WO2023005486A1 - 一种ebv复合抗原、树突状细胞疫苗及其应用 - Google Patents

一种ebv复合抗原、树突状细胞疫苗及其应用 Download PDF

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WO2023005486A1
WO2023005486A1 PCT/CN2022/099296 CN2022099296W WO2023005486A1 WO 2023005486 A1 WO2023005486 A1 WO 2023005486A1 CN 2022099296 W CN2022099296 W CN 2022099296W WO 2023005486 A1 WO2023005486 A1 WO 2023005486A1
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ebv
cells
dendritic cell
cell
cell vaccine
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刘慧宁
印泽
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上海恒赛生物科技有限公司
刘慧宁
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to an EBV composite antigen, a dendritic cell vaccine and their application in preparing medicines for EBV-related infectious diseases.
  • Epstein-Barr virus is a member of the genus Lymphotropic Virus of the Herpesviridae family. Its genetic material is DNA with a length of about 170 kb, encoding about 100 genes, and an important capsid antigen (viral capsid antigen, VCA), early antigen (early antigen, EA) and core antigen (nuclear antigen, NA) genes.
  • VCA viral capsid antigen
  • EA early antigen
  • NA core antigen
  • the population of the virus is generally susceptible and spreads widely all over the world. The infection rate of adults is as high as 95%. The virus can be carried for life, and the resulting disease has regional differences. EBV infection mostly occurs in childhood and adolescence.
  • BL Burkitts lymphoma
  • NPC nasopharyngeal carcinoma
  • NHL non-Hodgkin's lymphoma
  • gastric cancer breast cancer
  • IM infectious mononucleosis
  • EBV can specifically infect human and certain primate B cells in vivo or in vitro (in recent years, it has been found that it can also infect T lymphocytes, epithelial cells and natural killer cells, etc., and cause related diseases), and can stimulate infection.
  • Cells continue to grow and cause cells to be passed down indefinitely to achieve "immortalization", thus forming lymphoblastoid cell lines (LCLs), which are often used to study the occurrence and development of various diseases, making large-scale long-term It is possible to study the pathogenesis of certain diseases.
  • LCLs lymphoblastoid cell lines
  • Epstein-Barr virus has two infection modes: replicative infection and latent infection.
  • replicative infection viral DNA is transcribed, VCA and EA are expressed, and mature virus particles are produced, accompanied by lysis and death of host cells.
  • This mode of infection is mainly seen in Epstein-Barr virus Infectious diseases such as infectious mononucleosis.
  • latent infection period the expression of VCA and EA is inhibited, and EBNA, EBV-encoded small RNA (EBER) and latent membrane protein (latent membrane protein, LMP) are mainly expressed, and no new virus particles are produced.
  • EBER EBV-encoded small RNA
  • LMP latent membrane protein
  • Epstein-Barr virus latent infection virus expression products EB virus latent infection mainly expresses EBER, EBNA and LMP. There are 4 different gene expression types in cells latently infected by Epstein-Barr virus, and different latent infection types are related to different clinical malignant tumor diseases.
  • type I latent infection mainly occurs in tumor cells of endemic Burkitt lymphoma, and the virus products include EBNA-1 and EBER; type II latent infection involves nasopharyngeal carcinoma and Hodgkin's disease, and EBNA-1 appears in infected cells , LMP-1, LMP-2 and EBER and other viral products; type III latent infection is common in plasma cell lymphoma cells of immunosuppressed patients, and 6 kinds of EBNA, 3 kinds of LMP and 2 kinds of EBER in the latent infection period of the virus can be detected Detected; type IV latent infection occurs only in B lymphocytes of healthy virus carriers, these cells contain EBNA-1, LMP-2 and EBER-1.
  • IM Infectious mononucleosis
  • Epstein-Barr virus After the initial infection of Epstein-Barr virus, about 50% of people with normal immune function show typical infectious mononucleosis.
  • the pathological change is benign hyperplasia of lymphoid tissue, liver, spleen, myocardium, kidney, adrenal gland, lung and central nervous system can all be involved, manifested as abnormal lymphocyte infiltration.
  • the main manifestations are fever, angina, hepatomegaly, splenomegaly, enlarged lymph nodes, and abnormal lymphocytes in peripheral blood.
  • Hemophagocytic syndrome can be divided into primary and secondary. The latter is caused by Epstein-Barr virus infection and is called Epstein-Barr virus-associated hemophagocytic syndrome (EBV-HLH). Epstein-Barr virus infection causes abnormal activation and proliferation of CD8 + T lymphocytes, and activates macrophages, resulting in inflammatory cytokines such as interferon, tumor necrosis factor, soluble interleukin (interleukin, IL)-2 receptor, IL-1 , IL-6, IL-10, and macrophage colony-stimulating factor are produced and released in large quantities, resulting in hypercytokineemia (or cytokine storm), causing tissue cell proliferation and phagocytosis of self-blood cells.
  • cytokines such as interferon, tumor necrosis factor, soluble interleukin (interleukin, IL)-2 receptor, IL-1 , IL-6, IL-10, and macrophage colony-stimulating factor are produced and released in
  • Histopathology showed proliferation and infiltration of lymphocytes and histiocytes in all organs.
  • the disease manifests in 3 ways during the course of the disease: during the course of infectious mononucleosis, recurrent episodes during chronic active Epstein-Barr virus infection, and in patients with Epstein-Barr virus-positive NK/T-cell lymphoma.
  • the clinical manifestations are high fever, hepatomegaly, splenomegaly, enlarged lymph nodes, pancytopenia, abnormal liver function, significantly increased lactate dehydrogenase, significantly increased triacylglycerol, significantly increased ferritin, decreased fibrinogen, and diffuse intravascular coagulation etc.
  • Lymph node and bone marrow examination is characterized by phagocytosis of red blood cells and nucleated cells by histiocytes.
  • the prognosis of this disease is poor, and there is no safe and effective treatment method clinically. More than half of the patients die, and it is difficult to distinguish it from malignant histiocytosis clinically.
  • Epstein-Barr virus infection is currently considered to be an abnormal increase in anti-Epstein-Barr virus antibody and Epstein-Barr virus DNA.
  • Epstein-Barr virus can affect various types of lymphocytes in different parts and clone and proliferate, making the clinical manifestations of the disease various, such as persistent or intermittent fever, hepatomegaly, splenomegaly, and lymph node enlargement are prominent manifestations, and other symptoms include sore throat and lymph node tenderness , anemia, muscle pain, joint pain, vaccinia-like blisters and mosquito allergies, etc., can affect the blood system, central nervous system, digestive system and respiratory system. Related complications include hemophagocytic syndrome, leukemia, and NK/T-cell lymphoma.
  • Epstein-Barr virus infection and immunodeficiency diseases, such as systemic lupus erythematosus, multiple sclerosis, X-linked lymphoproliferative diseases and so on.
  • Systemic lupus erythematosus is a chronic autoimmune inflammatory disease that affects the skin, joints, kidneys, heart, lungs, nervous system and other organs of the body. The most common signs include erythema and arthritis, as well as fatigue and fever .
  • Multiple sclerosis is an immune-mediated demyelinating disease of the central nervous system, which is more common in young people and can cause severe reactions in the central nervous system.
  • X-linked lymphoproliferative disease is an X-linked combined immunodeficiency disease that is sensitive to Epstein-Barr virus infection. The defective gene mainly affects T lymphocytes and NK cells and affects the signal transduction of these cells.
  • Dendritic cells (Dendritic cells, DC) were discovered in 1973 by the Canadian scientist Ralph M. Steinman, who won the Nobel Prize in Medicine and Physiology in 2011.
  • DC is the professional antigen presenting cell (Antigen presenting cell, APC) with the most powerful body function known so far, which can efficiently ingest, process and present antigen, and is the only one found so far that can activate unsensitized naive T cells and immature DCs have a strong ability to migrate and absorb antigens, mature DCs can effectively activate naive T cells, and are at the center of initiating, regulating, and maintaining immune responses.
  • APC Antigen presenting cell
  • DC is an important type of innate immune cells and professional antigen-presenting cells. It plays a key regulatory role in the body's immune response and maintenance of autoimmune tolerance.
  • DC can effectively present antigen information to T cells, induce T cell activation and lead to a series of immune responses.
  • MHC molecules on the surface of DCs can bind to antigens to form peptide-MHC molecule complexes, presenting antigen signals to T cells, and costimulatory molecules (CD80/B7-1, CD86/B7-2, CD86/B7-2, CD86/B7-2, CD40, etc.) provide the second signal necessary for T cell activation, and at the same time, DC can also directly present antigen peptides to CD8 + T cells, and activate CD8 + T cells with the help of CD4 + T cells, and the activated DC can secrete a large amount of IL- 12.
  • DC can also Activation of perforin P granzyme B and FasL/Fas-mediated pathways enhances NK cytotoxicity to enhance the body's anti-tumor immune response, which is beneficial to tumor clearance and killing of related virus-infected cells.
  • DC itself can be used as a natural immune adjuvant to improve the immune ability of the body by secreting various cytokines, and can also enhance the immune response of various vaccines.
