EBV complex antigens, dendritic cell vaccines and uses thereof

Abstract

The present invention discloses an EBV complex antigen, a dendritic cell vaccine and its use in preparing a drug for treating EBV-related infections, which belongs to the technical field of biopharmaceuticals. The present invention stimulates the patient's own dendritic cells in vitro, loads them with lysates of various EBV-infected cells that have super-potent immunogenicity against EBV-related infections, and induces maturation in the presence of various cytokines and specific agonists to form a complete dendritic cell vaccine with the corresponding antigen, which is then returned to the human body to activate the immune system, generate cytotoxic T cells to kill EBV-infected cells, and exert immunological effects to improve the quality of patient vital activity. The production cycle of the dendritic cell vaccine is about one week, and it is short, low-cost, safe and has almost no side effects.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to EBV complex antigens, dendritic cell vaccines and their use in preparing drugs for treating EBV-associated infections. [Background technology]

[0002] Epstein-Barr virus (EBV) is a member of the lymphotropic virus genus of the Herpesviridae family. Its genetic material is DNA, approximately 170 kb in length, and encodes approximately 100 genes, including those for the essential capsid antigen (VCA), early antigen (EA), and nuclear antigen (NA). This virus is ubiquitous in humans and is widespread worldwide, with an infection rate of up to 95% in adults. The virus can persist for life, and the disease it causes varies geographically. EBV infection is common during childhood and adolescence, and after infection in the body, some cases develop into long-term latent infection, but it can also lead to the formation of various human malignancies and diseases, such as Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), gastric cancer, breast cancer, tumors that develop in immunocompromised patients, as well as chronic active EBV infection (CAEBV), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), and infectious mononucleosis (IM). EBV specifically infects B cells (although in recent years it has been shown to also infect T lymphocytes, epithelial cells, and natural killer cells, causing related diseases) of humans and certain primates both in vivo and ex vivo, stimulating the continuous proliferation of infected cells, leading to infinite cell passage and "immortalization," resulting in the formation of lymphoblastoid cell lines (LCLs). These cell lines are commonly used in research into the onset and progression of various diseases, making it possible to conduct large-scale, long-term studies on the pathogenesis of specific diseases. However, the pathogenesis of EBV is still poorly understood, and safe and effective treatments for EBV-related diseases have yet to be established.

[0003] EBV has two modes of infection: replicative and latent. During replicative infection, viral DNA is transcribed, VCA and EA are expressed, and mature virus particles are produced, resulting in host cell lysis and death. This mode of infection is primarily seen in EBV infections, such as infectious mononucleosis. During latent infection, VCA and EA expression is suppressed, and no new virus particles are produced. Instead, EBV expresses EBNA, EBV-encoded small RNA (EBER), and latent membrane protein (LMP). This mode of infection is primarily seen in EBV-associated malignancies. The viral products expressed during EBV latent infection are primarily EBER, EBNA, and LMP. Four distinct gene expression patterns exist in latently infected cells, and different latent infection patterns are associated with different clinical malignancies. Type I latent infection mainly occurs in tumor cells of endemic Burkitt lymphoma, and viral products include EBNA-1 and EBER. Type II latent infection is associated with nasopharyngeal carcinoma and Hodgkin's disease, and viral products such as EBNA-1, LMP-1, LMP-2, and EBER are detected in infected cells. Type III latent infection is commonly seen in plasma cell lymphoma cells of immunosuppressed patients, and six types of EBNA, three types of LMP, and two types of EBER are all detected during the latent viral infection phase. Type IV latent infection occurs exclusively in B lymphocytes of healthy virus carriers, and these cells contain EBNA-1, LMP-2, and EBER-1.

[0004] Infectious mononucleosis (IM) is an acute infection caused by the Epstein-Barr virus (EBV). Approximately 50% of immunocompetent individuals initially infected with EBV exhibit typical IM. The pathological changes are benign proliferation of lymphoid tissue, manifesting as abnormal lymphocytic infiltration, affecting the liver, spleen, myocardium, kidneys, adrenal glands, lungs, and central nervous system. Clinical symptoms include fever, pharyngitis, hepatomegaly, splenomegaly, lymphadenopathy, and atypical lymphocytosis in the peripheral blood. The prognosis for this disease is generally favorable, with a case fatality rate of 1-2%. Most patients die from complications, but a small percentage of patients experience prolonged disease and repeated recurrences, leading to chronic active EBV infection. It is currently believed that the immune response, in which T lymphocytes produce B lymphocytes in response to EBV infection, underlies the various clinical symptoms.

[0005] Hemophagocytic syndrome is divided into two types: primary and secondary. The latter is caused by EBV infection and is called EBV-associated hemophagocytic syndrome (EBV-HLH). EBV infection is a condition that affects CD8 +It induces abnormal activation and proliferation of T lymphocytes and activation of macrophages, resulting in the massive production and release of proinflammatory cytokines, such as interferon, tumor necrosis factor, soluble interleukin (IL)-2 receptor, IL-1, IL-6, IL-10, and macrophage colony-stimulating factor, resulting in hypercytokinemia (also known as a "cytokine storm"), which leads to proliferation of tissue cells and phagocytosis of autologous blood cells. Histopathologically, proliferation and infiltration of lymphocytes and tissue cells are observed in all organs. This disease manifests in three patterns: during the progression of infectious mononucleosis, as recurrent attacks during chronic active Epstein-Barr virus infection, and in patients with Epstein-Barr virus-positive NK / T-cell lymphoma. Clinical symptoms include high fever, hepatomegaly, splenomegaly, lymphadenopathy, complete blood count reduction, abnormal liver function, significantly elevated lactate dehydrogenase, significantly elevated triacylglycerol, significantly elevated ferritin, decreased fibrinogen, and disseminated intravascular coagulation. Lymph node and bone marrow examinations are characterized by phagocytosis of red blood cells and nucleated cells by histocytic cells. This disease has a poor prognosis, and there is no safe and effective clinical treatment. More than half of patients die, and it is very difficult to distinguish it from malignant histocytosis clinically.

