EBV tumor vaccine and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses an EBV (Epstein-Barr Virus) tumor vaccine and application thereof. The invention discloses a novel EBV vaccine antigen combination for the first time. The novel EBV vaccine antigen combination comprises BZLF1, EBNA1, EBNA3B and gHgL. The applicant carries out antigen modification on BZLF1, EBNA1 and EBNA3B, and safety modification is completed while antigen T cell epitopes are retained. A humanized mouse proves that the antigen combination has good safety, immunity and protection effects, and EBV infected cells can be killed by activating BZLF1, EBNA1 and EBNA3B antigen specific T cells, so that EBV tumors are effectively prevented. The vaccine of the antigen combination can be used for preventing EBV related tumors, such as lymphoma, lymphatic proliferative diseases after transplantation, nasopharynx cancer and gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an EBV tumor vaccine and application thereof. Background Art

[0002] Epstein-Barr virus (EBV) is a widely spread human virus that is closely related to the occurrence of a variety of malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, etc. It is also associated with infectious mononucleosis and multiple sclerosis. Although most people can carry the virus for a long time without symptoms under the control of the immune system after the initial infection with EBV, the persistent infection of EBV in the host may lead to the inability of the immune system to completely eliminate the virus, thus providing conditions for the virus to induce malignant transformation. Especially in individuals with impaired or suppressed immune function, such as patients receiving organ transplants or stem cell transplants, EBV reactivation will significantly increase the risk of lymphoproliferative diseases such as lymphoma.

[0003] According to statistics, there are about 200,000 new cases of EBV-related tumors each year in the world, including 78,100 cases of nasopharyngeal carcinoma, 28,600 cases of Hodgkin's lymphoma, 6,700 cases of Burkitt's lymphoma, 84,050 cases of gastric cancer, etc. Although EBV infection will bring a serious burden to the global medical and health system, there is still no effective vaccine and drug successfully marketed. Therefore, it is extremely important to develop a successful EBV vaccine to prevent tumors caused by EBV infection. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide an antigen composition.

[0005] The second aspect of the present invention aims to provide biological materials related to antigen compositions.

[0006] The purpose of the third aspect of the present invention is to provide the use of the antigen composition of the first aspect of the present invention or the biological material of the second aspect of the present invention in the preparation of a product.

[0007] The fourth aspect of the present invention aims to provide a product.

[0008] The fifth aspect of the present invention aims to provide a vaccine.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides an antigen composition, which comprises at least one of BZLF1, EBNA1, EBNA3B or gHgL;

[0011] Among them, the amino acid sequence of BZLF1 is as shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar function.

[0012] In some embodiments of the present invention, the amino acid sequence of EBNA1 is as shown in SEQ ID NO: 2, or an amino acid sequence of SEQ ID NO: 2 that is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0013] In some embodiments of the present invention, the amino acid sequence of EBNA3B is as shown in SEQ ID NO: 3, or an amino acid sequence shown in SEQ ID NO: 3 that is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0014] In some embodiments of the present invention, the amino acid sequence of gHgL is as shown in SEQ ID NO: 4, or an amino acid sequence shown in SEQ ID NO: 4 that is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0015] The antigens in the above-mentioned antigen composition have all undergone a certain degree of modification (BZLF1 and EBNA1 are carcinogenic, and their safety is improved while retaining their T cell epitopes to the maximum extent. The modifications are as follows: deleting the glycine-alanine repeat sequence, deleting the nuclear localization signal and mutating the amino acids to eliminate their potential effects; for EBNA3B, part of the nuclear localization signal is deleted; the full length of gL is connected to the full length of the extracellular domain of gH through (GGGGS)3). The modified antigen composition has good safety and immunogenicity, and can effectively prevent the occurrence of tumors caused by EBV infection.

[0016] The second aspect of the present invention provides a biological material related to the antigen composition, wherein the biological material is any one of a1) to a12):

[0017] a1) a nucleic acid molecule encoding the antigenic composition of the first aspect of the present invention;

[0018] a2) an expression cassette containing the nucleic acid molecule described in a1);

[0019] a3) a recombinant vector containing the nucleic acid molecule described in a1);

[0020] a4) a recombinant vector containing the expression cassette described in a2);

[0021] a5) a recombinant microorganism containing the nucleic acid molecule described in a1);

[0022] a6) a recombinant microorganism containing the expression cassette described in a2);

[0023] a7) a recombinant microorganism containing the recombinant vector described in a3);

[0024] a8) a recombinant microorganism containing the recombinant vector described in a4);

[0025] a9) a transgenic animal cell line containing the nucleic acid molecule described in a1);

[0026] a10) a transgenic animal cell line containing the expression cassette described in a2);

[0027] a11) a transgenic animal cell line containing the recombinant vector described in a3);

[0028] a12) a transgenic animal cell line containing the recombinant vector described in a4);

[0029] The transgenic animal cell line contains no reproductive material.

[0030] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0031] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a non-viral vector.

[0032] In some embodiments of the present invention, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0033] In some embodiments of the present invention, the non-viral vector comprises a plasmid vector.