  • the dendritic cells with vaccine function that carry relevant antigen information, Known as dendritic cell vaccine (DC vaccine).
  • Vaccine is a preventive or therapeutic biological product used for human vaccination, which plays an important role in the prevention, treatment, and control of the occurrence and prevalence of infectious diseases.
  • Vaccines containing only a single antigenic component are called “monovalent vaccines", which can only prevent one infectious disease or one type of pathogen infection; vaccines made by mixing more than two antigenic components in appropriate proportions are called “multivalent vaccines”.
  • valent vaccines or “combined vaccines”.
  • bivalent HPV vaccine can only prevent human papillomavirus 16 and 18,
  • these two types of viruses belong to "high-risk HPV", and 70% of cervical cancers are caused by these two types of viruses; the quadrivalent HPV vaccine can prevent 16, 18, 6 and 11 HPV infection; the nine-valent HPV vaccine can prevent 16, 18, Types 31, 33, 45, 52, 58, 6 and 11.
  • the development of multivalent vaccines has a history of nearly a hundred years. As early as the 1930s, people started research on multivalent vaccines.
  • the 3-valent influenza vaccine was first approved for use in the United States, followed by 6-valent pneumococcal vaccine, DPT-2 and DPT-3 vaccines, and 3-valent live attenuated oral polio vaccine. Results of clinical trials have shown that co-immunization with multivalent vaccines is often superior to multiple vaccinations with monovalent vaccines. When multivalent vaccines are immunized together, the immune effect is similar or better than that of monovalent vaccines, and the side effects of vaccines are not increased.
  • Patent 202011263782.X discloses an epitope of Epstein-Barr virus and its application.
  • the epitope of Epstein-Barr virus is highly immunogenic. It is transfected into dendritic cells by adeno-associated virus vector, and the antigen gene is in dendritic cells. It is expressed in the lymphoid cells, and presents the antigenic protein to T cells through direct or cross-presentation pathways, thereby inducing killer T cells that can specifically kill Epstein-Barr virus, which is of great significance in the field of treatment of EB antigen-positive diseases.
  • the object of the present invention is to provide an EBV composite antigen, a dendritic cell vaccine and applications thereof.
  • the present invention stimulates the patient's own dendritic cells in vitro, and loads various cell lysates (such as SNU-719, YCCEL1, GD1, B95-8, M81,
  • LCLs human immortalized B lymphoid blastoid cell line
  • the present invention provides an EBV composite antigen, which includes EBV strain-derived human immortalized B lymphoid blast cell line cell lysate or/and EBV positive infected cell lysate.
  • the human immortalized B lymphoid blast cell line is human immortalized B lymphoid blast cell derived from different Epstein-Barr virus strains such as GD1, B95-8, M81, HKNPC1-9, SNU-719 or/and YCCEL1
  • One or more combinations of cell lines (LCLs) are one or more combinations of C666-1, HNE1 or/and CCL85 and other EBV-infected cells.
  • said other EBV-infected cells are T cells, NK cells or B cells.
  • the present invention provides the application of the above-mentioned EBV composite antigen in the preparation of a dendritic cell vaccine.
  • the present invention provides a dendritic cell vaccine loaded with the above-mentioned EBV composite antigen.
  • the dendritic cell vaccine is a dendritic cell monovalent vaccine or a dendritic cell multivalent vaccine.
  • the dendritic cell vaccine is loaded with one, two or more than two kinds of cell lysate of immortalized B lymphoid blastoid cell line or/and EBV positive infected cell lysate.
  • the human immortalized B lymphoid blast cell line is a human immortalized B lymphoid blast cell line derived from different Epstein-Barr virus strains such as GD1, B95-8, M81, HKNPC1-9, SNU-719 or/and YCCEL1.
  • One or more combinations of cell line (LCLs) cells; the EBV-positive cells are one or more combinations of C666-1, HNE1 or/and CCL85 and other EBV-infected cells.
  • the amount of each cell used is 2.5 ⁇ 10 7 -2.5 ⁇ 10 9 .
  • the dendritic cell vaccine further comprises a first adjuvant or other cytokines for adjuvant therapy.
  • the first adjuvant is any one of PloyI:C, LPS or OK432; the other adjuvant therapy cytokines are TNF- ⁇ or IL-12.
  • the present invention provides the application of the above-mentioned EBV composite antigen or dendritic cell vaccine in the preparation of drugs for preventing and/or treating EBV-related infectious diseases.
  • EBV-related infectious diseases include but are not limited to infectious mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-related hemophagocytic lymphohistiocytosis (EBV-HLH) and Other EBV-related blood disorders.
  • IM infectious mononucleosis
  • CAEBV chronic active EBV infection
  • EBV-HLH EBV-related hemophagocytic lymphohistiocytosis
  • Other EBV-related blood disorders include but are not limited to infectious mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-related hemophagocytic lymphohistiocytosis (EBV-HLH) and Other EBV-related blood disorders.
  • the dendritic cell vaccine provided by the invention can stimulate the body's immune response to treat EBV-related infectious diseases, especially for infectious mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-related hemophagocytic Lymphohistiocytosis (EBV-HLH) and other EBV-related blood diseases have good curative effect and few side effects, and can effectively inhibit the division and proliferation of EBV-related infected cells for a long time and alleviate the disease process, and even achieve complete remission.
  • infectious mononucleosis IM
  • CAEBV chronic active EBV infection
  • EBV-HLH EBV-related hemophagocytic Lymphohistiocytosis
  • other EBV-related blood diseases have good curative effect and few side effects, and can effectively inhibit the division and proliferation of EBV-related infected cells for a long time and alleviate the disease process, and even achieve complete remission.
  • the antigen-sensitized population of dendritic cells that has been loaded with the corresponding antigen is an immunogenic composition.
  • the specific antigens include GD1, B95-8, M81, HKNPC1-9, SNU-719, YCCEL1 and other human immortalized B lymphoid blast cell line (LCLs) cell lysates derived from different EB virus strains and C666-1, HNE1, For cell lysates such as CCL85 and other EBV-infected cells, the specific amount of each cell is 2.5 ⁇ 10 7 -2.5 ⁇ 10 9 .
  • the dendritic cell vaccine loaded with EBV composite antigen can be a dendritic cell monovalent vaccine loaded with only one EBV-related infectious disease cell lysate or LCLs cell lysate, or It is a dendritic cell multivalent vaccine loaded with cell lysates or LCLs cell lysates of two EBV-related infectious diseases, and can also be loaded with three or even multiple cell lysates or LCLs cell lysates of EBV-related infectious diseases at the same time Dendritic cell multivalent vaccine.
  • the dendritic cell vaccine of the present invention may comprise the first adjuvant (Ploy (I:C), LPS, OK432, etc.) or other adjuvant therapeutic cytokines such as TNF- ⁇ , IL-12, etc. .
  • the dendritic cell vaccine is given 3-30 times by intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, intranodal, subcutaneous, or topical administration, with intervals of one or two weeks each time; The amount of cells reinfused each time is 1 ⁇ 10 6 -5 ⁇ 10 8 .
  • the present invention provides a drug for preventing and/or treating EBV-related infectious diseases, the drug comprising the above-mentioned dendritic cell vaccine.
  • Dendritic cell vaccine is a vaccine that can fight diseases by activating the patient's own immune system. Compared with the treatment of traditional EBV-related infectious diseases, it has the following advantages:
  • the dendritic cell vaccine described in the present invention is safer for treating EBV-related infectious diseases.
  • dendritic cell vaccine provides a new idea for the treatment of tumors. It directly targets immune cells in the body and kills cancer cells by activating the autoimmune system.
  • dendritic cell multivalent vaccine it will not cause direct damage, but instead Enhance the immune system; it can inhibit the evolution of cancer cells, the recurrence rate is low, and the side effects are significantly less than traditional chemotherapy and multi-target targeted drugs.
  • the mechanism of action of dendritic cell multivalent vaccine is to activate the immune system. Therefore, the most common side effects are only clinical grade I/II adverse reactions, such as fever, fatigue, dizziness, general muscle aches, lethargy, etc. Symptomatic treatment is enough, and it has a good clinical application prospect.
  • the selection of antigens in the dendritic cell multivalent vaccine of the present invention is more effective for treating EBV-related infectious diseases.
  • the dendritic cell multivalent vaccine of the present invention uses a variety of EBV cell lysates, including activating immune responses against cancer cells. All antigens load the EBV antigen information in the actual human body to the greatest extent, strengthen the diversity of antigens presented by dendritic cells, maximize the activation of human immune function, and induce stronger T cell responses, thereby improving its therapeutic effect.
  • the cell lysates selected for the dendritic cell multivalent vaccine of the present invention are derived from stable EBV cell lines and EBV-positive infected cells, without time-consuming and labor-intensive screening of EBV antigens in each patient, without HLA restrictions , a universal antigen.
  • the antigen is easy to prepare, the process is simplified, and the whole preparation cycle only takes 1 week, which saves several months compared with the preparation of the new antigen DC vaccine, and can achieve uniform quality of EBV lysate, eliminating the need for complicated antigen screening. It shows outstanding advantages in reducing costs and saving time.
