阅读说明：本技术 一种ebv复合抗原、树突状细胞疫苗及其应用 (EBV composite antigen, dendritic cell vaccine and application thereof ) 是由 刘慧宁 印泽 于 2021-07-29 设计创作，主要内容包括：本发明公开了一种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。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。(The invention discloses an EBV composite antigen, a dendritic cell vaccine and application thereof in preparation of medicines for treating EBV-related infectious diseases, and belongs to the technical field of biological medicines. The invention stimulates the dendritic cells of the patient in vitro, loads lysates of various EBV infected cells with super-strong immunogenicity aiming at EBV related infectious diseases, induces and matures under the conditions of various cell factors and specific agonists to form a complete dendritic cell vaccine with corresponding antigens, and the complete dendritic cell vaccine is infused back to a human body to activate an immune system, generate cytotoxic T cells, kill the EBV infected cells, play the immunological effect and improve the survival quality of the patient; and the preparation period of the dendritic cell vaccine is about 1 week, the time is short, the cost is low, the safety is realized, and almost no side effect is caused.)

1. An EBV composite antigen characterized by: the EBV composite antigen comprises EBV cell lysate of a human immortalized B lymphoid blast line derived from EB virus strain or/and EBV positive infected cell lysate.

2. The EBV composite antigen according to claim 1, characterized in that: the human immortalized B lymphoid blast cell line is one or more of GD1, B95-8, M81, HKNPC1-9, SNU-719 or/and YCCEL 1; the EBV positive infected cell is one or more of C666-1, HNE1 or/and CCL85 and EBV infected cell.

3. Use of the EBV complex antigen of any one of claims 1-2 in the preparation of a dendritic cell vaccine.

4. A dendritic cell vaccine, characterized by: the dendritic cell vaccine is loaded with the EBV composite antigen of any one of claims 1-2.

5. The dendritic cell vaccine of claim 4, wherein: the dosage of each cell in the EBV composite antigen is 2.5 multiplied by 10⁷-2.5×10⁹And (4) respectively.

6. The dendritic cell vaccine of claim 5, wherein: the dendritic cell vaccine further comprises a first adjuvant or cytokine for adjuvant therapy.

7. The dendritic cell vaccine of claim 6, wherein: the first adjuvant is any one of PloyI: C, LPS or OK 432; the cell factor for adjuvant therapy is TNF-alpha or IL-12.

8. Use of the EBV complex antigen according to any one of claims 1 to 2 or the dendritic cell vaccine according to any one of claims 4 to 7 for the preparation of a medicament for the prevention and/or treatment of an EBV-associated infectious disease.

9. Use according to claim 8, characterized in that: EBV-associated infectious diseases include, but are not limited to, infectious mononucleosis, chronic active EBV infection, EBV-associated hemophagocytic lymphohistiocytosis, and EBV-associated hematological diseases.

10. A medicament for preventing and/or treating an EBV-associated infectious disease, characterized by: the medicament comprises the dendritic cell vaccine of any one of claims 4-7.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an EBV composite antigen, a dendritic cell vaccine and application thereof in preparation of medicines for treating EBV-related infectious diseases.

Background

The Epstein-Barr virus (EBV) is a member of the genus lymphotropic virus of the family herpesviridae, the genetic material of which is DNA, is about 170kb in length, encodes about 100 genes, and has genes for important capsid antigen (VCA), Early Antigen (EA) and core antigen (NA). The virus is universally susceptible to people and widely spread all over the world, the infection rate of adults is as high as 95%, the virus can be carried for life, and the caused diseases have regional differences. EBV infection is most frequently occurring in young and adolescent stages, and after infecting the body, part of the infection develops long-term latent infection, but it may also result in the formation of various human malignant tumors and diseases, such as Burkitts Lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), gastric cancer, breast cancer, and tumors occurring in patients with immune deficiency, etc., as well as chronic active EBV infection (cae v), EBV-related hemophagocytic lymphocytosis (EBV-HLH), Infectious Mononucleosis (IM), etc. In addition, EBV can specifically infect B cells of human and some primate cells in vivo or in vitro (recently, it is found that it can also infect T lymphocytes, epithelial cells, natural killer cells, etc., and cause related diseases), and can stimulate the infected cells to continuously grow and cause the cells to be infinitely passaged to reach "immortalization" (immortalization), so as to form lymphoblast-Like Cell Lines (LCLs), which are often used for researching the occurrence and development of various diseases, so that the large-scale long-term research of pathogenesis of some diseases becomes possible. However, the understanding of the pathogenic mechanism of EBV is not completely clear, and no safe and effective treatment means exists for the EBV-related diseases.

EB virus has 2 modes of replicative and latent infection, during which viral DNA is transcribed, VCA and EA are expressed, mature viral particles are produced, and host cell lysis and death are accompanied, which is seen mainly in EB virus infectious diseases such as infectious mononucleosis. In the latent infection period, the expression of VCA and EA is inhibited, EBNA, EBV-encoded small RNA (EBER) and Latent Membrane Protein (LMP) are mainly expressed, no new virus particles are generated, and the infection mode is mainly found in EB virus related malignant tumors. The virus expression product of EB virus latent infection, EB virus latent infection mainly expresses EBER, EBNA and LMP. There are 4 different gene expression types in EB virus latently infected cells, and different latently infected types are related to different clinical malignant diseases. Among them, type I latent infection occurs mainly in tumor cells of endemic Burkitt's lymphoma, and the viral products are EBNA-1 and EBER; type II latent infection relates to nasopharyngeal carcinoma and Hodgkin's disease, and virus products such as EBNA-1, LMP-2, EBER and the like appear in infected cells; type III latent infection is common in plasma cell lymphoma cells of immunosuppressed patients, and 6 EBNA, 3 LMP and 2 EBER of virus latent infection period can be detected; latent infection type IV occurs only in B lymphocytes of healthy virus carriers, which contain EBNA-1, LMP-2 and EBER-1.

