Epstein-Barr virus (EBV) antigen composites and dendritic cell (DC)-based vaccine, and use thereof

Abstract

Epstein-Barr virus (EBV) antigen composites and a dendritic cell (DC)-based vaccine, and a use thereof in preparation of a drug for controlling an EBV-associated infectious disease are provided. Patient-derived DCs are stimulated in vitro, loaded with lysates of various types of EBV-infected cells with strong immunogenicity for EBV-associated infectious diseases, and induced into mature dendritic cells (mDCs) by various cytokines and specific agonists, so as to obtain a complete DC-based vaccine with corresponding antigens. The DC-based vaccine can be injected back into the patient to activate the immune system to produce cytotoxic T cells, thereby killing EBV-infected cells, exerting an immunological effect, and improving a life quality of the patient. In addition, the DC-based vaccine can be prepared in about one week with a low cost, is safe, and shows no obvious side effects.

Claims

1. A method for treating an Epstein-Barr virus (EBV)-associated infectious disease, comprising: administering to a subject a dendritic cell (DC)-based vaccine loaded with EBV antigen composites, wherein the EBV-associated infectious disease comprises infectious mononucleosis (IM), chronic active EBV (CAEBV) infection, and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH); the EBV antigen composites comprise lysates of human immortalized B lymphoblastoid cell lines (B-LCLs) B95-8-LCL, GD1-LCL, M81-LCL, HKNPC1-LCL to HKNPC9-LCL, SNU-719-LCL, YCCEL1-LCL, and lysates of EBV positive infected cells C666-1, HNE1, and EB-3; wherein a method for preparing the DC-based vaccine comprises: (1) induction of immature DC (imDC): transferring a CD14.sup.+ cell suspension to a well plate with 210.sup.6 cells/mL in each well, and then adding human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to a final concentration of 2,000 IU/mL and human recombinant interleukin (IL)-4 to a final concentration of 1,000 IU/mL to the well plate for induction to prepare imDC; (2) induction of mature DC (mDC): co-cultivating the EBV antigen composites with imDC in step (1), adding tumor necrosis factor (TNF)- to a final concentration of 2,000 IU/mL, LPS to a final concentration of 2 g/mL and Poly(I:C) to a final concentration of 1 g/mL to stimulate a maturation of imDC, wherein an amount of cells for producing each of the lysates is 310.sup.7; and (3) detection of induced mDCs: measuring an increase in cell surface markers CD11c, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC in the mDC compared to the imDC by flow cytometry (FCM).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows detection results of EBV loads in human immortalized B-LCLs;

(2) FIG. 2 shows the morphology of mDCs;

(3) FIG. 3 shows flow cytometry (FCM) results of expression levels of surface markers on DCs;

(4) FIG. 4 shows detection results of expression levels of IL-12p70 in DCs;

(5) FIG. 5 shows detection results of killing rates of CTL induced by in vitro stimulation;

(6) FIG. 6 shows detection results of EBV loads in LCLs after cocultivation; and

(7) FIG. 7 shows detection results of secreted IFN-7.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(8) The present disclosure is further described in detail below in conjunction with specific examples, and these examples are not intended to limit the present disclosure and are merely intended to illustrate the present disclosure. The experimental methods used in the examples are conventional, unless otherwise specified; the experimental methods without specified conditions in the examples are generally conducted under conventional conditions; and the materials, agents, or the like used in the examples are generally commercially available, unless otherwise specified.

Example 1. Isolation of PBMCs

(9) In this example, based on density differences among cell components in peripheral blood (the peripheral blood mainly includes platelets, mononuclear cells, granulocytes, and erythrocytes, where a density of the platelets is 1.030 kg/m.sup.3 to 1.035 kg/m.sup.3, a density of the mononuclear cells is 1.075 kg/m.sup.3 to 1.090 kg/m.sup.3, a density of the granulocytes is 1.092 kg/m.sup.3, and a density of the erythrocytes is 1.093 kg/m.sup.3), the Ficoll Paque Plus (GE Healthcare) solution (with a density of 1.075 kg/m.sup.3 to 1.089 kg/m.sup.3) was added to a peripheral blood sample, and then the density gradient centrifugation was conducted, so that different cell components were separated into different layers and thus the mononuclear cells could be quickly isolated from human peripheral blood.

(10) (1) Peripheral blood was collected from a vein of an EBV-infected patient into a centrifuge tube with an appropriate specification, and 4.5 mL of a Ficoll Paque Plus solution was added by a pipette to another two centrifuge tubes, respectively.