[0006] Chronic active EBV infection is currently considered to be a lymphoproliferative disorder associated with abnormally elevated levels of anti-EBV antibodies and elevated levels of EBV DNA, which can lead to lymphoma, virus-associated hemophagocytic syndrome, interstitial pneumonia, central nervous system lesions, and multiple organ failure. While the disease can occur at any age, it is most common in children and adolescents. Approximately 50% of patients die within five years of onset due to serious complications, including liver failure, myocarditis, coronary artery aneurysms, infection-associated hemophagocytic syndrome, and hematologic malignancies. Because EBV can replicate clonally and involve various types of lymphocytes in various locations, the clinical manifestations of the disease are diverse, with persistent or intermittent fever, hepatomegaly, splenomegaly, and lymphadenopathy being prominent symptoms, while other symptoms include sore throat, tender lymph nodes, anemia, myalgia, arthralgia, cowpox-like vesicles, and mosquito allergy, and can affect the hematologic, central nervous, gastrointestinal, and respiratory systems. Associated complications include hemophagocytic syndrome, leukemia, and NK / T-cell lymphoma. Currently, the diagnostic criteria proposed by Strauss in 1988 are still used internationally in many countries and regions: 1) symptoms persisting for more than six months after EBV infection and abnormal EBV antibody titers (anti-VCA-IgG ≥ 1:5120, anti-EA antibody ≥ 1:640, or EBNA antibody < 1:2), 2) major organ damage such as interstitial pneumonia, abnormal proliferation of bone marrow elements, retinitis, lymphadenitis, migratory hepatitis, and splenomegaly, and 3) EBV-DNA detection in damaged tissue or peripheral blood.With advances in medical technology, EBV DNA and RNA testing of tissue and peripheral blood, histopathology, and immunology have gradually been incorporated into the guidelines, but an aggressive and effective treatment has yet to be established.

[0007] Links between EBV infection and immunodeficiency disorders such as systemic lupus erythematosus, multiple sclerosis, and X-linked lymphoproliferative disorder (X-LDL) have also been reported. Systemic lupus erythematosus is a chronic inflammatory disease of the autoimmune system that affects the skin, joints, kidneys, heart, lungs, nervous system, and other organs. The most common symptoms include redness, arthritis, fatigue, and fever. Multiple sclerosis is an immune-mediated demyelinating disease of the central nervous system that predominates in young adults and can cause severe central nervous system reactions. It often presents as a relapsing-remitting disease with repeated episodes, gradually worsening the patient's condition and increasing disability. X-linked lymphoproliferative disorder (X-LDL) is an X-linked combined immunodeficiency disorder that is susceptible to EBV infection. The defective gene primarily functions in T lymphocytes and NK cells, affecting signaling in these cells.

[0008] Several studies have demonstrated that in the above-mentioned EBV-associated diseases, patients' dendritic cells are poorly differentiated, their numbers are reduced, their ability to recognize and present antigens is impaired, and their ability to activate early T cells is reduced, resulting in the body's inability to recognize and eliminate tumor cells. EBV infection is likely one of the factors that causes dendritic cell dysfunction in these patients. Restoring the number and function of dendritic cells in patients using specific technological methods can effectively treat the above-mentioned EBV-associated diseases. Dendritic cells (DCs) were discovered in 1973 by Canadian scientist Ralph M. Steinman, who won the 2011 Nobel Prize in Physiology or Medicine. They are named after the numerous dendrites or pseudopodial processes they exude upon maturation. Dendritic cells (hereinafter also referred to as "DCs") are antigen-presenting cells (APCs) with the strongest and most specialized biological functions known to date. They can efficiently take up, process, and present antigens, and are the only APCs currently discovered that can activate naive early T cells. Furthermore, immature DCs have a relatively strong ability to migrate and take up antigens, while mature DCs can effectively activate early T cells, making them central in the initiation, control, and maintenance of immune responses. Although their number is less than 1% of peripheral blood monocytes, dendritic cells (DCs) are an important type of innate immune cell and specialized antigen-presenting cell because they are rich in antigen-presenting molecules (e.g., MHC-I and MHC-II), costimulatory factors (CD80 / B7-1, CD86 / B7-2, CD40, etc.), and adhesion factors (ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-3) on their surface. They play an important regulatory role in activating the body's immune response and maintaining self-immune tolerance.

[0009] DCs, the most powerful antigen-presenting cells, can effectively present antigen information to T cells, inducing T cell activation and triggering a series of immune responses. MHC molecules on the surface of DCs bind to antigens to form peptide-MHC molecule complexes, which can present antigen signals to T cells. Costimulatory molecules (CD80 / B7-1, CD86 / B7-2, CD40, etc.) highly expressed on some dendritic cells provide a second signal required for T cell activation. At the same time, DCs deliver antigen peptides to CD8 + Direct presentation to T cells and CD4 + With T cell help, CD8 + DCs can also activate T cells. Activated DCs secrete large amounts of IL-12, IL-18, and chemotactic cytokines (CCK), promoting T cell proliferation and inducing MHC-I class-restricted CTL responses and MHC-II class-restricted CD4 + DCs can induce Th1 immune responses. Furthermore, DCs can activate perforin P-granzyme B and FasL / Fas-mediated pathways, enhancing the cytotoxic activity of NK cells and enhancing the host's antitumor immune response. This is advantageous for promoting tumor clearance and killing cells infected with relevant viruses. DCs themselves can act as natural immune adjuvants by secreting various cytokines, enhancing the host's immune system and enhancing immune responses to various vaccines. Dendritic cells that combine antigen-specific information with vaccine function are commonly referred to as dendritic cell vaccines (DC vaccines).

[0010] Vaccines are prophylactic or therapeutic biological products used in humans and play an important role in preventing, treating, and controlling the outbreak and spread of infectious diseases. Vaccines containing only a single antigen component are called "monovalent vaccines." Monovalent vaccines can only protect against one infectious disease or one type of pathogen infection. Vaccines containing two or more antigen components in appropriate proportions are called "polyvalent vaccines" or "combination vaccines." For example, there are over 100 types of human papillomaviruses, most of which only cause skin warts, but some can also cause cervical cancer. For example, bivalent HPV vaccines can only protect against high-risk HPV types 16 and 18, which account for 70% of cervical cancers. In addition, the quadrivalent HPV vaccine can protect against HPV types 16, 18, 6, and 11, while the 9-valent HPV vaccine can protect against HPV types 16, 18, 31, 33, 45, 52, 58, 6, and 11. The development of multivalent vaccines has a history of nearly 100 years, with research into multivalent vaccines beginning as early as the 1930s. The trivalent influenza vaccine was first approved for use in the United States in 1945, followed by the hexavalent pneumococcal vaccine, the diphtheria-tetanus combination vaccine, the diphtheria-pertussis-tetanus combination vaccine, and the trivalent live-attenuated oral polio vaccine. Clinical trials have shown that combined immunization with multivalent vaccines is often superior to multiple vaccinations with monovalent vaccines. Multivalent vaccine combinations provide equivalent or superior immunization efficacy to monovalent vaccines without increasing vaccine side effects.