Infectious Mononucleosis (IM) is an acute infectious disease of EB virus, and about 50% of people with normal immune function show typical infectious mononucleosis after the initial infection of EB virus. The pathological changes are benign hyperplasia of lymphoid tissues, and the liver, spleen, cardiac muscle, kidney, adrenal gland, lung and central nerves can all be affected, and are expressed as abnormal lymphocyte infiltration. Clinically, it is manifested by fever, angina, hepatomegaly, splenomegaly, lymphadenopathy and peripheral blood heteropolylymphocytosis. The disease is generally good in prognosis, the fatality rate is 1-2%, and patients mostly die from complications; however, the disease condition of very individual patients is prolonged and repeated, and the disease is converted into chronic active EB virus infection. It is currently believed that the immune response of T lymphocytes to generate B lymphocytes against infection by the EB virus is the basis for the development of various clinical symptoms.

Hemophagocytic syndrome can be divided into primary and secondary types, the latter being caused by EB virus infection and being called EB virusAssociated hemophagocytic syndrome (EBV-HLH). Infection with EB Virus causes CD8⁺T lymphocytes abnormally activate and proliferate and activate macrophages, leading to the production and release of inflammatory cytokines such as interferon, tumor necrosis factor, soluble Interleukin (IL) -2 receptor, IL-1, IL-6, IL-10, macrophage colony stimulating factor, etc., in large quantities, producing hypercytokinemia (also known as cytokine storm), causing the proliferation of tissue cells and the phagocytosis of autologous blood cells. Histopathology showed proliferation and infiltration of lymphocytes and histiocytes in all organs. The disease occurs in 3 ways during the course of the disease: occurring in 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 hyperpyrexia, hepatomegaly, splenomegaly, lymphadenopathy, pancytopenia, abnormal liver function, obvious increase of lactate dehydrogenase, obvious increase of triacylglycerol, obvious increase of ferritin, reduction of fibrinogen and the appearance of disseminated intravascular coagulation. Lymph node and bone marrow examinations are characterized by the appearance of phagocytosis of red blood cells and nucleated cells by tissue cells. The prognosis of the disease is poor, no safe and effective treatment means exists clinically, more than half of patients die, and the disease is difficult to be clinically distinguished from malignant histiocytosis.

Chronic active epstein-barr virus infection is currently considered to be a lymphoproliferative disease with abnormal anti-epstein-barr virus antibody increase and epstein-barr virus DNA increase, and the disease is easily developed into lymphoma, virus-related hemophagocytic syndrome, interstitial pneumonia and central nervous system lesion, and further into multi-organ failure. The disease can occur at any age, but occurs primarily in children and adolescents. About 50% of patients die within 5 years of morbidity due to serious complications such as liver failure, myocarditis, coronary aneurysm, infection-related hemophagocytic syndrome, and hematologic malignancies. EB virus can affect various types of lymphocytes at different parts and clone and proliferate, so that the clinical manifestations of the disease are various, persistent or intermittent fever, hepatomegaly, splenomegaly and lymph node are prominent manifestations, and other diseases have sore throat, lymph node tenderness, anemia, muscle soreness, arthralgia, cowpox-like blisters and mosquito allergy, and can affect the blood system, the central nervous system, the digestive system and the respiratory system. The related complications are hemophagocytic syndrome, leukemia, NK/T cell lymphoma and the like. Currently, the diagnostic criteria proposed by Straus in 1988 are still used in many countries and regions internationally: the EB virus infection begins to continue for more than 6 months, the antibody titer of the EB virus is abnormal (including that the anti-VCA-IgG is more than or equal to 1: 5120, the anti-EA antibody is more than or equal to 1: 640 or the EBNA antibody is less than 1: 2); ② major organs are damaged, such as interstitial pneumonia, bad hyperplasia of certain bone marrow components, retinitis, lymphadenitis, persistent hepatitis and splenomegaly; and detecting EBV-DNA in the damaged tissues and the peripheral blood. With the progress of medical technology, the DNA and RNA detection and histopathology of EB virus in tissues and periphery, immunology and the like are gradually brought into the guidelines, but still no active and effective treatment means exists.

In addition, EB virus infection has a certain relation with immunodeficiency diseases, such as systemic lupus erythematosus, multiple sclerosis, X-linked lymphoproliferative diseases and the like. Systemic lupus erythematosus is a chronic autoimmune inflammatory disease and affects the skin, joints, kidneys, heart, lungs, nervous system and other body organs, with the most common signs including erythema and arthritis, as well as fatigue and fever. Multiple sclerosis is an immune-mediated demyelinating disease of the central nerve, is mostly seen in young people, can cause severe reaction of the central nervous system, and is often expressed in recurrent relapsing and remitting courses of disease, so that patients are gradually worsened and disabled. The X-linked lymphoproliferative disease is an X-linked combined immunodeficiency disease sensitive to EB virus infection, and defective genes mainly involve T lymphocytes and NK cells and influence signal transduction of the cells.

Research shows that in the EBV related diseases, the dendritic cells of the patients have low differentiation degree, reduced quantity, impaired antigen recognition and presentation capacity and low activation capacity on initial T cells, and finally the organisms cannot recognize and remove tumor cells, EB virus infection is one of the causes for the impaired function of the dendritic cells of the related patients, and the EBV infectious related diseases can be effectively treated by recovering the quantity and the function of the dendritic cells of the patients by a certain technical means. Dendritic Cells (DCs) were named by 2011 nobel medical and physiological prize-taker canadian scientist Ralph m.steinman in 1973 because they mature to protrude a number of Dendritic-like or pseudopodoid processes. DC is the best known professional Antigen Presenting Cells (APC) with the strongest organism function, can efficiently take, process and present Antigen, and is the only APC which can activate unsensitized initial T cells discovered at present; and the immature DC has stronger abilities of transferring and absorbing antigens, and the mature DC can effectively activate initial T cells and is in a central link of starting, regulating and maintaining immune response. Although the number of the antigen is less than 1 percent of that of peripheral blood mononuclear cells, the antigen has abundant antigen presenting molecules (such as MHC-I and MHC-II), costimulatory factors (CD80/B7-1, CD86/B7-2, CD40 and the like) and adhesion factors (ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-3) and the like on the surface, so that the DC is an important natural immune cell and a professional antigen presenting cell and plays a key role in the processes of activating the immune response of the organism and maintaining autoimmune tolerance.