(11) (2) A blood sample was slowly added to an upper layer of the Ficoll solution along a wall of the centrifuge tube by a pipette, with 10 mL per centrifuge tube. Then the centrifuge tubes were centrifuged at room temperature and 800 g for 20 min.

(12) (3) The centrifuge tubes were taken out, in which the Ficoll Paque Plus isolated cells top to down, as follows: a plasma layer, a mononuclear cell layer, a Ficoll Paque Plus layer, a granulocyte and erythrocyte layer.

(13) (4) The mononuclear cell layer was carefully transferred to a 15 mL centrifuge tube, a phosphate buffered saline (PBS)/1% fetal bovine serum (FBS) solution was added to 14 mL, and a resulting mixture was pipetted up and down for thorough mixing and then centrifuged at room temperature and 800 g for 5 min.

(14) (5) A resulting supernatant was discarded, a bottom of the centrifuge tube was flicked to loosen cells, then 14 mL PBS/1% FBS solution was added to resuspend the cells, and a resulting mixture was pipetted up and down for thorough mixing and then centrifuged at room temperature and 700 g for 5 min.

(15) (6) A resulting supernatant was discarded, a bottom of the centrifuge tube was flicked to loosen cells, then 14 mL RPMI/10% FBS solution was added to resuspend the cells, and a resulting mixture was pipetted up and down for thorough mixing and then centrifuged at room temperature and 400 g for 5 min.

(16) (7) A resulting supernatant was discarded, a bottom of the centrifuge tube was flicked to loosen the cells, then 10 mL RPMI/10% FBS solution was added to resuspend the cells, and a resulting mixture was pipetted up and down for thorough mixing.

(17) (8) 10 L of a resulting cell suspension was transferred to a 1.5 mL centrifuge tube, and 90 L of an RPMI/10% FBS solution was added to dilute the cell suspension 10-fold. 10 L of a diluted cell suspension was taken and mixed with 10 L of Trypan Blue for staining, and the resulting mixture was added to a hemacytometer and counted under an inverted microscope.

(18) (9) The remaining cell suspension was centrifuged at room temperature and 700 g for 5 min, a resulting supernatant was discarded, and an appropriate amount of PBS/1% FBS was added for subsequent experiments.

Example 2. Construction of Human Immortalized B-LCLs Infected with EBV

(19) (1) 10 mL of a culture supernatant of B95-8 cells was transferred to a centrifuge tube and centrifuged at 2,000 rpm for 15 min, and a resulting supernatant was filtered.

(20) (2) The PBMCs prepared in Example 1 were resuspended in 2 mL of an RPMI1640/10% FBS medium.

(21) (3) 10 L of a resulting cell suspension was taken, diluted 10-fold with 90 L of RPMI/10% FBS, and then counted under a microscope. According to a counting result, a required volume of a filtered B95-8 supernatant was calculated, where every 110.sup.6 PBMCs corresponded to 1 mL of the filtered B95-8 supernatant.

(22) (4) PBMCs were collected and centrifuged at 1,000 rpm for 5 min, and a resulting PBMC supernatant was discarded.

(23) (5) According to a cell counting result, the PBMCs were resuspended with an appropriate amount of the filtered B95-8 supernatant to obtain a cell suspension in which a concentration of the PBMCs was 510.sup.5/500 L.

(24) (6) A sterile 96-well plate was prepared, and the suspension of PBMCs in the filtered B95-8 supernatant was added to the 96-well plate at 100 L/well.

(25) (7) The 96-well plate was incubated in a CO.sub.2 incubator for 24 h.

(26) (8) The 96-well plate was taken out, then 100 L of an R10 medium (RPMI1640/10% FBS, 1,000 U/mL penicillin, and 100 g/mL streptomycin) was added to each well, and a resulting mixture was pipetted up and down for thorough mixing.

(27) (9) The 96-well plate was further incubated in an incubator for 6 d, during which a cell status was observed every day to determine whether the cell status underwent the following lymphoblastoid changes: increased cell volume, enriched cytoplasm, spherical shape, aggregated distribution of small colonies, significantly increased cell masses at a bottom of the well, and yellowing medium.

(28) (10) After the 6-d cultivation was completed, the medium was changed every 3 d. The upper medium in each well was carefully removed, then 100 L of an R10 medium was added to each well, and the cells were further cultivated. When the medium turned yellow, the medium was changed timely, or the cells were dispensed into another 2 to 4 wells as required, and after the number of cells gradually increased, the cells were then combined and transferred to a 24-well plate, a 6-well plate, and a T25 flask in sequence.

