EBV-TARGETED ALLOGENEIC B CELL VACCINE AND PREPARATION METHOD THEREFOR

Abstract

The invention belongs to the field of biotechnology, and relates to an allogeneic B cell vaccine against various human-susceptible viruses and a preparation method therefor. The vaccine has anti-tumor and/or anti-viral preventive and/or therapeutic effects. Specifically, the present invention provides a B cell composition, wherein comprising an allogeneic B cell and a virus antigen, the B cell composition is irradiated with a certain dose of ionizing irradiation. The present invention also provides a B cell vaccine, comprising the above B cell composition. The invention also provides a preparation method for the B cell vaccine and a method and system for improving the antigen presentation ability of the B cell.

Claims

1.-19. (canceled)

20. A preparation method for a B cell composition, comprising the following steps: (1) collecting an allogeneic B cell that is diploid, the allogeneic B cell is subject to immortalization treatment, the immortalization treatment involves infecting the allogeneic B cell with Epstein-Barr virus or an antigen of Epstein-Barr virus; (2) culturing and expanding the allogeneic B cell, the allogeneic B cell carries an antigen of Epstein-Barr virus; (3) irradiating the allogeneic B cells obtained in the step (2) with ionizing irradiation lower than inactivation threshold, wherein the ionizing irradiation is one or more of X-ray, ray, and Co.sup.60 isotope, dose of the ionizing irradiation is about 10-200 Gy, dose rate of the ionizing irradiation is about 2-12 Gy/min; irradiation time is about 200-600 s.

21. The preparation method according to claim 20, wherein culture condition of the step (2) is: 37 C., 5% CO.sub.2, without serum or with serum.

22. The preparation method according to claim 20, wherein seeding density of the allogeneic B cell in the step (2) is 0.5-110.sup.6 cells/ml.

23. The preparation method according to claim 20, wherein number of generation of expansion of the allogeneic B cell in step (2) is greater than 100.

24. (canceled)

25. (canceled)

26. The preparation method according to claim 20, wherein the dose of the ionizing irradiation is about: 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 Gy, 85 Gy, 90 Gy, 95 Gy, 100 Gy, 105 Gy, 110 Gy, 115 Gy, 120 Gy, 125 Gy, 130 Gy, 135 Gy, 140 Gy, 145 Gy, 150 Gy, 155 Gy, 160 Gy, 165 Gy, 170 Gy, 175 Gy, 180 Gy, 185 Gy, 190 Gy, 195 Gy, or 200 Gy.

27. (canceled)

28. The preparation method according to claim 20, wherein the dose rate of the ionizing irradiation is about 3 Gy/min, 3.5 Gy/min, 4 Gy/min, 4.5 Gy/min, 5 Gy/min, 5.5 Gy/min, 6 Gy/min, 6.5 Gy/min, 7 Gy/min, 7.5 Gy/min, 8 Gy/min, 8.5 Gy/min, 9 Gy/min, 9.5 Gy/min, 10 Gy/min, 10.5 Gy/min, 11 Gy/min, 11.5 Gy/min or 12 Gy/min; the irradiation time is about 200 s, 225 s, 250 s, 275 s, 300 s, 325 s, 350 s, 375 s, 400 s, 425 s, 450 s, 475 s, 500 s, 525 s, 550 s, 575 s or 600 s.

29. The preparation method according to claim 20, wherein the allogeneic B cell is a peripheral blood lymphocyte obtained from healthy people.

30. (canceled)

31. The B cell vaccine prepared by the preparation method according to claim 20.

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. (canceled)

37. (canceled)

38. (canceled)

39.-43. (canceled)

44. The B cell composition according to claim 31, wherein an antigen of Epstein-Barr virus is presented on membrane surface of the allogeneic B cell and combined with an antigen-presenting molecule on the membrane surface.

45. The B cell composition according to claim 44, wherein the allogeneic B cell retains biological activity but is incapable of proliferation after being irradiated with the ionizing irradiation.