[0011] Traditional surgery, radiation therapy, and chemotherapy can cause damage to the patient's body; excessive radiation therapy and chemotherapy shorten the patient's survival time; and long-term drug treatment can lead to addiction, significantly reducing the patient's quality of life. Currently, there are no safe and effective clinical treatments for EBV-related infections, making the discovery of new therapeutic agents and therapies urgently necessary. Compared to traditional treatments, immunotherapy has the advantages of significant therapeutic efficacy and minimal side effects, gradually becoming a new cancer treatment method, among which DC vaccines are playing an increasingly important role. For example, Chinese Patent Application No. 201911127136.8 discloses a short peptide of an EBV-related antigen and its application. The short peptide has high affinity for MHC1 class and MHCII molecules on DC cells and can effectively exert antigen presentation, showing good potential for use as a polypeptide vaccine and a DC vaccine. Meanwhile, Chinese Patent Application No. 202011263782.X discloses an EB virus antigen epitope and its application. This EB virus antigen epitope is highly immunogenic, and is transformed into dendritic cells using an adeno-associated virus vector, the antigen gene is expressed in the dendritic cells, and the antigen protein is presented to T cells via a direct or cross-presentation pathway, thereby inducing killer T cells that can specifically kill the EB virus, and is of great significance in the field of treating EB antigen-positive diseases.

[0012] Although research into DC vaccines is currently gaining momentum, the therapeutic efficacy of monovalent DC vaccines is often limited. While various antigens and combinations are used to treat various diseases, single antigen messages are prone to immune escape from EBV-infected cells, resulting in ineffective immune cell killing and limited therapeutic efficacy. Furthermore, because many heterogeneous cells in the body secrete various cytokines that inhibit dendritic cell maturation, the number of dendritic cells present at disease sites is relatively low. Furthermore, even if dendritic cells lack potent tumor antigen stimulation, even if they elicit an antitumor immune response, they are unable to exert significant therapeutic effects in the host. Therefore, the development of dendritic cell vaccines using tumor-combined antigens is urgently needed for the treatment of EBV-associated infections. Summary of the Invention [Problem to be solved by the invention]

[0013] In view of the deficiencies of the prior art, it is an object of the present invention to provide an EBV complex antigen, a dendritic cell vaccine and uses thereof. The present invention involves stimulating a patient's own dendritic cells in vitro and loading them with various cell lysates (e.g., lysates of human immortalized B-lymphoblastoid cell lines (LCLs) derived from different EBV strains, such as SNU-719, YCCEL1, GD1, B95-8, M81, and HKNPC1-9, or lysates of EBV-positive infected cells, such as C666-1, HNE1, and CCL85) that have excellent immunogenicity against different EBV-related infected cells. Under the induction of various cytokines and specific agonists, these mature into dendritic cells, forming complete DC vaccines bearing the corresponding cancer antigens. These DC vaccines are then infused back into the human body to activate the immune system, stimulate innate immunity (such as the induction of NK cells), and stimulate lymphocytes to generate adaptive immune responses, thereby precisely killing EBV-infected cells and generating cytotoxic T cells to kill cancer cells, thereby achieving personalized therapy. Compared to radiation therapy and chemotherapy, the present invention is safe and has no side effects, and the preparation cycle of the dendritic cell vaccine is short, at about one week, and low cost. [Means for solving the problem]

[0014] To achieve the above objectives, the technical solution of the present invention is as follows:

[0015] In one aspect, the present invention provides an EBV complex antigen, said EBV complex antigen comprising a cell lysate of a human immortalized B lymphoblastoid cell line derived from an EB virus strain and / or a lysate of EBV-positive infected cells.

[0016] Specifically, the human immortalized B lymphoblastoid cell line is one or a combination of two or more of human immortalized B lymphoblastoid cell lines (LCLs) derived from different EBV strains, such as GD1, B95-8, M81, HKNPC1-9, SNU-719 and / or YCCEL1, and the EBV-positive infected cells are one or a combination of two or more of C666-1, HNE1 and / or CCL85 and other EBV-infected cells.

[0017] More specifically, said other EBV-infected cells are T cells, NK cells or B cells.

[0018] In another aspect, the present invention provides the use of the above-mentioned EBV complex antigen in the preparation of a dendritic cell vaccine.

[0019] In another aspect, the present invention provides a dendritic cell vaccine, wherein the dendritic cell vaccine is loaded with the above-mentioned EBV complex antigen.

[0020] Specifically, the dendritic cell vaccine is a monovalent dendritic cell vaccine or a polyvalent dendritic cell vaccine.

[0021] Specifically, the dendritic cell vaccine is loaded with cell lysates of one, two or more human immortalized B lymphoblastoid cell lines and / or lysates of EBV-positive infected cells.

[0022] More specifically, the human immortalized B lymphoblastoid cell line is one or a combination of two or more of human immortalized B lymphoblastoid cell lines (LCLs) derived from different EBV strains, such as GD1, B95-8, M81, HKNPC1-9, SNU-719 and / or YCCEL1, and the EBV-positive cells are one or a combination of two or more of C666-1, HNE1 and / or CCL85 and other EBV-infected cells.

[0023] Specifically, the amount of each cell used is 2.5 × 10 7 ~2.5×10 9 There are individuals.

[0024] Specifically, the dendritic cell vaccine further comprises a primary adjuvant or other therapeutically supporting cytokines.

[0025] More specifically, the first adjuvant is any one of Poly(I:C), LPS, and OK432, and the other therapeutic support cytokine is TNF-α or IL-12.

[0026] In another aspect, the present invention provides the use of the above-mentioned EBV multi-antigen or dendritic cell vaccine in the preparation of a medicament for the prevention and / or treatment of EBV-associated infections.

[0027] Specifically, the EBV-associated infections include, but are not limited to, infectious mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), and other EBV-associated blood diseases.