DCs, being the most potent antigen presenting cells, efficiently present antigenic information to T cells, inducing T cell activation leading to a range of immune responses. The MHC molecules on the surface of the DC can be combined with antigen to form a peptide-MHC molecule complex, an antigen signal is presented to T cells, co-stimulatory molecules (CD80/B7-1, CD86/B7-2, CD40 and the like) highly expressed by partial dendritic cells provide a second signal necessary for T cell activation, and the DC can also directly provide CD8 with the second signal⁺T cells present antigenic peptides, at CD4⁺Presentation of CD8 under T cell help⁺T cell activation, activated DC can secrete large amount of IL-12, IL-18, Chemotactic factor (CCK) and the like to activate T cell proliferation, and promote MHC-I restricted CTL response and MHC-II restricted CD4⁺A Th1 immune response; in addition, the DC can also activate the perforin P granzyme B and FasL/Fas mediated pathway to enhance NK cytotoxicity so as to enhance the anti-tumor immune response of the organism, thus being beneficial to tumor removal and killing related virus infected cells. The DC can be used as a natural immunologic adjuvantThe immune response of various vaccines can also be enhanced by secreting various cytokines to improve the immunity of the organism, and dendritic cells with relevant antigen information and vaccine functions are generally called dendritic cell vaccines (DC vaccines).

Vaccines (vaccines) are prophylactic or therapeutic biologics used for vaccination of humans, and are important for preventing, treating, and controlling the occurrence and prevalence of infectious diseases. Vaccines containing only a single antigenic component are known as "monovalent vaccines", which only prevent infection by one infectious disease or one type of pathogen; vaccines prepared by mixing two or more antigen components in a suitable ratio are called "multivalent vaccines" or "combination vaccines". For example, the human papillomavirus has more than 100 types, most of the human papillomavirus only causes skin warts, but some types of the human papillomavirus can cause cervical cancer, for example, a bivalent HPV vaccine can only prevent 16 and 18 types of the human papillomavirus, the two types of the virus belong to high-risk HPV, and 70 percent of cervical cancer is caused by the two types of the virus; tetravalent HPV vaccines can prevent HPV infection of types 16, 18, 6 and 11; the nine-valent HPV vaccine can prevent types 16, 18, 31, 33, 45, 52, 58, 6 and 11. The development of multivalent vaccines has been almost a century old, and as early as the last 30 centuries, studies on multivalent vaccines have begun. In 1945, 3-valent influenza vaccine was first approved for use in the united states, followed by the successive emergence of 6-valent pneumococcal vaccine, diphtheria and pertussis triple vaccines, and 3-valent oral poliomyelitis live attenuated vaccine. The results of clinical trials indicate that the use of multivalent vaccines in combination with immunization often outperforms multiple vaccinations with monovalent vaccines. When the multivalent vaccine is used for combined immunization, the immune effect is similar to or better than that of a monovalent vaccine, and the side effect of the vaccine is not increased.

Traditional operations and chemoradiotherapy have certain harm to the body of a patient, the survival time of the patient can be shortened due to excessive chemoradiotherapy, dependence can be generated due to long-term drug therapy, the life quality of the patient is seriously reduced, and a safe and effective treatment means does not exist in the clinic for treating the EBV-related infectious diseases at present, so that a new treatment medicine or method is urgently needed to be found. Compared with the traditional treatment method, immunotherapy is gradually becoming a new cancer treatment means due to the advantages of significant efficacy, small side effects and the like, wherein the DC vaccine plays more and more important roles. For example, patent 201911127136.8 discloses an EB virus-associated antigen short peptide and its application, wherein the short peptide has high affinity with MHC class I and MHC class II molecules on DC cells, and can effectively provide antigen presentation effect, and has potential of good polypeptide vaccine and DC vaccine. Patent 202011263782.X discloses an EB virus antigen epitope and application thereof, the EB virus antigen epitope shown by the EB virus antigen epitope has strong immunogenicity, the antigen gene is transfected into dendritic cells through an adeno-associated virus vector, the antigen gene is expressed in the dendritic cells, and antigen protein is presented to T cells through a direct or cross presentation way, so that killer T cells capable of specifically killing EB virus are induced, and the EB virus antigen epitope has important significance in the field of treatment of EB antigen positive diseases.

Currently, although there is an increasing research on DC vaccines, monovalent DC vaccines tend to have limited therapeutic efficacy. Although there are different types and combinations of antigens used to treat various diseases, a single antigenic message can easily cause the immune escape of EBV infected cells, the resulting immune response cannot successfully kill the infected cells, and the therapeutic effect is limited. In addition, in vivo, many heterogeneous cells can secrete a plurality of cytokines for inhibiting dendritic cell maturation, so that the number of dendritic cells existing in a disease part is relatively small, and the dendritic cell-induced anti-tumor immune response lack of strong tumor antigen stimulation cannot play a very significant therapeutic effect in a host, so that the construction of a dendritic cell vaccine adopting a tumor complex antigen for treating the EBV-related infectious disease is urgently needed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an EBV composite antigen, a dendritic cell vaccine and application thereof. The invention stimulates the dendritic cells of a patient in vitro, loads various cell lysates with super-strong immunogenicity aiming at different EBV related infected cells (such as lysates of human immortalized B lymphoid blast line (LCLs) cells of different EB virus strain sources such as SNU-719, YCCEL1, GD1, B95-8, M81, HKNPC1-9 and the like or lysates of EBV positive infected cells such as C666-1, HNE1, CCL85 and the like), becomes mature dendritic cells under the induction of various cytokines and specific agonists, forms a complete DC vaccine with corresponding cancer antigens, is infused back to a human body to activate an immune system, stimulates natural immunity (such as NK cells) and lymphocytes to generate an acquired immune response, generates cytotoxic T cells to kill cancer cells, and simultaneously kills EBV infected cells accurately to realize personalized treatment; compared with radiotherapy and chemotherapy, the preparation is safe and has no side effect; and the preparation period of the dendritic cell vaccine is about 1 week, the time is short, and the cost is low.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect, the invention provides an EBV complex antigen comprising a lysate of human immortalized B lymphoid blast line cells derived from an EB virus strain or/and a lysate of EBV-positively infected cells.

