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, specifically to EBV complex antigens, dendritic cell vaccines and their use in preparing drugs for treating EBV-related 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, is about 170 kb in length, and encodes about 100 genes, including the genes for the important capsid antigen (VCA), early antigen (EA), and nuclear antigen (NA). The virus is universally susceptible to human infection and is widespread worldwide, with infection rates reaching 95% in adults, the virus can persist for life, and there are regional differences in the disease it causes. 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 malignant tumors 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 patients with immunodeficiency, as well as chronic active EBV infection (CAEBV), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), and infectious mononucleosis (IM). In addition, EBV specifically infects human and certain primate B cells (in recent years, it has been found that EBV also infects T lymphocytes, epithelial cells, and natural killer cells, causing related diseases) either in vivo or in vitro, stimulating the continuous proliferation of infected cells and causing infinite cell passage, leading to "immortalization", resulting in the formation of lymphoblastoid cell lines (LCLs), which are commonly used in research into the onset and progression of various diseases, making it possible to conduct large-scale, long-term research into the onset mechanisms of certain diseases. However, the onset mechanism of EBV is still insufficient, and a safe and effective treatment for EBV-related diseases has not yet been established.

[0003] EBV has two modes of infection: replicative infection and latent infection. In replicative infection, viral DNA is transcribed, VCA and EA are expressed, and mature virus particles are produced, which are accompanied by host cell lysis and death. This type of infection is mainly seen in EBV infections such as infectious mononucleosis. In the latent infection stage, the expression of VCA and EA is suppressed, and new virus particles are not produced, and mainly EBNA, EBV-encoded small RNA (EBER), and latent membrane protein (LMP) are expressed. This type of infection is mainly seen in EBV-associated malignant tumors. The viral expression products during the latent infection stage of EBV are mainly EBER, EBNA, and LMP. There are four different gene expression types in cells latently infected with EBV, and different latent infection types are associated with different clinical malignant tumor diseases. Among them, type I latent infection mainly appears in the tumor cells of endemic Burkitt's lymphoma, and the viral products are EBNA-1 and EBER; type II latent infection is associated with nasopharyngeal carcinoma and Hodgkin's disease, and the infected cells contain viral products such as EBNA-1, LMP-1, LMP-2 and EBER; 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 stage; type IV latent infection occurs only 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 infectious disease caused by the EB virus. Approximately 50% of immunologically normal individuals who are initially infected with the EB virus show typical infectious mononucleosis. The pathological changes are benign proliferation of lymphoid tissue, affecting the liver, spleen, myocardium, kidneys, adrenal glands, lungs and central nervous system, and manifest as abnormal infiltration of lymphocytes. Clinical symptoms mainly include fever, pharyngitis, hepatomegaly, splenomegaly, lymphadenopathy and atypical lymphocytosis in peripheral blood. The prognosis of this disease is generally good, with a case fatality rate of 1-2%, and most patients die from complications, but in a very small proportion of patients, the disease progresses to chronic active EB virus infection with repeated recurrences. It is currently believed that the immune response in which T lymphocytes produce B lymphocytes in response to EB virus infection is the basis for the production of 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 CD8 +It induces abnormal activation and proliferation of T lymphocytes and activation of macrophages, resulting in the massive production and release of inflammatory cytokines, such as interferon, tumor necrosis factor, soluble interleukin (IL)-2 receptor, IL-1, IL-6, IL-10, and macrophage colony-stimulating factor, causing hypercytokinemia (also called "cytokine storm"), which leads to proliferation of tissue cells and phagocytosis of the patient's own blood cells. Histopathologically, proliferation and infiltration of lymphocytes and tissue cells are observed in all organs. The disease develops in three patterns: during the progression of infectious mononucleosis, as repeated attacks during chronic active EBV infection, and in patients with EBV-positive NK / T cell lymphoma. Clinical symptoms include high fever, hepatomegaly, splenomegaly, lymphadenopathy, complete blood cytopenia, abnormal liver function, significant increase in lactate dehydrogenase, significant increase in triacylglycerol, significant increase in ferritin, decrease in fibrinogen, and disseminated intravascular coagulation. Lymph node and bone marrow examinations are characterized by phagocytosis of red blood cells and nucleated cells by tissue cells. The disease has a poor prognosis, there is no safe and effective clinical treatment, more than half of the patients die, and it is very difficult to distinguish it from malignant histocytosis in clinical practice.