[0028] The present invention provides a dendritic cell vaccine capable of treating EBV-associated infections by stimulating the body's immune response. The dendritic cell vaccine of the present invention exhibits good therapeutic effects, particularly against infectious mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), and other EBV-associated blood diseases, with minimal side effects. It can efficiently suppress the division and proliferation of EBV-associated infected cells over the long term, attenuating the progression of the disease and ultimately achieving complete remission.

[0029] In some embodiments, the antigen-sensitized dendritic cell population is an immunogenic composition, and the dendritic cell population is loaded with a corresponding antigen, specifically, cell lysates of human immortalized B lymphoblastoid cell lines (LCLs) derived from different EBV strains, such as GD1, B95-8, M81, HKNPC1-9, SNU-719, and YCCEL1, or cell lysates of C666-1, HNE1, CCL85, and other EBV-infected cells, with the specific amount of each cell used being 2.5×10 7 ~2.5×10 9In yet another embodiment of the present invention, the dendritic cell vaccine loaded with an EBV complex antigen may be a monovalent dendritic cell vaccine loaded with only one type of cell lysate or LCL cell lysate of an EBV-associated infectious disease, or a polyvalent dendritic cell vaccine loaded with two types of cell lysates or LCL cell lysates of an EBV-associated infectious disease, or a polyvalent dendritic cell vaccine simultaneously loaded with three or more types of cell lysates or LCL cell lysates of an EBV-associated infectious disease.

[0030] In some embodiments, the dendritic cell vaccine of the present invention may contain a first adjuvant (such as Poly(I:C), LPS, or OK432) or other therapeutic adjuvant cytokines such as TNF-α and IL-12. The dendritic cell vaccine is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, intranodal, subcutaneously, or topically, 3 to 30 times at weekly or biweekly intervals, and the dendritic cell vaccine is administered at a dose of 1 x 10 per administration. 6 ~5×10 8 It is returned to the body in individual cell doses.

[0031] In another embodiment, the present invention provides a medicament for preventing and / or treating an EBV-associated infection, the medicament comprising the above-mentioned dendritic cell vaccine. [Effects of the Invention]

[0032] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0033] Dendritic cell vaccines aim to combat the disease by activating the patient's own immune system, and offer the following advantages over conventional treatments for EBV-associated infections:

[0034] 1) The dendritic cell vaccine of the present invention is safer for treating EBV-associated infections. Currently, surgery, chemotherapy, and radiation therapy kill virus-infected and cancer cells while also inflicting significant damage on the patient's body and significantly weakening the patient's own immune resistance. Furthermore, because tumors are heterogeneous, most anticancer drugs, especially new-generation molecular targeted drugs, are effective in only a minority of patients and are prone to developing drug resistance, resulting in a high recurrence rate. Compared to conventional chemotherapy and targeted therapy, the dendritic cell vaccine offers a new approach to tumor treatment. It directly targets the body's own immune cells and activates the patient's own immune system to kill cancer cells, thereby strengthening the immune system without directly damaging it. Furthermore, it can suppress the evolution of cancer cells, has a low recurrence rate, and overall has significantly lower side effects than conventional chemotherapy or multi-targeted molecular targeted drugs. For example, because the mechanism of action of dendritic cell multivalent vaccines is to activate the immune system, the most common side effects are limited to clinical grade I / II adverse reactions such as fever, fatigue, dizziness, generalized muscle pain, and drowsiness, making symptomatic treatment possible and making it promising for clinical application.

[0035] 2) The antigen selection of the dendritic cell polyvalent vaccine of the present invention is more effective in treating EBV-associated infections. While most current projects using DC vaccines to treat EBV-associated diseases use specific polypeptides as antigens, the dendritic cell polyvalent vaccine of the present invention uses a variety of EBV cell lysates containing all antigens that activate immune responses against cancer cells, and incorporates as much EBV antigen information as possible from the human body, enhancing the diversity of antigens presented by dendritic cells and maximizing activation of the human body's immune function, thereby inducing a stronger T cell response and improving therapeutic efficacy.

[0036] 3) The cell lysate used in the dendritic cell multivalent vaccine of the present invention is derived from a stable EBV cell line and EBV-positive infected cells, eliminating the need for time-consuming and laborious EBV antigen screening for each patient. It is a universal antigen without HLA restriction. This antigen is easy to prepare, the process is simplified, and the entire preparation cycle takes only about one week. This can reduce the preparation time by several months compared to the relatively new antigen DC vaccine. It also ensures uniform quality of the EBV lysate and eliminates the need for complex antigen screening processes, offering significant advantages in terms of cost and time savings.

[0037] 4) The dendritic cell polyvalent vaccine of the present invention exhibits a more sustained therapeutic effect and can effectively suppress the recurrence and metastasis of EBV-associated diseases for a long period of time. When the dendritic cell polyvalent vaccine is infused into a patient's body, a large number of memory T lymphocytes containing various EBV antigen information can be generated in the patient's body, and these can survive for several years to several decades. When exposed to the corresponding stimulus again, these T lymphocytes are quickly activated in the body and can kill EBV cells, thereby effectively preventing the recurrence and metastasis of EBV. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 shows the results of measuring the amount of EB virus in immortalized human B lymphocyte cell line LCLs cells. [Figure 2] FIG. 1 shows the morphology of mature dendritic cells. [Figure 3] FIG. 1 is a flow cytometry image showing the expression levels of marker molecules on the surface of dendritic cells. [Figure 4] FIG. 1 shows the results of measuring the expression level of IL-12p70 in dendritic cells. [Figure 5] FIG. 1 shows the results of measuring the rate of CTL-specific lymphocyte killing induced in vitro. [Figure 6] FIG. 1 shows the results of measuring the amount of EB virus in LCL cells after co-culture. [Figure 7]FIG. 1 shows the results of measuring the amount of interferon-γ secreted. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention will be described in more detail below with reference to specific examples, but the following examples are merely illustrative of the present invention and do not limit the present invention. Furthermore, unless otherwise specified, the experimental methods used in the following examples generally follow conventional conditions, and unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0040] Example 1: Isolation of peripheral blood mononuclear cells (PBMC) In this example, the density difference of each cellular component in peripheral blood (peripheral blood mainly contains cells such as platelets, mononuclear cells, granulocytes, and red blood cells. Among them, the platelet density is 1.030 to 1.035 kg / m 3 , mononuclear cell density 1.075~1.090kg / m 3 , granulocyte density 1.092kg / m 3 , red blood cell density 1.093 kg / m 3 ), peripheral blood samples were diluted with Ficoll (registered trademark) Paque Plus solution (GE Healthcare, density 1.075 to 1.089 kg / m 3 ) and then density gradient centrifugation to separate different cellular components enabled rapid isolation of mononuclear cells from human peripheral blood. Specifically, the procedure was as follows.

