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EBV (Epstein-Barr Virus) composite antigen, dendritic cell vaccine and application thereof

A technology of dendritic cells and complex antigens, applied in the field of biomedicine, can solve the problems of lack of anti-tumor immune response, killing of infected cells, immune escape, etc., and achieve good clinical application prospects, inhibit the evolution of cancer cells, and reduce immune resistance. Effect

Active Publication Date: 2021-10-22
SHANGHAI HENGSAI BIOLOGICAL TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are different antigen types and combinations used to treat various diseases, a single antigen information is likely to cause immune escape of EBV-infected cells, and the resulting immune response cannot successfully kill the infected cells, and the therapeutic effect is limited
In addition, because many heterogeneous cells in the body can secrete a variety of cytokines that inhibit the maturation of dendritic cells, the number of dendritic cells present in the disease site is relatively small, and there is a lack of dendritic cells strongly stimulated by tumor antigens. The induced anti-tumor immune response cannot play a very significant therapeutic effect in the host, so it is urgent to construct a dendritic cell vaccine using tumor complex antigens to treat EBV-related infectious diseases

Method used

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  • EBV (Epstein-Barr Virus) composite antigen, dendritic cell vaccine and application thereof
  • EBV (Epstein-Barr Virus) composite antigen, dendritic cell vaccine and application thereof
  • EBV (Epstein-Barr Virus) composite antigen, dendritic cell vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Peripheral Blood Mononuclear Cells PBMC Isolation

[0049] This embodiment is based on the difference in the density of each cell component in peripheral blood (peripheral blood mainly contains cells such as platelets, mononuclear cells, granulocytes, and red blood cells: the density of platelets is 1.030-1.035kg / m 3 , the mononuclear cell density is 1.075-1.090kg / m 3 , the granulocyte density is 1.092kg / m 3 , red blood cell density is 1.093kg / m 3 ), added in peripheral blood samples Paque Plus (GE Healthcare) solution (density 1.075-1.089kg / m 3 ), performing density gradient centrifugation to stratify different cell components, and can quickly separate mononuclear cells from human peripheral blood.

[0050] (1) Collect peripheral blood from the veins of EBV-infected patients, use centrifuge tubes of corresponding specifications, and use pipettes to add 4.5mL of Paque Plus solution.

[0051] (2) Use a pipette to draw blood samples and slowly inject th...

Embodiment 2

[0059] Embodiment 2: Immortalization human B lymphocyte line LCLs of construction Epstein-Barr virus strain infection

[0060] (1) Transfer 10 mL of B95-8 cell supernatant to a centrifuge tube, centrifuge at 2000 rpm for 15 min, and filter the supernatant.

[0061] (2) Resuspend the PBMC prepared in Example 1 in 2 mL of RPMI1640 / 10% FBS medium.

[0062] (3) Aspirate 10 μL of cell solution, add 90 μL RPMI / 10% FBS to dilute 10 times, and perform cell counting under a microscope. According to the counting results, calculate the required volume of B95-8 supernatant, where every 1×10 6 One PBMC cell corresponds to 1 mL of B95-8 supernatant.

[0063] (4) PBMC cells were collected, centrifuged at 1000 rpm for 5 min, and the PBMC supernatant was discarded.

[0064] (5) According to the cell counting results, add an appropriate amount of B95-8 cell supernatant to resuspend PBMC cells, so that the concentration of PBMC cells in the cell solution is 0.5×10 6 / 500 μL.

[0065] (6) Pr...

Embodiment 3

[0079] Embodiment 3: the preparation of EBV cell lysate

[0080]The repeated freezing and thawing 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.

[0081] (1) Set the water bath temperature to 37°C in advance.

[0082] (2) 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) (at least 3×10 7 ), centrifuged at 700 g for 5 min at room temperature to harvest the cells.

[0083...

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Abstract

The invention discloses an EBV (Epstein-Barr Virus) composite antigen, a dendritic cell vaccine and application of the dendritic cell vaccine in preparation of medicines for treating EBV-related infectious diseases, and belongs to the technical field of biological medicines. According to the invention, dendritic cells of a patient are stimulated in vitro, lysates of a plurality of EBV infected cells with superstrong immunogenicity for EBV-related infectious diseases are loaded, and the lysates are induced to mature under the conditions of a plurality of cytokines and specific agonists, so that a complete dendritic cell vaccine with corresponding antigens is formed; after being transfused back to a human body to activate an immune system, cytotoxic T cells are generated, EBV infected cells are killed, an immunological effect is exerted, and the life quality of a patient is improved; the dendritic cell vaccine has a preparation cycle of about 1 week, is short in time, low in cost, safe and almost free of side effects.

Description

technical field [0001] 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. Background technique [0002] Epstein-Barr virus (EBV) 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 capsidantigen, VCA ), early antigen (early antigen, EA) and core antigen (nuclear antigen, NA) genes. 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. After infecting the body, some develop into long-term latent infection, but it may also lead to the for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245A61K39/39A61K45/06A61P31/22A61P35/00A61P35/04
CPCA61K39/12A61K39/39A61K45/06A61P31/22A61P35/00A61P35/04A61K2039/5154Y02A50/30A61K35/15C12N5/0639A61K39/4622A61K2239/31A61K39/245A61K2239/38A61K39/4644A61K39/4615A61K39/464838
Inventor 刘慧宁印泽
Owner SHANGHAI HENGSAI BIOLOGICAL TECH CO LTD
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