46. A B cell vaccine, wherein comprising the B cell composition according to claim 31.

47. The B cell vaccine according to claim 46, wherein further comprising a coupling agent and/or an immune adjuvant.

48. The B cell vaccine according to claim 46, wherein dosage form of the B cell vaccine is subcutaneous injection, intradermal injection, local injection, intraperitoneal injection or intravenous injection.

49. Use of the B cell composition according to claim 31 in the preparation of an anti-tumor drug for activating anti-tumor immune response in vivo, wherein the anti-tumor drug is used to treat a tumor carrying Epstein-Barr virus.

50. The use according to claim 49, wherein the tumor carrying Epstein-Barr virus comprises one or more of nasopharyngeal carcinoma, lymphoma, lethal midline granuloma (midline malignant reticulosis), infectious mononucleosis and gastric cancer, the lymphoma comprises Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (lymphoma in African Children), diffuse large B-cell lymphoma and post-transplant lymphoma.

51. The use according to claim 49, wherein the anti-tumor drug is capable of activating an immune cell in a human body, and the immune cell includes T cell.

52. The use according to claim 49, wherein the anti-tumor drug up-regulates expression of related molecules involved in antigen presentation; the related molecules include CD36, SEC61A2, TAP1, SEC61A1, SEC61B or SEC61G.

53. The use according to claim 49, wherein the anti-tumor drug is capable of specifically recognizing an Epstein-Barr virus antigen.

54. Use of the B cell vaccine according to claim 46 in the preparation of an anti-tumor vaccine, wherein the anti-tumor vaccine is used to treat a tumor carrying Epstein-Barr virus.

55. The use according to claim 54, wherein the tumor carrying Epstein-Barr virus comprises one or more of nasopharyngeal carcinoma, lymphoma, lethal midline granuloma (midline malignant reticulosis), infectious mononucleosis and gastric cancer, the lymphoma comprises Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (lymphoma in African Children), diffuse large B-cell lymphoma and post-transplant lymphoma.

Description

DESCRIPTION OF DRAWINGS

[0068] To make the embodiments of the present invention or the technical solutions in the prior art clearer, the drawings required to be used in the description of the embodiments or the prior art will be briefly introduced below. It is obvious that the drawings described below are some embodiments of the present invention, and that other drawings can be obtained from these drawings for those of ordinary skill in the art without making inventive effort.

[0069] FIG. 1 shows a microscopic view of B cells in normal culture.

[0070] FIG. 2 shows a microscopic view of B cells in normal culture.

[0071] FIG. 3 shows a microscopic view of B cells in optimized culture.

[0072] FIG. 4 shows a statistical plot of B cells in optimized culture (horizontal coordinate: time; vertical coordinate: number of cells).

[0073] FIG. 5 shows a karyotype diagram of B cells.

[0074] FIG. 6 shows a statistical plot of live cells after irradiation (horizontal coordinate: time; vertical coordinate: number of cells).

[0075] FIG. 7 shows a statistical plot of total cells after irradiation (horizontal coordinate: time; vertical coordinate: number of cells).

[0076] FIG. 8 shows a statistical plot of antitumor effect of the vaccine (horizontal coordinate: time; vertical coordinate: tumor size).

[0077] FIG. 9 shows photographs of antitumor effect of the vaccine in the mouse model.

[0078] FIG. 10a shows significant changes in APC cross-presentation-related molecules in vaccine cells after irradiation.

[0079] FIG. 10b shows significant changes in APC cross-presentation-related molecules in vaccine cells after irradiation.

[0080] FIG. 11a shows significant changes in endogenous presentation-related molecules in vaccine cells after irradiation; FIG. 11b shows significant changes in endogenous presentation-related molecules in vaccine cells after irradiation.

[0081] FIG. 12a shows significant changes in exogenous presentation-related molecules in vaccine cells after irradiation; FIG. 12b shows significant changes in exogenous presentation-related molecules in vaccine cells after irradiation.

[0082] FIG. 13 shows that vaccine cells prepared on the basis of allogeneic B cells are more efficient in activating immune cells.

DETAILED DESCRIPTION

[0083] To make the objective, the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in combination with drawings. It is obvious that the described embodiments are some of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making inventive effort shall belong to the protection scope of the present invention.