(29) (11) A status of cells cultivated for 4 weeks was observed under a microscope, and it could be known that human immortalized B-LCLs infected with EBV were prepared.

(30) Normal B cells and the immortalized human B-LCL infected with EBV prepared in the present disclosure were tested by real-time fluorescent quantitative polymerase chain reaction (Q-PCR) to detect the expression of an EBNA1 gene, thereby reflecting the load or expression level of EBV.

(31) Detection of EBV viral loads (VLs) of cells: DNA was extracted with the MagMAX Viral Nucleic Acid Extraction Kit (Thermo A42352) and subjected to PCR with the EBV Real-TM Quant Kit (Sacace BioTechnologies Srl, Como, Italy), and 10 L of a sample was taken and tested by real-time quantitative PCR (EBV Real-TM Quant Kit) to detect the VLs of EBV. In this experiment, a coding region of the EBNA1 gene was selected as an amplification target, -actin was adopted as an internal reference gene, and the PCR was conducted according to instructions with a final volume of 25 L. Primer sequences were as follows:

(32) TABLE-US-00001 EBNA1-FP: 5-CCAGACAGCAGCCAATTGTC-3,asshowninSEQID NO:1; EBNA1-RP: 5-GGTAGAAGACCCCCTCTTAC-3,asshowninSEQID NO:2; -actin-FP: 5-CTCCATCCTGGCCTCGCTGT-3,asshowninSEQID NO:3; and -actin-RP: 5-GCTGTCACCTTCACCGTTCC-3,asshowninSEQID NO:4.

(33) Detection results of the VLs of EBV were shown in FIG. 1. EBV VLs of the normal B cells was almost undetectable, and EBV VLs of the human immortalized B-LCLs prepared in the present disclosure was much higher than EBV VLs of the normal B cells.

(34) Similarly, other EBV-infected cells such as GD1, B95-8, M81, HKNPC1-9, SNU-719, and YCCEL1 could be used to prepare human immortalized B-LCLs infected with corresponding EBVs according to the steps in this example, these cell lines were tested, and test results showed that EBV VLs of these cell lines were much higher than the EBV VLs of the normal B cells.

Example 3. Preparation of a Lysate of EBV-Infected Cells

(35) The repeated freezing-thawing method is a common mechanical lysis method, which usually consists of freezing and thawing. A principle of the method is as follows: The generation of intracellular ice particles and the increase of a salt concentration in the remaining cell solution cause swelling, so that a cell structure is broken and cells die, but the immunogenicity of the cells is retained. The freezing is usually conducted in liquid nitrogen or at 20 C., and the thawing can be conducted through heat shock in a water bath at 37 C., 50 C., 65 C., or 100 C., which is milder than the chemical lysis.

(36) (1) A temperature of a water bath was pre-set to 37 C.

(37) (2) Human immortalized B-LCL cells or EBV-positive infected cells (such as C666-1, HNE1, EB-3, or other EBV-infected T cells, NK cells, or B cells) (at least 310.sup.7 cells) were collected through centrifugation at room temperature and 700 g for 5 min.

(38) (3) A resulting supernatant was discarded, and resulting cells were resuspended in RPMI/10% FBS to obtain a cell suspension.

(39) (4) The cells were counted with trypan blue.

(40) (5) The cell suspension was centrifuged at room temperature and 700 g for 5 min, and a resulting supernatant was carefully removed.

(41) (6) Resulting cells were resuspended with RPMI/10% FBS in a 1 mL freezing tube, with a density of 510.sup.6/mL.

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

(43) (8) The cells were immediately thawed quickly and completely in a 37 C. water bath.

(44) (9) Steps (7) and (8) were repeated 4 times, that is, steps (7) and (8) were conducted 5 times in total.

(45) (10) A lysate of EBV-infected cells was stored in liquid nitrogen before use.

Example 4. Preparation of imDCs

(46) 1. Isolation of CD14.sup.+ Monocytes

(47) Methods for isolating CD14 monocytes include, but are not limited to, magnetic-activated cell sorting (MACS) in this example, CD14 negative selection and cell attachment. The isolation principle of MACS is based on the specific binding of antigens and antibodies. Human CD14 magnetic beads can specifically recognize and bind to human CD14.sup.+ cells among PBMCs, and the magnetic beads are indirectly coupled with biotin or dextran, so that the CD14.sup.+ cells can be separated under the affection of a high-intensity magnetic field. In this example, the EasySep CD14 positive selection kit was adopted.