  • the dendritic cell polyvalent vaccine of the present invention has a more durable therapeutic effect and can effectively inhibit the recurrence and metastasis of EBV-related diseases for a long time.
  • a large number of memory T lymphocytes containing a variety of EBV antigen information can be generated in the patient's body, and their existence lasts for several years to several decades.
  • it can be rapidly activated in the body to kill EBV cells and effectively prevent the recurrence and metastasis of EBV.
  • Figure 1 is a graph showing the detection results of EB virus load in the immortalized human B lymphocyte line LCLs cells.
  • Figure 2 is a morphological diagram of mature dendritic cells.
  • Figure 3 is a flow chart of the expression of marker molecules on the surface of dendritic cells.
  • Fig. 4 is a graph showing the detection results of the expression level of IL-12p70 in dendritic cells.
  • Fig. 5 is a graph showing the detection results of the killing rate of CTL-specific lymphocytes induced by stimulation in vitro.
  • Figure 6 is a graph showing the detection results of EB virus load in LCL cells after co-culture.
  • Fig. 7 is a graph showing the detection results of interferon gamma secretion.
  • peripheral blood mainly contains platelets, mononuclear cells, granulocytes, red blood cells and other cells: the density of platelets is 1.030-1.035kg/m 3 , and the density of mononuclear cells is 1.075- 1.090kg/m 3 , the density of granulocytes is 1.092kg/m 3 , the density of red blood cells is 1.093kg/m 3 ), added in peripheral blood samples Paque Plus (GE Healthcare) solution (with a density of 1.075-1.089kg/m 3 ) is subjected to density gradient centrifugation to separate different cell components, and can rapidly separate mononuclear cells from human peripheral blood.
  • Paque Plus GE Healthcare
  • Embodiment 2 Immortalization human B lymphocyte line LCLs of construction Epstein-Barr virus strain infection
  • Example 2 The PBMCs prepared in Example 1 were resuspended in 2 mL of RPMI1640/10% FBS medium.
  • PBMC cells were collected, centrifuged at 1000 rpm for 5 min, and the PBMC supernatant was discarded.
  • normal B cells and the immortalized human B lymphocyte line LCLs cells infected by the EB virus strain prepared by the application were respectively taken, and the immortalized human B lymphocytes prepared by the application were detected by real-time fluorescent quantitative PCR (Q-PCR).
  • Q-PCR real-time fluorescent quantitative PCR
  • EBNA1-FP 5'-CCAGACAGCAGCCAATTGTC-3', as shown in SEQ ID NO:1;
  • EBNA1-RP 5'-GGTAGAAGACCCCCTCTTAC-3', as shown in SEQ ID NO:2;
  • ⁇ -actin-FP 5'-CTCCATCCTGGCCTCGCTGT-3', as shown in SEQ ID NO:3;
  • ⁇ -actin-RP 5'-GCTGTCACCTTCACCGTTCC-3', as shown in SEQ ID NO:4.
  • Epstein-Barr virus The load or expression of Epstein-Barr virus is shown in Figure 1. Normal B cells can hardly detect the expression of Epstein-Barr virus, while the Epstein-Barr virus load of the immortalized human B lymphocyte line LCLs cells prepared by the present application is much higher than that of EB virus. higher than normal B cells.
  • EB virus cells such as GD1, B95-8, M81, HKNPC1-9, SNU-719, YCCEL1, etc. can be used to prepare immortalized human B lymphocytes infected by corresponding EB virus strains according to the steps of this example
  • the EB virus load of LCLs cells is much higher than that of normal B cells.
  • Embodiment 3 the preparation of EBV cell lysate
  • the repeated freeze-thaw method is a commonly used mechanical lysis method, usually consisting of two parts (freezing and thawing).
  • the principle is that the formation of ice particles in the cells and the increase in the salt concentration of the remaining cell fluid cause swelling, which breaks the cell structure and causes cell death, but retains the immunogenicity of the cells. Freezing is usually carried out in liquid nitrogen or on ice at -20°C, and thawing can be carried out in a water bath at 37°C, 50°C, 65°C or 100°C, which is milder than chemical lysis.
  • Collect immortalized human B lymphocyte line LCLs or EBV-positive infected cells such as C666-1 cells, HNE1, CCL85 or other EBV-infected T cells, NK cells or B cells
  • EBV-positive infected cells such as C666-1 cells, HNE1, CCL85 or other EBV-infected T cells, NK cells or B cells
  • the cells were resuspended in RPMI/10% FBS in a 1 mL cryovial with a density of 5 ⁇ 10 6 /mL.
  • step (7) and step (8) Repeat step (7) and step (8) 4 times, 5 times in total.
  • CD14 monocytes include but are not limited to CD14 + magnetic bead sorting in this example, CD14 negative selection, Miltenyi immune magnetic bead cell sorting (MACS), cell attachment and other methods can also be used.
  • the principle is based on the specific binding characteristics of antigens and antibodies.
  • the CD14 + magnetic bead sorting kit can specifically identify and bind CD14 + cells in PBMCs, and indirectly couple with magnetic beads through biotin or dextran.
  • the purpose of CD14 + cell separation is achieved under the action of a magnetic field.
  • the EasySep TM CD14 positive selection kit was used.
  • RapidSphere TM solution Add an appropriate amount of RapidSphere TM solution to the flow tube to a final concentration of 100 ⁇ L/mL, thoroughly mix by pipetting, and incubate at room temperature for 3 minutes.
  • step (9) Repeat step (7) and step (8) twice.
  • granulocyte-macrophage colony-stimulating factor gran ⁇ Locyte-macrophage colony stim ⁇ Lating factor, GM-CSF
  • GM-CSF gran ⁇ Locyte-macrophage colony stim ⁇ Lating factor
  • Interleukin-4 IL-4
  • IL-4 can inhibit the overgrowth of macrophages, reduce the expression of CD14 molecules on the cell surface, and induce the differentiation of CD14 + monocytes into iDCs.
  • Example 5 Loading EBV-infected cell lysate to prepare dendritic cell multivalent vaccine
  • Dendritic cell monovalent vaccine take the immortalized human B lymphocyte line LCLs cell lysate B95-8-LCL cell lysate prepared by B95-8 as an example):
  • lysis of T cells, NK cells, B cells infected with GD1-LCL, M81-LCL, HKNPC1-9-LCL, SNU-719-LCL, YCCEL1-LCL, C666-1, HNE1, CCL85 or other EBV Corresponding dendritic cell monovalent vaccines can be prepared from these products.
  • 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
  • EBV cell lysates such as cell lysates of LCLs
  • 2 ⁇ L of TNF- ⁇ final concentration was 2000IU/mL, Miltenyi, 170-076-103
  • 2 ⁇ L of LPS final concentration of 2 ⁇ g/ml, Sigma, L4391
  • Poly(I:C) 1 ⁇ g/mL, Sigma, P1530
  • Equal amounts of dendritic cells were mixed in the dendritic cell medium to prepare a dendritic cell multivalent vaccine loaded with EBV cell lysate anti
  • Example 1 Place the PBMC prepared in Example 1 at 37° C. in a 5% CO 2 incubator for 2 hours, collect the suspended cells, and prepare 1 mL of cell suspension;
  • the magnetic bead separation method can be used to separate T lymphocytes using CD3 + magnetic beads.
  • Cells were first incubated with anti-surface antigen monoclonal antibody for 12 minutes, and 107 cells were treated with 50 ⁇ L anti-CD3 monoclonal antibody. After washing, the cells were incubated with 100 ⁇ L biotin-labeled goat anti-mouse secondary antibody for 10 minutes. After washing, FITC-labeled streptavidin was added. 25 ⁇ L of Hetin, reacted for 8 minutes, and added biotin-labeled magnetic particles after washing (add 100 ⁇ L magnetic particles for those who added anti-CD3 monoclonal antibody) for 8 minutes.
  • T lymphocytes were obtained by immunomagnetic separation using a magnetized cell separator (MACS).
  • MCS magnetized cell separator
  • the dendritic cell monovalent vaccine, dendritic cell multivalent vaccine and normal mature dendritic cells prepared in Example 5 were resuspended in RPMI complete medium, respectively, and the density was adjusted to 2 ⁇ 10 5 cells/mL;
  • the autologous T lymphocyte suspension obtained in step 1 was adjusted to a density of 1.6 ⁇ 10 6 cells/mL with RPMI complete medium. Each group was added 1mL corresponding dendritic cells and T lymphocytes.
  • the content of IL-2 was 1000U/mL
  • the content of IL-12 was 1500U/mL
  • the content of Poly(I:C) was 10mg/mL
  • the content of TNF- ⁇ was 1000U/mL, etc. .
  • the expression of Epstein-Barr virus in LCL cells was detected by real-time fluorescent quantitative PCR, and the detection results were as shown in Figure 6.
  • T lymphocytes Ag-DC group
  • Control group Control group
  • the EB virus load in the medium was significantly lower
  • the EB virus load of the LCL in the Poly-DC group was lower than that of the Ag-DC group in each T cell effect target ratio, indicating that the dendritic cell multivalent vaccine
  • the stimulated T lymphocytes can effectively inhibit the expression of Epstein-Barr virus in LCL cells, kill LCL cells, and inhibit the proliferation of EBV-positive cells.