Specifically, the human immortalized B lymphoid mother cell line is one or more combinations of human immortalized B lymphoid mother cell line (LCLs) cells from different EB virus strains such as GD1, B95-8, M81, HKNPC1-9, SNU-719 or/and YCCEL 1; the EBV positive infected cell is one or more of C666-1, HNE1 or/and CCL85 and other EBV infected cells.

Further specifically, the other EBV-infected cells are T cells, NK cells or B cells.

On the other hand, the invention provides the application of the EBV composite antigen in preparing dendritic cell vaccines.

In still another aspect, the present invention provides a dendritic cell vaccine loaded with the EBV complex antigen described above.

Specifically, the dendritic cell vaccine is a dendritic cell monovalent vaccine or a dendritic cell multivalent vaccine.

Specifically, the dendritic cell vaccine is loaded with one, two or more than two human immortalized B lymphoid blast line cell lysates or/and EBV positive infection cell lysates.

More specifically, the human immortalized B lymphoid mother cell line is one or more combinations of human immortalized B lymphoid mother cell line (LCLs) cells from different EB virus strains such as GD1, B95-8, M81, HKNPC1-9, SNU-719 or/and YCCEL 1; the EBV positive cells are one or more combinations of C666-1, HNE1 or/and CCL85 and other EBV infected cells.

Specifically, the amount of each cell is 2.5X 10⁷-2.5×10⁹And (4) respectively.

In particular, the dendritic cell vaccine further comprises a first adjuvant or other therapeutic-aiding cytokine.

Further specifically, the first adjuvant is any one of PloyI, C, LPS or OK 432; the other auxiliary treatment cytokine is TNF-alpha or IL-12.

In another aspect, the invention provides an application of the EBV composite antigen or the dendritic cell vaccine in preparing a medicament for preventing and/or treating EBV-related infectious diseases.

Specifically, the EBV-associated infectious diseases include, but are not limited to, Infectious Mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-associated hemophagocytosis lymphohistiocytosis (EBV-HLH), and other EBV-associated hematologic diseases.

The dendritic cell vaccine capable of stimulating the immune response of an organism to treat the EBV-related infectious diseases, which is provided by the invention, particularly has good curative effect on Infectious Mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-related hemophagocytic lymphohistiocytosis (EBV-HLH) and other EBV-related blood diseases, has small side effect, can effectively inhibit the division and proliferation of EBV-related infected cells and alleviate the disease process for a long time, and even achieves the complete remission state.

In some embodiments, the antigen-primed dendritic cell population is an immunogenic composition, the dendritic cell population having been loaded with a corresponding antigen. The specific antigen comprises cell lysates of human immortalized B lymphoid blast line (LCLs) cell lysate and C666-1, HNE1, CCL85 and other EBV infected cells from different EB virus strains such as GD1, B95-8, M81, HKPPC 1-9, SNU-719 and YCCEL1, and the specific dosage of each cell is 2.5 v10⁷-2.5×10⁹And (4) respectively. In other aspects of the present invention, the dendritic cell vaccine loaded with the EBV complex antigen may be a dendritic cell monovalent vaccine loaded with only one cell lysate or LCLs cell lysate of the EBV-related infectious disease, or a dendritic cell multivalent vaccine loaded with two cell lysates or LCLs cell lysates of the EBV-related infectious disease, or a dendritic cell multivalent vaccine loaded with three or even more cell lysates or LCLs cell lysates of the EBV-related infectious disease at the same time.

In certain aspects, the dendritic cell vaccines of the present invention may comprise a first adjuvant (Ploy (I: C), LPS, OK432, etc.) or other adjunctive therapeutic cytokines such as TNF- α, IL-12, etc. The dendritic cell vaccine is administered 3-30 times, each one or two weeks apart, intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, intranodal, subcutaneously, or topically; the cell amount of the dendritic cell vaccine is 1 multiplied by 10 per time of back transfusion⁶-5×10⁸And (4) respectively.

In still another aspect, the present invention provides a medicament for preventing and/or treating an EBV-associated infectious disease, the medicament comprising the above dendritic cell vaccine.

Compared with the prior art, the invention has the advantages that:

a dendritic cell vaccine is a vaccine that serves the purpose of combating disease by activating the patient's own immune system. Compared with the traditional treatment of the EBV related infectious diseases, the method has the following advantages:

1. the dendritic cell vaccine provided by the invention is safer to treat EBV-related infectious diseases.

At present, no matter surgery, chemotherapy or radiotherapy, virus infected cells or cancer cells are killed, simultaneously, the body of a patient is greatly injured, and the autoimmune resistance of the patient is greatly reduced; moreover, due to the heterogeneity of tumors among patients, most anticancer drugs, especially the new generation of targeted drugs, are effective only in a small number of patients, and are prone to drug resistance, resulting in high recurrence rate of cancer. Compared with the traditional chemotherapy or targeted therapy, the dendritic cell vaccine provides a new idea for the treatment of tumors, the dendritic cell vaccine directly aims at immune cells in vivo, and the cancer cells are killed and killed by activating the autoimmune system, so that the dendritic cell vaccine can not cause direct damage but can enhance the immune system; the traditional Chinese medicine composition can inhibit the evolution of cancer cells, has low recurrence rate, has side effects which are obviously smaller than those of the traditional chemotherapy and are smaller than that of multi-target targeted medicines on the whole, for example, the dendritic cell multivalent vaccine has an action mechanism of activating an immune system, so that the most common side effects are only clinical grade I/II adverse reactions, such as fever, hypodynamia, dizziness, general muscle soreness, somnolence and the like, can be used for treating symptoms, and has good clinical application prospect.