[0034] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0035] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0036] The third aspect of the present invention provides use of the antigen composition of the first aspect of the present invention or the biomaterial of the second aspect of the present invention in preparing a product.

[0037] In some embodiments of the present invention, the product includes at least one of a drug (such as a vaccine), a reagent (such as a detection reagent, a diagnostic reagent), and a kit.

[0038] In some embodiments of the present invention, the function of the product is any one of (1) to (6):

[0039] (1) Prevent and / or treat EBV infection-related diseases;

[0040] (2) Preparation of EB virus antibodies;

[0041] (3) detecting BZLF1, EBNA1, EBNA3B and / or gHgL in EBV in the sample;

[0042] (4) Diagnosis of EB virus-specific neutralizing antibodies;

[0043] (5) triggering humoral-cellular immune responses;

[0044] (6) Inhibit tumor growth.

[0045] In some embodiments of the present invention, the EBV infection-related disease includes at least one of EBV-related B-cell lymphoma, nasopharyngeal carcinoma, chronic active EBV infection, hemophagocytic syndrome, EBV infection after hematopoietic stem cell transplantation or organ transplantation, lymphoproliferative disease and EBV-related gastric cancer.

[0046] In some embodiments of the present invention, the EBV infection specifically infects epithelial cells or B lymphocytes; preferably B lymphocytes.

[0047] The fourth aspect of the present invention provides a product, which includes the antigen composition of the first aspect of the present invention or the biological material of the second aspect of the present invention.

[0048] In some embodiments of the present invention, the product includes at least one of a drug, a reagent, and a kit.

[0049] In some embodiments of the present invention, when the product is a drug, the product further comprises a pharmaceutically acceptable excipient.

[0050] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an absorption promoter, a surfactant, an antioxidant, an excipient, a stabilizer, a flavoring agent, a sustained-release agent, a metal complexing agent, an inert gas, a preservative, a filler, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

[0051] In some embodiments of the present invention, the diluent includes but is not limited to: at least one of starch, sugar, cellulose, and inorganic salts. The wetting agent includes but is not limited to: at least one of water, ethanol, and propanol. The adhesive includes but is not limited to: at least one of starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol. The disintegrant includes but is not limited to: at least one of microcrystalline cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked povidone, surfactants (such as sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate, etc.), and effervescent disintegrants. The lubricant includes but is not limited to: at least one of talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micropowder silica gel, calcium carbonate, sodium bicarbonate, and polyethylene glycol. The color and flavor regulator includes but is not limited to: at least one of pigments, flavors, sweeteners, mucilage agents, and deodorants. The solvent includes but is not limited to: at least one of water, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, and vegetable oil (such as soybean oil, castor oil, peanut oil, blending oil, etc.). The solubilizer includes but is not limited to: at least one of Tweens, benzyl alcohols, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates. The cosolvent includes but is not limited to: at least one of organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, povidone, and glycerol. The emulsifier includes but is not limited to: at least one of Spans, Tweens, benzyl alcohols, benzyl alcohols, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxide, silicon dioxide, and bentonite. The suspending agent includes but is not limited to: at least one of glycerol, syrup, gum arabic, gum tragacanth, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, and thixotropic glue. The antioxidant includes but is not limited to: at least one of sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid and its esters. The metal complexing agent includes but is not limited to: at least one of disodium ethylenediaminetetraacetic acid and polycarboxylic acid compounds; the inert gas includes but is not limited to: at least one of nitrogen and carbon dioxide. The preservative includes but is not limited to: at least one of parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils. The filler includes at least one of starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose. The flavoring agent includes at least one of aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium. The local analgesic includes, but is not limited to, at least one of benzyl alcohol, chlorobutanol, lidocaine, and procaine.The pH regulator includes, but is not limited to, at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. The isotonic or isotonic regulator includes, but is not limited to, at least one of glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0052] In some embodiments of the present invention, the drug may further include a combination drug, which includes a drug for treating or preventing EBV infection-related diseases.

[0053] In this case, the at least two active ingredients may be administered together or separately and as part of the same pharmaceutical preparation or as different preparations.

[0054] In some embodiments of the present invention, the drug can be administered orally, parenterally (e.g., intravenously, intramuscularly, intraperitoneally), intracranially, intrathecally, intraarterially (e.g., via the carotid artery), rectally, cutaneously, nasally, vaginally, by inhalation, on the skin (patch), or ophthalmically, and the effective dose administered is a pharmaceutically acceptable dose.

[0055] In some embodiments of the present invention, the dosage form of the drug is selected from tablets (such as ordinary tablets, double-layer tablets, multi-layer tablets, sustained-release tablets, single-chamber controlled-release tablets, double-chamber controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally rapidly disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, patches, powders, suspensions, gels, emulsions, creams, granules, solutions, nanoparticles, drops, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing micropellets or small tablets, pH-dependent capsules containing micropellets or small tablets, gastrointestinal composite capsules), enemas, suppositories, granules, teas, injections, sprays and injections.