[0006] Chronic active EBV infection is currently considered to be a lymphoid tissue proliferative disorder associated with abnormally elevated levels of anti-EBV antibodies and elevated levels of EBV DNA, which may progress to lymphoma, virus-associated hemophagocytic syndrome, interstitial pneumonia, central nervous system lesions, and multiple organ failure. The disease can occur at any age, but is more prevalent in children and adolescents. Approximately 50% of patients die within 5 years of onset due to severe complications such as liver failure, myocarditis, coronary aneurysms, infection-associated hemophagocytic syndrome, and hematological malignancies. Because EBV can involve and replicate clonally in various types of lymphocytes at various sites, the clinical manifestations of the disease are diverse, prominently characterized by persistent or intermittent fever, hepatomegaly, splenomegaly, and lymphadenopathy, while other symptoms include sore throat, tender lymph nodes, anemia, myalgia, arthralgia, cowpox-like vesicles, and mosquito allergy, and may 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 persist for more than 6 months after EB virus infection, and EB virus antibody titers are abnormal (anti-VCA-IgG≧1:5120, anti-EA antibody≧1:640, or EBNA antibody<1:2), 2) damage to major organs such as interstitial pneumonia, abnormal proliferation of bone marrow components, retinitis, lymphadenitis, migratory hepatitis, and splenomegaly, and 3) EBV-DNA detected in damaged tissues and peripheral blood. With the advancement of medical technology, EB virus DNA and RNA tests for tissues and peripheral blood, histopathology, and immunology are gradually being incorporated into the guidelines, but an active and effective treatment has not yet been established.

[0007] In addition, there is also a link between EBV infection and immunodeficiency diseases such as systemic lupus erythematosus, multiple sclerosis, and X-linked lymphoproliferative disorder. 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 of the body, and 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, often presenting as a relapsing-remitting disease with repeated episodes of disease, leading to gradual deterioration of the patient's condition and severe disability. X-linked lymphoproliferative disorder is an X-linked combined immunodeficiency disorder that is susceptible to EBV infection, and the defective gene functions mainly in T lymphocytes and NK cells, affecting signaling in these cells.

[0008] Several studies have revealed that in the above EBV-related diseases, the patient's dendritic cells are poorly differentiated, their numbers are reduced, their ability to identify and present antigens is impaired, and their activation ability for 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 leads to the dysfunction of dendritic cells in the relevant patients, and the above EBV infection-related diseases can be effectively treated by restoring the number and function of the patient's dendritic cells through certain technical means. Dendritic cells (DCs) were discovered in 1973 by Canadian scientist Ralph M. Steinmann, who won the Nobel Prize in Physiology or Medicine in 2011, and are named after the fact that they protrude numerous dendrites or pseudopod-like processes during maturation. Dendritic cells (hereinafter also referred to as "DC") 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 discovered to date 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 the center of 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 of the abundant 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 strongest antigen-presenting cells, can effectively present antigen information to T cells, induce T cell activation, and trigger 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, and costimulatory molecules (CD80 / B7-1, CD86 / B7-2, CD40, etc.) highly expressed in 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 the help of T cells, CD8 + DCs can also activate T cells. Activated DCs secrete large amounts of IL-12, IL-18, and chemotactic cytokines (CCK), promoting the proliferation of T cells and inducing MHC-I class-restricted CTL responses and MHC-II class-restricted CD4 + It can induce Th1 immune responses. In addition, DCs can activate perforin P·granzyme B and FasL / Fas-mediated pathways, enhancing the cytotoxicity of NK cells to enhance the antitumor immune response of the body, which is advantageous in promoting tumor clearance and killing cells infected with related viruses. DCs themselves can act as natural immune adjuvants to enhance the immunity of the body by secreting various cytokines, and can also enhance the immune response to various vaccines. Dendritic cells that combine the information of relevant antigens and vaccine functions are usually called dendritic cell vaccines (DC vaccines).