  • the test results are shown in Figure 7, the T lymphocytes stimulated by dendritic cell monovalent vaccine (Ag-DC group) and the T lymphocytes stimulated by dendritic cell multivalent vaccine (Poly -DC group), both can produce a large amount of interferon ⁇ , which is significantly higher than that of the control group, and the content of interferon ⁇ secreted by T cells in the Poly-DC group is higher than that in the Ag-DC group, indicating that the tree loaded with EBV positive cell lysates
  • the multivalent vaccine of dendritic cells can more strongly stimulate the differentiation of T lymphocytes, secrete interferon gamma, and promote the body's ability to

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Abstract

提供了一种EBV复合抗原、树突状细胞疫苗及其在制备EBV相关感染性疾病药物中的应用，属于生物医药技术领域。通过在体外刺激患者自身的树突状细胞，负载具有针对EBV相关感染性疾病的超强免疫原性的多种EBV感染细胞的裂解物，并在多种细胞因子、特定激动剂情况下诱导成熟，形成完整的带有相应抗原的树突状细胞疫苗，回输到人体激活免疫系统，产生细胞毒性T细胞，杀伤EBV感染细胞，发挥免疫学效应，提高患者生存质量；且树突状细胞疫苗制作周期1周左右，时间短、成本低、安全并且几乎无副作用。

Description

一种EBV复合抗原、树突状细胞疫苗及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种EBV复合抗原、树突状细胞疫苗及其在制备EBV相关感染性疾病药物中的应用。
背景技术
EB病毒(Epstein-Barr virus，EBV)是疱疹病毒科嗜淋巴细胞病毒属的成员，其遗传物质为DNA，长度约为170kb，编码大约100个基因，有重要的衣壳抗原(viral capsid antigen,VCA)、早期抗原(early antigen,EA)和核心抗原(nuclear antigen,NA)的基因。该病毒人群普遍易感，在全世界广泛传播，成人的感染率高达95％，病毒可终身携带，且导致的疾病存在地域性差异。EBV的感染多发生于幼年和青春期，在感染机体后，部分发展为长期的潜伏感染，但也有可能导致人类多种恶性肿瘤和疾病的形成，如Burkitts淋巴瘤(BL)、鼻咽癌(NPC)、霍奇金淋巴瘤、非Hodgkin性淋巴瘤(NHL)、胃癌、乳腺癌和免疫功能缺陷患者发生的肿瘤等以及慢性活动性EBV感染(CAEBV)，EBV相关噬血淋巴组织细胞增生症(EBV-HLH)，传染性单核细胞增多症(IM)等。此外，EBV能专一地在体内或体外感染人及某些灵长类细胞B细胞(近年发现它亦可感染T淋巴细胞、上皮细胞及自然杀伤细胞等，并引发相关疾病)，能刺激感染细胞持续生长并引起细胞无限传代达到“永生(immortalization)”，从而形成淋巴母细胞样细胞系(lymphoblastoid cell lines，LCLs)，这类细胞系常用来研究多种疾病的发生发展，使得大规模长期研究某些疾病的发病机理成为可能。但是目前关于EBV致病机制的理解，还不完全清楚，针对EBV相关疾病尚无安全有效治疗手段。
EB病毒有复制性感染和潜伏感染2种感染方式，在复制性感染期间病毒DNA转录，表达VCA和EA，产生成熟病毒颗粒，并伴宿主细胞的溶解和死亡，这种感染方式主要见于EB病毒感染性疾病如传染性单核细胞增多症。在潜伏感染期VCA和EA的表达受到抑制，主要表达EBNA、EB病毒编码的小RNA(EBV-encoded small RNA,EBER)及潜伏膜蛋白(latent membrane protein,LMP)，不产生新的病毒颗粒，这种感染方式主要见于EB病毒相关恶性肿瘤。EB病毒 潜伏感染的病毒表达产物EB病毒潜伏感染主要表达EBER、EBNA及LMP。EB病毒潜伏感染的细胞有4种不同的基因表达类型，不同的潜伏感染类型与不同的临床恶性肿瘤性疾病有关。其中，Ⅰ型潜伏感染主要出现在地方性Burkitt淋巴瘤的肿瘤细胞中，病毒产物有EBNA-1和EBER；Ⅱ型潜伏感染涉及鼻咽癌和霍奇金病，被感染细胞中出现EBNA-1、LMP-1、LMP-2及EBER等病毒产物；Ⅲ型潜伏感染常见于免疫抑制患者的浆细胞性淋巴瘤细胞，病毒潜伏感染期的6种EBNA、3种LMP和2种EBER均可被检测到；Ⅳ型潜伏感染仅发生在健康的病毒携带者的B淋巴细胞中，这些细胞中含有EBNA-1、LMP-2及EBER-1。
传染性单核细胞增多症(IM)是EB病毒的急性感染性疾病，免疫功能正常的人初次感染EB病毒后，约50％表现为典型的传染性单核细胞增多症。其病理改变是淋巴组织的良性增生，肝、脾、心肌、肾、肾上腺、肺及中枢神经均可受累，表现为异常的淋巴细胞浸润。临床上以发热、咽峡炎、肝大、脾大、淋巴结大及外周血异型淋巴细胞增多为主要表现。该病预后一般良好，病死率为1-2％，患者多死于并发症；但极个别患者病情迁延，反复发作，转变为慢性活动性EB病毒感染。目前认为，T淋巴细胞产生抗EB病毒感染的B淋巴细胞的免疫反应是各种临床症状产生的基础。
噬血细胞综合征可分为原发和继发两类，后者由EB病毒感染引起，被称为EB病毒相关噬血细胞综合征(EBV-HLH)。EB病毒感染使CD8 +T淋巴细胞异常活化和增生，并激活巨噬细胞，导致炎症性细胞因子如干扰素、肿瘤坏死因子、可溶性白细胞介素(interleukin,IL)-2受体、IL-1、IL-6、IL-10和巨噬细胞集落刺激因子等大量产生并释放，产生高细胞因子血症(或称为细胞因子风暴)，引起组织细胞增生并吞噬自身血细胞。病理组织学表现为淋巴细胞和组织细胞在所有器官增生和浸润。该病在病程中以3种方式出现：在传染性单核细胞增多症病程中发生、在慢性活动性EB病毒感染过程中反复发作，以及发生在EB病毒阳性NK/T细胞淋巴瘤患者中。临床表现为高热、肝大、脾大、淋巴结大、全血细胞减少、肝功能异常、乳酸脱氢酶明显增高、三酰甘油明显增高、铁蛋白明显增高、纤维蛋白原降低以及出现弥散性血管内凝血等。淋巴结和骨髓检查的特点是出现红细胞和有核细胞被组织细胞吞噬的现象。本病预后差，临床上暂无安全有效的治疗手段，半数以上患者死亡，临床上很难与恶性组织细胞增多症鉴别。
慢性活动性EB病毒感染目前认为该病是一种异常抗EB病毒抗体增高和EB病毒DNA升高，易发展为淋巴瘤、病毒相关噬血细胞综合征、间质性肺炎、中枢神经系统病变，进而发展为多脏器衰竭的淋巴组织增生性疾病。该病可发生在任何年龄，但主要发生在儿童和青少年中。约50％的患者在发病5年内因为严重的并发症如肝衰竭、心肌炎、冠状动脉瘤、感染相关性噬血细胞综合征和血液系统恶性肿瘤而死亡。EB病毒可累及不同部位各种类型淋巴细胞并克隆增殖，使得该病的临床表现多种多样，持续或间断发热、肝大、脾大和淋巴结大为突出表现，其他尚有咽喉疼痛、淋巴结触痛、贫血、肌肉酸痛、关节痛、牛痘样水疱及蚊虫过敏等，可累及血液系统、中枢神经系统、消化系统及呼吸系统。相关的合并症有噬血细胞综合征、白血病和NK/T细胞淋巴瘤等。目前国际上许多国家和地区仍沿用1988年Straus提出的诊断标准：①EB病毒感染开始症状一直持续6个月以上，EB病毒抗体滴度异常(包括抗VCA-IgG≥1∶5120，抗EA抗体≥1∶640或EBNA抗体＜1∶2)；②主要脏器受损，如导致间质性肺炎、骨髓某成分的不良增生、视网膜炎、淋巴结炎、迁延性肝炎和脾大；③受损组织及外周血中检测到EBV-DNA。随着医疗技术的进步，组织和外周血EB病毒DNA、RNA检测和组织病理学、免疫学等也逐步纳入了指南，但依然是没有积极有效的治疗手段。