[0056] The fifth aspect of the present invention provides a vaccine, comprising the antigen composition of the first aspect of the present invention or the biological material of the second aspect of the present invention.

[0057] In some embodiments of the present invention, the vaccine includes a DNA vector vaccine and a viral vector vaccine. When the vaccine is used, the DNA vector vaccine is first inoculated, and the viral vector vaccine is used for booster immunization.

[0058] In some embodiments of the present invention, the DNA vector vaccine is constructed by comprising BZLF1, EBNA1, EBNA3B and / or gHgL as antigens and a DNA vaccine vector; the viral vector vaccine is constructed by comprising BZLF1, EBNA1, EBNA3B and / or gHgL as antigens and a viral vaccine vector.

[0059] In some embodiments of the present invention, the vaccine comprises a monovalent vaccine or a multivalent vaccine.

[0060] In some embodiments of the present invention, the DNA vector vaccine includes a DNA vector monovalent vaccine and a DNA vector multivalent vaccine, wherein the DNA vector monovalent vaccine is constructed by using BZLF1, EBNA1, EBNA3B or gHgL as antigens and DNA vaccine vectors; the DNA vector multivalent vaccine is obtained by mixing each DNA vector monovalent vaccine in a certain proportion (such as the mass ratio of each antigen). The DNA vaccine vector can be any common DNA vector in the art, such as pDRVI3.0.

[0061] In some embodiments of the present invention, the viral vector vaccine includes a viral vector monovalent vaccine and a viral vector multivalent vaccine, wherein the viral vector monovalent vaccine is constructed by using BZLF1, EBNA1, EBNA3B or gHgL as antigens and a viral vaccine vector respectively; the viral vector multivalent vaccine is obtained by mixing each viral vector monovalent vaccine in a certain proportion (such as the mass ratio of each antigen). The viral vaccine vector can be any common virus in the art, such as the Tiantan strain of poxvirus.

[0062] The beneficial effects of the present invention are:

[0063] The present invention provides a new EBV vaccine antigen combination, wherein EBNA1, EBNA3B and BZLF1 genes are targets of strong T cell responses, and EBV-infected positive cells can be identified and eliminated by inducing specific T cell immunity against these genes; specific antibodies induced by gHgL neutralize extracellular EBV viruses, and the four antigens are combined to form an EBV multivalent vaccine, which has good safety and immunogenicity, can significantly reduce EBV replication, and prevent the occurrence of EBV tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 To construct EBV DNA vector polyvalent vaccine and Tiantan strain poxvirus vector polyvalent vaccine; wherein, A is a schematic diagram of the construction of EBV monovalent DNA vaccine, B is a schematic diagram of the construction of EBV monovalent Tiantan strain vector vaccine, C is Western blotting proof that EBV monovalent DNA vector vaccine can express BZLF1, EBNA1, EBNA3B and gHgL, and D is Western blotting proof that EBV monovalent Tiantan strain vector vaccine can express BZLF1, EBNA1, EBNA3B and gHgL.

[0065] Figure 2The safety analysis of EBV polyvalent vaccine; A shows the weight change of mice in each group after BALB / c mice were immunized with EBV polyvalent vaccine, B shows the death of various mice after BALB / c mice were immunized with EBV polyvalent vaccine, C shows the normal appearance of organs in each group of mice after BALB / c mice were immunized with EBV polyvalent vaccine, D shows the HE staining of organs in each group of mice after BALB / c mice were immunized with EBV polyvalent vaccine, and E shows the residual status of Tiantan strain poxvirus in organs in each group of mice after BALB / c mice were immunized with EBV polyvalent vaccine. The survival rate curves were compared by log-rank test. The statistical analysis of body weight was performed by Mann-Whitney rank sum test (ns represents P ≥ 0.05; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001).

[0066] Figure 3 Figure 2 shows the humoral immune response of BALB / c mice induced by heterologous pre-boost vaccination with multi-antigen EBV vaccine; A is a schematic diagram of immunization grouping and immunization strategy (created using Biorender.com); B-E are ELISA tests for BZLF1 (B), EBNA1 (C), EBNA3B (D), and gH / gL (E)-specific IgG levels in the serum of mice vaccinated with EBV monovalent and multivalent vaccines at different time points (weeks 0, 6, 9, 12, 16, 20, 24, and 28); F-I are the antibody affinities for BZLF1 (F), EBNA1 (G), EBNA3B (H), and gH / gL (I) in the serum of mice vaccinated with EBV monovalent and multivalent vaccines at different time points (weeks 0, 6, 9, 12, 16, 20, 24, and 28); J and K are the neutralization titers of EBV against B cells (J) and epithelial cells (K). Neutralization titer was fitted using an S-shaped curve to calculate the half-maximal inhibition dilution (ID50). Each point represents the reciprocal half-maximal inhibition dilution (ID50) of a single mouse, and 0 means that no neutralization ability was detected at the dilution tested. Data are expressed as mean ± standard error (SEM). At the beginning of the experiment, n = 11 in the sham immunization group, n = 11 in the BZLF1 single antigen vaccine group, n = 11 in the EBNA1 single antigen vaccine group, n = 11 in the EBNA3B single antigen vaccine group, n = 11 in the gH / gL single antigen vaccine group, and n = 12 in the multi-antigen vaccine group. After the 9th week, there were 5 mice in each group. Statistical analysis was performed using the Mann-Whitney rank sum test (ns represents P ≥ 0.05; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001).