[0010] A vaccine is a preventive or therapeutic biological product used for inoculation into humans, and plays an important role in preventing, treating, and controlling the occurrence and spread of infectious diseases. A vaccine containing only a single antigen component is called a "monovalent vaccine", and a monovalent vaccine can only prevent one infectious disease or one type of pathogen infection, while a vaccine made by mixing two or more antigen components in an appropriate ratio is called a "polyvalent vaccine" or "combination vaccine". For example, there are more than 100 types of human papillomavirus, most of which only cause skin warts, but some human papillomaviruses can cause cervical cancer. For example, a bivalent HPV vaccine can only prevent human papillomaviruses 16 and 18, which belong to "high-risk HPV types", and 70% of cervical cancers are caused by these two virus types. In addition, the quadrivalent HPV vaccine can prevent HPV infections against types 16, 18, 6, and 11, while the 9-valent HPV vaccine can prevent HPV infections against types 16, 18, 31, 33, 45, 52, 58, 6, and 11. The development of polyvalent vaccines has a history of nearly 100 years, and research on polyvalent vaccines began as early as the 1930s. The trivalent influenza vaccine was first approved for use in the United States in 1945, and later the hexavalent pneumococcal vaccine, the diphtheria-tetanus combination, the diphtheria-pertussis-tetanus combination, and the trivalent live attenuated oral polio vaccine were approved one after another. Clinical trials have shown that combined immunity with polyvalent vaccines is often superior to multiple vaccinations with monovalent vaccines. When administered in combination with polyvalent vaccines, the immunity is equal to or greater than that of monovalent vaccines without increasing the side effects of the vaccines.

[0011] Traditional surgery, radiation therapy, and chemotherapy may damage the patient's body, excessive radiation therapy and chemotherapy may shorten the patient's survival time, and long-term drug therapy may cause dependency, significantly reducing the quality of patient survival. Currently, there is no safe and effective treatment for EBV-related infections in clinical practice, and it is urgent to find new therapeutic drugs and treatments. Compared with traditional treatments, immunotherapy has the advantages of remarkable therapeutic effect and small side effects, and is gradually becoming a new cancer treatment method, among which DC vaccines play an increasingly important role. For example, Chinese Patent Application No. 201911127136.8 discloses a short peptide of EB virus-related antigen and its application, which has high affinity for MHCI class and MHCII molecules on DC cells and can effectively exert antigen presentation effect, and has good potential for use as a polypeptide vaccine and DC vaccine. Meanwhile, Chinese Patent Application No. 202011263782.X discloses an EBV antigen epitope and its application. Such an EBV antigen epitope is highly immunogenic, and is transformed into dendritic cells using an adeno-associated virus vector, the antigen gene is expressed in 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 EBV, and is of great significance in the field of treatment of EBV antigen-positive diseases.

