Epstein-barr virus positive tumor mRNA vaccine and application thereof

Abstract

This invention discloses an EBV-positive tumor mRNA vaccine and its application. The EBV-positive tumor mRNA vaccine comprises a nucleic acid sequence encoding an antigen protein derived from EBV or a combination thereof, wherein the antigen protein is selected from (1) or (2): (1) the antigen protein comprises an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:149, SEQ ID NO:13, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10; (2) the antigen protein comprises an amino acid sequence having at least 90% sequence identity with the antigen protein defined in (1) and having comparable or better immunogenicity. The EBV-positive tumor mRNA vaccine provided by this invention can improve immunogenicity, significantly inhibit tumor development, and improve survival time, while also exhibiting high safety and broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an EB virus-positive tumor mRNA vaccine and its application. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck that originates from the mucosal epithelium of the nasopharynx.

[0003] Epstein-Barr virus (EBV) was first reported in 1964 as the cause of Burkitt lymphoma in African children and was the first virus discovered to be associated with human tumors.

[0004] Radiotherapy is currently the main treatment for nasopharyngeal carcinoma, and it is very effective for stage I nasopharyngeal carcinoma patients. However, because early symptoms of nasopharyngeal carcinoma are not obvious, more than 80% of patients are diagnosed at a locally advanced stage, posing a risk of local recurrence and distant metastasis, making radiotherapy alone less effective. For patients with recurrent or metastatic nasopharyngeal carcinoma, the current mainstream treatment is palliative chemotherapy and / or immunotherapy. However, although the combination of immunotherapy and chemotherapy has improved efficacy to some extent, a considerable number of patients still face failure of platinum-based chemotherapy; even if immune checkpoint PD-1 antibody therapy is effective for some patients, more than half of these patients will still relapse and progress within 12 months.

[0005] Epstein-Barr virus (EBV) antigen, as a novel antigen uniquely expressed by tumor cells, is considered the most ideal target for tumor neoantigen therapy. Therefore, tumor therapeutic products designed to target this neoantigen will enhance the body's T-cell immune response, targeting and killing tumor cells. Furthermore, its durable and immunologically memory-based activation offers hope for long-term prevention of tumor recurrence. Products targeting EBV antigen have entered clinical trials; however, to date, no therapeutic products specifically targeting EBV have been commercially available internationally. Summary of the Invention

[0006] Since existing technologies still require the development of therapeutic products targeting EB virus antigens, this invention provides an EB virus-positive tumor mRNA vaccine and its application. The product can enhance immunogenicity, significantly inhibit tumor development, improve survival time, and also has high safety.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions: The first aspect of the present invention provides an antigen protein derived from EB virus, said antigen protein being selected from (1) or (2): (1) The antigen protein comprises or has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 149, SEQ ID NO: 13, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 10; (2) The antigen protein comprises an amino acid sequence that has at least 90% sequence identity with the antigen protein defined in (1) and has comparable or better immunogenicity.

[0008] A second aspect of the present invention provides an antigen protein composition comprising one or more of the antigen proteins described in the first aspect.

[0009] In some embodiments of the present invention, the composition comprises two or three of the proteins described in the first aspect.

[0010] In some embodiments of the present invention, the antigen protein composition comprises: The amino acid sequences of the antigen proteins are as shown in SEQ ID NO: 8 and SEQ ID NO: 149, respectively; or, The amino acid sequences of the antigen proteins shown in SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and the amino acid sequences of the antigen proteins selected from any of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 149, and SEQ ID NO: 13; or An antigen protein with an amino acid sequence as shown in SEQ ID NO: 8, and an antigen protein selected from any of SEQ ID NO: 3, SEQ ID NO: 14, and SEQ ID NO: 13; or, The antigen protein with the amino acid sequence shown in SEQ ID NO: 10, and the antigen protein selected from any of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 149 and SEQ ID NO: 13.

[0011] A third aspect of the present invention provides an isolated nucleic acid that encodes an antigen protein as described in the first aspect.

[0012] In some embodiments of the present invention, the nucleic acid is RNA or DNA.

[0013] In some preferred embodiments of the present invention, the nucleic acid is mRNA.

[0014] In some embodiments of the present invention, the mRNA comprises a nucleotide sequence selected from any of the following: SEQ ID NO: 18-27, SEQ ID NO: 38-47, SEQ ID NO: 58-67, SEQ ID NO: 88-97, SEQ ID NO: 108-117, SEQ ID NO: 138-147, SEQ ID NO: 153-162, and SEQ ID NO: 163-172.

[0015] In some embodiments of the present invention, the mRNA further comprises nucleotide sequences encoding signal peptides, membrane anchoring segments, and / or lysosomal localization signals.

[0016] In some preferred embodiments of the present invention, the signal peptide is the signal peptide of LAMP1.

[0017] In some specific embodiments of the present invention, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 16.

[0018] In some preferred embodiments of the present invention, the membrane anchoring segment originates from the transmembrane region of LAMP1.

[0019] In some specific embodiments of the present invention, the amino acid sequence of the membrane anchoring segment is shown in SEQ ID NO: 15.

[0020] In some preferred embodiments of the present invention, the lysosomal localization signal is derived from the cytoplasmic tail of LAMP1.

[0021] In some specific embodiments of the present invention, the amino acid sequence of the lysosome localization signal is shown in SEQ ID NO:148.

[0022] In some embodiments of the present invention, the mRNA comprises a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 12.

[0023] In some specific embodiments of the present invention, the mRNA comprises any of the nucleotide sequences shown in SEQ ID NO: 28-37, SEQ ID NO: 48-57, SEQ ID NO: 68-77, SEQ ID NO: 78-87, SEQ ID NO: 98-107, SEQ ID NO: 118-127 and SEQ ID NO: 128-137.

[0024] In some embodiments of the present invention, the mRNA further comprises a 5'-cap structure, a 5'UTR, a 3'UTR, and / or Poly(A).

[0025] In some preferred embodiments of the present invention, the 5'-hat structure is of type CAP 0, type CAP I, or type CAP II.

[0026] In some preferred embodiments of the present invention, the Poly (A) comprises 25-400 adenosine monophosphates.

[0027] In some preferred embodiments of the present invention, the Poly (A) comprises 50-400 adenosine monophosphates, 50-300 adenosine monophosphates, 50-250 adenosine monophosphates, 60-250 adenosine monophosphates, or 100-150 adenosine monophosphates.

[0028] In some optional embodiments of the present invention, the mRNA further comprises a nucleotide sequence encoding a protein tag.

[0029] In some embodiments of the present invention, the mRNA includes modifications at one or more of the following locations: a 5' cap structure, a 5' UTR, an open reading frame, a 3' UTR, and Poly(A).

[0030] In some preferred embodiments of the present invention, the modification is selected from one or more of the following: pseudouridine modification, N1-methyl-pseudouridine modification, 5-methoxy-uridine modification, N6-methyl-adenosine modification, and 5-methyl-cytidine modification.

[0031] A fourth aspect of the present invention provides a nucleic acid composition comprising a nucleic acid encoding an antigen protein composition as described in the third aspect.

[0032] In some embodiments of the present invention, the encoding nucleic acid is mRNA as defined in the nucleic acid as described in the second aspect.

[0033] In some preferred embodiments of the present invention, the nucleic acid composition comprises two or three mRNAs.

[0034] In some preferred embodiments of the present invention, the mRNA comprises a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 7 and SEQ ID NO: 11, respectively.

[0035] In some preferred embodiments of the present invention, the mRNA comprises nucleotide sequences of the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 149, respectively.

[0036] In some further preferred embodiments of the present invention, the nucleic acid composition comprises two mRNAs, each mRNA comprising a nucleotide sequence shown in either SEQ ID NO: 78-87 and / or SEQ ID NO: 118-127.

[0037] In some specific embodiments of the present invention, the nucleic acid composition comprises nucleotide sequences as shown in SEQ ID NO: 82 and SEQ ID NO: 118.

[0038] In some further preferred embodiments of the present invention, the nucleic acid composition comprises two mRNAs, wherein the mRNAs respectively comprise the nucleotide sequences shown in any one of SEQ ID NO: 88-97 and / or any one of SEQ ID NO: 163-172.