[0067] Figure 4Heterologous booster immunization with a multi-antigen EBV vaccine induces a cellular immune response in BALB / c mice. At 9 and 28 weeks after the first immunization, splenocytes were stimulated with BZLF1, EBNA1, and EBNA3B peptide pools (5 μg / mL) and gH / gL protein (5 μg / mL), respectively, and flow cytometry was used to detect the production of IFN-γ, TNF-α, or IL-2 by CD8+ and CD4+ T cells. The percentages of BZLF1 (A), EBNA1 (B), EBNA3B (C), and gH / gL-specific (D) CD8+ and CD4+ T cells producing IFN-γ, TNF-α, and IL-2 are shown. Each point represents an individual mouse. Data are presented as mean ± standard error of the mean. Control group n = 6, BZLF1 single antigen vaccine n = 6, EBNA1 single antigen vaccine n = 6, EBNA3B single antigen vaccine n = 6, gH / gL single antigen vaccine n = 6, and multi-antigen vaccine n = 7. At week 28, 5 mice were included in each group. Other statistical analyses were performed using the Mann-Whitney rank-sum test (ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0068] Figure 5 The EBV multivalent vaccine elicited BZLF1, EBNA1, and EBNA3B-specific T cell responses in humanized mice. Humanized mice were vaccinated with DNA vaccine (5 μg of each antigen) and rTTV vaccine (1.25×10 2PFU), followed by a two-week interval EBV polyvalent DNA vector vaccine prime-boost immunization schedule. Two weeks after the last immunization, peripheral blood was collected and stimulated with BZLF1, EBNA1 and EBNA3B peptide pools and gH / gL proteins. Among them, A is the proportion of antigen-specific CD8+ cells producing IFN-γ, TNF-α and IL-2 after BZLF1 stimulation, B is the proportion of antigen-specific CD8+ cells producing IFN-γ, TNF-α and IL-2 after EBNA1 stimulation, C is the proportion of antigen-specific CD8+ cells producing IFN-γ, TNF-α and IL-2 after EBNA3B stimulation, D is the proportion of antigen-specific CD8+ cells producing IFN-γ, TNF-α and IL-2 after gH / gL stimulation, E is the proportion of antigen-specific CD4+ cells producing IFN-γ, TNF-α and IL-2 after BZLF1 stimulation, F is the proportion of antigen-specific CD4+ cells producing IFN-γ, TNF-α and IL-2 after EBNA1 stimulation, G is the proportion of antigen-specific CD4+ cells producing IFN-γ, TNF-α and IL-2 after EBNA3B stimulation, and H is the proportion of antigen-specific CD4+ cells producing IFN-γ, TNF-α and IL-2 after gH / gL stimulation. The control group, n = 6, and the vaccine group, n = 8. Data are presented as mean ± standard error (SEM), and other statistical analyses were performed using the Mann-Whitney rank sum test (ns represents P ≥ 0.05; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001).

[0069] Figure 6 EBV DNA vector polyvalent vaccine and Tiantan strain poxvirus vector polyvalent vaccine can protect humanized mice from lethal doses of EBV challenge; A is the experimental flow chart, B is the body weight of humanized mice after challenge, C is the survival rate of humanized mice after challenge, D is the tumor incidence rate of humanized mice after challenge, E is the copy number of EBV DNA in humanized mice after challenge (each row represents a single mouse), and F is the peripheral blood hCD19 + hCD45 + and hCD20 + hCD45 + B cells, G is the DNA copy number in the spleen at the end of the experiment (each dot represents a separate mouse), H is the hCD45 + hCD19 + and hCD45 + hCD20 + B cell count / 10 4cells (each dot represents a single mouse), I is the number of hCD19 in the peritoneal fluid of mice at the end of the experiment + hCD20 + B cell percentage (each dot represents an individual mouse), J is hCD8 + hCD137 + The frequency of cells (each dot represents an individual mouse), K is the hCD4 + hCD137 + The frequency of cells (each dot represents an individual mouse), L is the hCD19 measured in peritoneal fluid at necropsy + hCD24 - The frequency of hCD38high cells (each dot represents a separate mouse). In this figure, the data schematics of the uninfected control group, empty vector + EBV, and vaccine + EBV are represented in gray, blue, and red, respectively. In E and G to L, the uninfected control group n = 3, the empty vector + EBV n = 11, and the vaccine + EBV n = 11. In J to L, the uninfected control group n = 3, the empty vector + EBV n = 8, and the vaccine + EBV n = 7. Survival curves were compared using the log-rank test, and tumor incidence (%) was compared using the two-tailed Fisher's test. Data are expressed as mean ± standard error (SEM), and other statistical analyses were performed using the Mann-Whitney rank sum test (ns represents P ≥ 0.05; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001).