[0012] Currently, research on DC vaccines is becoming more and more active, but the therapeutic effect of monovalent DC vaccines is often limited. Although there are many types and combinations of different antigens used to treat various diseases, a single antigen message is likely to cause immune escape from EBV-infected cells, and as a result, the immune response cannot successfully kill the infected cells, and the therapeutic effect is limited. In addition, since many heterogeneous cells in the body may secrete various cytokines that inhibit the maturation of dendritic cells, the number of dendritic cells present at the disease site is relatively small, and even if an anti-tumor immune response is induced in dendritic cells that lack strong tumor antigen stimulation, it cannot exert a significant therapeutic effect in the host body. Therefore, it is urgent to construct a dendritic cell vaccine using tumor complex antigens, which is expected to be used for the treatment of EBV-related 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 stimulates the patient's own dendritic cells in vitro, loads them with various cell lysates (e.g., lysates of human immortalized B-lymphoblastoid cell lines (LCLs) cells derived from different EBV strains, such as SNU-719, YCCEL1, GD1, B95-8, M81, HKNPC1-9, etc., or lysates of EBV-positive infected cells, such as C666-1, HNE1, CCL85, etc.) that have excellent immunogenicity against different EBV-related infected cells, and matures them under the induction of various cytokines and specific agonists to form complete DC vaccines carrying corresponding cancer antigens, which are then returned to the human body to activate the immune system and stimulate natural immunity (such as the induction of NK cells), as well as stimulate lymphocytes to generate adaptive immune responses, thereby precisely killing EBV-infected cells and generating cytotoxic T cells to kill cancer cells, thereby realizing personalized treatment. Compared with 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 (approximately one week), and is low cost. [Means for solving the problem]

[0014] In order 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 positively 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 positively 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 embodiment, the present invention provides a dendritic cell vaccine, said dendritic cell vaccine being loaded with the above EBV complex antigens.

[0020] Specifically, the dendritic cell vaccine is a monovalent dendritic cell vaccine or a multivalent 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 positively 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 of the cells used is 2.5 × 10 7 ~2.5×10 9 There are 10 pieces.

[0024] Specifically, the dendritic cell vaccine further comprises a first adjuvant or other adjuvant 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 embodiment, the present invention provides the use of an above-mentioned EBV complex antigen or dendritic cell vaccine in the preparation of a medicament for the prevention and / or treatment of an EBV-associated infection.

[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 stimulating the body's immune response to treat EBV-related infections. 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-related blood diseases, with minimal side effects, and can efficiently suppress the division and proliferation of EBV-related infected cells over the long term, attenuate the progression of the disease, and ultimately achieve complete remission.

[0029] In some embodiments, the antigen-sensitized dendritic cell population is an immunogenic composition, and the dendritic cell population is loaded with the 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, YCCEL1, and cell lysates of C666-1, HNE1, CCL85, and other EBV-infected cells, and the specific amount of each cell used is 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 multivalent dendritic cell vaccine loaded with two types of cell lysates or LCL cell lysates of an EBV-associated infectious disease, or a multivalent dendritic cell vaccine loaded with three or more types of cell lysates or LCL cell lysates of an EBV-associated infectious disease simultaneously.

[0030] In some embodiments, the dendritic cell vaccine of the present invention can include a first adjuvant (e.g., Poly(I:C), LPS, OK432, etc.) or other therapeutic adjuvant cytokines such as TNF-α and IL-12. The dendritic cell vaccine is administered 3 to 30 times at weekly or biweekly intervals by intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, intranodal, subcutaneous, or local administration, and the dendritic cell vaccine is administered at a dose of 1 x 10 per administration. 6 ~5×10 8 The individual cells are then administered back into the body.

[0031] In another embodiment, the present invention provides a medicament for preventing and / or treating an EBV-associated infection, said medicament comprising the above-mentioned dendritic cell vaccine. Effect 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 according to the present invention is safer in treating EBV-related infections. In other words, at present, surgery, chemotherapy, and radiation therapy kill virus-infected cells and cancer cells while at the same time causing great damage to the patient's body and greatly reducing the patient's own immune resistance. In addition, because tumors are heterogeneous for each patient, most anticancer drugs, especially new generation molecular targeted drugs, are effective only in a small number of patients and are prone to developing drug resistance, resulting in a high recurrence rate of cancer. Compared with conventional chemotherapy and targeted therapy, the dendritic cell vaccine provides a new idea for tumor treatment, directly targeting immune cells in the body and activating the patient's own immune system to kill cancer cells, thereby strengthening the immune system without causing direct damage. Furthermore, the evolution of cancer cells can be suppressed, the recurrence rate is low, and from an overall perspective, side effects are significantly lower than those of conventional chemotherapy and multi-targeted molecular targeted drugs. For example, because the mechanism of action of the dendritic cell multivalent vaccine 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, making it promising for clinical application.