[0041] 1) Peripheral blood was collected from the vein of an EBV-infected individual and placed in an appropriate centrifuge tube. 4.5 mL of Ficoll® Paque Plus solution was added to each of two new centrifuge tubes using a pipette.

[0042] 2) The blood sample was drawn up with a pipette, and the upper layer of Ficoll solution was slowly poured into each centrifuge tube, 10 mL per tube, along the wall. The tubes were centrifuged at room temperature at 800 g for 20 minutes.

[0043] 3) The centrifuge tube was removed and the sample was separated into four layers: plasma, mononuclear cells, Ficoll solution, red blood cells, and granulocytes.

[0044] 4) The mononuclear cells were carefully aspirated and transferred to a 15 mL centrifuge tube, and the volume was adjusted to 14 mL with PBS / 1% FBS solution. After mixing uniformly by pipetting, the tube was centrifuged at room temperature at 800 g for 5 minutes.

[0045] 5) The supernatant was removed, and the bottom of the tube was gently tapped to loosen the cells. 14 mL of PBS / 1% FBS solution was then added to resuspend the cells, which were then mixed by pipetting and centrifuged at 700 g for 5 minutes at room temperature.

[0046] 6) The supernatant was removed, and the bottom of the tube was gently tapped to loosen the cells. 14 mL of RPMI / 10% FBS solution was added to resuspend the cells, and the mixture was mixed by pipetting. The mixture was then centrifuged at 400 g for 5 minutes at room temperature.

[0047] 7) The supernatant was removed, and the bottom of the tube was gently tapped to loosen the cells. 10 mL of RPMI / 10% FBS solution was then added to resuspend the cells, and the mixture was mixed evenly by pipetting.

[0048] 8) 10 μL of the cell solution was aspirated and transferred to a new 1.5 mL centrifuge tube, and 90 μL of RPMI / 10% FBS solution was added to dilute it 10-fold. 10 μL of the diluted cell solution was aspirated, and 10 μL of trypan blue staining solution was added, then the tube was placed on a hemocytometer and the number of cells was counted using an inverted microscope.

[0049] 9) The mixture was centrifuged at 700 g for 5 minutes at room temperature, the supernatant was removed, and an appropriate amount of PBS / 1% FBS was added to prepare it for subsequent testing.

[0050] Example 2: Construction of immortalized human B lymphocyte cell lines (LCLs) infected with EB virus strains 1) 10 mL of the B95-8 cell supernatant was aspirated and transferred to a centrifuge tube, centrifuged at 2000 rpm for 15 minutes, and the supernatant was filtered.

[0051] 2) The PBMCs prepared in Example 1 were resuspended in 2 mL of RPMI1640 / 10% FBS medium.

[0052] 3) 10 μL of cell solution was aspirated and diluted 10-fold with 90 μL of RPMI / 10% FBS, and the number of cells was counted under a microscope. Based on the counting results, the required volume of B95-8 supernatant was calculated, and 1 × 10 6 Each PBMC cell corresponds to 1 mL of B95-8 supernatant.

[0053] 4) PBMC cells were collected and centrifuged at 1000 rpm for 5 minutes, and the PBMC supernatant was discarded.

[0054] 5) Based on the cell count, the concentration of PBMC cells in the cell solution was 0.5 × 10 6 An appropriate amount of B95-8 cell supernatant was added to resuspend the PBMC cells at a volume of 500 μL.

[0055] 6) A sterilized 96-well plate was prepared, and 100 μL of the PBMC cell solution resuspended in B95-8 was transferred to each well of the 96-well plate.

[0056] 7) The 96-well plate was cultured in a CO2 incubator for 24 hours.

[0057] 8) The 96-well plate was removed, and 100 μL of R10 medium (RPMI1640 / 10% FBS, penicillin 1000 IU / mL, streptomycin 100 μg / mL) was added to each well, followed by pipetting to mix evenly.

[0058] 9) The 96-well plate was placed in an incubator and cultured for a further 6 days. The cells were observed daily to determine whether they showed any changes in cell morphology, such as an increase in cell volume, fullness of cytoplasm, spheroidization, distribution of small colony aggregates, lymphoblastoid changes with obvious enlargement of the cell population at the bottom of the well, and a change in the color of the medium to yellow.

[0059] 10) After 6 days of culture, the medium was replaced every 3 days. After carefully aspirating and discarding the upper layer of culture medium from each well, 100 μL of R10 medium was added to each well and the cells in the wells were continued to be cultured. Carefully, the medium was replaced as needed when the cell solution turned yellow. Alternatively, the cells were divided into 2 to 4 new wells and cultured. As the cell numbers increased, the cells were collected and transferred to 24-well plates, 6-well plates, and T25 flasks, respectively.

[0060] 11) After 4 weeks of culture, the cells were observed under a microscope, and immortalized human B lymphocyte cell line LCLs cells infected with EB virus strain were prepared.

[0061] Meanwhile, normal B cells and immortalized human B lymphocyte cell line LCLs cells infected with the EB virus strain prepared in this application were taken, and the expression status of the EBNA1 gene in the immortalized human B lymphocyte cell line LCLs cells prepared in this application was detected using real-time fluorescent quantitative PCR (Q-PCR), thereby reflecting the amount or expression status of the EB virus.

[0062] The EBV viral load (VL) of cells was measured as follows: DNA extraction and PCR polymerase chain reaction were performed using the MagMAX viral nucleic acid extraction kit (Thermo A42352) and the EBV Real-™ Quant kit (Sacace BioTechnologies Srl, Como, Italy). EBV quantification was performed on 10 μL samples using real-time quantitative PCR (EBV Real-™ Quant kit). In this experiment, the coding region of the EBNA1 gene was used as the amplification target, and β-actin was used as the internal reference gene. The polymerase chain reaction was performed in a final volume of 25 μL according to the instructions in the attached manual.

[0063] Among them, the primer sequences were as follows: EBNA1-FP: 5'-CCAGACAGCAGCCAATTGTC-3' (SEQ ID NO: 1), EBNA1-RP: 5'-GGTAGAAGACCCCCCTTAC-3' (SEQ ID NO: 2), β-actin-FP: 5'-CTCCATCCTGGCCTCGCTGT-3' (SEQ ID NO: 3), β-actin-RP: 5′-GCTGTCACCTCACCGTTCC-3′ (SEQ ID NO: 4).