(48) (1) A PBMC suspension was transferred to a 5 mL FAC tube.

(49) (2) An appropriate amount of a selection cocktail solution was added to the FAC tube with a final concentration of 100 L/mL, and a resulting mixture was pipetted up and down for thorough mixing and then incubated at room temperature for 10 min.

(50) (3) Preparation of magnetic beads: a RapidSphere solution was vortexed for 30 s, so that the magnetic beads were dispersed evenly.

(51) (4) An appropriate amount of a RapidSphere solution was added to the FAC tube with a final concentration of 100 L/mL, and a resulting mixture was pipetted up and down for thorough mixing and then incubated at room temperature for 3 min.

(52) (5) An appropriate amount of PBS/2% FBS solution with 1 mM EDTA was added to the FAC tube until a total volume was 2.5 mL, and a resulting mixture was pipetted up and down for thorough mixing.

(53) (6) The FAC tube was vertically inserted into the EasySep magnet and incubated for 3 min at room temperature.

(54) (7) A magnet was placed invertedly, and a cell solution flowing out from the FAC tube was collected into a 15 mL centrifuge tube, where the magnet was placed invertedly for 3 s, and the tube should not be shaken or a liquid on a wall of the tube should not be totally removed.

(55) (8) The magnet was placed upright and then the FAC tube was taken out.

(56) (9) Steps (7) and (8) were repeated two times.

(57) (10) 2 mL of RPMI/10% FBS was added to the FAC tube to resuspend cells, and the cells were counted with Trypan Blue.

(58) 2. Induction of CD14.sup.+ Monocytes to Produce imDCs

(59) In vitro, granulocyte-macrophage colony-stimulating factor (GM-CSF) can promote the survival of imDCs and induce the massive proliferation of imDCs. IL-4 can inhibit the overgrowth of macrophages, reduce the expression of CD14 on cells, and induce the differentiation of CD14.sup.+ monocytes into iDCs.

(60) (1) In a clean bench, a CD14.sup.+ cell suspension was transferred by a pipette to each well in a 6-well plate with a concentration of 210.sup.6 cells/mL, and then 1 L of human recombinant GM-CSF (final concentration: 2,000 IU/mL, Miltenyi, 170-076-112) and 1 L of human recombinant IL-4 (final concentration: 1,000 IU/mL, Miltenyi, 170-076-101) were added to the 6-well plate.

(61) (2) The 6-well plate was placed on a surface of the clean bench, then gently shaken three times back and forth and three times left and right to make the cells dispersed evenly, and incubated in a cell incubator at 37 C. and 5% CO.sub.2 for 3 d.

(62) (3) The 6-well plate was taken out from the incubator, and then 2 mL of RPMI1640/10% FBS, 1 L of human recombinant GM-CSF (final concentration: 2,000 IU/mL, Miltenyi, 170-076-112), and 1 L of human recombinant IL-4 (final concentration: 1,000 IU/mL, Miltenyi, 170-076-101) were added to the 6-well plate in a clean bench.

(63) (4) The plate was incubated in an incubator at 37 C. and 5% CO.sub.2 for 2 d to obtain imDCs.

Example 5. Loading of a Lysate of EBV-Infected Cells to Prepare a Multivalent DC-Based Vaccine

(64) (1) Preparation of a Monovalent DC-Based Vaccine (Take Human Immortalized B-LCLs Prepared with B95-8 Named B95-8-LCL as an Example):

(65) imDCs were co-cultivated with the lysate of B95-8-LCL for 6 h, then 2 L of TNF- (final concentration: 2,000 IU/mL, Miltenyi, 170-076-103), 2 L of LPS (final concentration: 2 g/mL, Sigma, L4391), and 1 L of Poly(I:C) (1 g/mL, Sigma, P1530) were added to stimulate the maturation of DCs, and resulting mDCs were prepared into the monovalent DC-based vaccine, which was denoted as Ag-DC.

(66) Similarly, corresponding monovalent DC-based vaccines could be prepared with lysates of GD1-LCL, M81-LCL, HKNPC1-9-LCL, SNU-719-LCL, YCCEL1-LCL, C666-1, HNE1, EB-3, or other EBV-infected T cells, NK cells, and B cells.