[0039] A fifth aspect of the present invention provides a recombinant expression vector comprising a nucleic acid as described in the third aspect or a nucleic acid composition as described in the fourth aspect.

[0040] A sixth aspect of the present invention provides a transformant comprising a nucleic acid as described in the third aspect or a recombinant expression vector as described in the fourth aspect.

[0041] A seventh aspect of the present invention provides a pharmaceutical composition comprising an antigen protein as described in the first aspect, an antigen protein composition as described in the second aspect, a nucleic acid as described in the third aspect, a nucleic acid composition as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, or a transformant as described in the sixth aspect.

[0042] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipients.

[0043] In some preferred embodiments of the present invention, the pharmaceutical composition is a vaccine comprising (i) a nucleic acid as described in the third aspect or a nucleic acid composition as described in the fourth aspect; and (2) a delivery vector.

[0044] In some preferred embodiments of the present invention, when the vaccine comprises the nucleic acid composition, the mass ratio of the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 11 is 40-50%:50-60%, for example, 40%:60%, 41%:59%, 42%:58%, 43%:57%, 44%:56%, 45%:55%, 46%:54%, 47%:53%, 48%:52%, 49%:51%, or 50%:50%.

[0045] In some preferred embodiments of the present invention, when the vaccine comprises the nucleic acid composition, the mass ratio of the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 149 is 40-50%: 50-60%, for example 40%:60%, 41%:59%, 42%:58%, 43%:57%, 44%:56%, 45%:55%, 46%:54%, 47%:53%, 48%:52%, 49%:51%, or 50%:50%.

[0046] In some preferred embodiments of the present invention, the delivery carrier is a lipid nanoparticle, an ionic lipid complex, a lipid polymer complex, a polymer nanoparticle, an inorganic nanoparticle, a cationic nanoemulsion, or an exosome.

[0047] In some further preferred embodiments of the present invention, the lipid nanoparticles are composed of cationic lipids, cholesterol, phospholipids, and lipid conjugates.

[0048] In some further preferred embodiments of the present invention, the lipid nanoparticles are composed of RL151, cholesterol, DSPC, and DMG50-PEG2000; In some further preferred embodiments of the present invention, the molar ratio of RL151:cholesterol:DSPC:DMG50-PEG2000 is (30~60): (20~40): (10~30): (1~5).

[0049] In some specific embodiments of the present invention, the molar ratio of RL151:cholesterol:DSPC:DMG50-PEG2000 is 50:38.5:10:1.5.

[0050] The eighth aspect of the present invention provides a drug kit comprising an antigen protein as described in the first aspect, an antigen protein composition as described in the second aspect, a nucleic acid as described in the third aspect, a nucleic acid composition as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, or a transformant as described in the sixth aspect, or a drug composition as described in the seventh aspect, and other drugs for the prevention, relief, and / or treatment of tumors.

[0051] In some embodiments of the present invention, the other drugs for preventing, alleviating and / or treating tumors are immunosuppressants, cytotoxic drugs, tumor vaccines, antibodies, peptides and / or chemotherapeutic agents.

[0052] In some specific embodiments of the present invention, the chemotherapeutic agent is paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine, temozolomide, irinotecan and / or carboplatin.

[0053] In some specific embodiments of the present invention, the antibody is a PD-1 antibody.

[0054] The ninth aspect of the present invention provides a method for preparing an antigen protein as described in the first aspect or an antigen protein composition as described in the second aspect, the method comprising culturing a transformant as described in the sixth aspect under conditions suitable for the expression of an antigen protein derived from EB virus.

[0055] The tenth aspect of the present invention provides the use of an antigen protein as described in the first aspect, an antigen protein composition as described in the second aspect, a nucleic acid as described in the third aspect, a nucleic acid composition as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, or a transformant as described in the sixth aspect in the preparation of a medicament for the prevention, mitigation, and / or treatment of diseases caused by EB virus and / or EB virus-positive diseases.

[0056] In some embodiments of the present invention, the disease is a tumor that highly expresses EBNA1, Zta and / or LMP2A.

[0057] In some preferred embodiments of the present invention, the tumor is nasopharyngeal carcinoma, Burkitt lymphoma, gastric cancer, or Hodgkin lymphoma.

[0058] The eleventh aspect of the present invention provides a method for preventing, alleviating, and / or treating diseases caused by and / or with EBV positivity, the method comprising administering to a subject an antigen protein as described in the first aspect, an antigen protein composition as described in the second aspect, a nucleic acid as described in the third aspect, a nucleic acid composition as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, or a transformant as described in the sixth aspect.

[0059] In some embodiments of the present invention, the disease is a tumor that highly expresses EBNA1, Zta and / or LMP2A.

[0060] In some preferred embodiments of the present invention, the tumor is nasopharyngeal carcinoma, Burkitt lymphoma, gastric cancer, or Hodgkin lymphoma.

[0061] The twelfth aspect of the present invention provides an antigen protein as described in the first aspect, an antigen protein composition as described in the second aspect, a nucleic acid as described in the third aspect, a nucleic acid composition as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, or a transformant as described in the sixth aspect, for the prevention, mitigation, and / or treatment of diseases caused by and / or positive for EB virus.

[0062] In some embodiments of the present invention, the disease is a tumor that highly expresses EBNA1, Zta and / or LMP2A.

[0063] In some preferred embodiments of the present invention, the tumor is nasopharyngeal carcinoma, Burkitt lymphoma, gastric cancer, or Hodgkin lymphoma.

[0064] In the bivalent seedlings of this invention: Antigens: Composed of two mRNA sequences, NE7 and NE15, where NE7 is a tandem expression of truncated variants of Zta and EBNA1, and NE15 is a standalone truncated variant of LMP2A. This design removed amino acids 1-65, 81-409, and 618-645 of EBNA1, amino acids 175-194 of Zta, and amino acids 1-119 of LMP2A. The N-terminus of the NE7 antigen is the LAMP1 signal peptide sequence, and the C-terminus is the lysosomal localization signal of the LAMP1 transmembrane and intracellular regions. The C-terminus of the NE15 antigen is the LAMP1 lysosomal signal region.

[0065] Sequence design: Codon optimization was performed. 5'UTR, 3'UTR, and poly(A) were used.

[0066] LNP-mRNA preparation: uridine (U) is converted to pseudouridine (Ψ), with the two mRNAs having a mass ratio of 1:1. The lipid component of LNP is a cationic lipid.

[0067] Example of administration: Epstein-Barr virus-positive nasopharyngeal carcinoma. Intramuscular injection, in combination with immune checkpoint inhibitors such as PD-1 antibodies.

[0068] Advantages of this invention: It improves the potential therapeutic effect in two ways: antigen selection and sequence design; and the use of mRNA technology.

[0069] Advantages in antigen selection: In the development and progression of nasopharyngeal carcinoma, the following three EBV viral proteins play key roles: EBNA1 regulates EBV gene replication and enhances viral gene transcription; LMP2A has a profound impact on the maintenance of latent EBV infection, affecting various cellular processes, including proliferation, survival, invasion, and motility; Zta is a direct early transcription factor that activates lytic EBV replication, indirectly promoting the development of EBV-related malignancies (such as nasopharyngeal carcinoma, lymphoma, or gastric cancer) by activating the viral lysis cycle and regulating host oncogenic pathways (such as cell cycle arrest and pro-inflammatory cytokine expression). These molecules are expressed in EBV-related tumors, exhibit important activity in tumorigenesis and development, and have no similar proteins in normal individuals, demonstrating specificity and making them highly promising targets for the treatment of EBV-related cancers. This vaccine is designed to cover antigens in both the latent and lysis phases, enhancing the breadth of the immune response and anti-tumor efficacy. When the immune response to any antigen fluctuates or is locally limited, the immune response to other antigens can provide compensatory protection.

[0070] In particular, the sequence design advantages of the bivalent vaccine regimen: (1) Two mRNA sequences containing three EB virus antigens. In NE7, Zta and EBNA1 are tandemly linked, while in NE15, LMP2a is the only antigen. All three antigens were truncated to varying degrees to improve their safety. Specifically, amino acids 1-65, 81-409, and 618-645 of EBNA1 were removed, amino acids 175-194 of Zta were removed, and amino acids 1-119 and 471-497 of LMP2A were removed.