[0064] The results of detecting the amount or expression status of EB virus are shown in Figure 1. While EB virus expression was barely detected in normal B cells, it was confirmed that the amount of EB virus in the immortalized human B lymphocyte cell line LCLs cells prepared in this application was much higher than that in normal B cells.

[0065] Similarly, by preparing other EB virus cells such as GD1, B95-8, M81, HKNPC1-9, SNU-719, and YCCEL1 according to the procedure of this example, immortalized human B lymphocyte cell line LCLs cells infected with the corresponding EB virus strains could be obtained, and the amount of EB virus in these cells was detected, confirming that it was much higher than that in normal B cells.

[0066] Example 3: Preparation of EBV cell lysate Repeated freezing and thawing is a commonly used form of mechanical lysis and typically consists of two parts: freezing and thawing. The principle is that as ice particles form within the cells, the salt concentration of the remaining cellular fluid increases, causing lysis and swelling, destroying the cell structure and killing the cells while preserving their immunogenicity. Freezing is typically performed in liquid nitrogen or on ice at -20°C, and thawing can be achieved by heat shock in a water bath at 37°C, 50°C, 65°C, or 100°C, which is gentler than chemical lysis. In this example, the procedure was specifically performed as follows.

[0067] 1) The temperature of the water bath was preset to 37°C.

[0068] 2) Collect immortalized human B lymphocyte cell line (LCLs) or EBV-positive infected cells (e.g., C666-1 cells, HNE1, CCL85, or other EBV-infected T cells, NK cells, or B cells) (at least 3 × 10 7 The cells were collected by centrifugation at 700 g for 5 minutes at room temperature.

[0069] 3) The supernatant was removed and the cells were resuspended in RPMI / 10% FBS.

[0070] 4) The number of cells was counted using trypan blue.

[0071] 5) The mixture was centrifuged at room temperature at 700 g for 5 minutes, then slowly stopped and the supernatant was removed.

[0072] 6) RPMI / 10% FBS was used to culture 5 x 10 cells. 6 The cells were resuspended in 1 mL freezing tubes to a density of 1 mL / mL.

[0073] 7) The cells were frozen in liquid nitrogen for 20 seconds.

[0074] 8) The cells were immediately thawed quickly and completely in a 37°C water bath.

[0075] The above-mentioned processes 7) and 8) were repeated four times, for a total of five times.

[0076] 10) The EBV cell lysate was stored in liquid nitrogen for future use.

[0077] Example 4: Preparation of immature dendritic cells I) CD14 + Mononuclear cell isolation and acquisition In this example, CD14 was used to separate and extract CD14 mononuclear cells. + Although magnetic bead sorting was used, other methods such as CD14 negative sorting, Melteny immunomagnetic cell sorting (MACS), and cell adhesion sorting can also be used. The principle is based on the specific binding properties of antigens and antibodies, and CD14+ Magnetic bead sorting kit for CD14 in PBMCs + It can specifically identify and bind to cells, and can be indirectly bound to magnetic beads via biotin or dextran, and then stimulated by the action of a high-strength magnetic field to identify CD14 + In this example, the EasySep TM A CD14 positive sorting kit was used. Specifically, the procedure was as follows.

[0078] 1) The PBMC cell suspension was transferred to a 5 mL tube for flow cytometry measurement.

[0079] 2) An appropriate amount of Selection Cocktail solution was added to a flow cytometry measurement tube to give a final concentration of 100 μL / mL, mixed thoroughly by pipetting, and incubated at room temperature for 10 minutes.

[0080] 3) Prepare magnetic beads and use RapidSphere TM The solution was vortexed for 30 seconds to uniformly distribute the magnetic bead particles.

[0081] 4) Place RapidSphere in a tube for flow cytometry measurement. TM An appropriate amount of the solution was added so that the final concentration was 100 μL / mL, mixed thoroughly by pipetting, and incubated at room temperature for 3 minutes.

[0082] 5) An appropriate amount of PBS / 2% FBS containing 1 mM EDTA was added to a tube for flow cytometry measurement so that the total volume was 2.5 mL, and the mixture was mixed thoroughly by pipetting.

[0083] 6) Place tubes for flow cytometry measurement into EasySep TM The plate was inserted vertically into the magnet and incubated at room temperature for 3 minutes.

[0084] 7) The magnet was inverted and the cell fluid flowing out of the flow cytometry measurement tube was collected in a 15 mL centrifuge tube. The magnet was held inverted for 3 seconds, taking care not to shake the tube or completely absorb the fluid from the tube wall.

[0085] 8) The magnet was placed in the upright position and the tube for flow cytometry measurement was removed.

[0086] 9) The above steps 7) and 8) were repeated twice.

[0087] 10) 2 mL of RPMI / 10% FBS was added to a tube for flow cytometry measurement to resuspend the cells, and the cell number was counted using trypan blue.

[0088] II) CD14 + Induction and generation of immature dendritic cells by monocytes In vitro, granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes the survival of immature dendritic cells (imDCs) and can induce their massive proliferation. On the other hand, interleukin-4 (IL-4) suppresses the excessive proliferation of macrophages, reduces the expression of CD14 molecules on the cell surface, and promotes the proliferation of CD14. + Induce differentiation of mononuclear cells into iDCs.

[0089] 1) CD14 + Cell solution was added at 2 x 10 cells per well. 6 The cells were transferred to a 6-well plate using a pipette in a clean bench so that the concentration was 1 cell / mL. 1 μL of human recombinant GM-CSF (final concentration 2000 IU / mL, Miltenyi product number 170-076-112) and 1 μL of human recombinant IL-4 (final concentration 1000 IU / mL, Miltenyi product number 170-076-101) were then added to the 6-well plate.

[0090] 2) The 6-well plate was placed on a clean bench and gently shaken three times back and forth to evenly distribute the cells. The cells were then cultured in an incubator at 37°C with 5% CO2 for three days.

[0091] 3) The 6-well plate was removed from the incubator, and 2 mL of RPMI1640 / 10% FBS, 1 μL of human recombinant GM-CSF (final concentration 2000 IU / mL, Miltenyi 170-076-112), and 1 μL of human recombinant IL-4 (final concentration 1000 IU / mL, Miltenyi 170-076-101) were added to the 6-well plate in a clean bench.

