Immune-activating recombinant oncolytic virus and use thereof

Abstract

Disclosed in the present application are an immune-activating recombinant oncolytic virus and use thereof, relating to the field of biomedicine. A genome of the immune-activating recombinant oncolytic virus comprises a first nucleotide sequence for encoding an HBsAg molecule, a second nucleotide sequence for encoding an IL-15 molecule, and a third nucleotide sequence for encoding a sushi domain of IL-15Ra. According to the present application, by inserting the first nucleotide sequence for encoding the HBsAg molecule, the second nucleotide sequence for encoding the IL-15 molecule, and the third nucleotide sequence for encoding the sushi domain of IL-15Ra into the genome of the immune-activating recombinant oncolytic virus, the cure rate of tumors is improved while tumor recurrence is prevented.

Description

An immune-activated recombinant oncolytic virus and its application Technical Field

[0001] This application relates to the field of biomedicine, and in particular to an immune-activated recombinant oncolytic virus and its applications. Background Technology

[0002] Liver cancer is one of the major challenges threatening human life and health, with hepatocellular carcinoma (HCC) being the most common form. Its development is associated with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. In my country, 85% of patients with primary HCC have a history of HBV infection. Previous treatments for HBV-positive liver cancer primarily focused on the cancer cells themselves. In recent years, immunotherapy has emerged, primarily exerting its anti-tumor effect by regulating the body's own immune system, bringing revolutionary progress to liver cancer treatment. 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. As an oncolytic virus, 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] However, due to the high HBV DNA levels in HBV-positive liver cancer patients and the hepatocyte necrosis and regeneration caused by liver inflammation, DNA mutations and instability are induced, resulting in a high recurrence rate of HBV-positive liver cancer. Summary of the Invention

[0006] In view of the shortcomings of the above-mentioned related technologies, this application provides an immune-activated recombinant oncolytic virus and its application. This application improves the cure rate of tumors while reducing the recurrence rate and preventing tumor recurrence by inserting a first nucleotide sequence encoding HBsAg, a second nucleotide sequence encoding IL-15, and a third nucleotide sequence encoding the sushi domain of IL-15Ra into the genome of the immune-activated recombinant oncolytic virus.

[0007] Firstly, the immune-activated recombinant oncolytic virus provided in this application adopts the following technical solution:

[0008] An immune-activated recombinant oncolytic virus, the genome of which includes a first nucleotide sequence encoding an HBsAg molecule, a second nucleotide sequence encoding an IL-15 molecule, and a third nucleotide sequence encoding a sushi domain of IL-15Ra.

[0009] Preferably, the first nucleotide sequence is derived from HBV genotype A, B, C, or D.

[0010] Preferably, the first nucleotide sequence is as shown in SEQ ID NO:1.

[0011] Preferably, the second nucleotide sequence is as shown in SEQ ID NO:2.

[0012] Preferably, the third nucleotide sequence is as shown in SEQ ID NO:3.

[0013] By employing the above-mentioned technical approach, HBV-positive tumor cells are infected with an immune-activated recombinant oncolytic virus, resulting in lysis and the release of large amounts of tumor antigens. This recruits more immune cells to infiltrate the tumor tissue, activating an anti-tumor immune response. Simultaneously, the expression of HBV surface antigen activates HBV-specific T cells, thereby enhancing the killing effect on residual HBV-positive cells not infected by VSV. Furthermore, the expression of IL-15 facilitates the activation of T cells and NK cells and mediates their proliferation and survival. In addition, it activates, maintains, and expands CD8+ memory T cells, further promoting the survival of memory CD8 cells and thus preventing the recurrence of HBV-positive tumors. Through these combined effects, the cure rate of tumors is improved while preventing tumor recurrence.

[0014] Preferably, the first nucleotide sequence and the second nucleotide sequence are linked by a nucleotide sequence encoding a P2A cleavage peptide, the amino acid sequence of which is shown in SEQ ID NO:4, and the second nucleotide sequence and the third nucleotide sequence are linked by a nucleotide sequence encoding a linker peptide, the amino acid sequence of which is shown in SEQ ID NO:5.

[0015] Preferably, the vector of the immune-activated recombinant oncolytic virus includes vesicular stomatitis virus, wherein the 51st amino acid of the M gene of the vesicular stomatitis virus undergoes an M->R mutation.