[0035] 2) The antigen selection of the dendritic cell multivalent vaccine of the present invention is more effective in treating EBV-related infections. Most current projects using DC vaccines to treat EBV-related diseases use specific polypeptides as antigens, but the dendritic cell multivalent vaccine of the present invention uses a variety of EBV cell lysates containing all antigens that activate immune responses against cancer cells, incorporates the maximum amount of EBV antigen information in the actual human body, enhances the diversity of antigens presented by dendritic cells, and maximizes the activation of the immune function of the human body to induce a stronger T cell response, thereby improving the therapeutic effect.

[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, which does not require time-consuming and laborious screening of EBV antigens for each patient, has no HLA restriction, and is a versatile antigen. This antigen is easy to prepare, the process is simplified, and the entire preparation cycle takes only about one week, which can reduce the time required for preparing a relatively new antigen DC vaccine by several months, and also achieves uniform quality of the EBV lysate and eliminates the need for complicated antigen screening processes, which has the excellent advantages of reducing costs and time.

[0037] 4) The dendritic cell polyvalent vaccine according to the present invention shows a more sustained therapeutic effect and can effectively suppress the recurrence and metastasis of EBV-related diseases for a long period of time. When the dendritic cell polyvalent vaccine is reintroduced into the patient's body, it can generate a large number of memory T lymphocytes containing various EBV antigen information in the patient's body, and the survival time of these T lymphocytes can range from several years to several decades. When exposed to the corresponding stimulus again, these T lymphocytes can be rapidly activated in the body and kill EBV cells, thereby effectively preventing the recurrence and metastasis of EBV. [Brief description 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. [Diagram 2] FIG. 1 shows the morphology of mature dendritic cells. [Diagram 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. [Diagram 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 PREFERRED EMBODIMENTS

[0039] The present invention will be described in more detail below based on specific examples, but the following examples are merely illustrative of the present invention and do not limit the present invention. Furthermore, in the experimental methods used in the following examples, unless otherwise specified, the specific conditions are generally in accordance with 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 stratify different cellular components, enabling rapid isolation of mononuclear cells from human peripheral blood.

[0041] 1) Peripheral blood was collected from the vein of an EBV-infected individual and placed in an appropriate centrifuge tube, and 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 the Ficoll solution was slowly poured along the wall of the centrifuge tube (10 mL per tube). The tube was 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, from top to bottom.

[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, then 14 mL of PBS / 1% FBS solution was added to resuspend the cells, pipette to mix, 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, then 14 mL of RPMI / 10% FBS solution was added to resuspend the cells, pipette to mix, and 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, then 10 mL of RPMI / 10% FBS solution was added to resuspend the cells and pipetted to mix evenly.

[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 solution 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 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 total volume of 500 μL.

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

[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 every day to see 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 group 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 once every 3 days. After carefully sucking and discarding the upper layer of culture fluid from each well, 100μL of R10 medium was added to each well and the cells in the wells were continued to be cultured. It was noted that the medium was replaced at the appropriate time when the cell fluid turned yellow, or the cells were divided into 2 to 4 new wells as necessary and cultured. When the number of cells gradually increased, they were collected and transferred to a 24-well plate, a 6-well plate, and a T25 flask in that order.

[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 the present application were taken, and the expression status of the EBNA1 gene in the immortalized human B lymphocyte cell line LCLs cells prepared in the present application was detected using real-time fluorescent quantitative PCR (Q-PCR) to reflect the amount or expression status of the EB virus.