[0092] 4) The cells were placed in an incubator at 37°C with 5% CO2 and cultured for 2 days to prepare immature dendritic cells.

[0093] Example 5: Preparation of a dendritic cell multivalent vaccine by loading with EBV-infected cell lysate 1) A monovalent dendritic cell vaccine (using a cell lysate of immortalized human B lymphocyte cell line LCLs cells B95-8-LCL prepared from B95-8 as an example) was prepared as follows.

[0094] Immature dendritic cells were co-cultured with B95-8-LCL cell lysate for 6 hours, and then 2 μL of TNF-α (final concentration 2000 IU / mL, Miltenyi 170-076-103), 2 μL of LPS (final concentration 2 μg / mL, Sigma L4391), and 1 μL of Poly(I:C) (1 μg / mL, Sigma P1530) were added to stimulate dendritic cell maturation. A monovalent dendritic cell vaccine was prepared and designated Ag-DC.

[0095] Similarly, monovalent dendritic cell vaccines could be prepared using lysates of GD1-LCL, M81-LCL, HKNPC1-9-LCL, SNU-719-LCL, YCCEL1-LCL, C666-1, HNE1, CCL85, or other EBV-infected T cells, NK cells, or B cells.

[0096] 2) Preparation of dendritic cell polyvalent vaccine Dendritic cells were co-cultured with different EBV cell lysates, including those from EBV-infected B lymphocytes, T cells, NK cells, C666-1, HNE1, CCL85, GD1-LCL, M81-LCL, HKNPC1-9-LCL, SNU-719-LCL, YCCEL1-LCL, or B95-8-LCL, for 6 hours. Dendritic cell maturation was stimulated by the addition of 2 μL of TNF-α (final concentration 2000 IU / mL, Miltenyi 170-076-103), 2 μL of LPS (final concentration 2 μg / mL, Sigma-Aldrich L4391), and 1 μL of Poly(I:C) (1 μg / mL, Sigma-Aldrich P1530). A polyvalent dendritic cell vaccine loaded with antigens from EBV cell lysate was prepared by mixing equal amounts of multiple dendritic cells loaded with EBV cell antigen information in a dendritic cell culture medium, and designated Poly-DC (in this example, a polyvalent dendritic cell vaccine prepared from EBV-infected B lymphocytes and B95-8-LCLs cell lysate was used as an example).

[0097] 3) The morphology of mature dendritic cells was observed. As shown in Figure 2, when a culture dish of mature dendritic cells was observed under an optical microscope (10x objective), it was confirmed that mature dendritic cells grew by adhering to the wall, that the protrusions on the cell surface increased and became longer, that they exhibited a long, radial pattern, and that they had a clear dendritic morphology. At high densities, multiple cells adhered to each other to form a mesh-like structure.

[0098] 4) Surface molecular markers of immature and mature dendritic cells, including CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC, were measured using flow cytometry. The results are shown in Figure 3. The expression of molecules such as CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC on the surface of mature dendritic cells was higher than that on immature dendritic cells, demonstrating that dendritic cells were matured upon induction. (Iso represents the flow cytometry graph of the corresponding antibody isotype control; imDC represents the flow cytometry graph of surface molecules on immature dendritic cells; mDC represents the flow cytometry graph of surface molecules on mature dendritic cells.)

[0099] 5) Culture supernatants of immature and mature dendritic cells were collected and the expression of IL-12p70 in dendritic cells was measured using ELISA. The results are shown in Figure 4. Immature dendritic cells secreted very little IL-12p70, but when induced to become mature dendritic cells, the expression and secretion of IL-12p70 was enhanced.

[0100] Experimental Example 1 I) Preparation of T lymphocytes 1) The PBMCs prepared in Example 1 were placed in an incubator at 37°C and 5% CO 2 and allowed to stand for 2 hours, after which the floating cells were collected to prepare 1 mL of a cell suspension.

[0101] 2) The cell suspension was added to a nylon wool column preheated to 37°C, and the column was placed flat. 200 μL of preheated RPMI1640 containing 10% FBS was added, the column was sealed, and the column was incubated at 37°C for 2 hours.

[0102] 3) The nylon wool column was washed with RPMI1640 containing 10% FBS at a flow rate of approximately 1 mL / min, and 10 mL of the first eluted cell suspension, which was enriched for T cells and NK cells, was collected.

[0103] 4) The cells were centrifuged at 700 g for 5 minutes at room temperature to collect the cells in the lower layer. The cell number was counted and the cell concentration was adjusted to 1 × 10 in RPMI 1640 complete medium containing 80 IU / mL of IL-2. 7 The concentration was adjusted to cells / mL for later use.

[0104] Alternatively, magnetic bead separation can be used to identify CD3 + The beads can be used to isolate T lymphocytes. Specifically, the cells are first incubated with anti-surface antigen monoclonal antibodies for 12 minutes, then incubated with 10 7 50 μL of anti-CD3 monoclonal antibody was used for each cell. After washing, the cells were incubated with 100 μL of biotin-conjugated goat anti-mouse secondary antibody for 10 minutes. After washing, 25 μL of FITC-conjugated streptavidin was added and incubated for 8 minutes. After washing, biotin-conjugated magnetic particles (100 μL of magnetic particles was added for anti-CD3 monoclonal antibody) were added and incubated for 8 minutes. After each reaction step, the cells were washed with 1 mL of PBS containing 1% bovine serum albumin and centrifuged at 2000 rpm for 10 minutes. T lymphocytes were isolated by immunomagnetic separation using a magnetic cell separator (MACS).

[0105] II) Induction of CTL cells by in vitro stimulation The dendritic cell monovalent vaccine, the dendritic cell polyvalent vaccine, and the normal mature dendritic cells prepared in Example 5 were each resuspended in RPMI complete medium, and 2 × 10 5 The autologous T lymphocyte suspension isolated in step I) above was adjusted to a density of 1.6 × 10 cells / mL using RPMI complete medium. 6 The density was adjusted to 100 cells / mL, and 1 mL of the corresponding dendritic cells and T lymphocytes was added to each set.