2. The dendritic cell multivalent vaccine provided by the invention is more effective in treating EBV related infectious diseases by selecting the antigen.

The DC vaccine is used for treating EBV related diseases at present, a specific section of polypeptide is mostly selected as an antigen, and the dendritic cell multivalent vaccine of the invention selects various EBV cell lysates, contains all antigens for activating immune response aiming at cancer cells, loads EBV antigen information in an actual human body to the maximum extent, strengthens the diversity of presented antigens of dendritic cells, activates the immune function of the human body to the maximum extent, can induce stronger T cell reaction, and thus improves the treatment effect.

3. The cell lysate selected by the dendritic cell multivalent vaccine is derived from a stable EBV cell line and EBV positive infected cells, the EBV antigen of each patient does not need to be screened with time and labor, and the dendritic cell multivalent vaccine has no HLA limitation and belongs to a universal antigen. The antigen is easy to prepare, the process is simplified, the whole preparation period only needs 1 week, the preparation time of the new antigen DC vaccine is saved by several months compared with the preparation of the new antigen DC vaccine, the quality uniformity of EBV lysate can be realized, the complex antigen screening process is omitted, and the obvious advantages are shown in the aspects of cost reduction and time saving.

4. The dendritic cell multivalent vaccine has a more lasting treatment effect, and can effectively inhibit the recurrence and transfer of EBV related diseases for a long time. The dendritic cell multivalent vaccine can generate a large number of memory T lymphocytes containing various EBV antigen information in a patient body after being returned to the patient body, and the existence time of the memory T lymphocytes can reach several years to several decades. When meeting the corresponding stimulation again, the medicine can be quickly activated in vivo to kill EBV cells, and the recurrence and metastasis of EBV are effectively prevented.

Drawings

FIG. 1 is a graph showing the result of detecting EB virus load in LCLs of an immortalized human B lymphocyte cell line.

FIG. 2 is a morphology of mature dendritic cells.

FIG. 3 is a flow chart showing the expression level of dendritic cell surface marker molecules.

FIG. 4 is a graph showing the results of measuring the expression level of IL-12p70 in dendritic cells.

FIG. 5 is a graph showing the results of the CTL-specific lymphocyte killing rate measurement induced by in vitro stimulation.

FIG. 6 is a graph showing the results of EB virus load detection in LCL cells after co-culture.

FIG. 7 is a graph showing the results of measurement of the secretion amount of interferon-. gamma..

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Example 1: peripheral blood mononuclear cell PBMC isolation

This example is based on the difference in the density of each cellular component in peripheral blood (peripheral blood mainly contains cells such as platelets, mononuclear cells, granulocytes, erythrocytes, etc.: the density of platelets is 1.030-1.035kg/m³The density of the mononuclear cells is 1.075-1.090kg/m³The density of the granulocytes is 1.092kg/m³The density of red blood cells is 1.093kg/m³) Adding into peripheral blood samplePaque Plus (GE Healthcare) solution (density 1.075-1.089 kg/m)³) Performing density gradient separationThe core can separate different cell components into layers, and can rapidly separate the mononuclear cells from the peripheral blood of a human body.

(1) Peripheral blood was collected from veins of EBV-infected patients, and 4.5mL of each of the blood was added to each of two new centrifuge tubes using a pipette with the corresponding centrifuge tube sizePaque Plus solution.

(2) The blood sample was pipetted and the top layer of Ficoll solution was slowly injected along the tube wall of the centrifuge tube at 10mL per tube. Centrifuge at 800g for 20min at room temperature.

(3) Taking out the centrifuge tube, dividing the sample into four layers, and sequentially preparing plasma, mononuclear cells, Ficoll solution, red blood cells and granulocytes from top to bottom.

(4) The monocytes were carefully pipetted into a 15mL centrifuge tube, filled to 14mL with PBS/1% FBS solution, pipetted and mixed well. Centrifuge at 800g for 5min at room temperature.

(5) Remove supernatant, flick the bottom of the tube to loosen the cells, add 14mL PBS/1% FBS solution to resuspend the cells, pipette and mix, centrifuge at room temperature at 700g for 5 min.

(6) Remove supernatant and flick the bottom of the tube to loosen the cells. Add 14mL RPMI/10% FBS solution heavy suspension cell suction and mix, room temperature, 400g centrifugation for 5 min.

(7) The supernatant was removed and the bottom of the tube was flicked to loosen the cells. Add 10mL RPMI/10% FBS solution to resuspend the cells, and blow and mix well.

(8) Pipetting 10. mu.L of cell sap into a new 1.5mL centrifuge tube, and adding 90. mu.L of RPMI/10% FBS solution to dilute 10 times; 10 μ L of diluted cell liquid was aspirated, 10 μ L of Trypan Blue was added to the diluted cell liquid for staining, and the diluted cell liquid was added to a hemocytometer and counted under an inverted microscope.

(9) Centrifuge at 700g for 5min at room temperature, remove supernatant and add appropriate amount of PBS/1% FBS for subsequent experiments.

Example 2: construction of immortalized human B-lymphocyte cell lines LCLs infected with EB Virus strains

(1) 10mL of B95-8 cell supernatant was transferred to a centrifuge tube, centrifuged at 2000rpm for 15min, and the supernatant was filtered.

(2) The PBMCs prepared in example 1 were resuspended in 2mL RPMI 1640/10% FBS medium.

(3) 10 μ L of cell fluid was aspirated, diluted 10-fold with 90 μ L RPMI/10% FBS, and cell counts were performed under a microscope. From the counting results, the volume of the supernatant of B95-8 required was calculated every 1X 10⁶Each PBMC cell corresponds to 1mL of B95-8 supernatant.

(4) PBMC cells were collected, centrifuged at 1000rpm for 5min and PBMC supernatant discarded.