[0062] The amount of EBV virus (Viral Load, VL) carried by the cells was measured as follows. DNA extraction and PCR polymerase chain reaction were performed using MagMAX viral nucleic acid extraction kit (A42352, Thermo) and EBV Real-TM Quant kit (Sacace BioTechnologies Srl, Como, Italy), and EBV was quantified using real-time quantitative PCR (EBV Real-TM Quant Kit) for 10 μL of sample. In this experiment, the coding region of the EBNA1 gene was used as the amplification target, β-actin was used as the internal reference gene, and the polymerase chain reaction was performed in a final volume of 25 μL according to the procedure indicated in the attached instruction 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' (sequence number 4).

[0064] The results of detecting the amount or expression status of EB virus are shown in Figure 1. It was confirmed that, while EB virus expression was hardly detected in normal B cells, 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 following the procedure of this example using other EB virus cells such as GD1, B95-8, M81, HKNPC1-9, SNU-719, and YCCEL1, 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 the cells was detected, confirming that it was far higher than that in normal B cells.

[0066] Example 3: Preparation of EBV cell lysate Repeated freezing and thawing is commonly used as a form of mechanical lysis, and usually consists of two parts: freezing and thawing. The principle is that as ice particles are formed inside the cells, the salt concentration of the remaining cell fluid increases, causing lysis and swelling, destroying the cell structure and killing the cells, while preserving the immunogenicity of the cells. Freezing is usually performed in liquid nitrogen or on ice at -20°C, and thawing can be performed 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 specific procedure was 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), respectively (at least 3 × 10 7 The cells were harvested 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 cell number was counted using trypan blue.

[0071] 5) Centrifuge at room temperature at 700 g for 5 minutes, slowly stop the centrifuge, and remove the supernatant.

[0072] 6) Using RPMI / 10% FBS, cells were cultured at 5 × 10 6 The cells were resuspended in 1 mL cryotubes to a density of 100 / 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 steps 7) and 8) were repeated four times for a total of five times.

[0076] 10) EBV cell lysates were stored in liquid nitrogen for future use.

[0077] Example 4: Preparation of immature dendritic cells I) CD14 + Isolation and acquisition of mononuclear cells In this example, CD14 was used to separate and extract CD14 mononuclear cells. + Although the magnetic bead sorting method was used, other methods such as CD14 negative sorting, Meltenyi immunomagnetic bead cell sorting (MACS), and cell adhesion method can also be used. The principle is based on the specific binding property of antigens and antibodies, and CD14+ Magnetic bead sorting kit for CD14 in PBMCs + The cells can be specifically identified and bound, and can be indirectly bound to magnetic beads via biotin or dextran, and then stimulated with a high-strength magnetic field to express CD14 + The objective of this embodiment is to separate cells. 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 so that the final concentration was 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 disperse 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 flow cytometry measurement tube 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 in EasySep TM The plate was inserted vertically into the magnet and incubated at room temperature for 3 minutes.

[0084] 7) Invert the magnet and collect the cell liquid flowing out of the flow cytometry measurement tube into a 15 mL centrifuge tube. Hold the magnet inverted for 3 seconds, taking care not to shake or completely absorb the liquid 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 massive proliferation of imDCs. 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 suppresses the proliferation of CD14. + Induce differentiation of mononuclear cells into iDCs.

[0089] 1) CD14 + Cell solution was diluted to 2 x 10 per well. 6 The cells were transferred to a 6-well plate in a clean bench using a pipette so that the cells were concentrated to 100 cells / mL. Then, 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.

[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 a 37°C, 5% CO2 incubator 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 a 37°C, 5% CO2 incubator and cultured for 2 days to prepare immature dendritic cells.

[0093] Example 5: Preparation of a dendritic cell multivalent vaccine by loading 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] B95-8-LCL cell lysate was used to co-culture with immature dendritic cells 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, and a monovalent dendritic cell vaccine was prepared and designated Ag-DC.

[0095] Similarly, corresponding 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, and B cells.