[0070] Figure 7 The multi-antigen EBV vaccine can reduce the replication and tissue damage of EBV in humanized mice; A is the result of liver sectioning and EBER staining of hematoxylin and eosin, human CD20 (hCD20) and EBV, B is the result of tumor sectioning and EBER staining of hematoxylin and eosin, human CD20 (hCD20) and EBV, and the scale bar shows 200μm.

[0071] Figure 8 DNA vector polyvalent vaccine and Tiantan strain poxvirus vector polyvalent vaccine can reduce EBV replication in humanized mice. Spleen tissue was embedded and tested for tissue integrity by hematoxylin and eosin, immunohistochemistry for the distribution of human CD20 (hCD20) positive cells, and hybridization for EBV encoded RNA (EBER). The scale bar shows 200 μm. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below through specific examples.

[0073] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0074] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0075] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0076] Example 1 Construction of EBV monovalent vaccine

[0077] The present invention selects the M81 strain of EBV as the source of antigenic genes. Since the antigens (BZLF1 and EBNA1) selected by the present invention are carcinogenic, the above antigens are modified for safety while retaining their T cell epitopes to the maximum extent. It is roughly as follows: by deleting the glycine-alanine repeat sequence, deleting the nuclear localization signal and mutating the amino acids to eliminate their potential effects. At the same time, part of the nuclear localization signal of EBNA3B is also deleted, and the full length of gL is connected to the full length of the extracellular domain of gH through (GGGGS)3.

[0078] Amino acid sequence of the modified BZLF1 antigen:

[0079] MEPLPQGQLTAYHVSAAPTGSWFPAPQPAPENAYQAYAAPQLFPVSDITQNQLTNQAGGEAPQPGDNSTVQPAAAVVLACPGANQEQQLADIGAPQPAPAAAPARRTRKPLQ PESLEESDSELEIKRYKNRVASRKSRAKFKHLLQHYREVASAKSSENDRLRLLLKQMSPSLDV (SEQ ID NO: 1).

[0080] Amino acid sequence of the modified EBNA1 antigen:

[0081] MFHPVGDADYFEYLQEGGPDGEPDVPPGAIEQGPTDDPGAGPATGPRGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRVLLARSHVERTTEEGNWVAGVFVYGGSKTSLYNLRRGI ALAVPQCRITPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLKDAIKDLVMTKPAPTCNIKVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDEGEEGQE(SEQ ID NO:2).

[0082] Amino acid sequence of the modified EBNA3B antigen:

[0083] MKKAWLSRAQQADAGGASGSEDPPDYGDQGNVTQVESEPISPEIGPFELSAASEDDPQSGPVEENLDAAAREEEEPHEQEHNGGDDPLDVHTRQPRFVDVNPTQAPVIQLVHAVYDSMLQSDLRPLGSLFLEQNLNIEEFIWMCMTVRHRCQAIRKKPLWKLLSSCRSWRMGYRTHNLKVNSFESGGDNVHPVLVTATLGCDEGTRHATTYSAGIVQIPRISDQNQKIETAFLMARRARSLSAERYTLFFDLVSSGNTLYAIWIGLGTKNRVSFIEFVGWLCKKDHTHIREWFRQCTGRPKAAKPWLRAHPVAIPYDDPLTNEEIDLAYARGQAMNIEAPRLPDDPIIVEDDDESEEIEAESDEEEDKSGMESLKNIPQTLPYNPTVYGRPAVFNRKSDAKSTKKCRAIVTDFSVINAIEEEHRKKKAARTEQPRATPESQAPTVVLQRPPTQQEPGPAGPLSVQARLEPWQPLPGPQVTTVLLHEESMQGVQVHGSMLDLLEKDDEVMEQRVMATLLPPVPQQPRAGRRGPCVFTGDLGIESDEPASTEPVHDQLLPAPGPDPLEIQPLTSPNTSQLSSSAPSCAQTPWPVVQPSQTPDDPTKQSRPPETAAPRQWPMPLRPIPMRPLRMQPIPFNHPVGPTPHQTPQVEITPYKPTWAQIGHIPYQPTPTGPATMLLRQWAPATMKIPPRAPTPMSPPEVPPVPRQRPRGAPTPTPPPQVPPVPRQRPRGAPTPTPPPQVLPTPMQLALRAPAGQQGPTKQILRQLLTGGVKKGRPSLKLQAALERQAAAGWQPSPGSGTSDKIVRAPIFYPPVLQPIQVMGQGGSPTAMAASAVTQAPTEYTRERRGVGPMPPTDIEAYTEPEMPHGGGSHSPVVILENVSRGQQQTLECGGTAKQERDMLGLGDIAVSSPSSSETSNDE(SEQ ID NO:3).