此外EB病毒感染与免疫缺陷性疾病还存在一定的联系，如系统性红斑狼疮、多发性硬化症、X-连锁淋巴组织增生性疾病等。系统性红斑狼疮是一种慢性自身免疫系统发炎的疾病，并会影响皮肤、关节、肾、心、肺、神经系统和身体的其他器官，最常见的征兆包括红斑和关节炎，以及疲劳和发烧。多发性硬化是一种免疫介导中枢神经脱髓鞘性疾病，多见于年轻人，可引起中枢神经系统的严重反应，常表现为反复出现的复发缓解病程，使患者病情逐渐加重而致残。X-连锁淋巴组织增生性疾病是一种对EB病毒感染敏感的X连锁联合免疫缺陷性疾病，缺陷的基因主要累及T淋巴细胞和NK细胞，影响这些细胞的信号转导。
有研究表明以上EBV相关疾病，其患者的树突状细胞分化程度较低，数量减少，识别及呈递抗原的能力受损，对初始T细胞的激活能力低下，最终导致机体无法识别和清除肿瘤细胞，EB病毒感染极有可能是导致相关患者的树突状细胞功能受损的原因之一，通过一定技术手段恢复患者树突状细胞的数量和功能， 就能有效治疗上述EBV感染性相关疾病。树突状细胞(Dendritic cells,DC)由2011年诺贝尔医学及生理学奖获得者加拿大科学家Ralph M.Steinman于1973年发现，因其成熟时伸出许多树突样或伪足样突起而得名。DC是目前所知的机体功能最强的专职抗原递呈细胞(Antigen presenting cells,APC)，能高效地摄取、加工处理和呈递抗原，是目前发现的唯一能激活未致敏的初始型T细胞的APC；且未成熟的DC具有较强的迁移和摄取抗原的能力，成熟DC可有效激活初始T细胞，处于启动、调控、并维持免疫应答的中心环节。其数量虽然不足外周血单核细胞的1％，但其表面具有丰富的抗原递呈分子(如MHC-Ⅰ和MHC-Ⅱ)、共刺激因子(CD80/B7-1、CD86/B7-2、CD40等)和粘附因子(ICAM-1、ICAM-2、ICAM-3、LFA-1、LFA-3)等，故DC是一类重要的天然免疫细胞和专职性抗原提呈细胞，在激活机体免疫应答及维持自身免疫耐受过程中发挥着关键性的调控作用。
DC作为最强的抗原呈递细胞，能有效地把抗原信息呈递给T细胞，诱导T细胞活化而导致一系列免疫应答。DC表面的MHC分子能与抗原结合，形成肽-MHC分子复合物，将抗原信号呈递给T细胞，部分树突状细胞高表达的共刺激分子(CD80/B7-1、CD86/B7-2、CD40等)提供T细胞活化所必须的第二信号，同时DC也能直接向CD8 +T细胞呈递抗原肽，在CD4 +T细胞辅助下使CD8 +T细胞活化，活化的DC可以大量分泌IL-12、IL-18、趋化因子(Chemotactic Cytokines，CCK)等激活T细胞增殖，以及启动MHC-I类限制性CTL反应和MHC-Ⅱ类限制性的CD4 +Th1免疫应答；此外，DC还可激活穿孔素P颗粒酶B和FasL/Fas介导的途径增强NK细胞毒作用来增强机体抗肿瘤免疫应答，利于肿瘤清除以及杀伤相关病毒感染细胞。DC本身能作为一种天然的免疫佐剂通过分泌各种细胞因子来提高机体免疫能力，也可增强各种疫苗的免疫反应，通常将带有相关抗原信息的具有疫苗功能的树突状细胞，称为树突状细胞疫苗(DC疫苗)。
疫苗(Vaccine)是用于人体接种的预防或治疗性生物制品，对于预防、治疗、控制传染病的发生和流行具有重要作用。只含有单一的抗原成分的疫苗被称为“单价疫苗”，单价疫苗只能预防一种传染病或一种类型的病原体感染；两种以上抗原成分按照适当比例混合制成的疫苗称为“多价疫苗”或“联合疫苗”。如人乳头瘤病毒有100多个分型，大部分只引起皮肤疣，但有一些型的人乳头瘤 病毒可引起宫颈癌，如二价HPV疫苗只能预防人乳头瘤病毒的16和18，这两型病毒属于“高危型HPV”，70％的宫颈癌由这两型病毒引起；四价HPV疫苗可预防16、18、6和11型HPV感染；九价HPV疫苗可预防16、18、31、33、45、52、58、6和11型。多价疫苗的发展已经有将近百年的历史，早在上世纪30年代，人们即开始了有关多价疫苗的研究。1945年，3价流感疫苗在美国最早获准使用，随后6价肺炎球菌疫苗、白破二联及百白破三联疫苗和3价口服脊髓灰质炎减毒活疫苗等相继问世。临床试验的结果表明，使用多价疫苗联合免疫往往优于使用单价疫苗多次接种。多价疫苗联合免疫时，免疫效果与单价疫苗相似或者更佳，而且未增加疫苗的副反应。
传统的手术、放化疗对病人身体存在一定伤害，过度放化疗会缩短患者的生存时间，长期的药物治疗可能产生依赖性，严重降低患者生存质量，且EBV相关感染性疾病目前在临床上没有安全有效的治疗手段，急需寻找新的治疗药物或方法。与传统的治疗方法相比，免疫治疗由于其疗效显著、副作用小等优势，正逐渐成为新的癌症治疗手段，其中DC疫苗正发挥越来越重要的作用。如专利201911127136.8公开了一种EB病毒相关抗原短肽及其应用，该短肽具有与DC细胞上MHC I类和MHC II分子高度的亲和力，并能有效地使其起到抗原提呈作用，具备良好的多肽疫苗及DC疫苗的潜力。专利202011263782.X则公开了一种EB病毒抗原表位及其应用，其所示的EB病毒抗原表位免疫原性强，通过腺相关病毒载体转染入树突状细胞，抗原基因在树突状细胞中表达，通过直接或交叉提呈的途径将抗原蛋白提呈至T细胞，从而诱导出能够特异性杀伤EB病毒的杀伤性T细胞，在EB抗原阳性疾病的治疗领域具有重要意义。
目前，对于DC疫苗的研究虽然越来越多，但单价DC疫苗治疗疗效往往有限。虽然有不同的抗原类型及组合形式用以治疗各种疾病，但单一的抗原信息容易造成EBV侵染的细胞产生免疫逃逸，引起的免疫反应无法成功对感染细胞造成杀伤，治疗效果有限。另外在体内由于很多异质性的细胞可以分泌多种抑制树突状细胞成熟的细胞因子，使存在于疾病部位的树突状细胞数量相对较少，而缺少强烈肿瘤抗原刺激的树突状细胞诱导的抗肿瘤的免疫反应也无法在宿主体内起到非常显著的治疗效果，因此亟需构建采用肿瘤复合抗原的树突状细胞疫苗来治疗EBV相关感染性疾病。
发明内容
针对现有技术的不足，本发明的目的是提供一种EBV复合抗原、树突状细胞疫苗及其应用。本发明通过在体外刺激患者自身的树突状细胞，负载具有针对不同EBV相关感染细胞的超强免疫原性的多种细胞裂解物(如SNU-719，YCCEL1，GD1，B95-8，M81，HKNPC1-9等不同EB病毒株来源的人永生化B淋巴样母细胞系(LCLs)细胞的裂解物或C666-1，HNE1、CCL85等EBV阳性感染细胞的裂解物)，并在多种细胞因子、特定激动剂诱导下成为成熟的树突状细胞，形成完整的带有相应癌抗原的DC疫苗，回输到人体激活免疫系统，刺激天然免疫(如诱导NK细胞)和刺激淋巴细胞产生获得性免疫反应，产生细胞毒性T细胞杀伤癌细胞，一同精准杀伤EBV感染细胞，实现个性化治疗；相较于放化疗，安全无副作用；且树突状细胞疫苗制备周期为1周左右，时间短，成本低。
为实现上述目的，本发明的技术方案如下：
一方面，本发明提供了一种EBV复合抗原，所述的EBV复合抗原包括EB病毒株来源的人永生化B淋巴样母细胞系细胞裂解物或/和EBV阳性感染细胞裂解物。
具体地，所述的人永生化B淋巴样母细胞系为GD1、B95-8、M81、HKNPC1-9、SNU-719或/和YCCEL1等不同EB病毒株来源的人永生化B淋巴样母细胞系(LCLs)细胞中的一种或多种组合；所述的EBV阳性感染细胞为C666-1、HNE1或/和CCL85及其他EBV感染细胞中的一种或多种组合。
进一步具体地，所述的其他EBV感染细胞为T细胞、NK细胞或B细胞。
另一方面，本发明提供了上述EBV复合抗原在制备树突状细胞疫苗中的应用。
又一方面，本发明提供一种树突状细胞疫苗，所述的树突状细胞疫苗负载有上述EBV复合抗原。
具体地，所述的树突状细胞疫苗为树突状细胞单价疫苗或树突状细胞多价疫苗。
具体地，所述的树突状细胞疫苗负载一种、两种或两种以上的人永生化B淋巴样母细胞系细胞裂解物或/和EBV阳性感染细胞裂解物。
进一步具体地，所述的人永生化B淋巴样母细胞系为GD1，B95-8，M81，HKNPC1-9，SNU-719或/和YCCEL1等不同EB病毒株来源的人永生化B淋巴样母细胞系(LCLs)细胞中的一种或多种组合；所述的EBV阳性细胞为C666-1、HNE1或/和CCL85及其他EBV感染细胞中的一种或多种组合。