[0096] 2) Preparation of polyvalent dendritic cell vaccine 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-LCLs, were co-cultured with dendritic cells for 6 h, 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. Multiple dendritic cells loaded with EBV cell antigen information were mixed in equal amounts in a dendritic cell culture medium to prepare a polyvalent dendritic cell vaccine loaded with antigens from EBV cell lysate, 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 the culture dish of mature dendritic cells was observed under an optical microscope (10x objective lens), 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 shape, and that at high density, multiple cells adhered to each other to form a mesh-like structure.

[0098] 4) Surface molecular markers of immature and mature dendritic cells, CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC, were measured using flow cytometry. The results are shown in Figure 3, where 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 of immature dendritic cells, demonstrating that dendritic cells were matured by induction (Iso represents the flow cytometry graph of the corresponding antibody isotype control, imDC represents the flow cytometry graph of the surface molecules of immature dendritic cells, and mDC represents the flow cytometry graph of the surface molecules of 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, and it was found that immature dendritic cells secrete 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. with 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 that had been warmed to 37°C, and the column was laid flat. 200 μL of pre-warmed RPMI1640 containing 10% FBS was then 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 cell suspension that was first eluted and was rich in 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 number of cells was counted, and the cell concentration was adjusted to 1 × 10 in RPMI 1640 complete medium containing 80 IU / mL IL-2. 7 The concentration was adjusted to 1 / mL for future use.

[0104] Alternatively, magnetic bead separation was used to detect CD3 + The beads can be used to separate T lymphocytes. Specifically, the cells are first incubated with anti-surface antigen monoclonal antibodies for 12 minutes, then incubated for 10 minutes. 7 50 μL of anti-CD3 monoclonal antibody was used for each cell, and the cells were washed and then incubated with 100 μL of biotin-labeled goat anti-mouse secondary antibody for 10 minutes. After washing, 25 μL of FITC-labeled streptavidin was added and reacted for 8 minutes. After washing, biotin-labeled magnetic particles (100 μL of magnetic particles was added to the anti-CD3 monoclonal antibody-containing cells) were added and reacted for 8 minutes. After each reaction step, 1 mL of PBS containing 1% bovine serum albumin was added to wash the cells, and the cells were centrifuged at 2000 rpm / min for 10 minutes. T lymphocytes were obtained 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 each was diluted to 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 10 cells / mL, and 1 mL of the corresponding dendritic cells and T lymphocytes were added to each set.

[0106] All the above experimental groups were supplemented with the same supplementary cytokines, such as IL-2 at 1000 U / mL, IL-12 at 1500 U / mL, Poly(I:C) at 10 mg / mL, and TNF-α at 1000 U / mL. After 2 weeks of culture in a 37°C, 5% CO2, temperature-controlled, humidified incubator, IL-2 was added at a final concentration of 30 U / mL, and 2 × 10 5 The corresponding dendritic cell vaccine was added and the cells were subsequently cultured for one week, and on the 21st day, the cells were harvested 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, suspended in RPMI1640 complete medium, and the cell concentration was adjusted to separate them 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 Each well was added with 2 × 10 cells as effector cells, and 2 × 10 cells were added with 2 × 10 cells as target cells. 4Each well was added with 100 μL of LCL cells, with the final volume being 200 μL. At the same time, a control group was also prepared with blank culture medium without cells, and each group was prepared in parallel with 5 wells. After 24 hours, the released effector cells were removed from each well, and the wells were washed twice with PBS. Then, 100 μL of a reagent containing 20 μL of CCK8 was added to each well, and the wells were further cultured for 2 hours. The optical density (OD) at 450 nm was measured using a microplate reader, and the specific lymphocyte killing rate (%) was calculated. The measurement results are shown in Figure 5. In vitro, the killing activity of recovered cells against LCL cells was measured for T lymphocytes stimulated with dendritic cell polyvalent vaccine (Poly-DC group) compared with T lymphocytes stimulated only with LCL cell lysate-loaded dendritic cell monovalent vaccine (Ag-DC group) or a control group. Both the Poly-DC group and the Ag-DC group were able to effectively kill LCL cells and inhibit their proliferation. It was also found that the Poly-DC group of T lymphocytes stimulated with dendritic cell polyvalent vaccine showed stronger killing ability against LCL cells, and the killing effect was more pronounced the more T lymphocytes there were.