[0084] The embodiments are for the purpose of better illustration of the present invention, but the contents of the present invention are not limited to the embodiments. Therefore, non-essential improvements and adjustments of the embodiments made by those skilled in the art according to the above-mentioned contents of the present invention still belong to the protection scope of the present invention.

[0085] Unless otherwise specified, the term include and comprise and their grammatical variations thereof are used to indicate open-ended or inclusive language, such that they include enumerated technical features but also allow for the inclusion of additional technical features that are not enumerated.

[0086] As used herein, the term about (for example, used for indicating dose rate of ionizing irradiation or irradiation time values), typically means +/5% of the stated value, more typically +/4% of the stated value, more typically +/3% of the stated value, more typically, +/2% of the stated value, even more typically +/1% of the stated value, and even more typically +/0.5% of the stated value.

[0087] Throughout this application, embodiments of this invention may be presented with reference to a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc.; as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0088] Cell immortalization in the present invention refers to a process in which cells are cultured in vitro using genetic changes or various external stimulating factors to avoid the normal senescence and death processes of cells, thereby achieving long-term subculture and infinite division and proliferation. The currently used methods of cell immortalization include the use of radioactive elements, telomere-telomerase activation, and viral gene transfection, etc.

[0089] An allogeneic B cell in the present invention refers to a B cell derived from different individuals of humans or the same species of animals. For example, the B cells described in the present invention are peripheral blood B cells derived from different healthy people. The experimental results of the present invention show that the vaccine derived from allogeneic B cells from different people has the same good safety and anti-tumor immune response as vaccines derived from autologous B cells of a patient, but the vaccine in the present invention has more ways to obtain and is more convenient to prepare.

Embodiment 1

[0090] The characteristics of the vaccine cells of the present invention are as follows: the vaccine is irradiated B cells, most of which are living cells, hardly proliferate, but can stimulate anti-tumor cell immune response.

[0091] Before irradiation, the vaccine cells are immortalized diploid B cells under serum-free culture conditions (normal culture condition is under 20% serum), which can be expanded to reach large quantities and can present EBV and other antigens.

[0092] The B cells used to prepare the vaccine of the present invention can carry EBV antigens in various ways: (i) B cells are the natural host of EBV, if contacting EBV, B cells are infected with EBV which can also make B cells achieve immortalization; (ii) B cells can also be genetically engineered to express specific EBV antigens.

[0093] Before irradiation, B cells were cultured at 37 C., 5% CO.sub.2 under serum-free culture conditions with a cell seeding density of 0.5110.sup.6 cells/ml. B cells mainly grew in clusters in suspension, with a small part grew in a single scattered state. Antenna-like protrusions can be seen on the edge of B cells. See FIG. 1 and FIG. 2 for details.

[0094] B cells are cultured according to the above-mentioned optimized culture conditions provided by the present invention. After culture, the B cells are in a more obvious clonal growth, and can be expanded by more than 100 folds in two weeks. See FIG. 3 and FIG. 4 for details. FIG. 4 is a statistical plot of optimized culture of B cells. After 16 days of culture, the number of cells increased significantly. See Table 1 for details.

TABLE-US-00001 TABLE 1 Statistical table of the number of B cells Culture time (days) Cell number (10.sup.7) 0 1.4 2 2 4 4.2 6 6.4 8 11.47 10 23.57 12 43.76 14 87.9 16 163.9

[0095] Please note that according to actual needs, the culture conditions of B cells before irradiation can also contain a certain amount of serum. In addition, the expansion fold of B cells can also be adjusted, for example, the expansion of more than 40 folds can be followed by irradiation treatment.

Embodiment 2

[0096] Karyotype analysis of B cells for the preparation of the vaccine of the present invention: take cultured B cells (expansion >40 generations, preferably expansion >100 generations) on a glass slide to prepare observation slices, digest with trypsin for 2-3 minutes, and rinse with 0.9% normal saline to stop the enzymatic action of trypsin, then stain with Giemsa stain solution for 10-15 minutes, rinse and dry the slides, and perform karyotype analysis under a microscope to observe whether there are any abnormalities in their morphology. The analyzed B cells were of normal diploid karyotype, and the karyotype of the cells submitted for inspection was: 46, XY. See FIG. 5 for details. In addition, from the perspective of principle action of the vaccine, B cells with a karyotype of 46, XX also have similar immune activation capabilities and can also be used to make vaccine cells of the present invention.