(5) According to the cell counting result, adding a proper amount of B95-8 cell supernatant to resuspend the PBMC cells so that the concentration of the PBMC cells in the cell fluid is 0.5X 10⁶/500μL。

(6) A sterile 96-well plate was prepared and the resuspended PBMC cell fluid from B95-8 was transferred to the 96-well plate at a rate of 100. mu.L per well.

(7) Place 96-well plate in CO₂The incubator is used for 24 h.

(8) The 96-well plate was removed and 100. mu. L R10 of medium (RPMI 1640/10% FBS, 1000IU/mL penicillin, 100. mu.g/mL streptomycin) was added to each well and pipetted well.

(9) The 96-well plate was placed in an incubator and cultured for another 6 days, and the cell state was observed every day. Observing whether the cell morphology exhibits lymphoblastoid changes: the cell volume is increased, the cytoplasm is rich and spherical, the small colony is gathered and distributed, the cell mass at the bottom of the hole is obviously increased, and the color of the culture medium turns yellow.

(10) After 6 days of culture, the medium was changed every 3 days. Carefully sucking the culture solution on the upper layer of each well and discarding, then adding 100 mu L R10 culture medium into each well to culture the cells in the wells, timely replacing the culture medium when the cell fluid turns yellow, or separating the cells into new 2-4 wells to culture according to the needs, and combining and transferring the cells into a 24-well plate, a 6-well plate and a T25 bottle in sequence when the cell number gradually increases.

(11) And (4) performing microscopic examination on the cells cultured for 4 weeks to observe the cell state, and preparing the immortalized human B lymphocyte cell line LCLs infected by the EB virus strain.

Meanwhile, normal B cells and the EBNA1 gene expression condition in the immortalized human B lymphocyte cell line LCLs cells infected by the EB virus strain prepared by the method are respectively taken, and the EBNA1 gene expression condition in the immortalized human B lymphocyte cell line LCLs cells prepared by the method is detected by using real-time fluorescent quantitative PCR (Q-PCR), so that the load or the expression condition of the EB virus is reflected.

Cell EBV Viral Load number (Viral Load, VL) assay: DNA extraction and PCR polymerase chain reaction were performed using MagMAX Virus nucleic acid extraction Kit (Thermo A42352) and EBV Real-TM Quant Kit (Sacace Biotechnologies Srl, Como, Italy), and EBV quantification was performed on 10. mu.L of the samples using Real-time quantitative PCR (EBV Real-TM Quant Kit). In the experiment, the coding region of the EBNA1 gene is selected as an amplification target, the beta-actin gene is selected as an internal reference gene, the final volume of the polymerase chain reaction is 25 mu L, and the reaction is operated according to the instruction. Wherein the primer sequences are as follows:

EBNA 1-FP: 5'-CCAGACAGCAGCCAATTGTC-3', as shown in SEQ ID NO: 1;

EBNA 1-RP: 5'-GGTAGAAGACCCCCTCTTAC-3', as shown in SEQ ID NO: 2;

beta-actin-FP: 5'-CTCCATCCTGGCCTCGCTGT-3', as shown in SEQ ID NO. 3;

beta-actin-RP: 5'-GCTGTCACCTTCACCGTTCC-3', as shown in SEQ ID NO. 4.

The test results of the load or expression condition of the EB virus are shown in figure 1, the expression of the EB virus can not be detected by normal B cells, and the EB virus load of the immortalized human B lymphocyte line LCLs prepared by the method is far higher than that of the normal B cells.

Similarly, other EB virus cells such as GD1, B95-8, M81, HKNPC1-9, SNU-719, YCCEL1 and the like can be adopted to prepare the corresponding EBV strain-infected immortalized human B lymphocyte cell line LCLs according to the steps of the embodiment, and the EB virus load is detected to be much higher than that of normal B cells.

Example 3: preparation of EBV cell lysate

The repeated freeze-thaw method, which is a common mechanical lysis method, generally consists of freezing and thawing two parts (freezing and thawing). The principle is that the formation of ice granules in cells and the increase of salt concentration of residual cell sap cause swelling, break the cell structure and kill the cells, but the immunogenicity of the cells is remained. Freezing is usually carried out on liquid nitrogen or ice at-20 deg.C, and thawing can be carried out by heat shock in a water bath at 37 deg.C, 50 deg.C, 65 deg.C or 100 deg.C, which is milder than chemical lysis.

(1) The temperature of the water bath was previously set to 37 ℃.

(2) Separately collecting LCLs or EBV positive infected cells (such as C666-1 cells, HNE1, CCL85 or other EBV-infected T cells, NK cells or B cells) (at least 3 × 10)⁷One), 700g cells were harvested by centrifugation at room temperature for 5 min.

(3) The supernatant was removed and resuspended in RPMI/10% FBS.

(4) Cells were counted using trypan blue.

(5) Centrifuging at 700g for 5min at room temperature, slowly braking, and removing supernatant.

(6) Resuspend cells in 1mL cryovial with RPMI/10% FBS at a density of 5X 10⁶/mL。

(7) The cells were frozen in liquid nitrogen for 20 s.

(8) Cells were immediately thawed rapidly and completely in a 37 ℃ water bath.

(9) Repeating the steps (7) and (8) 4 times for 5 times.

(10) EBV cell lysates were stored in liquid nitrogen prior to use.

Example 4: preparation of immature dendritic cells

1. Separation of CD14⁺Monocyte cell

The separation and extraction method of CD14 monocyte includes, but is not limited to, the method using CD14 in the present embodiment⁺The magnetic bead sorting can also adopt methods such as CD14 negative sorting, Meitian and whirly immunomagnetic bead cell sorting (MACS), cell attachment and the like. The principle is based on the specific binding characteristics of antigen-antibody, CD14⁺The magnetic bead sorting kit can specifically recognize and bind to CD14 in PBMC⁺Cells are indirectly coupled with magnetic beads through biotin or glucan and reach CD14 under the action of a high-intensity magnetic field⁺Cell isolation purposes. EasySep is selected for use in this example^TMCD14 positive selection kit.

(1) The PBMC cell suspension was transferred to a 5mL flow tube.