[0071] In addition to containing the target antigen, the two mRNA sequences mentioned above also include an N-terminal signal peptide sequence, a C-terminal LAMP1 transmembrane region, and / or an intracellular region. The N-terminal signal peptide of LAMP1 guides the antigen protein to the endoplasmic reticulum lumen, the C-terminal transmembrane region anchors the antigen to the lysosomal membrane, and the intracellular region of LAMP1 contains an 11-amino acid short peptide, including a conserved YXXΦ motif (Φ represents a hydrophobic amino acid), which is a key signal for lysosomal targeting. When the antigen is sorted and transported to the lysosome, it is degraded into polypeptides, binds to MHC class II molecules, and is presented to the cell surface, activating CD4. + T cells induce adaptive immune responses. After partial degradation in lysosomes, antigens can escape into the cytoplasm, where they are processed by the proteasome and presented to MHC class I molecules, activating CD8. + T cells (cytotoxic T cell response).

[0072] Advantages of mRNA technology: Previously, most therapeutic vaccines for nasopharyngeal carcinoma used adenovirus technology, peptide technology, and DNA technology. Compared with these technology platforms, mRNA technology has obvious advantages and therefore has a broader clinical application prospect. (1) Compared with DNA technology, it is safer. mRNA is only expressed in the cytoplasm, without the risk of genome integration, and there is a natural metabolic degradation mechanism in the body. (2) mRNA-LNP can achieve efficient and stable delivery and efficient expression in the body. It has adjuvant activity, activates natural immune pathways such as TLR, and can also induce and activate stronger adaptive immunity, enhance T cell infiltration, and reshape the tumor immune microenvironment. (3) It can encode a large number of antigens. Through the optimization of the mRNA expression framework, a single mRNA molecule can theoretically express up to dozens of tandem neoantigen epitopes or multiple long-sequence protein antigens at the same time, activating a broader spectrum of cellular and humoral immunity. (4) The preparation cycle is short. mRNA is synthesized through in vitro transcription, without the risk of contamination from pathogens in biosynthesis; at the same time, compared with other technologies, its production line is more versatile.

[0073] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0074] The reagents and raw materials used in this invention are all commercially available.

[0075] The positive and progressive effects of this invention are as follows: This invention provides an EB virus-positive tumor mRNA vaccine and its application. The product can improve immunogenicity, significantly inhibit tumor development, and improve survival time, while also exhibiting high safety. Attached Figure Description

[0076] Figure 1A This is a schematic diagram of the antigen protein of a therapeutic vaccine for EB virus-positive nasopharyngeal carcinoma.

[0077] Figure 1B The design, design method, and bivalent vaccine combination scheme for EB virus-positive nasopharyngeal carcinoma therapeutic vaccines.

[0078] Figure 2A To detect the expression of NE7 protein by Western blotting.

[0079] Figure 2B The transfection positivity rate of NE15.

[0080] Figure 2C The results of MFI transfection of NE15.

[0081] Figure 3A Number of IFN-γ spots activated by the EBNA1 vaccine NE1 and T-cell enhanced NE2 in wild-type C57BL / 6J.

[0082] Figure 3B The number of IL-2 spots activated by NE1 and NE2 in wild C57BL / 6J.

[0083] Figure 3C IFN-γ+TNF-α+CD8+ T cells activated by NE1 and NE2.

[0084] Figure 3D IFN-γ+TNF-α+CD4+ T cells activated by NE1 and NE2.

[0085] Figure 3E CD69+CD137+CD8+ T cells activated by NE1 and NE2.

[0086] Figure 4A The number of IFN-γ spots activated by the LMP2A vaccine NE3 and T-cell enhanced NE4 in wild-type mouse C57BL / 6J.

[0087] Figure 4BThe number of IL-2 spots activated by NE3 and NE4 in wild C57BL / 6J.

[0088] Figure 4C IFN-γ+TNF-α+ CD8+ T cells activated by NE3 and NE4 in wild-type C57BL / 6J.

[0089] Figure 4D IFN-γ+TNF-α+ CD4+ T cells activated by NE3 and NE4 in wild-type C57BL / 6J.

[0090] Figure 4E CD69+CD137+CD8+ T cells activated by NE3 and NE4 in wild-type C57BL / 6J.

[0091] Figure 5A The number of IFN-γ spots activated by Zta's vaccine NE5 and T-cell enhanced NE6 in wild-type C57BL / 6J.

[0092] Figure 5B The number of IL-2 spots activated by NE5 and NE6 in wild C57BL / 6J.

[0093] Figure 5C IFN-γ+TNF-α+ CD8+ T cells activated by NE5 and NE6 in wild-type C57BL / 6J.

[0094] Figure 5D IFN-γ+TNF-α+ CD4+ T cells activated by NE5 and NE6 in wild-type C57BL / 6J.

[0095] Figure 5E CD69+CD137+CD8+ T cells activated by NE5 and NE6 in wild-type C57BL / 6J.

[0096] Figure 6A Number of IFN-γ spots activated by the EBNA1 and Zta fusion vaccine (NE7-NE10) in wild-type C57BL / 6J mice.

[0097] Figure 6B The number of IL-2 spots activated by NE7-NE10 in wild-type mouse C57BL / 6J.

[0098] Figure 6C These are IFN-γ+TNF-α+ CD8+ T cells activated by NE7-NE10 in wild-type C57BL / 6J.

[0099] Figure 6DThese are IFN-γ+TNF-α+ CD4+ T cells activated by NE7-NE10 in wild-type C57BL / 6J.

[0100] Figure 7A The number of IFN-γ spots activated by the mutant / truncated LMP2A vaccines NE15, NE16, and NE17 in wild-type C57BL / 6J mice.

[0101] Figure 7B The number of IL-2 spots activated by NE15, NE16, and NE17 in wild-type mouse C57BL / 6J.

[0102] Figure 7C IFN-γ+TNF-α+ CD8+ T cells activated in wild-type C57BL / 6J for NE15, NE16, and NE17.

[0103] Figure 7D IFN-γ+TNF-α+ CD4+ T cells activated in wild-type C57BL / 6J for NE15, NE16, and NE17.

[0104] Figure 8A The number of IFN-γ spots activated by the EBV bivalent vaccine (NE7+NE15) in wild-type mouse C57BL / 6J.

[0105] Figure 8B IFN-γ+TNF-α+CD8+ T cells activated by the EBV bivalent vaccine (NE7+NE15) in wild-type mouse C57BL / 6J.

[0106] Figure 8C IFN-γ+TNF-α+CD4+ T cells activated by the EBV bivalent vaccine (NE7+NE15) in wild-type mouse C57BL / 6J.

[0107] Figure 8D CD69+CD137+CD8+ T cells activated by the EBV bivalent vaccine (NE7+NE15) in wild-type mouse C57BL / 6J.

[0108] Figure 8E and Figure 8F The results show the humoral immune response of the EBV bivalent vaccine (NE7+NE15) in wild-type mice C57BL / 6J.

[0109] Figure 9A The number of IFN-γ spots activated by the EBV bivalent vaccine (NE7+NE15) in HLA-A2.1 transgenic mice.

[0110] Figure 9BThe IFN-γ+TNF-α+CD8+ T cells activated by the EBV bivalent vaccine (NE7+NE15) in HLA-A2.1 transgenic mice.

[0111] Figure 9C The IFN-γ+TNF-α+CD4+ T cells activated by the EBV bivalent vaccine (NE7+NE15) in HLA-A2.1 transgenic mice.

[0112] Figure 9D CD69+CD137+CD8+ T cells activated by the EBV bivalent vaccine (NE7+NE15) in HLA-A2.1 transgenic mice.

[0113] Figure 10A This is a schematic diagram of the NE7 monovalent vaccine and its combination with PD-1 antibody in the B16-Zta-EBNA1 tumor model.

[0114] Figure 10B The study investigated the inhibitory effects of NE7 monovalent vaccine and its combination with PD-1 antibody on tumors in the B16-Zta-EBNA1 tumor model.

