradient needs to be established for horizontal ultracentrifugation of the virus. Add 3 mL of 55% sucrose solution, 3 mL of 40% sucrose solution, and 3 mL of 25% sucrose solution sequentially to a horizontal centrifuge tube, along with 23 mL of virus suspension and 13 mL of 1× TNF buffer. Add the solutions slowly, ensuring clear separation between layers. After balancing the horizontal centrifuge tube, ultracentrifuge at the lowest possible speed for 2 hours. After horizontal centrifugation, aspirate the purified virus (located at the 25% and 40% sucrose separation points) using a 1 mL syringe and transfer it to a 1.5 mL EP tube. Transfer the virus solution to a 25 mL ultracentrifuge tube, fill with 1× TNF buffer, and ultracentrifuge vertically for 3 hours. After centrifugation, resuspend the virus in 300 μL of 1× TNF buffer, aliquot into 6 tubes, and store at -80°C.

[0048] Example 2

[0049] This embodiment uses oncolytic virus rVSV M51R The treatment of HBV-positive hepatocellular carcinoma in mice using -C is described in detail.

[0050] This embodiment utilizes the rVSV constructed in Example 1. M51R The efficacy evaluation of -C in the treatment of HBV-positive liver cancer utilizes a viral vector system to express the HBV core antigen, thereby activating a specific anti-tumor immune response. Specific implementation cases involve treatment using viral antigens expressed via a specific purified recombinant non-segmental, negative-strand RNA viral vector.

[0051] (1) Culture of H22-DE-HBV tumor cell line and establishment of subcutaneous hepatocellular carcinoma tumor-bearing model and immunotherapy

[0052] The HBV-positive murine hepatocellular carcinoma cell line H22-DE-HBV was obtained, and the specific construction method is as follows: First, two transfection plasmids were obtained, namely pWPI-tet-off and pCMV-DE-HBV (the construction of pWPI-tet-off and pCMV-DE-HBV plasmids can be found in 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 pCMV-DE-HBV plasmid is to perform a point mutation at the start codon of e antigen on the basis of pCMV-HBV plasmid, so that it cannot form HBV e antigen. Only after the transfected cells circularize to form complete cccDNA can e antigen be formed. First, linearize the plasmids. Then, digest the two plasmids with ScaI restriction endonuclease and perform gel recovery. After linearization, pCMV-DE-HBV showed only one band, so gel recovery was performed directly. After single digestion of pWPI-tet-off plasmid, two bands were observed, one approximately 7800 bp and the other approximately 4300 bp. The band of approximately 7800 bp was recovered from the gel. Two linearized plasmids were simultaneously transfected into H22 cells. 48 hours after transfection, H22 cells were killed using 1640 medium supplemented with puromycin. After stabilization for 10 days, H22-DE-HBV polyclonal cell lines were obtained. Subsequently, H22-DE-HBV monoclonal cell lines were further obtained and seeded at a density of 0-1 cells / 96-well plate. After 14 days of cell growth, clear cell clusters were observed. Wells containing only one cell cluster were selected, labeled, and a small amount of supernatant was collected for HBV S and e antigen detection. From the confirmed stable linear HBV integration cell lines, Dox-regulated cell lines were further selected. After 10 days of Dox treatment, changes in HBV S and e antigen expression and HBV DNA expression were detected. Specific results are shown in [link to results]. Figure 5 A. Finally, the H22-DE-HBV cell line, which is regulated by doxycycline (Dox) and stably integrates linear HBV, was successfully obtained. Simultaneously, it was treated with an equal dose of rVSV. M51RThree cell groups were simultaneously infected with GFP: the H22 group, the H22-DE-HBV group, and the H22-DE-HBV+DOX-regulated group. After 24 hours of infection, the GFP positivity rate of the cells was measured. The results showed that HBV did not affect the oncolytic virus rVSV. M51R -GFP replication capacity. Three cell types were infected with different doses of the virus, and cell viability was assessed using the CCK8 assay after 48 hours. The results showed that linear HBV integration did not affect the oncolytic virus rVSV. M51R -GFP's oncolytic ability, see details in [link to results]. Figure 5 B.

[0053] H22-DE-HBV cells were cultured in 1640 complete medium. When the cell density reached 70-80%, tumor cells were inoculated. The cells were observed before inoculation, and their growth rate was maintained in the exponential growth phase. One day before inoculation, the outer right thigh of the mice was shaved using a power shaver. Six- to eight-week-old male C57BL / 6 mice were selected as tumor-bearing mice. H22-DE-HBV tumor cells were resuspended in PBS, and the cell resuspension density was 5 × 10⁶ cells / mL. 6 200 μL of tumor cells were subcutaneously injected into the groin area on the outer thigh of each mouse. Tumors generally formed in about 6 days. The tumor size was measured, and the mice were grouped after statistical analysis. The tumor volume in mice generally remained at 70 mm². 3 On day 6, mice with appropriately sized tumors in the HBV-positive liver cancer model were divided into three groups: a saline group, a group receiving oncolytic virus rVSV, and a group receiving saline. M51R -GFP group, and oncolytic virus rVSV M51R Group C. On day 6, the virus was injected intratumorally using a 1 mL insulin syringe, with a dosage of 1 × 10⁻⁶. 8 PFU (50 μL) was administered intratumorally on days 8 and 10, following the previous treatment plan. Tumor size was measured every other day, and tumor volume was calculated using the formula: V = 0.52 × L × W. 2 (L = length, W = width), until the tumor volume reaches 1500 mm. 3 According to the humane endpoint of the experiment, the mice were euthanized by cervical dislocation.