Embodiment 3

[0097] Virus detection of vaccine cells of the present invention: use cell DNA extraction kit to extract B cell DNA or RNA, use real-time fluorescence quantitative PCR to perform hepatitis B virus (HBV-DNA) quantitative detection, hepatitis C virus (HCV-RNA) quantitative detection, human Papillomavirus (HPV16/18-DNA) qualitative detection, human papillomavirus (HPV6/11-DNA) qualitative detection, parvovirus (B19-DNA) qualitative detection, cytomegalovirus (CMV-DNA) quantitative detection and Epstein-Barr virus (EB DNA) quantitative detection. The results showed that the quantitative detection result of HBV-DNA in B cells was <5.00E+02 IU/mL, which was lower than the lowest limit of detection of 5.00E+02 IU/mL; the quantitative detection result of HCV-RNA was <5.00E+02 IU/mL, which was lower than the lowest limit of detection of 5.00E+02 IU/mL; the qualitative detection result of human papillomavirus (HPV16/18-DNA) was negative (); the qualitative test detection of human papillomavirus (HPV6/11-DNA) was negative (); the qualitative detection result of parvovirus (B19-DNA) was negative (); the quantitative detection result of cytomegalovirus (CMV-DNA) was <1.00E+03 IU/mL, which was lower than the lowest limit of detection of 1.00E+03 IU/mL; the quantitative detection result of Epstein-Barr virus (EB DNA) was 6.17E+06 Copies/mL.

[0098] The above detection results show that the B cells used to prepare the present invention do not carry other viruses except EBV.

Embodiment 4

[0099] Preparation of vaccine cells by irradiation, and the characteristics of vaccine cells after irradiation:

[0100] After suspension in the serum-free medium according to the above method, B cells were irradiated using the irradiation instrument RS-2000 Biological Irradiator, and the irradiation conditions were as follows:

[0101] Irradiation dose: Irradiation was carried out at about 10-200 Gy. Specifically, the irradiation dose can be chosen from about 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 Gy, 85 Gy, 90 Gy, 95 Gy, 100 Gy, 105 Gy, 110 Gy, 115 Gy 120 Gy, 125 Gy, 130 Gy, 135 Gy, 140 Gy, 145 Gy, 150 Gy, 155 Gy, 160 Gy, 165 Gy, 170 Gy, 175 Gy, 180 Gy, 185 Gy, 190 Gy, 195 Gy, or 200 Gy.

[0102] Irradiation dose rate: about 3-12 Gy/min. Specifically, the irradiation dose rate is about 3 Gy/min, 3.5 Gy/min, 4 Gy/min, 4.5 Gy/min, 5 Gy/min, 5.5 Gy/min, 6 Gy/min, 6.5 Gy/min, 7 Gy/min, 7.5 Gy/min, 8 Gy/min, 8.5 Gy/min, 9 Gy/min, 9.5 Gy/min, 10 Gy/min, 10.5 Gy/min, 11 Gy/min, 11.5 Gy/min, or 12 Gy/min.

[0103] Irradiation time: about 200-600 s. Specifically, the irradiation time is about 200 s, 225 s, 250 s, 275 s, 300 s, 325 s, 350 s, 375 s, 400 s, 425 s, 450 s, 475 s, 500 s, 525 s, 550 s, 575 s, or 600 s.

[0104] Energy for irradiation: 160 KV (kilovoltage); current intensity for irradiation: 25 mA.

[0105] Other conditions for irradiation: no filter, adding reflector.

[0106] The morphology of B cells was normal after irradiation, and the living cells accounted for about 80%. The number of living cells and the total number of cells remained unchanged after 4-5 days of continuous culture, indicating that B cells had no obvious proliferation ability after irradiation. See FIG. 6 and FIG. 7 for details. Among them, FIG. 6 is a statistical plot of live cells after irradiation, and the specific values are shown in Table 2. FIG. 7 is a statistical plot of total cells after irradiation, and the specific values are shown in Table 3.