[0115] Figure 10C Survival curves of mice using NE7 monovalent vaccine and in combination with PD-1 antibody in the B16-Zta-EBNA1 tumor model.

[0116] Figure 11A The inhibitory effects of monovalent vaccine NE7 and bivalent vaccine (NE7+NE15) on tumors in the TC-1-EBNA1-Zta tumor model were investigated.

[0117] Figure 11B Survival curves of mice in the TC-1-EBNA1-Zta tumor model after inoculation with monovalent NE7 and bivalent (NE7+NE15) vaccines.

[0118] Figure 12A The inhibitory effects of the monovalent vaccine NE15 and the bivalent EBV vaccine (NE7+NE15) on tumors in the B16-LMP2A tumor model were investigated.

[0119] Figure 12B Survival curves of mice in the B16-LMP2A tumor model after inoculation with monovalent NE15 vaccine and bivalent EBV vaccine (NE7+NE15).

[0120] Figure 13 The results of pMHC tetramer detection after transfection of human PBMCs with NE7, NE15 and EBV bivalent vaccines.

[0121] Figure 14The results of Elispot assay using EBNA1 peptide restimulation were obtained on day 10 after transfection of PBMCs with the NE7 and EBV bivalent vaccine.

[0122] Figure 15 PBMCs were transfected with NE7, NE15 and EBV bivalent vaccines, and Elispot assays were performed after restimulation with LMP2A or Zta peptide.

[0123] Figure 16A In vitro killing effect of NE7 and EBV bivalent vaccine transfected PBMCs on target cells A375-Luc-EGFP.

[0124] Figure 16B In vitro killing effect of NE15 and EBV bivalent vaccine transfected PBMCs on target cells A375-Luc-EGFP.

[0125] Figure 17A To evaluate the toxicological safety of rats immunized with EBV bivalent vaccine.

[0126] Figure 17B Immunogenicity evaluation of the EBV bivalent vaccine (number of activated IFN-γ spots in rats). Detailed Implementation

[0127] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0128] Example 1: mRNA Sequence Design and Optimization

[0129] This invention provides an mRNA vaccine for EBV-positive nasopharyngeal carcinoma containing two mRNA sequences, NE7 and NE15. NE7 is a tandem combination of Zta and EBNA1, and NE15 is a standalone LMP2A. Amino acids 1-65, 81-409, and 618-645 of EBNA1 were removed; amino acids 175-194 of Zta were removed; and amino acids 1-119 and 471-497 of LMP2A were removed. For EBNA1, Zta, and NE7 (Zta-EBNA1), the N-terminus of the antigen is a LAMP1 signal peptide sequence, and the C-terminus contains the lysosomal localization signal of the LAMP1 transmembrane and intracellular regions. For LMP2A, only the intracellular region of LAMP1 was added to the C-terminus. The coding region was codon-optimized with a GC content of 56-60%. It also includes a 5'UTR, a 3'UTR, and a poly(A) region. See [link to vaccine antigen design and construction] for details. Figure 1A For vaccine numbering, design methods, and bivalent vaccine combinations, please refer to... Figure 1B .

[0130] Example 2 Preparation of lipid nanoparticles

[0131] The mRNAs of NE7 and NE15 from Example 1 were ionized (cationic) at low pH and coated into nanoparticles by two auxiliary lipids, DSPC (distearylphosphatidylcholine, catalog number: B90536, manufacturer: Nippon Seika Co., Ltd.) and cholesterol (catalog number: C00373, manufacturer: Nippon Seika Co., Ltd.), and PEGylated lipid (DMG-PEG2000, catalog number: M-DMG-2000, purchased from: JenKem). An aqueous solution of the mRNA was prepared by mixing the mRNA dissolved in ultrapure water with a 1:1 volume ratio of 100 mM (millimoles per liter, or mmol / L) citrate buffer at pH 4.0. The ratio of four lipid components (e.g., cationic lipid RL151 (catalog number: W211-YB211202, purchased from Zhejiang Shenzhou Pharmaceutical Co., Ltd.): cholesterol: DSPC: DMG-PEG2000 = 50: 38.5: 10: 1.5) was adjusted and dissolved in 99.5% ethanol to form a lipid solution. The mRNA and lipid solution were mixed in a NanoAssemblr microfluidic mixing system (manufacturer: Precision Nanosystems) at a volume mixing ratio of H2O: EtOH = 3:1 and a constant total flow rate of 12 mL / min to obtain lipid nanoparticles (LNPs) containing mRNA. After dialysis, concentration, filtration, and storage, the lipid nanoparticles yielded a therapeutic mRNA vaccine for EBV-positive nasopharyngeal carcinoma. NE7 had a particle size of 82 nM, a PDI of 0.05, and an encapsulation efficiency of 93.9%. The particle size of NE15 was 79 nM, the PDI was 0.03, and the encapsulation efficiency was 95.5%. The bivalent vaccine (NE7+NE15) had a particle size of 72 nM, a PDI of 0.09, and an encapsulation efficiency of 96.5%. The mass ratio of the two strands of NE7 and NE15 was determined to be 49%:51% by qPCR (using a 7500 real-time quantitative PCR system, manufacturer: Thermofisher).

[0132] Example 3: Protein expression of LNP

[0133] The protein expression level of LNP in HEK293A cells was tested. Figures 2A-2C ).

[0134] For NE7 ( Figure 2A): 2 and 5 µg of LNP were incubated with HEK293T cells in the wells of a 12-well plate for 20 hours. Cells were collected and NE7 protein expression was detected by Western blotting. Cells were collected and washed once with PBS (phosphate-balanced saline), and then lysed with RIPA lysis buffer (strong) (manufacturer: Absin; catalog number: Abs9229) containing protease inhibitors, 1 mM PMSF, and 10 mM DTT. All samples were mixed with SDS (sodium dodecyl sulfate) loading buffer, separated in 4–20% gradient SDS gels, and transferred to PVDF membranes (polyvinylidene fluoride membranes) using the Trans-Blot Turbo transfer system (a versatile protein transfer system, purchased from BioRad). The PVDF membranes were blocked with PBST (phosphate-buffered saline) containing 5% skim milk powder and then incubated with primary antibody (manufacturer: Santa Cruz Biotechnology; catalog number: sc-53904). The signal was detected using HRP-conjugated secondary antibody (manufacturer: Santa Cruz Biotechnology; catalog number: sc-516102) and an enhanced chemiluminescence (ECL) detection system.

[0135] For NE15 ( Figure 2B and Figure 2C ): Set up transfection gradient concentrations (ng) of NE15: 1, 10, 50, 100, 150, 250, 500, 1000, and 2000. Incubate HEK293A cells with each gradient concentration of LNP in 12-well plates for 20 hours. Collect cells and detect NE15 protein expression by flow cytometry. Collect cells and wash once with PBS (phosphate-balanced saline), add the live / dead dye FVS780 (manufacturer: BD Horizon; catalog number: 565388), and stain at room temperature for 15 minutes. After washing, stain again with anti-LMP2A antibody cross-linked with the fluorescent labeling group AF647-NHS (manufacturer: Thermofisher; catalog number: A88068), and incubate at room temperature for 25 minutes. After washing twice more, detect the transfection rate using flow cytometry and calculate the EC50 (conversion rate of the vaccine). Figure 2B ) is 293.4 ng / mL (1 mL / well, R 2 =0.9995), the MFI ratio (NE15 / negative control) is approximately 3 times at 2000 ng / well. Figure 2C )).

[0136] Example 4: Animal vaccination, cellular immunogenicity detection, and specific IgG enzyme-linked immunosorbent assay.

[0137] C57BL / 6 mice were vaccinated: Six- to eight-week-old female mice (purchased from Spifort (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized with 20 μg of an EB virus-positive nasopharyngeal carcinoma therapeutic vaccine (hereinafter referred to as "immunization"). A second and third dose were administered on days 7 and 14 post-immunization to enhance the immune response. The number of mice in each group was 6 (n=6).