具体地，所述的每种细胞的用量为2.5×10 7-2.5×10 9个。
具体地，所述树突状细胞疫苗还包含第一佐剂或其他辅助治疗的细胞因子。
进一步具体地，所述的第一佐剂为PloyI:C，LPS或OK432中任一种；所述的其他辅助治疗的细胞因子为TNF-α或IL-12。
又一方面，本发明提供了上述EBV复合抗原或树突状细胞疫苗在制备预防和/或治疗EBV相关感染性疾病药物中的应用。
具体地，所述的EBV相关感染性疾病包括但不限于传染性单核细胞增多症(IM)、慢性活动性EBV感染(CAEBV)、EBV相关噬血性淋巴组织细胞增生症(EBV-HLH)以及其他EBV相关血液性疾病。
本发明提供的能刺激机体免疫应答来治疗EBV相关感染性疾病的树突状细胞疫苗，特别是针对传染性单核细胞增多症(IM)、慢性活动性EBV感染(CAEBV)、EBV相关噬血性淋巴组织细胞增生症(EBV-HLH)以及其他EBV相关血液性疾病，具有良好疗效，且副作用小，能长期高效抑制EBV相关感染细胞的分裂增殖和减轻疾病进程，甚至达到完全缓解状态。
在一些实施方案的抗原致敏的树突状细胞群属免疫原性组合物，所述树突状细胞群已被相应抗原负载。具体的抗原包含GD1，B95-8，M81，HKNPC1-9，SNU-719，YCCEL1等不同EB病毒株来源的人永生化B淋巴样母细胞系(LCLs)细胞裂解物和C666-1，HNE1，CCL85及其它EBV感染细胞等细胞裂解物，每种细胞具体用量在2.5×10 7-2.5×10 9个。在本发明的另一些方面中，载有EBV复合抗原的树突状细胞疫苗，可以是只负载一种EBV相关感染性疾病的细胞裂解物或LCLs细胞裂解物的树突状细胞单价疫苗，或是负载两种EBV相关感染性疾病的细胞裂解物或LCLs细胞裂解物的树突状细胞多价疫苗，也可同时负载三种，甚至多种EBV相关感染性疾病的细胞裂解物或LCLs细胞裂解物的树突状细胞多价疫苗。
在某些方面，本发明所述的树突状细胞疫苗可以包含第一佐剂(Ploy(I:C)，LPS，OK432等)或其他辅助治疗的细胞因子如TNF-α、IL-12等。树突状细胞疫苗以经静脉内、皮内、肿瘤内、肌肉内、腹膜内、结内、皮下或局部施用被给予3-30次，每次间隔一周或两周；树突状细胞疫苗每次回输细胞量在1×10 6-5×10 8个。
又一方面，本发明提供了一种预防和/或治疗EBV相关感染性疾病的药物，所述的药物包含上述树突状细胞疫苗。
与现有技术相比，本发明的积极和有益效果在于：
树突状细胞疫苗是一种通过激活患者自身的免疫系统达到对抗疾病目的的疫苗。与传统EBV相关感染性疾病的治疗相比较，有以下优点：
1、本发明所述的树突状细胞疫苗治疗EBV相关感染性疾病更加安全。
现在无论手术、化疗还是放疗，在杀死病毒感染细胞或癌细胞的同时都极大伤害病人身体，大大降低患者自身免疫抵抗力；而且由于每个病人肿瘤异质性的不同，所以绝大多数抗癌药，尤其是新一代的靶向药物，都只对一小部分病人有效，且容易产生抗药性，导致癌症复发率很高。相对传统化疗或靶向治疗，树突状细胞疫苗为肿瘤的治疗提供了新的思路，其在体内直接针对的是免疫细胞，通过激活自身免疫系统杀伤癌症细胞，它不会造成直接损伤，反而增强免疫系统；可以抑制癌细胞进化，复发率低，且副作用整体而言显著小于传统的化疗，也小于多靶点的靶向药物，比如树突状细胞多价疫苗作用机制是激活免疫系统，因此最常见的副作用仅为临床I/II级不良反应，如发热、乏力、头晕、全身肌肉酸痛、嗜睡等，对症处理即可，具有良好的临床应用前景。
2、本发明所述的树突状细胞多价疫苗对抗原的选择对于治疗EBV相关感染性疾病更为有效。
目前用DC疫苗治疗EBV相关疾病的项目，多是选用特定的一段多肽作为抗原，而本发明的树突状细胞多价疫苗选用的是多种EBV细胞裂解物，包含激活免疫应答针对癌细胞的全部抗原，最大程度的负载实际人体中的EBV抗原信息，加强了树突状细胞呈递抗原的多样性，最大限度激活人体免疫功能，可诱导更强的T细胞反应，从而提高其治疗效果。
3、本发明所述的树突状细胞多价疫苗所选用的细胞裂解物来源于稳定的EBV细胞系和EBV阳性感染细胞，无需耗时耗力筛选每个患者的EBV抗原，没有HLA限制性，属于通用抗原。此抗原易于制备，工艺简化，整个制备周期仅需1周，较新抗原DC疫苗的制备节约几个月的时间，并可实现EBV裂解物的质量均一，免去了复杂的抗原筛选过程，在降低成本、节约时间方面显示了突出的优势。
4、本发明所述的树突状细胞多价疫苗治疗效果更为持久，能长期有效抑制EBV相关疾病复发和转移。树突状细胞多价疫苗在回输患者体内后可使患者体内产生数量庞大且含有多种EBV抗原信息的记忆T淋巴细胞，其存在时间长达几年至几十年。当再次遇到相应刺激后，可迅速在体内活化，杀伤EBV细胞，有效防止EBV的复发和转移。
附图说明
图1为永生化人B淋巴细胞系LCLs细胞中的EB病毒载量检测结果图。
图2为成熟树突状细胞形态图。
图3为树突状细胞表面标志分子表达量流式图。
图4为树突状细胞IL-12p70的表达量检测结果图。
图5为体外刺激诱导的CTL特异性淋巴细胞杀伤率检测结果图。
图6为共培养后LCL细胞中EB病毒载量检测结果图。
图7为干扰素γ分泌量检测结果图。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：外周血单核细胞PBMC分离
本实施例基于外周血中各细胞成分密度的差异性(外周血主要含有血小板、单个核细胞、粒细胞、红细胞等细胞：血小板密度为1.030-1.035kg/m 3，单个核细胞密度为1.075-1.090kg/m 3，粒细胞密度为1.092kg/m 3，红细胞密度为1.093kg/m 3)，在外周血样品中加入
Figure PCTCN2022099296-appb-000001
Paque Plus(GE Healthcare)溶液(密 度为1.075-1.089kg/m 3)，进行密度梯度离心，使不同细胞成分分层，能快速从人体外周血中分离得到单核细胞。
(1)从EBV感染者的静脉采集外周血，用相应规格的离心管，使用移液管在两个新的离心管中分别加入4.5mL的
Figure PCTCN2022099296-appb-000002
Paque Plus溶液。
(2)使用移液管吸取血样并沿着离心管管壁缓慢地注入Ficoll溶液上层，每管10mL。室温，800g离心20min。
(3)取出离心管，样品分为四层，从上到下依次为血浆、单核细胞、Ficoll溶液、红细胞与粒细胞。
(4)小心吸取单核细胞转移至一个15mL离心管中，加入PBS/1％FBS溶液补齐至14mL，吸打混匀。室温，800g离心5min。
(5)去上清，轻弹管底部使细胞松散，加入14mL PBS/1％FBS溶液重悬细胞吸打混，室温，700g离心5min。
(6)去上清，轻弹管底部使细胞松散。加入14mL RPMI/10％FBS溶液重悬细胞吸打混，室温，400g离心5min。
(7)去除上清，轻弹管底部使细胞松散。加入10mL RPMI/10％FBS溶液重悬细胞，吹打混匀。
(8)吸取10μL细胞液至一个新的1.5mL离心管，加入90μL RPMI/10％FBS溶液稀释10倍；吸取10μL稀释细胞液，加入10μL Trypan Blue染色后加至血球计数板，在倒置显微镜下计数。
(9)室温，700g离心5min，去除上清，加入适量PBS/1％FBS用于后续试验。
实施例2：构建EB病毒株感染的永生化人B淋巴细胞系LCLs
(1)取10mL B95-8细胞上清液转移至离心管中，2000rpm离心15min，过滤上清液。
(2)将实施例1制备的PBMC重悬于2mL RPMI1640/10％FBS培养基中。
(3)吸取10μL细胞液，加入90μL RPMI/10％FBS稀释10倍，在显微镜下进行细胞计数。根据计数结果，计算所需B95-8上清液的体积，其中每1×10 6个PBMC细胞对应1mL B95-8上清液。
(4)收集PBMC细胞，1000rpm离心5min，弃去PBMC上清液。
(5)根据细胞计数结果，加入适量B95-8细胞上清液重悬PBMC细胞，使细胞液中PBMC细胞浓度为0.5×10 6/500μL。
(6)准备一个无菌96孔板，将B95-8重悬的PBMC细胞液以每孔100μL的标准转移至96孔板中。
(7)将96孔板置于CO 2培养箱培养24h。