[0084] Amino acid sequence of the modified gHgL antigen:

[0085] MWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASNLSPKNGSNQLVISRCANGNLNVVSFSILIKRSSSALTSHLRELLTTLESLYGSFSVEDLFGANLNRYAWHRGGGGGGSGGGGSGGGGSASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLN (SEQ ID NO:4)。

[0086] The modified antigens of BZLF1, EBNA1, EBNA3B and gHgL were inserted into the SacII and SaII restriction sites of the DNA vaccine vector pDRVI3.0, and four separate DNA vector vaccines were successfully constructed by sequencing. At the same time, the antigens of BZLF1, EBNA1, EBNA3B and gHgL were inserted into the HindIII and SmaI restriction sites of the shuttle plasmid pSC65-GFP, and the pSC65-GFP shuttle plasmid with the antigen gene was homologously recombined with the Tiantan strain poxvirus genome, and the antigen gene was integrated into the tyrosine kinase gene (TK) of the Tiantan strain poxvirus. Then, the DNA vector vaccine with each antigen was transfected or the Tiantan strain vector vaccine was infected with 293T cells, and the protein was collected for protein detection after 3 days. Western Blotting was used to prove that the constructed multivalent vaccine can express BZLF1, EBNA1, EBNA3B and gHgL.

[0087] Western blotting results Figure 1 As shown, the results showed that monovalent vaccines of each antigen were successfully constructed.

[0088] Example 2 Safety Testing of EBV Multivalent Vaccine

[0089] In order to test the safety and immunogenicity of the multivalent vaccine, this example initially conducted relevant experiments on BALB / c mice.

[0090] Four-week-old female BALB / c mice were selected as the immunization objects and received three DNA vector polyvalent vaccines (the monovalent DNA vector vaccine dose of each antigen was 50 μg, and the ratio of each antigen was 1:1:1:1) and one Tiantan strain poxvirus vector polyvalent vaccine (the monovalent Tiantan strain poxvirus vaccine dose of each antigen was 1.25×10 6 PFUs, each antigen ratio 1:1:1:1) booster immunization, immunization method is intramuscular injection, immunization interval is 2 weeks. The weight change and survival rate of mice before and after immunization were recorded. In the third week after Tiantan strain vector vaccine immunization, mice were killed and brain, heart, liver, spleen, lung and kidney were collected, part of which was used for DNA extraction, and part of which was paraffin embedded for HE staining.

[0091] The results showed that after mice were immunized with EBV polyvalent vaccine (i.e. DNA vector polyvalent vaccine and Tiantan strain poxvirus monovalent vaccine), the weight of the mice did not decrease ( Figure 2 No death occurred ( Figure 2 Middle B). HE staining showed that the morphology of each organ tissue was normal ( Figure 2 C), semi-quantitative PCR showed that there was no residual Tiantan strain poxvirus in any tissue or organ ( Figure 2The above results show that the constructed EBV DNA vector vaccine and Tiantan strain poxvirus vector vaccine have good safety.

[0092] Example 3 Immunity detection of EBV multivalent vaccine

[0093] After evaluating the safety of EBV multivalent vaccine (i.e., DNA vector multivalent vaccine and Tiantan strain poxvirus multivalent vaccine) in immunizing BALB / c mice, we further evaluated whether the multivalent vaccine could activate the immune system response and whether there would be immune interference between the four antigens.

[0094] The immunization dose and immunization procedure are consistent with the safety test experiment in Example 3. The immunization grouping and detailed process are as follows: Figure 3 As shown in A. Blood was collected at weeks 0, 6, 9, 12, 16, 20, 24, and 28, and the mice were killed at weeks 9 and 28 to obtain spleens. Mice immunized with empty DNA vaccine and wild-type Tiantan poxvirus served as control groups. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the IgG antibody titers of BZLF1, EBNA1, EBNA3B, and gH / gL in the serum of mice vaccinated with EBV monovalent and polyvalent vaccines at different time points (weeks 0, 6, 9, 12, 16, 20, 24, and 28).

[0095] The results showed that 2 weeks after the third immunization with the multivalent DNA vector vaccine, antigen-specific antibodies against BZLF1, EBNA1, EBNA3B and gH / gL were detected in the mouse serum ( Figure 3 (B-E). Three weeks after booster immunization with the Tiantan strain poxvirus vector polyvalent vaccine, higher titers of antigen-specific antibodies were detected in the mouse serum ( Figure 3 In addition, antigen-specific antibodies could still be detected in the serum of mice immunized with EBV multivalent vaccine 5 months later ( Figure 3 When the four antigens were administered alone or in combination, the antibody titer against each antigen ( Figure 3 B~E) and antibody affinity ( Figure 3 The F-I) in the samples remained at similar levels.

[0096] gH / gL is an important component of the core fusion mechanism required for EBV to infect B cells and epithelial cells. A variety of monoclonal antibodies targeting gHgL have been identified that can effectively block EBV infection of B cells and epithelial cells. The present invention uses EBV multivalent vaccine or EBV gHgL monovalent vaccine to immunize mice, and the immunized serum can prevent EBV from infecting Akata B cells and HNE1 epithelial cells ( Figure 3 J and K).