[0016] Preferably, the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are located between the G gene and the L gene of the vesicular stomatitis virus.

[0017] Secondly, the application of an immune-activated recombinant oncolytic virus in the preparation of a tumor therapeutic vaccine, as provided in this application, adopts the following technical solution:

[0018] The application of an immune-activated recombinant oncolytic virus in the preparation of a therapeutic tumor vaccine, wherein the tumors include HBV-positive liver cancer, HBV-positive diffuse large B-cell lymphoma, HBV-positive ovarian cancer, HBV-positive endometrial cancer, HBV-positive cervical cancer, HBV-positive pancreatic cancer, and HBV-positive biliary tract cancer.

[0019] Thirdly, the tumor therapeutic vaccine provided in this application adopts the following technical solution:

[0020] A cancer therapeutic vaccine whose active ingredient includes an immune-activated recombinant oncolytic virus.

[0021] In summary, this application includes at least one of the following beneficial technical effects:

[0022] 1. The immune-activated recombinant oncolytic virus rVSV of this application M51R Compared with the control group, -S-IL15-Ra significantly inhibited tumor growth and improved the survival time of tumor-bearing mice. By inserting the gene encoding hepatitis B virus surface antigen into an attenuated oncolytic virus vector, and infecting HBV-positive liver cancer cells to express large amounts of hepatitis B virus surface antigen and cytokine IL-15 and IL-15Ra complex, it enhanced antigen delivery to antigen-presenting cells, promoted MHC I and MHC II processing and presentation, enhanced tumor-specific cellular immune response, improved the clearance of tumor cells by cytotoxic T cells, improved the cure rate of tumors while reducing the recurrence rate and preventing tumor recurrence.

[0023] 2. Mice were given immune-activated recombinant oncolytic virus rVSV. M51R The tumor cure rate of -S-IL15-Ra was 37.5%, which was much higher than the tumor cure rate of mice that received the virus as a control group.

[0024] 3. This application utilizes an immune-activated recombinant oncolytic virus (rVSV). M51R Mice successfully treated with -S-IL15-Ra showed significant resistance to tumor attack even after three months, demonstrating the effectiveness of the immune-activated recombinant oncolytic virus rVSV described in this application. M51R -S-IL15-Ra helps patients develop long-term anti-tumor immune memory, thereby reducing the recurrence rate and preventing tumor recurrence in mice. Attached Figure Description

[0025] Figure 1 shows the immune-activated recombinant oncolytic virus rVSV. M51R -S-IL15-Ra modification diagram.

[0026] Figure 2 shows the construction of the viral packaging plasmid pXN2-S-IL15-Ra. Specifically, Figure 2 shows the pXN2-M51R-S-IL15-Ra pattern (Figure 2A) and its enzyme digestion verification results (Figure 2B).

[0027] Figure 3 shows the immune-activated recombinant oncolytic virus rVSV. M51R Construction and identification of -S-IL15-Ra. Figure 3A shows the immune-activated recombinant oncolytic virus rVSV. M51RVero cells were infected with -S-IL15-Ra, and the expression of the cytokine complexes IL-15 and IL-15Ra was verified by Western blotting; Figure 3B shows the immune-activated recombinant oncolytic virus rVSV. M51R Vero cells were infected with S-IL15-Ra, and the expression of hepatitis B virus surface protein was verified by ELISA.

[0028] Figure 4 shows the immune-activated recombinant oncolytic virus rVSV. M51R Construction and identification of -S-IL15-Ra. Figure 4A shows a schematic diagram of the experimental procedure; Figure 4B shows the tumor volume of different cytokine treatment groups in the HBV-positive subcutaneous tumor-bearing model of hepatocellular carcinoma; Figure 4C shows the tumor weight of different cytokine treatment groups in the HBV-positive subcutaneous tumor-bearing model of hepatocellular carcinoma.

[0029] Figure 5 shows the tumor therapeutic vaccine rVSV. M51R Evaluation of the therapeutic effect of -S-IL15-Ra on HBV-positive hepatocellular carcinoma. Figure 5A shows a schematic diagram of the experimental procedure; Figure 5B shows the tumor volume of different treatment groups in the subcutaneous tumor-bearing model of HBV-positive hepatocellular carcinoma.