(2) An appropriate amount of selection cocktail solution was added to a flow tube to a final concentration of 100. mu.L/mL. Fully sucking, uniformly mixing and incubating at room temperature for 10 min.

(3) Preparing magnetic beads, and mixing RapidSphere^TMThe solution was vortexed for 30s to uniformly disperse the magnetic bead particles.

(4) Adding a proper amount of RapidSphere into a flow pipe^TMThe solution was brought to a final concentration of 100. mu.L/mL, pipetted thoroughly and mixed, and incubated at room temperature for 3 min.

(5) An appropriate amount of PBS/2% FBS with 1mM EDTA solution was added to the flow tube to a total volume of 2.5mL, and the mixture was thoroughly pipetted and mixed.

(6) Vertical insertion of flow tube into EasySep^TMmagnet, incubate for 3min at room temperature.

(7) The magnet was inverted and the flow tube effluent cell fluid was collected into a 15mL centrifuge tube, keeping the magnet inverted for 3s without shaking or blotting the fluid on the tube wall.

(8) The magnet is placed right and the flow tube is taken out.

(9) Repeating the step (7) and the step (8) for 2 times.

(10) 2mL of RPMI/10% FBS-resuspended cells were added to the flow tube and Trypan Blue cells were counted.

2、CD14⁺Monocyte induction generation of immature dendritic cells

In vitro, granulocyte-macrophage colony stimulating factor (GM-CSF) can promote the survival of immature dendritic cells (imdcs) and induce the mass proliferation of imdcs. Interleukin-4 (IL-4) can inhibit excessive growth of macrophage, reduce cell surface expression CD14 molecule, and induce CD14⁺Monocytes differentiate towards idcs.

(1) Pipette off CD14 in clean bench⁺Cell sap was transferred to six-well plates at 2 × 10 per well⁶Mu.l of human recombinant GM-CSF (final concentration of 2000IU/mL, Miltenyi, 170-.

(2) Placing the six-hole plate on the surface of a superclean workbench, and slightly shaking the six-hole plate for 3 times respectively to disperse and homogenize the cellsAnd (4) homogenizing. Placing in a cell culture box at 37 deg.C and 5% CO₂And (5) culturing for 3 d.

(3) The six well plates were removed from the incubator and 2mL of RPMI 1640/10% FBS, 1. mu.L of human recombinant GM-CSF (final concentration 2000IU/mL, Miltenyi, 170-.

(4) Placing in a cell culture box at 37 deg.C and 5% CO₂And culturing for 2d to prepare the immature dendritic cells.

Example 5: preparation of dendritic cell multivalent vaccine by loading EBV infected cell lysate

(1) Dendritic cell monovalent vaccine (taking B95-8-LCL cell lysate of immortalized human B lymphocyte cell line LCLs prepared by B95-8 as an example):

co-culturing the immature dendritic cells with B95-8-LCL cell lysate, and after 6 hours of co-culture, adding 2. mu.L of TNF-alpha (final concentration of 2000IU/mL, Miltenyi, 170-076-one 103), 2. mu.L of LPS (final concentration of 2. mu.g/mL, Sigma, L4391), and 1. mu.L of Poly (I: C) (1. mu.g/mL, Sigma, P1530) to stimulate the dendritic cells to mature, thus preparing a dendritic cell monovalent vaccine, which is marked as Ag-DC;

similarly, corresponding dendritic cell monovalent vaccines can be prepared by using GD1-LCL, M81-LCL, HKPC 1-9-LCL, SNU-719-LCL, YCCEL1-LCL, C666-1, HNE1, CCL85 or other EBV infected T cells, NK cells and B cell lysates.

(2) Dendritic cell multivalent vaccine:

taking EBV-infected B lymphocytes, T cells, NK cells, cell lysates of C666-1, HNE1, CCL85, GD1-LCL, M81-LCL, HKNPC1-9-LCL, SNU-719-LCL, YCCEL1-LCL, or B95-8-LCLs and other different EBV cell lysates, co-culturing the EBV cell lysates with dendritic cells for 6 hours, respectively, then adding 2 uL of TNF-alpha (final concentration of 2000IU/mL, Miltenyi, 170-, as Poly-DC (this example takes the dendritic cell polyvalent vaccine prepared from EBV-infected B lymphocytes and B95-8-LCLs cell lysate as an example);

(3) and observing the morphology of the mature dendritic cells. As shown in FIG. 2, when the mature dendritic cell culture dish was observed under an optical microscope (10X objective lens), adherent growth of the mature dendritic cells was observed, and the number of protrusions on the cell surface increased and became longer, and the cell surface appeared in a long radial shape with a distinct dendritic form, and a plurality of cells were connected to each other at a high density to form a network-like structure.

(4) The molecular markers of the surface molecules of the immature dendritic cells and the mature dendritic cells, namely CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR and HLA-ABC, are detected by flow cytometry. As shown in FIG. 3, the molecular expressions of the mature dendritic cells, such as CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR and HLA-ABC, are higher than those of the immature dendritic cells, and high expression is shown, so that the dendritic cells are proved to be induced to mature (Iso represents the flow pattern of corresponding antibody isotype control, imDC represents the flow pattern of the surface molecules of the immature dendritic cells, and mDC represents the flow pattern of the surface molecules of the mature dendritic cells).

(5) Immature dendritic cells and culture supernatants of mature dendritic cells were separately collected and tested for expression of IL-12p70 by ELISA. As shown in FIG. 4, it was found that the immature dendritic cells hardly secreted IL-12p70, and that the expression and secretion of IL-12p70 were enhanced after the cells were induced to mature dendritic cells.

Experimental example 1

1. T lymphocyte preparation

(1) PBMC prepared in example 1 were incubated at 37 ℃ with 5% CO₂After the incubator is used for 2 hours, collecting suspension cells and preparing 1mL of cell suspension;

(2) adding the cell suspension into a nylon hair column incubated at 37 ℃, flatly placing the column, adding 200 mu L of pre-warmed RPMI1640 containing 10% FBS for sealing, and standing and incubating for 2h at 37 ℃;

(3) washing nylon wool column with 10% FBS-containing RPMI1640 at about lmL/min, collecting 10mL of initially washed cell suspension rich in T cells and NK cells;

(4) centrifuge at 700g for 5min at room temperature and collect the bottom layer cells. Count and adjust cell concentration to 1 xl 0⁷one/mL of the cells were put in RPMI1640 complete medium containing 80IU/mL of IL-2 for use.