[0138] On Day 21, after euthanizing the mice, the spleen was aseptically separated, ruptured, and mononuclear cells were obtained using lymphocyte separation medium (Shanghai Dako Biotechnology Co., Ltd., catalog number: 7211011). Cellular immunoassay was then performed using the following methods: (1) Elispot detection of the number of cellular spots of target antigen-specific IFN-γ and IL-2 According to 2×10 per hole 5 Cells were seeded onto mouse IFN-γ Elispot and IL-2 Elispot plates, and an antigen protein overlapping peptide library was added. The peptides contained 15 amino acid sequences, with each peptide sequence shifted by 4 amino acids, ultimately covering the entire antigen sequence. Phorbol ester (PMA) was added to the positive control wells, DMSO of the same concentration was added to the negative control wells (NC), and the SARS-CoV-2 spike S1 peptide (S1) was added to the irrelevant peptide group. The plates were then incubated in a 5% CO2 incubator at 37°C for 20 hours. Detection was performed according to the Elispot kit instructions (manufacturer: Mabtech Inc.; catalog number: 3321-4AST-10), and images were taken using an ELISA imager.

[0139] (2) Flow cytometry detection of target antigen-specific T cell response

[0140] According to 1×10 per hole 6 Cells were seeded into standard 96-well cell culture plates and incubated with peptides or controls for 1 hour. Monensin (manufacturer: BD; catalog number: 554724) was added as an inhibitor, and the plates were placed in a 5% CO2 incubator at 37°C for 5 hours before staining. First, surface antibodies against CD3, CD4, and CD8 were used for staining. Then, the cells were fixed and permeabilized. Finally, intracellular factors IFNγ and TNFα were stained. The resulting cells were analyzed using a Cytek flow cytometer. The percentages of IFNγ and TNFα in CD4+ and CD8+ cells were obtained by gating the cells.

[0141] (3) Flow cytometry detection of target antigen-specific T cell activation markers

[0142] According to 2×10 per hole 5Cells were seeded into 96-well V plates, and a peptide library was added to a final concentration of 2 μg / mL. After 20-24 hours, CD3, CD4, CD8, CD137, and CD69 were stained and identified by flow cytometry. The positive proportions of activated CD8+ cytotoxic cells were indicated by CD3+, CD8+, CD69+, and CD137+.

[0143] (4) Specific IgG enzyme-linked immunosorbent assay

[0144] The specific antibody reactions of EBNA1 and Zta proteins in the serum of mice immunized with the vaccine were determined by enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated overnight at 4°C with 50 µL of coating buffer containing 50 ng / well of recombinant EBNA1 (manufacturer: Abcam, catalog number: ab138345) or Zta antigen (manufacturer: Targetmol, catalog number: TMPH-00548). The plates were blocked at room temperature for 1 hour with 3% skim milk. The immunized mouse serum was diluted 3000-fold (for Zta antibody detection) or 100-fold (for EBNA1 antibody detection) as initial concentrations, and then serially diluted 2-fold in PBS buffer for a total of 8 dilutions, incubated at room temperature for 2 hours. To determine the specific antibody reaction, the plates were coated with goat anti-mouse IgG HRP (manufacturer: Luoyang Baitong, catalog number: C030205). Incubate at C for 1 hour, then develop color using a tetramethylbenzidine (TMB) substrate solution (Invitrogen, catalog number 00-4201-56). After approximately 10 minutes, terminate the color development reaction with 1 M sulfuric acid, and measure the absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific, model: Varioskan LUX 3020.0).

[0145] 4.1 Immunogenicity of EBNA1, Zta, and LMP2A monovalent vaccines in mouse C57BL / 6J

[0146] Each antigen corresponds to two versions of the vaccine: EBNA1 vaccine is NE1 and NE2, LMP2A vaccine is NE3 and NE4, and Zta vaccine is NE5 and NE6. Among them, NE1, NE3 or NE5 only encode the corresponding antigen, while NE2, NE4 or NE6 encode the corresponding antigen and T cell enhanced design (incorporating the lysosomal localization signal LAMP1).

[0147] (1) Immunogenicity of EBNA1 ( Figures 3A-3E ): Detected by ELISPOT ( Figure 3A and 3BThe number of IFN-γ and IL-2 spots activated by the T-cell enhanced NE2 vaccine were 2.31 times and 2.28 times that of NE1, respectively. Intracellular cytokine staining ( Figure 3C and 3D The results showed that NE2 activated 18.9% of IFN-γ+TNF-α+CD8+ T cells, a 2.27-fold increase compared to NE1 (8.33%); simultaneously, NE2 activated approximately 1% of IFN-γ+TNF-α+CD4+ T cells, stronger than NE1 (approximately 0%, almost no activation). Cell surface marker analysis ( Figure 3E The study confirmed that NE2 activated 30.8% of CD69+CD137+CD8+ T cells, which was 1.79 times higher than NE1 (17.2%), highlighting the immunogenic advantages of the T cell-enhanced design.

[0148] (2) Immunogenicity of LMP2A Figures 4A-4E ): LMP2A vaccine NE4 (T-cell enhanced) passed ELISPOT testing ( Figure 4A and 4B The results showed that the number of activated IFN-γ and IL-2 spots was higher in the NE3 group than in the NE3 group, and both were significantly better than in the Buffer control group. Intracellular cytokine staining ( Figure 4C and 4D The results showed that the proportions of IFN-γ+TNF-α+ CD8+ T cells activated by NE3 and NE4 were similar (2.54% vs 2.89%), but neither activated CD4+ cells, indicating that the immune response specifically depends on CD8+ T cells. Cell surface marker analysis ( Figure 4E Further studies revealed that the proportion of CD69+CD137+CD8+ T cells activated by NE4 (11.5%) was significantly higher than that of NE3 (6.34%) by 81%, highlighting the immunogenic advantages of the T cell-enhanced design.

[0149] (3) Immunogenicity of Zta ( Figures 5A-5E ): The number of IFN-γ spots and IL-2 spots activated by the T-cell enhanced Zta vaccine NE6 were 2.22 times and 2.33 times that of NE5, respectively, confirming that this design is suitable for Zta antigen ( Figure 5A and 5B Intracellular cytokine staining ( Figure 5C and 5D This confirmed that both NE5 and NE6 activated only CD4+ T cells (NE6: 0.79% IFN-γ+TNF-α+ CD4+ cells, 2.26 times that of NE5 (0.35%)), and did not activate CD8+ T cells, indicating immunogenicity-dependent CD4+ T cell activation. Cell surface marker analysis ( Figure 5EThe study found no significant difference in the proportion of CD69+CD137+CD8+ T cells activated by NE5 / NE6 (1.26% vs 1.37%), suggesting that the Zta antigen has a weak CD8+ T cell activation potential.

[0150] Overall, all three EBV antigens, EBNA1, LMP2A, and Zta, activated an immune response in mouse C57BL / 6J. EBNA1 was the most potent, simultaneously activating both strong CD8+ and CD4+ T cells. LMP2A was the next most potent, primarily activating CD8+ T cells. Zta was the least potent, primarily acting on CD4+ T cells. Furthermore, the T-cell-enhancing design significantly improved the immunogenicity of all three antigens.

[0151] 4.2 Immunogenicity of EBNA1 and Zta fusion antigen NE7

[0152] Two fusion sequences (NE8 / NE10) and corresponding T-cell enhanced vaccines (NE7 / NE9) were constructed targeting EBNA1 and Zta antigen. Figures 6A-6D Elispot detection () Figure 6A and 6B The total number of IFN-γ and IL-2 spots activated by NE7, i.e., the sum of EBNA1 and Zta-specific spots, was superior to other tandem designs and monoantigen vaccines (NE2 / NE6), indicating that the tandem structure retains the immunogenicity of dual antigens. Intracellular cytokine staining ( Figure 6C and 6D All tandem designs activated IFN-γ+TNF-α+ CD8+ T cells (EBNA1 peptide library) and CD4+ T cells (EBNA1 / Zta peptide library). NE7 activated 10% of double-positive CD8+ T cells and 0.69% of double-positive CD4+ T cells.