TABLE-US-00002 TABLE 2 Statistical table of living cells Culture time (days) Cell number (10.sup.6) 0 3.38 1 3.69 2 3.42 3 3.37 4 3.21

TABLE-US-00003 TABLE 3 Statistical table of total cell number Culture time (days) Cell number (10.sup.6) 0 4.3 1 4.95 2 4.73 3 5.02 4 4.96

[0107] B cells used to prepare the vaccine were irradiated at least once according to the above conditions before cryopreservation. During cryopreservation, cryopreservation solution (BioLife Sulution, USA) was used to freeze vaccine cells (i.e. irradiated cells) at 110.sup.7 cells/ml.

Embodiment 5

[0108] Tumorigenicity assay of vaccine cells: 6-8 weeks old, BALB/c nude mice, females, 10, were used for the experiments, kept in a standard SPF-level animal room, with room temperature maintained at 25 C. and free access to food and water during the experiments. After irradiation, vaccine cells were washed twice with PBS and resuspended in serum-free culture medium to a final concentration of 10.sup.8 /ml. BALB/c nude mice were inoculated subcutaneously with 100 ul of vaccine cell suspension of 110.sup.7 cells/ml. After subcutaneous inoculation of vaccine cells, nude mice were observed weekly for 9 weeks for survival and for tumor formation at the inoculation site. No nude mice had subcutaneous xenograft tumor formation, indicating that the present vaccine cells were non-tumorigenic.

Embodiment 6

[0109] Antitumor effect: well-grown human B cells carrying EBV antigens were taken and irradiated to prepare vaccine cells according to the above experimental parameters, and then the vaccine cells were resuspended with serum-free culture medium to a final concentration of 510.sup.7 /ml, and 200 ul of vaccine cell suspension (110.sup.7 cells) were subcutaneously injected into the groin of BALB/c mice (6-8 weeks old, female) at multiple points for 1, 2, and 4 weeks, respectively. At week 5, BALB/c mice that had been immunized three times were executed, and the spleens were removed after aseptic treatment. Splenic lymphocytes were isolated and resuspended in RPMI-1640 medium at a concentration of 10.sup.8 /ml. 150 ul of spleen lymphocyte suspension (containing 1.510.sup.7 lymphocytes) was injected via tail vein into tumor-bearing mice that had been inoculated with nasopharyngeal carcinoma cells C666-1 (EBV+) for three injections. Tumors were measured every two days, tumor volumes were calculated, and tumor growth curves were plotted.

[0110] The results showed that the tumor growth of mice in the adoptive immune cell therapy group was significantly slower than that of the control group and began to gradually decrease on day 10 after inoculation, and all tumors in the treatment group regressed on day 20. The details are shown in FIGS. 8 and 9. FIG. 8 shows a statistical plot of the anti-tumor effect of the vaccine, and the specific values are shown in Table 4.

TABLE-US-00004 TABLE 4 Statistical table of anti-tumor effect of the vaccine Tumor volume of experimental group (mm.sup.3) Tumor volume of control group (mm.sup.3) time Individual Individual Individual Individual Individual Individual Individual Individual Individual Individual Individual (day) 1 2 3 4 5 6 1 2 3 4 5 8 77.15 15.32 26.64 54.32 32.81 41.43 51.60 44.80 50.10 27.74 20.18 10 72.90 15.74 22.57 55.94 34.33 40.79 99.39 66.93 67.59 31.59 18.78 13 26.02 33.28 25.13 28.68 24.38 25.47 219.16 174.26 193.47 58.75 57.02 15 13.62 11.64 8.29 5.24 6.66 7.98 195.31 200.34 207.06 115.14 61.71 17 9.16 8.72 8.09 4.56 2.35 2.96 201.03 180.91 190.08 123.51 98.17 20 4.22 2.75 8.59 2.14 3.08 2.96 205.20 254.53 253.37 158.41 146.12 23 1.08 5.01 1.00 1.10 0.77 0.75 299.22 317.22 248.69 216.17 196.75