(8)取出96孔板，在每孔中继续加入100μL R10培养基(RPMI1640/10％FBS，1000IU/mL青霉素，100μg/mL链霉素)吸打混匀。
(9)将96孔板置于培养箱继续培养6天，每天观察细胞状态。观察细胞形态是否呈现淋巴母细胞样改变：细胞体积增大，胞质丰富，呈球状，呈现小集落聚集分布，孔底细胞团明显增大，培养基颜色变黄。
(10)6天培养完成后，每隔3天更换一次培养基。小心地吸取每孔上层培养液弃去，然后每孔加入100μL R10培养基培养孔内细胞，注意细胞液变黄时及时更换培养基，或是根据需要分至新的2-4孔中培养，待细胞数慢慢变多，依次合并转入24孔板，6孔板，T25瓶中。
(11)对达到4周培养时长的细胞进行镜检观察细胞状态，制备得到EB病毒株感染的永生化人B淋巴细胞系LCLs细胞。
同时，分别取正常B细胞和本申请制备得到的EB病毒株感染的永生化人B淋巴细胞系LCLs细胞，通过利用实时荧光定量PCR(Q-PCR)检测本申请制备得到的永生化人B淋巴细胞系LCLs细胞中的EBNA1基因表达情况，来反映EB病毒的载量或表达情况。
细胞EBV病毒载量数(Viral Load,VL)测定：使用MagMAX病毒核酸提取试剂盒(Thermo A42352)和EBV Real-TM Quant试剂盒(Sacace BioTechnologies Srl，Como，意大利)进行DNA提取和PCR聚合酶链反应，用实时定量PCR(EBV Real-TM Quant Kit)对10μL的样品进行EBV定量。本实验选择EBNA1基因编码区作为扩增靶点，β-actin作为内参基因，聚合酶链反应在最终体积为25μL，反应按照说明书操作。其中，引物序列如下：
EBNA1-FP：5’-CCAGACAGCAGCCAATTGTC-3’，如SEQ ID NO:1所示；
EBNA1-RP：5’-GGTAGAAGACCCCCTCTTAC-3’，如SEQ ID NO:2所示；
β-actin-FP：5’-CTCCATCCTGGCCTCGCTGT-3’，如SEQ ID NO:3所示；
β-actin-RP：5’-GCTGTCACCTTCACCGTTCC-3’，如SEQ ID NO:4所示。
EB病毒的载量或表达情况检测结果如图1所示，正常B细胞几乎检测不到EB病毒的表达，而本申请制备得到的永生化人B淋巴细胞系LCLs细胞的EB病毒载量远远高于正常B细胞。
同理，可采用其他EB病毒细胞如GD1，B95-8，M81，HKNPC1-9，SNU-719，YCCEL1等，按照本实施例的步骤制备得到相应的EB病毒株感染的永生化人B淋巴细胞系LCLs细胞，检测其EB病毒载量也远远高于正常B细胞。
实施例3：EBV细胞裂解物的制备
反复冻融法，是一种常用的机械裂解方式，通常由冷冻和解冻两部分组成(freezing and thawing)。原理是由于细胞内冰粒形成和剩余细胞液的盐浓度增高引起溶胀，使细胞结构破碎，使细胞死亡，但是又保留有细胞的免疫原性。冷冻通常在液氮或-20℃冰上进行，解冻可以在37℃、50℃、65℃或100℃水浴中进热休克，比化学裂解温和。
(1)预先设置水浴温度为37℃。
(2)分别收集永生化人B淋巴细胞系LCLs或EBV阳性感染细胞(如C666-1细胞、HNE1、CCL85或其他EBV感染的T细胞、NK细胞或B细胞)(至少3×10 7个)，700g室温离心5min收获细胞。
(3)去除上清液并重悬于RPMI/10％FBS中。
(4)用台盼蓝计数细胞。
(5)700g室温离心5min，缓慢制动，去除上清液。
(6)用RPMI/10％FBS重新悬浮细胞于1mL冻存管中，密度为5×10 6/mL。
(7)将细胞置于液氮中冷冻20s。
(8)立刻将细胞在37℃水浴中快速完全解冻。
(9)重复4次步骤(7)和步骤(8)，共5次。
(10)使用前将EBV细胞裂解物储存在液氮中。
实施例4：未成熟树突状细胞的制备
1、分离获取CD14 +单核细胞
CD14单核细胞的分离提取方法包括但不限于本实施例中采用CD14 +磁珠分选，还可以采用CD14阴选，美天旎免疫磁珠细胞分选(MACS)，细胞贴附等 方法。其原理是基于抗原抗体的特异性结合特点，CD14 +磁珠分选试剂盒可以特异性识别结合PBMC中的CD14 +细胞，并通过生物素或葡聚糖间接与磁珠偶联，在高强度磁场作用下达到CD14 +细胞分离目的。本实施例选用EasySep TMCD14阳选试剂盒。
(1)将PBMC细胞悬液转移至5mL流式管。
(2)在流式管中加入适量selection cocktail溶液至终浓度为100μL/mL。充分吸打混匀，室温孵育10min。
(3)准备磁珠，将RapidSphere TM溶液涡旋30s使磁珠颗粒均匀分散。
(4)在流式管中加入适量RapidSphere TM溶液至终浓度为100μL/mL，充分吸打混匀，室温孵育3min。
(5)在流式管中加入适量PBS/2％FBS with 1mM EDTA溶液至总体积为2.5mL，充分吸打混匀。
(6)将流式管垂直插入EasySep TMmagnet，室温孵育3min。
(7)倒置磁铁，收集流式管流出的细胞液至15mL离心管，保持磁铁倒置状态3s，不要摇晃或吸干管壁上的液体。
(8)将磁铁正置，取出流式管。
(9)重复2次步骤(7)和步骤(8)。
(10)在流式管中加入2mL RPMI/10％FBS重悬细胞，Trypan Blue细胞计数。
2、CD14 +单核细胞诱导产生未成熟树突状细胞
在体外，粒细胞-巨噬细胞集落刺激因子(granμLocyte-macrophage colony stimμLating factor,GM-CSF)能促进未成熟树突状细胞(imDC)的存活，诱导imDC大量增殖。白细胞介素4(Interleukin-4,IL-4)能抑制巨噬细胞的过度生长，降低细胞表面表达CD14分子，诱导CD14 +单核细胞向iDC分化。
(1)在超净工作台内使用移液管吸取CD14 +细胞液转移至六孔板中，每孔2x10 6个/mL，往六孔板中继续加入1μL人重组GM-CSF(终浓度为2000IU/mL，Miltenyi，170-076-112)和1μL人重组IL-4(终浓度为1000IU/mL，Miltenyi，170-076-101)。
(2)将六孔板置于超净工作台台面上，前后左右各轻轻晃动3次使细胞分散均匀。置于细胞培养箱中37℃，5％CO 2培养3d。
(3)从培养箱取出六孔板，在超净工作台内向六孔板中继续加入2mL RPMI1640/10％FBS、1μL人重组GM-CSF(终浓度为2000IU/mL，Miltenyi，170-076-112)和1μL人重组IL-4(终浓度为1000IU/mL，Miltenyi，170-076-101)。
(4)置于细胞培养箱中37℃，5％CO 2培养2d，制备得到未成熟树突状细胞。
实施例5：负载EBV感染细胞裂解物制备树突状细胞多价疫苗
(1)树突状细胞单价疫苗(以B95-8制备得到的永生化人B淋巴细胞系LCLs细胞B95-8-LCL细胞裂解物为例)：
用B95-8-LCL细胞裂解物与未成熟树突状细胞共培养，共培养6小时后，再加入2μL的TNF-α(终浓度为2000IU/mL，Miltenyi，170-076-103)，2μL的LPS(终浓度为2μg/mL，Sigma，L4391)，和1μL的Poly(I:C)(1μg/mL，Sigma，P1530)刺激树突状细胞成熟，制备成树突状细胞单价疫苗，记为Ag-DC；
同理，用GD1-LCL，M81-LCL，HKNPC1-9-LCL，SNU-719-LCL，YCCEL1-LCL，C666-1，HNE1，CCL85或其他EBV感染的T细胞、NK细胞、B细胞的裂解物可制备得到相应的树突状细胞单价疫苗。
(2)树突状细胞多价疫苗：
取EBV感染的B淋巴细胞、T细胞、NK细胞，C666-1，HNE1，CCL85、GD1-LCL、M81-LCL、HKNPC1-9-LCL、SNU-719-LCL、YCCEL1-LCL、或B95-8-LCLs的细胞裂解物等不同的EBV细胞裂解物，分别与树突状细胞共培养6小时后，再加入2μL的TNF-α(终浓度为2000IU/mL，Miltenyi，170-076-103)，2μL的LPS(终浓度为2μg/ml，Sigma，L4391)，和1μL的Poly(I:C)(1μg/mL，Sigma，P1530)刺激树突状细胞成熟，将多个负载有EBV细胞抗原信息的树突状细胞等量混合在树突状细胞培养基中，制备成负载EBV细胞裂解物抗原的树突状细胞多价疫苗，记为Poly-DC(本实施例以EBV感染的B淋巴细胞和B95-8-LCLs细胞裂解物制备的树突状细胞多价疫苗为例)；