[0153] The dose-efficiency advantage of this design is that, at the same dose, the number of monoantigen moles in NE7 is only about half that of monoantigen vaccines (NE2 / NE6), but the level of immune activation is comparable, confirming that it achieves simultaneous activation of dual-antigen T cells without affecting the independent immunogenicity of EBNA1 / Zta. This design overcomes the limitation of Zta antigen relying solely on CD4+ T cells (see NE5 / NE6 data), and the tandem design achieves the joint activation of EBNA1-specific CD8+ T cells and dual-antigen CD4+ T cells.

[0154] 4.3 Immunogenicity of LMP2A truncated antigen NE15

[0155] This experiment compared the effects of four LMP2A variants (NE4: wild-type T-cell enhancer; NE17: mutant; NE16: mutant T-cell enhancer; NE15: truncated T-cell enhancer) on activating antigen-specific CD8+ and CD4+ T cells in C56BL / 6J mice. NE16 and NE17, by mutating the N-terminus Y74, Y85, and Y112 of LMP2A to F, lost their substrate-binding ability. Elispot assay (...) Figure 7A and 7B The results showed that NE15-induced mean values ​​of IFN-γ and IL-2 spots were significantly higher than those of other variants (some differences were statistically significant). Intracellular cytokine staining ( Figure 7C The results showed that there was no significant difference in the proportion of IFN-γ and TNF-α double-positive CD8+ T cells activated among all variants (mean 4.5-5%); mean 4.5-5%; mean 4.5-5%; and among all variants, only NE15 could activate trace amounts of IFN-γ and TNF-α double-positive CD4+ T cells. Figure 7D Therefore, NE15, as a truncated variant, can maintain high T cell activation levels, highlighting its superior design and supporting its potential application value in immunotherapy.

[0156] The final version of the vaccine is an EBV bivalent vaccine, which is a mixed package of NE7 and NE15 mRNAs, namely NE7V5 (SEQ ID NO: 82) and NE15V1 (SEQ ID NO: 118), with a mass ratio of 1:1.

[0157] 4.3 Immunogenicity of EBV bivalent vaccine in C57BL / 6J WT mice

[0158] Immunogenicity assays for C57BL / 6J WT mice immunized with EBV bivalent vaccine (20 μg) are shown in the table below. Figures 8A-8F Elispot shows ( Figure 8A EBNA1, LMP2A, and Zta activated 3613, 1583, and 944 IFN-γ spots per million cells, respectively. ICS showed ( Figure 8B and Figure 8C EBNA1 and LMP2A both activated approximately 6.0% of CD8+ IFNγ+ and TNFα+ T cells. EBNA1 and Zta activated 2.5% and 1.9% of CD4+ IFNγ+ and TNFα+ T cells, respectively. AIM experiments showed ( Figure 8D EBNA1 and LMP2A activated 8.2% and 12.2% of CD8+CD69+CD137+ antigen-specific T cells, respectively. Zta still failed to activate CD8+ specific T cells.

[0159] Specific IgG enzyme-linked immunosorbent assay showed that EBNA1 and Zta could also activate humoral immunity, with total antititer titers of approximately 300 and 6000, respectively. Figure 8E and Figure 8F 4.4 Immunogenicity of EBV bivalent vaccine in human HLA2.1 mice

[0160] Immunogenicity assay of human HLA2.1 mice immunized with EBV bivalent vaccine (20 μg) is shown in [link to relevant documentation]. Figures 9A-9D Elispot shows ( Figure 9A EBNA1, LMP2A, and Zta activated 326, 4379, and 1233 IFN-γ spots per million cells, respectively. ICS showed ( Figure 9B and Figure 9C LMP2A alone activated 6.4% of IFN-γ and TNF-α double-positive CD8+ T cells, while EBNA1 and Zta failed to effectively activate IFN-γ and TNF-α double-positive CD8+ T cells. EBNA1, Zta, and LMP2A activated 0.3%, 0.3%, and 0.12% of IFN-γ and TNF-α double-positive CD4+ T cells, respectively. AIM experiments showed ( Figure 9D LMP2A activated 34.9% of CD8+CD69+CD137+ antigen-specific T cells, significantly higher than 3% for EBNA1. Zta, however, failed to activate CD8+ specific T cells.

[0161] Example 5: In vivo pharmacodynamic evaluation of an EB virus-positive nasopharyngeal carcinoma therapeutic vaccine in mice

[0162] This study used B16 cell lines overexpressing Zta-EBNA1 or LMP2a for pharmacodynamic evaluation. The B16 cell line, derived from spontaneous melanomas behind the ears of C57BL / 6 mice, is widely used to assess tumorigenesis and antitumor immune responses.

[0163] 1. Evaluation of the antitumor efficacy of NE7 monovalent vaccine and its combination with PD-1 antibody in the B16-Zta-EBNA1 tumor model.

[0164] according to Figure 10A As shown, on Day 0, C57BL / 6J mice were subcutaneously inoculated with B16-EBNA1-Zta cells in logarithmic growth phase, 1×10⁻⁶ cells. 5 / each. NE7 vaccine 20 μg or vaccine buffer was administered on Day 5, Day 9, Day 12, and Day 15. PD-1 antibody (manufacturer: Biolegend; catalog number: 135250) 200 μg or isotype control (manufacturer: Biolegend; catalog number: 400574) was administered intraperitoneally on Day 6, Day 10, Day 13, and Day 16. Tumor size was measured every 2-3 days using the commonly used formula V=L×W. 2 The tumor volume was calculated using a ratio of 2, and the survival curve was observed.

[0165] Figure 10B The results showed that on Day 16, NE7 achieved a 59.9% tumor inhibition rate compared to the PBS group. On Day 18, the combination of NE7 alone and PD-1 antibody showed the most significant inhibition of tumor growth, significantly stronger than either NE7 or PD-1 antibody alone (p values ​​less than 0.0001). Mouse survival data indicated ( Figure 10C From day 16, mice in the PBS group began to die; by day 26, the survival rate in the combination therapy group was 62.5%, higher than the PD-1 monoclonal antibody group, and significantly higher than the NE7 group and PBS (P=0.0231 relative to NE7; P=0.0093 relative to PBS). These data demonstrate that NE7, a tumor treatment product targeting EB virus, can produce a significant tumor-suppressing effect when used alone, and that its combination with a PD-1 antibody can achieve a significant tumor-suppressing effect, effectively prolonging animal survival, providing a theoretical basis for subsequent clinical combination therapy design.

[0166] 2. Evaluation of the antitumor efficacy of monovalent vaccine NE7 and bivalent vaccine (NE7+NE15) in the TC-1-EBNA1-Zta tumor model.

[0167] according to Figure 11A As shown, on Day 0, C57BL / 6 mice were subcutaneously inoculated with TC-1-EBNA1-Zta cells in logarithmic growth phase, 5 × 10⁶ cells per cell line. 5 / animal. On Day 7, Day 10, Day 14, and Day 17, patients were administered 20 μg of RQ2400 vaccine, 10 μg of the monovalent NE7 vaccine, or a vaccine buffer. At a dose of 20 μg, the tumor inhibition rate of the bivalent vaccines (NE7+NE15)-20 μg and NE7-10 μg on Day 19 was 42%, with p-values ​​less than 0.0001. On Day 24, the survival curve ( Figure 11B The results showed that the bivalent vaccine significantly improved the survival rate of mice, with significance levels of 0.0064 and 0.0272, respectively. This indicates that immunization of mice with the bivalent vaccine can achieve a significant tumor-suppressing effect and effectively prolong animal survival, providing a theoretical basis for the design of subsequent clinical trials.

[0168] 3. Evaluation of the antitumor efficacy of the monovalent NE15 vaccine and the bivalent EBV vaccine in the B16-LMP2A tumor model.

[0169] according to Figure 12A As shown, on Day 0, C57BL / 6 mice were subcutaneously inoculated with B16-LMP2a cells in logarithmic growth phase, 2 × 10⁶ cells per cell line. 5 / animal. EBV bivalent vaccine 20 μg, NE15 monovalent vaccine 10 μg, or vaccine buffer were administered on Day 4, Day 7, Day 10, and Day 14. The tumor inhibition rates of EBV bivalent vaccine 20 μg and NE15 vaccine 10 μg on Day 19 were 76.0% and 68.6%, respectively, with p-values ​​less than 0.0001. On day 25, the survival curve ( Figure 12B The results showed that the EBV bivalent vaccine and NE15 vaccine groups significantly improved the survival rate of mice, with significance levels of p=0.0002 and 0.002, respectively. This indicates that immunization of mice with the bivalent vaccine, like the monovalent NE15 vaccine, can achieve a significant tumor-suppressing effect and effectively prolong animal survival, providing a theoretical basis for the design of subsequent clinical trials.