[0030] Figure 6 shows the tumor therapeutic vaccine rVSV. M51R Evaluation of the effect of S-IL15-Ra on the secondary inoculation of tumor cells in mice cured with S-IL15-Ra. Figure 6A shows a schematic diagram of the experimental procedure; Figure 6B shows the tumor volume of different treatment groups in the HBV-positive subcutaneous hepatocellular carcinoma tumor model; Figure 6C shows the survival rate of different treatment groups in the HBV-positive subcutaneous hepatocellular carcinoma tumor model. Embodiments of the present invention

[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 any additional cooling or heating treatment. Generally, the room temperature is controlled between 10 and 30°C, preferably between 15 and 25°C.

[0035] Example 1

[0036] Example 1 of this application improves an immune-activated recombinant oncolytic virus rVSV. M51R The modified S-IL15-Ra is shown in Figure 1. The specific steps of its construction process are as follows:

[0037] 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 then cloned into the plasmid pXN2-GFP (which contains the full-length VSV genome and 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 prepare the plasmid pXN2-M51R-GFP (its construction can be found in reference 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.). Next, we obtained the HBsAg 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, HBsAg, mouse IL-15 and mouse IL-15Ra sequences, homologous recombination upstream and downstream primers were designed, and restriction enzyme sites Xho I and Nhe I were added upstream and downstream, respectively, and an HA tag sequence was added downstream. The primers are shown in Table 1 below.

[0038]

[0039] Amplification of HBsAg-P2A-mIL15-ra: The target fragment of the HBsAg-P2A-mIL15-Ra gene was synthesized at Shanghai Sangon Biotech Co., Ltd. and loaded into the universal vector pUC19-HBsAg-P2A-mIL15-Ra. Using this vector as a template, PCR amplification was performed under the following conditions: The PCR reaction system consisted of pUC19-HBsAg-P2A-mIL15-Ra: 1 μL; pXN2-M51R-S-mIL15-Ra-F: 1 μL; pXN2-M51R-S-mIL15-Ra-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 performing 1% agarose gel electrophoresis on the PCR products, the gel imaging system was used to capture images, the target band was cut, and DNA was recovered from the gel according to the instructions of the Axygen gel recovery kit.

[0040] 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.

[0041] The target gene was ligated to the vector using the following ligation system: HBsAg target gene fragment: 3.5 μL; pXN2-M51R-GFP enzyme digestion product: 2.5 μL; 2×Basic assembly mix: 6 μL, and the reaction was carried out at 50℃ for 30 min.

[0042] 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-S-mIL15-ra plasmid in small quantities according to the instructions of the AXYGEX plasmid mini-extraction kit.

[0043] 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.

[0044] In this embodiment, the DNA sequence of the surface antigen of hepatitis B virus type D was found, and primers were designed using plasmid AAV-HBV1.3 as a template. The S-HBs gene fragment was obtained by PCR. The target gene was cloned into 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 shown in Figure 2A. The digestion results were verified by double digestion with Xho I and Nhe I and sequencing. The digestion results are shown in Figure 2B. Lane 4 is the plasmid with correct digestion. The digested vector fragment is larger than 10kb, and the inserted target gene is 1476bp.

[0045] Four plasmids were prepared for packaging recombinant vesicular stomatitis virus: pXN2-M51R-GFP / pXN2-M51R-S / pXN2-M51R-S-mIL15-Ra, pP, pN, and pL (plasmids pP, pN, and pL 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-T7 / 5 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 (its construction 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.) was added to 10 mL of DMEM medium to infect BSR cells for 2 h. After infecting BSR cells, the vesicular stomatitis virus (VSV) packaging plasmid pXN2-M51R-GFP / pXN2-M51R-S / pXN2-M51R-S-mIL15-Ra (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. After 24 hours, cytopathic effects were observed; if the cells became rounded, the in vitro packaging of VSV virus was successful. A mutant attenuated VSV strain was rescued from BSR cells using reverse genetics. M51R-GFP, rVSV M51R -S-IL15-Ra. Then, using rVSV... M51R Vero cells were infected with S-IL15-Ra. Western blotting and ELISA experiments were used to verify that the super cytokine complexes IL-15, IL-15Ra and S-HBsAg proteins were efficiently expressed in the lysate and supernatant of infected cells. The specific results are shown in Figures 3A and 3B.