(67) (2) Preparation of a Multivalent DC-Based Vaccine:

(68) DCs were co-cultivated with each of lysatates of different EBV-infected cells such as EBV-infected B lymphocytes, T cells, and NK cells, C666-1, HNE1, EB-3, GD1-LCL, M81-LCL, HKNPC1-9-LCL, SNU-719-LCL, YCCEL1-LCL, or B95-8-LCL for 6 h, then 2 L of TNF- (final concentration: 2,000 IU/mL, Miltenyi, 170-076-103), 2 L of LPS (final concentration: 2 g/ml, Sigma, L4391), and 1 L of Poly(I:C) (1 g/mL, Sigma, P1530) were added to stimulate the maturation of DCs, and resulting multiple types of DCs loaded with EBV-infected cell antigen were mixed in equal amounts in a DC medium to obtain the multivalent DC-based vaccine loaded with lysate antigens of EBV-infected cells, which was denoted as Poly-DC (in this example, a multivalent DC-based vaccine prepared with lysates of EBV-infected B lymphocytes and B95-8-LCL cells was taken as an example).

(69) (3) Observation of morphology of mDCs as shown in FIG. 2, a culture dish of mDCs was placed under an optical microscope (10 objective lens) and observed, and it could be seen that the mDCs grew adherently, and had increased long protrusions radially distributed on their surfaces, indicating an obvious dendritic shape; and at a high density, various cells were connected to each other to form a network structure.

(70) (4) Surface markers CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC on imDCs and mDCs were detected by FCM. Detection results were shown in FIG. 3 (where Iso represents an FCM pattern of a corresponding antibody isotype control, imDC represents an FCM pattern of molecules on the surface of imDCs, and mDC represents an FCM pattern of molecules on the surface of mDCs). It can be seen from this figure that expression levels of molecules CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC on the surface of mDCs were higher than that on the surface of imDCs, indicating that imDCs had been induced into mDCs.

(71) (5) Culture supernatants of imDCs and mDCs were collected, and the expression of IL-12p70 secreted by DCs was detected by enzyme-linked immunosorbent assay (ELISA).

(72) Detection results were shown in FIG. 4, and it could be seen from this figure that imDCs secreted almost no IL-12p70, and after imDCs were induced to mature, the secretion of IL-12p70 were enhanced.

Experimental Example 1

(73) 1. Preparation of T Lymphocytes

(74) (1) The PBMCs prepared in Example 1 were cultivated in an incubator at 37 C. and 5% CO.sub.2 for 2 h, and then suspended cells were collected and prepared into 1 mL of a cell suspension.

(75) (2) The cell suspension was added to a nylon wool fiber column incubated at 37 C., the column was laid flat, then 200 L of pre-warmed 10% FBS-containing RPMI 1640 was added for sealing, and then the column was statically incubated at 37 C. for 2 h.

(76) (3) The nylon wool fiber column was subjected to elution with 10% FBS RPMI 1640 at a flow rate of about 1 mL/min, and 10 mL of a cell suspension obtained at the beginning was collected, which contained abundant T cells and NK cells.

(77) (4) The cell suspension was centrifuged at room temperature and 700 g for 5 min, and a resulting cell pellet was collected, counted and adjusted to a concentration of 110.sup.7 cells/mL, and placed in 80 IU/mL IL-2-containing RPMI1640 complete medium for later use.

(78) Alternatively, the magnetic bead separation method could be used, that is, T lymphocytes could be isolated through CD3.sup.+ magnetic beads. Cells were first incubated with a surface antigen monoclonal antibody (mAb) for 12 min (50 L of CD3 mouse mAb was used for every 10.sup.7 cells), then washed and incubated with 100 L of a biotin-labeled goat anti-mouse secondary antibody for 10 min, then washed and incubated with 25 L of FITC-labeled streptavidin for 8 min, and then washed and incubated with 100 L of biotin-labeled magnetic beads for 8 min. After the above reactions were completed, 1 mL of 1% BSA-containing PBS was added for washing, and a resulting mixture was centrifuged at 2,000 r/min for 10 min. T lymphocytes were isolated through immunomagnetic separation of a magnetic cell separator (MACS).

(79) 2. Induction of CTLs Through In Vitro Stimulation

(80) The monovalent DC-based vaccine and the multivalent DC-based vaccine prepared in Example 5 and normal mDCs each were resuspended in an RPMI complete medium, with a cell density adjusted to 210.sup.5 cells/mL. The autologous T lymphocyte suspension isolated in step 1 was adjusted with an RPMI complete medium to a cell density of 1.610.sup.6/mL. 1 mL of each of corresponding DCs and T lymphocytes was added in each group.