[0170] Example 6: Pharmacodynamic evaluation of EB virus-positive nasopharyngeal carcinoma therapeutic vaccine in human PBMCs

[0171] 6.1 Immunogenicity testing of EBV-positive nasopharyngeal carcinoma therapeutic vaccine in human PBMCs

[0172] PBMCs were transfected with NE7, NE15, and a bivalent vaccine. A series of cytokines were added to promote DC differentiation and maturation, present antigenic epitopes, and initialize naïve T cells, inducing them to become antigen-specific CD8+ and CD4+ memory T cells or effector T cells. Immunogenicity testing was performed as follows: (1) The proportion of epitope-specific T cells to CD3+CD8+ T cells was determined by staining PBMCs with pMHC-tetramer containing EBV epitopes and corresponding HLA types. Among them, EBNA1 (FVYGGSKTSL, SEQ ID NO: 150) corresponds to HLA-C 03:03, LMP2A (FLYALALLL, SEQ ID NO: 151) corresponds to HLA-A 02:01, Zta (EPLPQGQLTAY, SEQ ID NO:152) corresponds to HLA-B 35:01. (For example) Figure 13As shown: PBMCs were transfected with NE7, NE15 and EBV bivalent vaccines, which initialized and stimulated the production of Tetramer-positive specific T cells targeting EBNA1, LMP2A and Zta, respectively. The positive clusters were clearly separated, with high specificity and a positive rate more than 2 times higher than that of the negative control group.

[0173] (2) PBMCs were restimulated with peptides containing EBNA1, Zta, and LMP2A, and the number of spots of antigen-specific T cells secreting IFN-γ was detected using Elispot. Cells were plated at 100,000 cells per well. Figure 14 As shown, after transfection of human PBMCs with NE7 and EBV bivalent vaccines, the EBNA1 peptide library and its HLA-C0303 epitope peptide both stimulated 1.5-2 times the number of IFN-γ spots compared to the DMSO group. This experiment also tested irrelevant peptides, which showed low background signal. According to... Figure 15 As shown, transfection of human PBMCs with NE7, NE15, and EBV bivalent vaccines, followed by the LMP2A peptide library and its HLA-A0201 epitope peptide, all stimulated a 5-14 fold increase in the number of IFN-γ spots compared to the DMSO group, with very low background signal. Simultaneously, the Zta peptide library and Zta-HLA-B3501 epitope peptide also stimulated a 2.5-5 fold increase in the number of IFN-γ spots compared to the DMSO group. This example suggests that EBV peptide incubation of human PBMCs can induce antigen-specific T cell signaling.

[0174] 6.2 In vitro killing assay of EB virus-positive nasopharyngeal carcinoma therapeutic vaccine in human PBMCs

[0175] Add EBV peptide library (15 mer) or HLA-A to target cells A375-Luc-EGFP. 0201 and HLA-C The 0303-specific epitope peptide (15 mer) was used to mix PBMC cells transfected with the vaccine with target cells at a ratio of PBMC:target cells = 10:1 and cultured for 24 hours. Then, lysis buffer, ATP and substrate were added. The amount of light emitted by the Luciferase-catalyzed substrate in the live target cells was detected to determine the proportion of live cells and the in vitro killing rate of target cells.

[0176] according to Figure 16A As shown, PBMCs transfected with NE7 or EBV bivalent vaccines achieved killing rates of 68.4% and 70.2% respectively against target cells incubated with a 5 μg / mL EBNA1 peptide library. Meanwhile, PBMCs transfected with NE7 showed improved killing rates against LMP2A-HLA-A... The 02:01 peptide (15 mer, containing the precise epitope FLYALALLL, SEQ ID NO: 151) did not produce non-specific killing of target cells after incubation. According to... Figure 16B As shown, PBMCs transfected with NE15 and EBV bivalent vaccines achieved killing rates of 44.7% and 56.5%, respectively, against target cells incubated with a 2 μg / mL LMP2A peptide library; and against HLA-A cells incubated with a 2 μg / mL LMP2A peptide library. Target cells incubated with the 02:01 epitope peptide (15 mer, containing the precise epitope FLYALALLL, SEQ ID NO: 151) achieved killing rates of 46.7% and 61.1%, respectively. Simultaneously, PBMCs transfected with NE15 showed improved killing rates against EBNA1-HLA-C. The 03:03 peptide (15 mer, containing the precise epitope FVYGGSKTSL, SEQ ID NO: 151) did not produce non-specific killing of target cells after incubation. Furthermore, we observed no non-specific killing of target cells by the EBNA1 peptide library and LMP2A-HLA-A. The 02:01 peptide (15 mer, containing the precise epitope FLYALALLL, SEQ ID NO: 151) also had a concentration gradient set up, and it was observed that the specific killing effect of the vaccine transfected PBMCs on target cells was positively correlated with the concentration of the antigen-specific peptide (library). Figure 16A and 16B The results show a significant dose-response effect. These results confirm that the NE7, NE15, and EBV bivalent vaccines can activate antigen-specific T cells and kill target cells in the human system, providing a theoretical basis for subsequent clinical trials.

[0177] Example 7: Safety evaluation of a therapeutic vaccine for EB virus-positive nasopharyngeal carcinoma

[0178] The safety of an EBV-positive nasopharyngeal carcinoma therapeutic vaccine (bivalent vaccine; i.e., a mixture of NE7 and NE15) was evaluated in SD rats (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). Five groups of SD rats were included: a high-dose group (117 µg / dose), a medium-dose group (35 µg / dose), a low-dose group (12 µg / dose), a blank liposome group, and a PBS group, with 20 rats per group (n=20 per sex). The vaccine was administered via single / multi-point intramuscular injection in the right hind limb (gastrocnemius and quadriceps femoris muscles), once every week for 4 weeks, for a total of 5 administrations. The day of the first administration was designated as D0. Ten animals (5 per sex) from each group were dissected 3 days after the last administration (D31) to evaluate toxicity during the administration period; the remaining 10 surviving animals (5 per sex) were dissected after a 2-week recovery period (D42) to evaluate the reversibility and delay of toxicity. During the experiment, animals underwent clinical observation (including local observation and scoring of drug administration), weight, food intake, clinical pathology (blood cell count, blood biochemistry), immunological indicators (immune cell phenotype, cytokines), cellular immunity, gross anatomy, organ weighing, and histopathological examination.

[0179] Results showed no animal deaths or near-death experiences during the experiment. Animals in all bivalent vaccine dose groups exhibited changes associated with blank liposomes, acute phase reactions, and / or bivalent vaccine-induced immune responses, all common reactions to mRNA vaccines. These changes showed recovery or a recovery trend two weeks after drug withdrawal (D42). Figure 17A The no-observed-adverse-effect level (NOAEL) for the EBV bivalent vaccine was 117 μg / rat, equivalent to 28 times the clinically intended dose of 1 mg / human (rats weighing 0.25 kg and humans weighing 60 kg). Specific activation of EBNA1 and Zta was detected 2 weeks (D42) after the third EBV bivalent vaccine immunization using IFN-γ Elispot assay. Figure 17B ).