[0097] In order to further test the persistent response of T cells, in this example, mice were killed at the 3rd week (i.e., the 9th week after the first immunization) and the 22nd week (i.e., the 9th week after the first immunization) after immunization with the Tiantan strain poxvirus vector multivalent vaccine, and the spleen cells of the mice were collected and specifically stimulated with the peptide library (5 μg / mL) of BZLF1, EBNA1, EBNA3B and gH / gL protein (5 μg / mL). The antigen-specific CD8+ cells expressing type 1 (Th1) immune response cytokines (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) in spleen cells were detected. + T cells and CD4 + The frequencies of T cells (i.e., CD8+ and CD4+ T cells producing IFN-γ, TNF-α, or IL-2) were measured using flow cytometry.

[0098] The results showed that compared with the control group, the DNA vector multivalent vaccine prime immunization / Tiantan strain poxvirus vector vaccine booster immunization could induce more BZLF1, EBNA1, EBNA3B and gH / gL-specific CD8 + and CD4 + T cells ( Figure 4 Among these four antigens, the frequency of BZLF1-specific T cells was higher ( Figure 4 When the four antigens were used alone or in combination, the BZLF1, EBNA1, EBNA3B, and gH / gL-specific T cell responses remained at similar levels ( Figure 4 A to D), indicating that there is no cellular immune interference between the antigens, and no humoral immune interference between the four antigens was observed under the current vaccination dose and immunization program.

[0099] Example 4 Detection of the protective effect of EBV multivalent vaccine

[0100] This example further uses a humanized mouse model to evaluate the protective effect of the EBV multivalent vaccine in preventing B cell lymphoma caused by high-dose EBV infection. The details are as follows:

[0101] Humanized mice derived from human PBMCs (peripheral blood mononuclear cells) were constructed and immunized with EBV polyvalent DNA vector vaccine (DNA vaccine dose of each antigen was 5 μg, and the ratio of each antigen was 1:1:1:1) for the first time (by intraperitoneal injection of vaccine). Two weeks later, EBV Tiantan strain poxvirus vector polyvalent vaccine (Tiantan strain poxvirus vaccine dose of each antigen was 1.25×10 2After the booster immunization, peripheral blood was collected and stimulated with BZLF1, EBNA1 and EBNA3B peptide libraries and gH / gL proteins, and antigen-specific T cell responses were detected by flow cytometry.

[0102] Figure 5 Antigen-specific CD8 cells that produce IFN-γ, TNF-α, and IL-2 have been shown + and CD4 + The proportion of T cells and CD8 + ( Figure 5 A to D) and CD4 + ( Figure 5 The combined frequencies of multiple cytokines produced by E-H T cells in the humanized mice were shown in Table 1. The results showed that the EBV multivalent vaccine could activate the specific T cell responses of BZLF1, EBNA1 and EBNA3B in the peripheral blood of humanized mice.

[0103] Two weeks after the booster immunization, EBV challenge experiments were conducted in humanized mice by intraperitoneal injection of polyvalent DNA vector vaccine (5 μg per antigen) and Tiantan strain poxvirus vector polyvalent vaccine (1.25×10 per antigen). 2 PFUs) with an immunization interval of two weeks. Two weeks after immunization with Tiantan strain poxvirus vector polyvalent vaccine, Akata EBV was intraperitoneally injected into humanized mice at a dose of 1×10 5 Raji infection units. Uninfected mice (uninfected control) were not vaccinated with vaccine or virus; mice in the control group were vaccinated with empty DNA vector vaccine and wild-type Tiantan strain poxvirus and then challenged with virus; mice in the vaccine group were vaccinated with DNA vector polyvalent vaccine and Tiantan strain poxvirus vector polyvalent vaccine and then challenged with virus. After challenge, the weight, survival rate, tumor incidence, EBV DNA copy number and peripheral blood hCD19 of humanized mice were recorded. + hCD45 + and hCD20 + hCD45 + B cells (specific experimental procedures as follows Figure 6 (shown in A).

[0104] The results showed that compared with the vaccine group, the body weight of humanized mice in the control group decreased significantly in the third week after the challenge ( Figure 6 In the control group, 10 out of 11 mice died and developed lymphoma within seven weeks after the challenge. Figure 6 C and D), human B cells (CD45 + CD19 + CD20 +) content and EBV DNA copy number increased significantly in the third week after infection ( Figure 6 E and F), EBV DNA copy number and human B cells (CD45 + CD19 + CD20 + ) were significantly higher than those in the vaccine group ( Figure 6 Human B cells (CD45 + CD19 + CD20 + ) were significantly higher than those in the vaccine group ( Figure 6 Further analysis revealed that activated CD8 + T cells (CD45 + CD8 + CD137 + ) and activated CD4 + T cells (CD45 + CD8 + CD137 + ) was significantly lower than that in the vaccine group ( Figure 6 In J and K), human plasma cells (CD45 + CD19 + CD24 - CD38 high ) was significantly higher than that in the vaccine group ( Figure 6 Middle L).