(81) The same helper cytokines were added in each of the above experimental groups, with an IL-2 content of 1,000 U/mL, an IL-12 content of 1,500 U/mL, a Poly(I:C) content of 10 mg/mL, and a TNF- content of 1,000 U/mL. Cells were cultivated for 2 weeks in a constant-temperature and constant-humidity incubator at 37 C. and 5% CO.sub.2, IL-2 was added with a final concentration of 30 U/mL, then 210.sup.5 corresponding DCs were added in each group for secondary stimulation, and the cells were further cultivated for one week and harvested on day 21 to obtain CTLs.

(82) 3. Detection of a Killing Activity of T Cells Stimulated by a DC-Based Vaccine for EBV-Infected Cells

(83) The cells obtained in step 2 were centrifuged and resuspended in an RPMI1640 complete medium, a cell concentration was adjusted, and the cells were added as effector cells to a 96-well culture plate at 410.sup.5 cells/well, 210.sup.5 cells/well, and 110.sup.5 cells/well, so as to set three experimental groups with different effector-target ratios. 210.sup.4 LCLs were added as target cells to each well, with a final volume of 200 L. A blank control group without cells was set. Each of the above groups was conducted for 5 repetitive times. 24 h later, free effector cells in each well were removed, the plate was washed twice with PBS, 100 L of a reagent including 20 L of CCK8 was added to each well, and the cells were further cultivated for 2 h. The absorbance (OD) at 450 nm was detected by a microplate reader, and a killing rate (%) of specific lymphocytes was calculated. Detection results were shown in FIG. 5. In vitro, T lymphocytes stimulated by the multivalent DC-based vaccine (Poly-DC group) was compared with T lymphocytes stimulated by the monovalent DC-based vaccine only loaded with a lysate of LCLs (Ag-DC group) or the control group; and in terms of a killing activity of tested cells for LCLs, both the Poly-DC group and the Ag-DC group could effectively kill LCLs and inhibit the proliferation of LCLs, and the poly-DC group of T lymphocytes stimulated by the multivalent DC-based vaccine exhibited a stronger killing ability against LCLs, where the more the T lymphocytes, the more significant the killing effect.

(84) The expression of EBV in LCLs was detected by real-time fluorescent quantitative PCR, and detection results were shown in FIG. 6. T lymphocytes stimulated by the multivalent DC-based vaccine (Poly-DC group) was compared with T lymphocytes stimulated by the monovalent DC-based vaccine loaded with a lysate of LCLs (Ag-DC group) or the control group; and in terms of an EBV load in LCLs in each group, EBV loads in LCLs in the Poly-DC group and the Ag-DC group were significantly lower than that in the control group, and an EBV load in LCLs at each T cell effector-target ratio in the Poly-DC group was lower than that in the Ag-DC group, indicating that the T lymphocytes stimulated by the multivalent DC-based vaccine could effectively inhibit EBV gene expression of LCLs, kill LCLs, and inhibit the proliferation of EBV-positive cells.

(85) 4. In Vitro Detection of IFN- Secretion

(86) The effector CTLs and LCLs in each group obtained in step 2 were mixed in a U-bottom 96-well plate according to an effector-target ratio of 20:1 and cultivated for 72 h, and a content of IFN- in a culture supernatant was detected with an IFN- ELISA kit according to instructions. Detection results were shown in FIG. 7. T lymphocytes stimulated by the monovalent DC-based vaccine (Ag-DC group) and T lymphocytes stimulated by the multivalent DC-based vaccine (Poly-DC group) could produce a large amount of IFN-, and a content of IFN- in each of the two groups was significantly higher than that in the control group; and a content of IFN- secreted by T cells in the Poly-DC group was higher than that in the Ag-DC group, indicating that the multivalent DC-based vaccine loaded with lysates of EBV-positive cells could intensely stimulate the differentiation of T lymphocytes to secrete IFN- and promote the anti-EBV infection ability of the body.

(87) The above examples merely represent several implementations of the present disclosure, and the descriptions thereof are specific and detailed, but these examples should not be construed as limiting the patent scope of the present disclosure. It should be noted that those of ordinary skill in the art can further make several variations and improvements without departing from the concept of the present disclosure, and all of these fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope defined by the claims.