[0106] All the above experimental groups were supplemented with the same supplementary cytokines, such as IL-2 (1000 U / mL), IL-12 (1500 U / mL), Poly(I:C) (10 mg / mL), and TNF-α (1000 U / mL). After 2 weeks of culture in a 37°C, 5% CO2, temperature- and humidity-controlled incubator, IL-2 was added to a final concentration of 30 U / mL. Furthermore, 2 × 10 5 The corresponding dendritic cell vaccine was added and the cells were cultured for another week, and on the 21st day, the cells were collected to obtain CTL cells.

[0107] III) Measurement of killing activity of T cells stimulated with dendritic cell vaccine against EBV-infected cells The cells obtained in step II) above were centrifuged and then suspended in RPMI1640 complete medium. The cell concentration was adjusted and the cells were divided into three experimental groups with different effector-to-target ratios (E / T ratios). Each group was plated in a 96-well plate at 4 × 10 cells per well. 5 , 2 × 10 5 , 1×10 5 1 x 10 cells were added to each well as effector cells. 2 x 10 cells were added to each well as target cells. 4LCL cells were added to the wells for a final volume of 200 μL. A control group consisting of blank culture medium without cells was also set up. Five wells of each group were set up in parallel. After 24 hours, the released effector cells were removed from each well and washed twice with PBS. Then, 100 μL of a reagent containing 20 μL of CCK8 was added to each well and the wells were incubated for another 2 hours. The optical density (OD) at 450 nm was measured using a microplate reader, and the specific lymphocyte killing rate (%) was calculated. The results are shown in Figure 5. The in vitro assay of the killing activity of recovered T lymphocytes stimulated with the dendritic cell polyvalent vaccine (Poly-DC group) against LCL cells was compared with T lymphocytes stimulated only with the LCL cell lysate-loaded monovalent dendritic cell vaccine (Ag-DC group) or a control group. Both the Poly-DC and Ag-DC groups were able to effectively kill LCL cells and inhibit their proliferation. Furthermore, the Poly-DC group of T lymphocytes stimulated with the dendritic cell polyvalent vaccine exhibited stronger killing ability against LCL cells, and the killing effect was more pronounced with increasing numbers of T lymphocytes.

[0108] EBV expression in LCL cells was detected by real-time fluorescent quantitative PCR. The results are shown in Figure 6. EBV levels in LCL cells from T lymphocytes stimulated with the dendritic cell polyvalent vaccine (Poly-DC group) were compared with those stimulated with the LCL cell lysate-loaded monovalent vaccine (Ag-DC group) or the control group. Measurement of EBV levels in LCL cells from each group revealed significantly lower EBV levels in both the Poly-DC and Ag-DC groups compared with the control group. Furthermore, the EBV levels in LCL cells from the Poly-DC group were lower than those in the Ag-DC group at all T cell effector-to-target ratios. These results suggest that T lymphocytes stimulated with the dendritic cell polyvalent vaccine effectively inhibit EBV expression in LCL cells, killing them and suppressing the proliferation of EBV-positive cells.

[0109] IV) Measurement of interferon-γ secretion in vitro Each set of CTL effector cells and LCL cells obtained in step II above were mixed in a U-bottom 96-well plate at an effector-to-target (E / T) ratio of 20:1 and cultured for 72 hours. The IFN-γ content in the culture supernatant was measured using an interferon-γ enzyme-linked immunoassay kit according to the manufacturer's instructions. The results are shown in Figure 7. Both T lymphocytes stimulated with the dendritic cell monovalent vaccine (Ag-DC group) and T lymphocytes stimulated with the dendritic cell polyvalent vaccine (Poly-DC group) produced significantly higher levels of interferon-γ than the control group. Furthermore, the interferon-γ content secreted by T cells in the Poly-DC group was higher than that of the Ag-DC group. This suggests that the dendritic cell polyvalent vaccine loaded with EBV-positive cell lysate can more strongly stimulate T lymphocyte differentiation, enhance interferon-γ secretion, and promote the body's resistance to EBV infection.

[0110] The above-described examples merely describe some embodiments of the present invention in more detail and should not be construed as limiting the scope of the claims of the present invention. Needless to say, those skilled in the art will understand that various modifications and variations are possible within the scope of the present invention, and all such modifications and variations are within the scope of the present invention. Therefore, the scope of patent protection of the present invention is defined by the appended claims.

Claims

1. 1. Use of a dendritic cell vaccine loaded with an EBV complex antigen in the preparation of a medicament for preventing and / or treating an EBV-associated infection, comprising: The EBV-associated infections include infectious mononucleosis, chronic active EBV infection, EBV-associated hemophagocytic lymphohistiocytosis, and EBV-associated blood disorders; the EBV complex antigen comprises an EBV cell lysate of a human immortalized B lymphoblastoid cell line derived from an EBV virus strain and a lysate of EBV-positive infected cells, the EBV virus strain of the human immortalized B lymphoblastoid cell line is derived from B95-8, and the EBV-positive infected cells are EBV-infected B lymphocytes; The method for preparing the dendritic cell vaccine comprises: (1) CD14 ＋ The cell solution was added at 2 x 10 per well. ６ an imDC induction step of transferring the cells to a well plate so that the cells reach a final concentration of 1000 IU / mL, and adding human recombinant GM-CSF and human recombinant IL-4 to the well plate at a final concentration of 2000 IU / mL and 1000 IU / mL, respectively, to prepare immature dendritic cells (imDCs); (2) co-culturing the immature dendritic cells obtained in step (1) with an EBV complex antigen, and then stimulating the maturation of the dendritic cells by adding TNF-α at a final concentration of 2000 IU / mL, LPS at a final concentration of 2 μg / mL, and Poly(I:C) at a final concentration of 1 μg / mL to prepare a mature dendritic cell vaccine, wherein the amount of each type of cell used in the EBV complex antigen is 2.5×10 ７ ~2.5 x 10 ９ and a step of inducing mature dendritic cells (mDCs), which are individual cells; Use of a dendritic cell vaccine, characterized in that the mature dendritic cells obtained in step (2) show high expression of surface molecular markers, CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC, as measured by flow cytometry, and have enhanced expression and secretion of IL-12p70 as measured by ELISA.

2. The use of the dendritic cell vaccine according to claim 1, wherein the dendritic cell vaccine further comprises a first adjuvant or a cytokine for therapeutic adjuvant.

3. The use of a dendritic cell vaccine according to claim 2, wherein the first adjuvant is any one of Poly(I:C), LPS, or OK432, and the therapeutic support cytokine is TNF-α or IL-12.

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