[0105] In order to study the protective effect of EBV multivalent vaccine at the tissue level, the present embodiment dissected and collected the lymph nodes, spleen and liver of the animals. The results showed that 10 of the 11 humanized mice in the control group developed tumors ( Figure 6 In the middle (D), the liver and abdominal tissues showed obvious changes. Morphologically, the liver of the mice in the control group was significantly enlarged, and tumors were observed ( Figure 7 In contrast, the livers of mice immunized with the EBV multivalent vaccine and those of mice not infected had normal liver size and no tumors occurred ( Figure 7 In the middle (A), tumors were observed in the abdominal cavity of mice in the control group, whereas no tumors were observed in the abdominal cavity of mice in the vaccine group or the uninfected group ( Figure 7 (B). The liver and tumors were sectioned and stained for hematoxylin and eosin (H&E), human CD20 (hCD20), and EBV. In situ hybridization for EBERs and immunohistochemistry (IHC) for hCD20 confirmed the presence of typical hCD20 in the liver and peritoneum of control humanized mice. + EBER + B-cell lymphoma ( Figure 7(A and B). hCD20 + EBER + B-cell lymphomas are abundant and widely distributed throughout the tissue sections ( Figure 7 In contrast, hCD20 was rarely found in the livers of humanized mice immunized with the multi-antigen vaccine. + EBER + cell( Figure 7 Meanwhile, in the spleen sections of humanized mice in the control group, several hCD20 + EBER + B cell clusters, whereas these clusters were rare in mice treated with the multiantigen vaccine and in uninfected controls ( Figure 8 ).

[0106] The above results show that in the humanized mouse model, the EBV multivalent vaccine can significantly reduce EBV replication and prevent EBV-induced B cell lymphoma.

[0107] In summary, EBV multivalent vaccine immunized humanized mice, activated EBNA1, EBNA3B and BZLF1 antigen-specific T cells, recognized and killed EBV-infected B cells, and effectively inhibited the transformation and proliferation of B cells, thereby preventing the occurrence of lymphoma caused by EBV infection. The four antigens were combined to form an EBV multivalent vaccine, which has good safety and immunogenicity, and can effectively prevent the occurrence of EBV tumors.

[0108] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An antigen composition, characterized in that The antigen composition includes at least one of BZLF1, EBNA1, EBNA3B or gHgL; Among them, the amino acid sequence of BZLF1 is as shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar function.

2. The antigen composition according to claim 1, characterized in that The amino acid sequence of EBNA1 is as shown in SEQ ID NO: 2, or an amino acid sequence of SEQ ID NO: 2 that is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions; and / or The amino acid sequence of EBNA3B is as shown in SEQ ID NO: 3, or an amino acid sequence of SEQ ID NO: 3 which has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions; and / or The amino acid sequence of gHgL is as shown in SEQ ID NO: 4, or an amino acid sequence shown in SEQ ID NO: 4 which is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

3. A biological material related to the antigen composition according to claim 1 or 2, wherein the biological material is any one of a1) to a12): a1) a nucleic acid molecule encoding the antigen composition according to claim 1 or 2; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic animal cell line containing the nucleic acid molecule described in a1); a10) a transgenic animal cell line containing the expression cassette described in a2); a11) a transgenic animal cell line containing the recombinant vector described in a3); a12) a transgenic animal cell line containing the recombinant vector described in a4); The transgenic animal cell line contains no reproductive material.

4. Use of the antigen composition according to claim 1 or 2 or the biomaterial according to claim 3 in preparing a product.

5. The use according to claim 4, characterized in that: The product includes at least one of a drug, a reagent, and a kit; Preferably, the function of the product is any one of (1) to (6): (1) Prevent and / or treat EBV infection-related diseases; (2) Preparation of EB virus antibodies; (3) detecting BZLF1, EBNA1, EBNA3B and / or gHgL in EBV in the sample; (4) Diagnosis of EB virus-specific neutralizing antibodies; (5) triggering humoral-cellular immune responses; (6) Inhibit tumor growth.

6. The use according to claim 5, characterized in that: The EBV infection-related diseases include at least one of EBV-related B-cell lymphoma, nasopharyngeal carcinoma, chronic active EBV infection, hemophagocytic syndrome, EBV infection after hematopoietic stem cell transplantation or organ transplantation, lymphoproliferative disease and EBV-related gastric cancer.

7. A product, characterized in that The product comprises the antigen composition according to claim 1 or 2 or the biological material according to claim 3, Preferably, the product comprises at least one of a drug, a reagent, and a kit.

8. A vaccine comprising the antigen composition according to claim 1 or 2 or the biological material according to claim 3.

9. The vaccine according to claim 8, characterized in that The vaccines include DNA vector vaccines and viral vector vaccines.

10. The vaccine according to claim 9, characterized in that The DNA vector vaccine is constructed by comprising BZLF1, EBNA1, EBNA3B and / or gHgL as antigens and a DNA vaccine vector; the viral vector vaccine is constructed by comprising BZLF1, EBNA1, EBNA3B and / or gHgL as antigens and a viral vaccine vector.

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