[0111] Please note that the above experiment is different from using vaccine cells to treat cancer patients. In order to test whether human immune cells can activate mouse T cells in the mouse model, and then kill human cancer cells in the mouse model, the vaccine cells of the present invention were first injected into BALB/c mice with a healthy immune system to activate T cells in the BALB/c mice, and then the activated T cells were injected into tumor-bearing mice (with a flawed immune system) that have been inoculated with human tumor cells. In the treatment of a cancer patient with a healthy immune system, the vaccine cells can be injected directly into the patient's body to activate T cells in the patient's body, and then eliminate the tumor/cancer cells (in addition to nasopharyngeal carcinoma, it also includes: various lymphomas (Hodgkin's lymphoma (HD), non-Hodgkin's lymphoma (NHD), Burkitt's lymphoma (lymphoma in African children), diffuse large B-cell lymphoma and post-transplant lymphoma, etc.), lethal midline granuloma (midline malignant reticulosis), infectious mononucleosis, and gastric cancers) cells carrying EBV virus antigens.

[0112] In addition, the above experiment based on the mouse model also shows that although the vaccine cells of the present invention come from human, they can still activate the immune system of micein other words, the vaccine cells of the present invention can function in heterogeneous organisms, then they should also be possible to activate and have an effect in the immune system of allogeneic individuals (different individuals of same species (Homo sapiens)).

[0113] FIG. 13 further shows that vaccine cells prepared based on allogeneic B cells can not only activate T cells in the allogeneic individuals, but even have a better effectin the experiments in FIG. 13, the B cells used to prepare vaccine cells were taken from individual 01, after co-cultivating vaccine cells from 01 with mononuclear cells (PBMC) or lymphocytes (PBL) in the peripheral blood of individuals 02, 03, and 04, the number of activated lymphocytes was significantly higher than that activated in individual 01. See Table 5 for values of positive spots of individuals vaccinated in FIG. 13.

TABLE-US-00005 TABLE 5 Number of positive spots Individual number of spots 01 45 76 58 02 187 191 234 03 127 146 184 04 163 152 168

Embodiment 7

[0114] Antigen Presentation Assay of Irradiated B Cells

[0115] Divide the vaccine cells being cultured into 2 parts, one part of 510.sup.6 vaccine cells was centrifuged at 600 g for 5 minutes, the supernatant was discarded, then resuspended in 1 ml of ice TRIzol, repeated pipetting until the liquid became clear, and then frozen at 80 C. The other one contained 1.510.sup.7 vaccine cells. After -ray irradiation, it's divided into 3 parts on average. Each part contained 510.sup.6 vaccine cells, and continued to culture in the incubator, and centrifuged at 600 g for 5 minutes after 6 hours, 12 hours and 24 hours respectively. The supernatant was discarded, and 1 ml of ice TRIzol was added to resuspend vaccine cells. Perform repeated pipetting until the liquid became clear, and then freeze them at 80 C. The results of RNA-Seq of next-generation high-throughput sequencing of vaccine cells (transport on dry ice) are as follows: [0116] 1) Analyze the changes of B cell transcriptome before and after irradiation by RNA-seq of high-throughput transcriptome sequencing. The related molecules of B cells involved in antigen presentation after irradiation (for example, CD36, SEC61A2, TAP1, SEC61A1, SEC61B, SEC61G) expression levels were up-regulated: except for CD36, the expression levels of other molecules showed up-regulation at the measurement points of 6 hours and 12 hours, and showed a down-regulation trend at the measurement point of 24 hours; expression levels of CD36 showed a trend of gradual up-regulation at the measurement points of 6 hours, 12 hours and 24 hours (FIG. 10). [0117] 2) Flow cytometry detection of changes of MHC-I, MHC-II, CD80, CD86, CD40, CD54 and other antigen presentation-related molecules on the surface of B cells before and after irradiation (FIG. 11, FIG. 12). In FIG. 11, the expression of ASB2-UBE2C molecule showed a downward trend over time; the expression of HLA-F-FBXO44 molecule showed an upward trend over time. In FIG. 12, the expression of KIF11-KIF23 molecule showed a downward trend (but with fluctuations) over time, and the expression of HLA-DMB-HLA-DOA molecule showed an upward trend over time (but the expression level at the measurement point of 24 hours was lower than that at the measurement point of 12 hours). [0118] 3) The expression of intracellular cytokines such as Granzyme B, IFN-, and IL2 of T cells educated by B cells after irradiation detected by Flow cytometry indirectly proves that the function of the educated CTLs (Cytotoxic T Cells). [0119] 4) As shown in FIG. 13, the Elispot assay of the IFN- secretion of T cells indirectly proves that the function of the educated CTLs.