[0046] 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 (VSV). M51R -GFP, rVSV M51R VSV (-S-IL15-Ra) was used to infect Vero cells at an MOI of 0.01. Since VSV can spread via aerosols, VSV-infected cells needed to be cultured separately to prevent cross-contamination. Cell morphology was observed 24 hours after infection. After successful infection of Vero cells with recombinant VSV, the cells became rounded. When the vast majority of cells were rounded and more than 50% of the cells were floating, the amplified VSV was collected, centrifuged at 3000 rpm for 20 min, and cell debris was discarded.

[0047] 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× TNE buffer.

[0048] 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 2 mL of virus suspension and 1 mL of 1× TNF buffer. Add the solutions slowly, ensuring clear separation between the 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 and aliquot into 6 tubes to obtain the immune-activated recombinant oncolytic virus rVSV. M51R -S-IL15-Ra, store at -80℃.

[0049] Testing and Inspection

[0050] (1) Screening for highly effective cytokines in an HBV hepatocellular carcinoma model

[0051] H22-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 hair on the outer right thigh of mice was shaved using a power shaver. Six- to eight-week-old male C57BL / 6 mice were selected as tumor-bearing mice. H22-HBV tumor cells were resuspended in PBS at a density of 5 × 10⁶ / mL. 200 μL of tumor cells were subcutaneously inoculated into the groin area on the outer thigh of each mouse. Tumors generally formed in about 6 days. Tumor size was measured, and the results were statistically analyzed and the mice were grouped. The tumor volume in mice generally remained around 70 mm². 3 On day 6, mice with suitable tumor size in the HBV-positive hepatocellular carcinoma model were divided into 5 groups: PBS group, mIL2 group, mIL7 group, mIL12 group, and mIL15 group. Cytokines were administered intraperitoneally every other day for a total of 5 times, with a dosage of 2 μg per administration. The specific experimental procedure is shown in Figure 4A. The tumor volume of different cytokine treatment groups in the HBV-positive hepatocellular carcinoma subcutaneous tumor-bearing model is shown in Figure 4B, and the tumor weight of different cytokine treatment groups in the HBV-positive hepatocellular carcinoma subcutaneous tumor-bearing model is shown in Figure 4C.

[0052] (2) Establishment of subcutaneous tumor-bearing model of positive liver cancer and immunotherapy

[0053] Six- to eight-week-old male C57BL / 6 mice were selected to bear tumors. H22-HBV tumor cells were resuspended in PBS at a density of 5 × 10⁶ / mL. 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. Tumor size was measured, and the results were statistically analyzed before grouping the mice. The tumor volume in mice generally remained around 70 mm². 3 On day 6, mice with appropriately sized tumors in the HBV-positive liver cancer model were divided into three groups: PBS group, oncolytic virus VSV group, and oncolytic virus group. M51R -GFP group, oncolytic virus VSV M51R -S group, and immune-activated recombinant oncolytic virus rVSV M51R -S-IL15-Ra group. On day 6, the virus was injected intratumorally using a 1 mL insulin syringe at a dose of 1 × 10⁸ PFU (50 μL). On days 8 and 10, the same intratumoral administration was administered as before. Tumor size was recorded 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 Mice were euthanized by cervical dislocation according to the humane endpoint of the experiment. A schematic diagram of the experimental procedure is shown in Figure 5A. The tumor volume of different treatment groups in the HBV-positive subcutaneous hepatocellular carcinoma tumor model is shown in Figure 5B.

[0054] (3) rVSV M51R -S-IL15-Ra Cures Mice and Prevents Tumor Recurrence Experiment

[0055] rVSV M51R Mice successfully treated with S-IL15-Ra were vaccinated a second time with 5×10⁻⁶ IL-15 after three months. 5 H22-HBV tumor cells were used, and the same number of untreated C57BL / 6 mice of the same age were used as the control group. The experimental flowchart is shown in Figure 6A. The tumor volume of different treatment groups in the HBV-positive subcutaneous hepatocellular carcinoma tumor model is shown in Figure 6B. The survival rate of different treatment groups in the HBV-positive subcutaneous hepatocellular carcinoma tumor model is shown in Figure 6C.

[0056] Results Analysis

[0057] The following detailed description of this application is based on the experimental results provided in Figures 2-6.

[0058] Referring to Figure 2, the recombinant plasmid map of pXN2-M51R-HBsAg is shown in Figure 2A. The digestion results were verified by double digestion with Xho I and Nhe I and sequencing, as shown in Figure 2B. Lane 4 is the plasmid that was digested correctly. The digested vector fragment is larger than 10kb and the inserted target gene is 1476bp.