[0108] Meanwhile, the expression of EBV in LCL cells was detected by real-time fluorescent quantitative PCR, and the detection results are shown in Figure 6. When the amount of EBV in LCL cells of T lymphocytes stimulated with dendritic cell polyvalent vaccine (Poly-DC group) was measured compared with T lymphocytes stimulated with LCL cell lysate-loaded dendritic cell monovalent vaccine (Ag-DC group) or the control group, it was found that the amount of EBV in LCL cells of both Poly-DC and Ag-DC groups was significantly lower than that of the control group, and the amount of EBV in LCL cells of the Poly-DC group was lower than that of the Ag-DC group at all T cell effector-to-target ratios, suggesting that T lymphocytes stimulated with dendritic cell polyvalent vaccine can effectively inhibit the expression of EBV in LCL cells, kill LCL cells, and suppress the proliferation of EBV-positive cells.

[0109] IV) Measurement of interferon-γ secretion in vitro The CTL effector cells and LCL cells of each set obtained in step II) above were mixed in a U-bottom 96-well plate according to an effector-to-target ratio (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 procedure in the instruction manual, and the measurement results were as shown in Figure 7. It was found that the T lymphocytes stimulated with the dendritic cell monovalent vaccine (Ag-DC group) and the T lymphocytes stimulated with the dendritic cell polyvalent vaccine (Poly-DC group) were both able to produce a significantly higher amount of interferon-γ than the control group, and the content of interferon-γ secreted by the T cells of the Poly-DC group was higher than that of the Ag-DC group, suggesting that the dendritic cell polyvalent vaccine loaded with EBV-positive cell lysate can more strongly stimulate the differentiation of T lymphocytes, enhance the secretion of interferon-γ, and promote the body's resistance to EBV infection.

[0110] The above-mentioned examples merely describe some embodiments of the present invention in more detail and should not be interpreted as limiting the scope of the claims of the present invention. Needless to say, those skilled in the art can understand that various modifications and changes are possible within the scope of the present invention, and all of them are within the scope of the present invention. Therefore, the scope of patent protection of the present invention shall be subject to the scope of the attached claims.

Claims

Use of dendritic cell vaccine prepared using EBV complex antigen in the preparation of a drug for preventing and / or treating EBV-related infectious diseases, wherein the EBV-related infectious diseases include infectious mononucleosis, chronic active EBV infection, EBV-related hemophagocytic lymphohistiocytosis, and EBV-related blood diseases, the EBV complex antigen includes 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 B95-8, and the EBV-positive infected cells are EBV-infected B lymphocytes, the method for preparing the dendritic cell vaccine is (1) An imDC induction step of transferring a CD14+ cell solution to a well plate so that there are 2×106 cells / mL per well, adding 2000 IU / mL of human recombinant GM-CSF and 1000 IU / mL of human recombinant IL-4 to the well plate to prepare immature dendritic cells (imDC), and (2) a step of co-culturing with the immature dendritic cells obtained in step (1) using the EBV complex antigen, and then adding 2000 IU / mL of TNF-α, 2 μg / mL of LPS, and 1 μg / mL of Poly(I:C) to stimulate the maturation of dendritic cells to prepare a mature dendritic cell vaccine, wherein the usage amount of various cells in the EBV complex antigen is 2.5×107 to 2.5×109 cells, a mature dendritic cell (mDC) induction step, Characterized by comprising, the use of dendritic cell vaccine.

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 assistance.

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

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