[0120] The above experimental results show that after irradiation, the antigen presentation ability of B cells is improved. Therefore, the present invention can be used to efficiently present not only EBV virus antigens, but also can efficiently present antigens of viruses such as human herpes virus, HIV virus, and hepatitis virus to prepare a B cell-virus antigen composition to activate the immune response against the corresponding virus in vivo and produce a specific antiviral effect.

[0121] B cells have the ability to present antigens, and can present EBV antigens to the cell surface, and exist on the cell membrane in the form of antigen peptide-HLA complexes combined with HLA (Human Leukocyte Antigen) molecules. Although existing studies have shown that B cells transformed by EBV virus can activate T cells in vitro and educate T cells to become CTL cells that can specifically recognize EBV antigens which can produce obvious anti-tumor effects after expansion to reach large quantities and infusion back into tumor patients. However, it is difficult to promote and apply in clinical practice because of cumbersome operation, long cycle and high cost in this process.

[0122] For example, adoptive infusion therapy of CTLs targeting EBV antigens in the clinical trial stage in the prior art includes the in vitro preparation of T lymphocytes (LCL-CTL) that can recognize EBV antigens and then infusion back of the LCL-CTL cells to the patient for treatment. In some clinical trials, the use of LCL-CTL has produced good therapeutic effects, achieving 30-50% complete recover (CR) for relapsed and refractory lymphoma. However, the disadvantages of this treatment are also very obvious: (i) due to infusion back of T cells that are activated in vitro, the whole process takes a long time, and many patients cannot wait for such a long time or lose the therapeutic window; (ii) due to kill EBV-infected cancer cells by infusion back of LCL-CTL, rejection can only be prevented by individualized cell preparation and treatment, and multiple rounds of stimulation are required to generate a sufficient number of CTL cells, which is technically cumbersome; (iii) CTL cells from some patients cannot grow sufficiently to complete the treatment.

[0123] The innovation of the present invention is to use irradiated B cells as a vaccine to immunize patients, and exert the antigen presentation ability of B cells in the human body, thereby activating the immune response in vivo and producing anti-tumor effectsthis avoids the problem of long cycle of in vitro culture, education, and expansion of T cells.

[0124] In addition, although individualized therapy (that is, using the patient's own B cells to prepare the vaccine) can be adopted in the present invention, it is also technically feasible to use allogeneic B cells to prepare the vaccine to educate T cells in the patient's bodybecause the T cells are educated in vivo, human body will naturally not reject the activated T cells in vivo; while the allogeneic B cells may be more conducive to the activation of the human immune system. In fact, in the present invention, allogeneic B cells have a better effect on educating T cells, and will produce better anti-tumor effects. Therefore, the present invention can also be prepared into a standardized cell line, which may have better therapeutic effect, and be feasible to large-scale production.

[0125] It should be noted that the term include, comprise or any variant thereof is intended to encompass nonexclusive inclusion so that a process, method, article or device including a series of elements includes not only those elements but also other elements which have been not listed definitely or an element(s) inherent to the process, method, article or device. Moreover, the expression comprising a(n) . . . in which an element is defined will not preclude presence of an additional identical element(s) in a process, method, article or device comprising the defined element(s) unless further defined.

[0126] The embodiments of the present invention are described above with reference to the accompanying drawings, but the present invention is not limited to the aforementioned specific embodiments. The aforementioned embodiments are merely illustrative and not limiting. For those of ordinary skill in the art, many forms can be made under the teaching of present invention without departing from the spirit of the present invention and the scope of the claims, all of which shall fall within the protection scope of the present invention.