[0059] Referring to Figure 3, Figure 3A shows the immune-activated recombinant oncolytic virus rVSV. M51R Vero cells were infected with -S-IL15-Ra, and the expression of the cytokine complexes IL-15 and IL-15Ra was verified by Western blotting. Figure 3B shows the expression of the immune-activated recombinant oncolytic virus rVSV. M51R Vero cells were infected with S-IL15-Ra, and the expression of hepatitis B virus surface protein was verified by ELISA.

[0060] Referring to Figure 4, as shown in Figure 4B, both cytokines IL12 and IL15 exhibited good anti-tumor effects, showing significant inhibition in both tumor volume and tumor weight compared to the PBS group.

[0061] Referring to Figure 5, as shown in Figure 5B, after three consecutive injections every other day, rVSV M51R -S-IL15-Ra and control virus VSV M51R Compared with GFP, it significantly inhibited tumor growth and improved the survival time of tumor-bearing mice. Analysis showed that on day 30 after viral therapy, the PBS treatment group and the VSV treatment group... M51R The tumor cure rate in the -GFP treatment group was 0, and rVSV M51R The tumor cure rate in the -S treatment group was 12.5%, while the rate in mice receiving rVSV was significantly lower. M51R The cure rate of S-IL15-Ra tumors was 37.5%, which was much higher than the other three groups. This was attributed to the important role played by the insertion of hepatitis B surface antigen and cytokines into attenuated vesicular stomatitis virus in the anti-mouse tumor response.

[0062] Referring to Figure 6, rVSV M51R In an experiment on the prevention of tumor recurrence in mice cured by S-IL15-Ra, after secondary inoculation with H22-HBV cells, the tumor volume of wild-type mice gradually increased, and the survival rate gradually decreased after 20 days post-secondary inoculation, while rVSV... M51R -S-IL15-Ra cured mice showed significant resistance to tumor attacks and did not develop tumors, with a survival rate that remained at 100%.

[0063] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An immunostimulatory recombinant oncolytic virus, characterized in that: The immune-activated recombinant oncolytic virus genome includes a first nucleotide sequence encoding the HBsAg molecule, a second nucleotide sequence encoding the IL-15 molecule, and a third nucleotide sequence encoding the sushi domain of IL-15Ra.

2. The immunostimulatory recombinant oncolytic virus of claim 1, wherein: The first nucleotide sequence is derived from HBV genotype A, B, C, or D.

3. The immunostimulatory recombinant oncolytic virus of claim 1, wherein: The first nucleotide sequence is shown in SEQ ID NO:

1.

4. The immunostimulatory recombinant oncolytic virus of claim 1, wherein: The second nucleotide sequence is shown in SEQ ID NO:

2.

5. The immunostimulatory recombinant oncolytic virus of claim 1, wherein: The third nucleotide sequence is shown in SEQ ID NO:

3.

6. The immunostimulatory recombinant oncolytic virus of claim 1, wherein: The first nucleotide sequence and the second nucleotide sequence are linked by a nucleotide sequence encoding a P2A cleavage peptide, the amino acid sequence of which is shown in SEQ ID NO:

4. The second nucleotide sequence and the third nucleotide sequence are linked by a nucleotide sequence encoding a linker peptide, the amino acid sequence of which is shown in SEQ ID NO:

5.

7. The immunostimulatory recombinant oncolytic virus of claim 1, wherein: The vector of the immune-activated recombinant oncolytic virus includes vesicular stomatitis virus, wherein the 51st amino acid of the M gene of the vesicular stomatitis virus undergoes an M->R mutation.

8. The immunostimulatory recombinant oncolytic virus of claim 7, wherein: The first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are located between the G gene and the L gene of the vesicular stomatitis virus.

9. Use of the immunostimulatory recombinant oncolytic virus according to any one of claims 1 to 8 for the manufacture of a therapeutic vaccine against tumors, characterized in that: The tumors include HBV-positive liver cancer, HBV-positive diffuse large B-cell lymphoma, HBV-positive ovarian cancer, HBV-positive endometrial cancer, HBV-positive cervical cancer, HBV-positive pancreatic cancer, and HBV-positive biliary tract cancer.

10. A tumor therapeutic vaccine, characterized by: Its active ingredient includes the immune-activated recombinant oncolytic virus as described in any one of claims 1-8.

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