(3)观察成熟树突状细胞形态。如图2所示，成熟的树突状细胞培养皿置于光学显微镜(10X物镜)下观察，可以看到成熟树突状细胞贴壁生长，细胞表 面突起增多变长，呈长条放射状，有明显树突状形态，在高密度时多个细胞相互连接，形成网状型结构。
(4)利用流式细胞术检测未成熟树突状细胞与成熟树突状细胞表面分子标志CD11c，CD14，CD40，CD80，CD83，CD86，HLA-DR，HLA-ABC。检测结果如图3所示，成熟树突状细胞其表面CD11c，CD14，CD40，CD80，CD83，CD86，HLA-DR，HLA-ABC等分子表达高于未成熟树突状细胞，呈现高表达，证实树突状细胞已被诱导成熟(Iso代表相应抗体同型对照的流式图谱，imDC代表未成熟树突状细胞表面分子的流式图谱，mDC代表成熟树突状细胞表面分子的流式图谱)。
(5)分别取未成熟树突状细胞与成熟树突状细胞培养上清液，利用ELISA检测树突状细胞IL-12p70的表达。检测结果如图4所示，发现未成熟树突状细胞几乎不分泌IL-12p70，而诱导成为成熟的树突状细胞后，其IL-12p70的表达分泌增强。
实验例1
1、T淋巴细胞制备
(1)将实施例1制备的PBMC置于37℃，5％CO 2培养箱2h后，收集悬浮细胞，制备成1mL细胞悬液；
(2)将细胞悬液加入37℃温育的尼龙毛柱，平放柱体，加入200μL预温的含10％FBS的RPMI 1640，用于封口，37℃静置孵育2h；
(3)用含10％FBS的RPMI 1640清洗尼龙毛柱，流速大约为lmL/min，收集最初冼下的10mL细胞悬液，富含T细胞和NK细胞；
(4)室温700g离心5min，收集底层细胞。计数并调整细胞浓度至1×l0 7个/mL，置于含80IU/mL IL-2的RPMI1640完全培养基中备用。
或者可采用磁珠分离法，利用CD3 +磁珠分离T淋巴细胞。细胞先与抗表面抗原的单抗孵育12min，10 7个细胞用50μL抗CD3单抗，细胞经洗涤后与生物素标记的100μL羊抗鼠二抗孵育10min，洗涤后加入FITC标记的链霉亲和素25μL，反应8min，洗涤后加生物素标记的磁颗粒(加抗CD3单抗者加100μL磁颗粒)反应8min。上述每步反应后，均加入1mL的含1％牛血清白蛋白的PBS 洗涤，2000r/min离心10min。使用磁化细胞分离器(MACS)作免疫磁性分离，得到T淋巴细胞。
2、体外刺激诱导CTL细胞
用RPMI完全培养基分别重悬实施例5制备的树突状细胞单价疫苗、树突状细胞多价疫苗与正常成熟的树突状细胞，分别调整密度到2×10 5个/mL；将上述步骤1分离得到的自体T淋巴细胞悬液，用RPMI完全培养基调整密度到1.6×10 6个/mL。每组各加入1mL相应的树突状细胞和T淋巴细胞。
以上实验组均加入同样的辅助细胞因子，IL-2含量为1000U/mL、IL-12含量为1500U/mL、Poly(I:C)含量为10mg/mL和TNF-α含量为1000U/mL等。在37℃、5％CO 2恒温恒湿培养箱中培养2周后，加入终浓度为30U/mL的IL-2，以及各组再加入2×10 5个相应的树突状细胞疫苗进行二次刺激，继续培养一周，于第21天收集细胞，得到CTL细胞。
3、树突状细胞疫苗刺激的T细胞对EBV感染细胞的杀伤活性检测
将上述步骤2得到的细胞离心后用RPMI1640完全培养基悬浮，调整细胞浓度，分三个不同效靶比实验组，每组分别每孔4×10 5、2×10 5、1×10 5加入96孔培养板作效应细胞；向每孔中加入2×10 4个LCL细胞作为靶细胞，终体积为200μL。同时设置无细胞的空白培养液对照组，均设5复孔。24h后吸去各孔中游离的效应细胞，PBS洗涤2次，每孔各加入含20μL CCK8试剂100μL，继续培养2h，酶标仪检测450nm处吸光度值(OD)，计算特异性淋巴细胞杀伤率(％)。检测结果如图5所示，在体外，接受树突状细胞多价疫苗刺激的T淋巴细胞(Poly-DC组)与只接受负载LCL细胞裂解物的树突状细胞单价疫苗刺激的T淋巴细胞(Ag-DC组)或对照组(Control组)相比，通过检测收集的细胞对LCL细胞的杀伤活性发现，Poly-DC组和Ag-DC组都能有效的杀伤LCL细胞，抑制LCL细胞的增殖，且树突状细胞多价疫苗刺激的T淋巴细胞Poly-DC组在针对LCL细胞时表现出更强的杀伤能力，且T淋巴细胞越多，杀伤效果越明显。
同时通过实时荧光定量PCR检测LCL细胞中EB病毒的表达，检测结果如图6所示，树突状细胞多价疫苗刺激的T淋巴细胞(Poly-DC组)与负载LCL细胞裂解物的树突状细胞单价疫苗刺激的T淋巴细胞(Ag-DC组)或对照组(Control组)相比，通过检测各组LCL细胞的EB病毒载量发现，Poly-DC组 和Ag-DC组的LCL细胞中EB病毒载量相较于对照组明显降低，且Poly-DC组中的LCL在各个T细胞效靶比的EB病毒载量都低于Ag-DC组，说明经树突状细胞多价疫苗刺激的T淋巴细胞能有效抑制LCL细胞中EB病毒的表达，杀伤LCL细胞，抑制EBV阳性细胞的增殖。
4、体外检测干扰素γ的分泌
将上述上述步骤2得到的各组CTL效应细胞和LCL细胞，按照效靶比20：1混合于U型底96孔板中培养72h后，使用干扰素γ酶联免疫试剂盒，按照说明书流程检测培养上清中IFN-γ的含量，检测结果如图7所示，树突状细胞单价疫苗刺激的T淋巴细胞(Ag-DC组)与树突状细胞多价疫苗刺激的T淋巴细胞(Poly-DC组)，均可产生大量干扰素γ，显著高于对照组，且Poly-DC组的T细胞分泌的干扰素γ含量高于Ag-DC组，说明负载有EBV阳性细胞裂解物的树突状细胞多价疫苗能更为强烈地刺激T淋巴细胞的分化，分泌干扰素γ，促进机体的抗EB病毒感染能力。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Figure PCTCN2022099296-appb-000003
Figure PCTCN2022099296-appb-000004
Figure PCTCN2022099296-appb-000005

Claims (10)

  1. 一种EBV复合抗原，其特征在于：所述的EBV复合抗原包括EB病毒株来源的人永生化B淋巴样母细胞系EBV细胞裂解物或/和EBV阳性感染细胞裂解物。
  2. 根据权利要求1所述的EBV复合抗原，其特征在于：所述的人永生化B淋巴样母细胞系为GD1、B95-8、M81、HKNPC1-9、SNU-719或/和YCCEL1中的一种或多种组合；所述的EBV阳性感染细胞为C666-1、HNE1或/和CCL85及EBV感染细胞中的一种或多种组合。
  3. 权利要求1-2任一项所述的EBV复合抗原在制备树突状细胞疫苗中的应用。
  4. 一种树突状细胞疫苗，其特征在于：所述的树突状细胞疫苗负载有权利要求1-2任一项所述的EBV复合抗原。
  5. 根据权利要求4所述的树突状细胞疫苗，其特征在于：所述的EBV复合抗原中每种细胞的用量为2.5×10 7-2.5×10 9个。
  6. 根据权利要求5所述的树突状细胞疫苗，其特征在于：所述树突状细胞疫苗还包含第一佐剂或辅助治疗的细胞因子。
  7. 根据权利要求6所述的树突状细胞疫苗，其特征在于：所述第一佐剂为PloyI:C、LPS或OK432中的任一种；所述的辅助治疗的细胞因子为TNF-α或IL-12。
  8. 权利要求1-2任一项所述的EBV复合抗原或权利要求4-7任一项所述的树突状细胞疫苗在制备预防和/或治疗EBV相关感染性疾病药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于：EBV相关感染性疾病包括但不限于传染性单核细胞增多症、慢性活动性EBV感染、EBV相关噬血性淋巴组织细胞增生症以及EBV相关血液性疾病。
  10. 一种预防和/或治疗EBV相关感染性疾病的药物，其特征在于：所述的药物包含权利要求4-7任一项所述的树突状细胞疫苗。
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