[0054] Oncolytic virus rVSV M51R -C's pharmacological properties, such as Figure 6 As shown, after three consecutive injections every other day, rVSV M51R -C and control virus rVSV M51R Compared with GFP, it significantly inhibited tumor growth and improved the survival time of tumor-bearing mice. Analysis showed that on day 14 after viral treatment, the average tumor volume in the saline treatment group was 1075 mmHg. 3 rVSVM51R The average tumor volume in the GFP treatment group was approximately 496 mm. 3 Left and right, while mice received rVSV M51R -C treatment average volume is approximately 276 mm. 3 Furthermore, by comparing survival rates, it was found that rVSV M51R The complete remission rate in the -C treatment group was 33%, which was significantly higher than that in the saline treatment group and the rVSV group. M51R -GFP treatment group.

[0055] (2) Establishment of a liver cancer ascites tumor model and immunotherapy

[0056] To establish a mouse model of HBV-positive hepatocellular carcinoma ascites tumor, 6-8 week old male C57BL / 6 mice bearing the tumor were selected. H22-DE-HBV tumor cells were resuspended in PBS, and the cell resuspension density was 5×10⁻⁶. 6 Tumor cells were injected intraperitoneally at a rate of 200 μL / mL into each mouse. On day 5, mice with appropriately sized tumors in the HBV-positive liver cancer model were divided into three groups: a saline treatment group, an rVSV treatment group, and a control group. M51R -GFP group and rVSV M51R Group C. The virus was injected intraperitoneally with a 1mL syringe on the same day, at a dose of 1×10⁻⁶. 8 PFU was administered intraperitoneally on days 7 and 9, following the same treatment as day 5. Mice were weighed daily to monitor weight changes. Mice were considered dead when their weight exceeded 30g.

[0057] Comparing the body weight of mice in different treatment groups, the average body weight of the saline treatment group was 26.6g, and the rVSV... M51R The average body weight in the -GFP treatment group was 24.7g, and the rVSV... M51R The average weight in the -C treatment group was 22.1 kg. After rVSV... M51R -C treatment significantly inhibited the formation of ascites, and statistical analysis of the 40-day survival rate of mice showed that rVSV M51R The -C treatment group achieved a survival rate of 75%, significantly higher than that of rVSV. M51R -GFP group and saline group. These results suggest that the insertion of hepatitis B core antigen into attenuated vesicular stomatitis virus plays an important role in anti-tumor activity in mice. See below for details. Figure 7 .

[0058] This invention designs and develops an attenuated vector system (rVSV) capable of rapidly and efficiently chimeric expression of hepatitis B core antigen. M51RThis invention addresses a long-standing unresolved issue both domestically and internationally by using gene editing to embed the nucleotide sequence encoding the hepatitis B virus core antigen into a modified viral expression vector. In specific eukaryotic cells, a stably attenuated viral system expressing the chimeric hepatitis B virus core antigen is obtained through recombination. Furthermore, rVSV, capable of efficiently expressing the hepatitis B virus core antigen in tumor tissues, is screened for this purpose. M51R -C oncolytic virus was used to further evaluate the efficacy of this oncolytic virus in HBV-positive liver cancer models in mice, providing a new technical solution and option for developing therapeutic products for solid tumors.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a recombinant oncolytic virus in the preparation of a therapeutic tumor vaccine, characterized in that: The tumor described is HBV-positive liver cancer; The recombinant oncolytic virus is a recombinant attenuated vesicular stomatitis virus carrying at least one HBV antigen gene; the HBV antigen gene is the HBcAg gene. The attenuated vesicular stomatitis virus mentioned is a vesicular stomatitis virus with an M->R mutation at amino acid position 51 of the M gene; The nucleotide sequence of the HBcAg gene is shown in SEQ ID NO.1; The HBcAg gene is located between the G and L genes of vesicular stomatitis virus.

2. The application according to claim 1, characterized in that: The recombinant attenuated vesicular stomatitis virus further comprises one or more of the following genes: (1) Reporter genes; (2) Genes delivered to target cells or tissues; (3) Immune checkpoint inhibitory antibodies; (4) Immunostimulatory genes; (5) Tumor-associated antigens used for tumor inoculation.

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