Alternatively, magnetic bead separation method using CD3 can be used⁺Magnetic beads isolate T lymphocytes. Incubating the cells with monoclonal antibody against surface antigen for 12min, 10⁷The cells are washed by 50 mu L of anti-CD 3 monoclonal antibody, incubated with 100 mu L of goat anti-mouse secondary antibody labeled by biotin for 10min, washed, added with 25 mu L of FITC-labeled streptavidin and reacted for 8min, and washed, added with biotin-labeled magnetic particles (added with 100 mu L of anti-CD 3 monoclonal antibody) and reacted for 8 min. After each reaction, 1mL of PBS containing 1% bovine serum albumin was added for washing, and the mixture was centrifuged at 2000r/min for 10 min. T lymphocytes were obtained by immunomagnetic separation using a magnetized cell separator (MACS).

2. In vitro stimulation and induction of CTL cells

The dendritic cell monovalent vaccine, the dendritic cell multivalent vaccine and the normal mature dendritic cells prepared in example 5 were resuspended in RPMI complete medium, and the density was adjusted to 2X10⁵Per mL; adjusting the density of the autologous T lymphocyte suspension obtained in the step 1 to 1.6X 10 by using RPMI complete medium⁶one/mL. 1mL of the corresponding dendritic cells and T lymphocytes were added to each group.

The same helper cytokines were added to each of the above experimental groups, with an IL-2 content of 1000U/mL, an IL-12 content of 1500U/mL, a Poly (I: C) content of 10mg/mL, a TNF-. alpha.content of 1000U/mL, and so on. At 37 deg.C, 5% CO₂After 2 weeks in a constant temperature and humidity incubator, IL-2 was added to a final concentration of 30U/mL, and 2X10 was added to each group⁵And (3) performing secondary stimulation on the corresponding dendritic cell vaccine, continuing to culture for one week, and collecting cells on day 21 to obtain CTL cells.

3. Detection of killing activity of T cells stimulated by dendritic cell vaccine on EBV infected cells

Centrifuging the cells obtained in step 2, suspending the cells in RPMI1640 complete medium, and adjusting the cellsConcentration, divided into three experimental groups with different effective target ratios, each group is respectively 4 multiplied by 10 per hole⁵、2×10⁵、1×10⁵Adding 96-well culture plate as effector cell; adding 2X10 to each well⁴One LCL cell served as the target cell with a final volume of 200. mu.L. Meanwhile, blank culture solution control groups without cells are arranged, and 5 multiple holes are arranged. After 24h, absorbing free effector cells in each well, washing with PBS for 2 times, adding 100 mu L of CCK8 reagent containing 20 mu L into each well, continuously culturing for 2h, detecting the absorbance value (OD) at 450nm by using an enzyme-labeling instrument, and calculating the specific lymphocyte killing rate (%). As shown in fig. 5, in vitro, compared to T lymphocytes (Ag-DC group) or Control group (Control group) which were stimulated by dendritic cell multivalent vaccine, the T lymphocytes (Poly-DC group) which were stimulated by dendritic cell multivalent vaccine only receive LCL cell lysate-loaded dendritic cell monovalent vaccine, it was found that both the Poly-DC group and Ag-DC group can effectively kill LCL cells and inhibit proliferation of LCL cells by detecting killing activity of collected cells on LCL cells, and the T lymphocytes Poly-DC group stimulated by dendritic cell multivalent vaccine showed stronger killing ability against LCL cells, and the more T lymphocytes, the more killing effect.

Meanwhile, the expression of EB virus in LCL cells is detected through real-time fluorescent quantitative PCR, the detection result is shown in figure 6, compared with T lymphocytes stimulated by dendritic cell multivalent vaccine (Poly-DC group) and dendritic cell monovalent vaccine loaded with LCL cell lysate (Ag-DC group) or a Control group (Control group), EB virus load of LCL cells in the Poly-DC group and Ag-DC group is obviously reduced compared with the Control group through detecting EB virus load of LCL cells in each group, and EB virus load of LCL in each T cell effective target ratio in the Poly-DC group is lower than that in the Ag-DC group, so that the T lymphocytes stimulated by the dendritic cell multivalent vaccine can effectively inhibit the expression of EB virus in the LCL cells, kill the LCL cells and inhibit the proliferation of EBV positive cells.

4. In vitro detection of secretion of interferon gamma

And (3) mixing the CTL effector cells and the LCL cells obtained in the step 2 according to an effective target ratio of 20: 1, mixing the mixture in a U-shaped bottom 96-well plate, culturing for 72 hours, and detecting the content of IFN-gamma in culture supernatant by using an interferon gamma enzyme linked immunosorbent assay kit according to the flow of a specification, wherein the detection result is shown in figure 7, T lymphocytes (Ag-DC group) stimulated by dendritic cell monovalent vaccine and T lymphocytes (Poly-DC group) stimulated by dendritic cell multivalent vaccine can both generate a large amount of interferon gamma which is obviously higher than that of a control group, and the content of interferon gamma secreted by the T cells of the Poly-DC group is higher than that of the Ag-DC group, which indicates that the dendritic cell multivalent vaccine loaded with EBV positive cell lysate can more strongly stimulate the differentiation of the T lymphocytes, secrete the interferon gamma and promote the anti-EB virus infection capacity of an organism.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

Sequence listing

<110> Shanghai Hengsai Biotechnology Ltd

<120> EBV composite antigen, dendritic cell vaccine and application thereof

<130> 20210713

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

ccagacagca gccaattgtc 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

ggtagaagac cccctcttac 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

ctccatcctg gcctcgctgt 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

gctgtcacct tcaccgttcc 20