[0180] The following are the antigen protocols involved in this invention (Table 1): Table 1 Sequence information of the antigen scheme of the present invention

[0181]

[0182]

[0183] Antigen sequence of NE4: MGSLEMVPMGAGPPSPGGDPDGDDGGNNSQYPSASGSSGNTPTPPNDEERESNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVCLPVIVAPYLFWLAAIAASCFTASVSTVVSATGLALSLLLLAAVANSSAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSILFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGMMFLACLVVLIVDAVLQLSPLLGAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKVLLARLFLYALALLLLASALTAGGSILQTNFKSLSSTEFIPHLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMSLGGLLTMVAGAVWLTVMTNTLLSAWILTAGFLIFLIGFALFGVI(SEQ ID NO: 14)

[0184] Signal peptide: MAAPGSARRPLLLLLLLLLLGLMHCASA(SEQ ID NO: 16)

[0185] Transmembrane region and intracellular region of LAMP1: SMLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI(SEQ ID NO:17)

[0186] Transmembrane region of LAMP1: SMLIPIAVGGALAGLVLIVLIAYLVG(SEQ ID NO: 15); Intracellular region of LAMP1: RKRSHAGYQTI(SEQ ID NO: 148); The antigen sequence of the LMP2A truncated body (Truncated LMP2A), namely NE15: MNPVCLPVIVAPYLFWLAAIAASCFTASVSTVVSATGLALSLLLLAAVANSSAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSILFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGMMFLACLVVLIVDAVLQLSPLLGAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKVLLARLFLYALALLLLASALTAGGSILQTNFKSLSSTEFIPHLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMSLGGLLTMVAGAVWLTVMTNTLLSAWILTAGFLIFLIGFALFGVI (SEQ ID NO: 149) Coding sequence 1 of NE1 (NE1V1) SEQ ID NO: 18: AUGCACAGAGACGGCGUGAGAAGACCCCAGAAGAGGCCCAGCUGCAUCUUCCACCCUGUGGGAGAUGCCGACUAUUUCGAGUACCUGCAGGAGGGAGGACCUGAUGGAGAACCAGAUGUGCCACCUGGAGCCAUUGAACAGGGCCCAACAGAUGAUCCUGGAGAAGGACCAUCUACAGGACCUAGAGGACAGGGAGACGGAGGAAGAAGAAAGAAAGGCGGCUGGUUUGGCAAACACAGGGGACAGGGCGGCAGCAAUCCCAAGUUUGAGAACAUCGCCGAAGGCCUGAGAGUGCUGCUGGCCAGAAGCCAUGUGGAGAGAACAACCGAGGAAGGCAAUUGGGUGGCCGGAGUGUUUGUGUAUGGAGGAAGCAAGACCAGCCUGUACAACCUGAGAAGGGGCAUUGCUCUGGCUGUGCCACAGUGUAGAAUCACCCCUCUGUCUAGGCUGCCAUUUGGAAUGGCUCCUGGACCUGGACCACAGCCAGGCCCUCUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUUUUCGCCGAGGUGCUGAAGGAUGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGAUGGCGUGGAUCUGCCUCCUUGGUUUCCUCCCAUG Coding sequence 2 of NE1 (NE1V2) SEQ ID NO: 19: AUGCACAGAGACGGCGUGAGAAGACCCCAGAAGAGGCCCAGCUGCAUCUUCCACCCUGUGGGAGAUGCCGACUAUUUCGAGUACCUGCAGGAGGGAGGACCUGAUGGAGAACCAGAUGUGCCACCUGGAGCCAUUGAACAGGGCCCAACAGAUGAUCCUGGAGAAGGACCAUCUACAGGACCUAGAGGACAGGGAGACGGAGGAAGAAGAAAGAAAGGCGGCUGGUUUGGCAAACACAGGGGACAGGGCGGCAGCAAUCCCAAGUUUGAGAAUAUCGCCGAGGGCCUGAGAGUGCUGCUGGCCAGAAGCCAUGUGGAGAGAACAACCGAGGAAGGCAAUUGGGUGGCCGGAGUGUUUGUGUAUGGAGGAAGCAAGACCAGCCUGUACAACCUGAGAAGGGGCAUUGCUCUGGCUGUGCCACAGUGUAGAAUCACCCCUCUGUCUAGGCUGCCAUUUGGAAUGGCUCCUGGACCUGGACCACAGCCAGGCCCUCUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUUUUCGCCGAGGUGCUGAAAGACGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGAUGGCGUGGAUCUGCCUCCUUGGUUUCCUCCCAUG Coding sequence 3 of NE1 (NE1V3) SEQ ID NO: 20: AUGCACAGAGACGGCGUGAGAAGACCCCAGAAGAGGCCCAGCUGCAUCUUUCACCCCGUGGGCGAUGCCGACUAUUUCGAGUACCUGCAGGAGGGAGGACCAGAUGGCGAACCAGAUGUGCCACCUGGAGCUAUUGAACAGGGCCCAACAGAUGAUCCUGGAGAAGGACCAUCUACAGGACCUAGAGGACAGGGAGACGGAGGAAGAAGAAAGAAAGGCGGCUGGUUUGGCAAACACAGGGGACAGGGCGGCAGCAAUCCCAAGUUUGAGAAUAUCGCCGAGGGCCUGAGAGUGCUGCUGGCCAGAAGCCAUGUGGAGAGAACAACCGAGGAAGGCAAUUGGGUGGCCGGAGUGUUUGUGUAUGGAGGAAGCAAGACCAGCCUGUACAACCUGAGAAGGGGCAUUGCUCUGGCUGUGCCACAGUGUAGAAUCACCCCUCUGUCUAGGCUGCCAUUUGGAAUGGCUCCUGGACCUGGACCACAGCCAGGCCCUCUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUUUUCGCCGAGGUGCUGAAAGACGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGAUGGCGUGGAUCUGCCUCCUUGGUUCCCUCCCAUG Coding sequence 4 of NE1 (NE1V4) SEQ ID NO: 21: AUGCACAGAGACGGCGUGAGAAGACCCCAGAAGAGGCCCAGCUGCAUCUUUCACCCCGUGGGCGAUGCCGAUUAUUUCGAGUACCUGCAGGAGGGAGGACCAGAUGGCGAACCAGAUGUGCCACCUGGAGCUAUUGAACAGGGCCCAACAGAUGAUCCUGGAGAAGGACCAUCUACAGGACCUAGAGGACAGGGAGACGGAGGAAGAAGAAAGAAAGGCGGCUGGUUUGGCAAACACAGGGGACAGGGCGGAAGCAAUCCCAAGUUUGAGAACAUCGCCGAGGGCCUGAGAGUGCUGCUGGCCAGAAGCCAUGUGGAGAGAACAACCGAGGAAGGCAAUUGGGUGGCCGGAGUGUUUGUGUAUGGAGGAAGCAAGACCAGCCUGUACAACCUGAGAAGGGGCAUUGCUCUGGCUGUGCCACAGUGUAGAAUUACCCCUCUGUCUAGGCUGCCCUUUGGAAUGGCCCCUGGACCUGGACCACAGCCAGGCCCUCUGAGAGAAAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUUUUCGCCGAGGUGCUGAAAGACGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGAUGGCGUGGAUCUGCCUCCUUGGUUCCCUCCCAUG Coding sequence 5 of NE1 (NE1V5) SEQ ID NO: 22: AUGCACAGAGACGGCGUGAGAAGACCACAGAAGAGGCCCAGCUGCAUCUUUCAUCCCGUGGGCGAUGCCGACUAUUUCGAGUAUCUGCAGGAGGGAGGACCAGAUGGCGAACCAGAUGUGCCACCUGGAGCCAUUGAACAGGGCCCUACAGAUGAUCCUGGAGAAGGACCAUCUACAGGACCUAGAGGACAGGGAGACGGAGGAAGAAGAAAGAAAGGCGGCUGGUUUGGCAAGCACAGGGGACAGGGCGGAAGCAAUCCCAAGUUUGAGAACAUCGCUGAGGGCCUGAGAGUGCUGCUGGCCAGGAGCCAUGUGGAAAGAACAACCGAGGAAGGCAAUUGGGUGGCCGGAGUGUUUGUGUAUGGAGGAAGCAAGACCAGCCUGUACAACCUGAGAAGGGGCAUUGCUCUGGCUGUGCCACAGUGUAGAAUUACCCCUCUGUCUAGGCUGCCCUUUGGAAUGGCCCCUGGACCUGGACCACAGCCAGGCCCUCUGAGAGAGAGCAUUGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUUUUCGCCGAGGUGCUGAAAGACGCCAUCAAGGACCUGGUGAUGACCAAGCCUGCCCCUACCUGUAACAUCAAGGUGACCGUGUGUAGCUUUGACGAUGGCGUGGAUCUGCCUCCUUGGUUCCCUCCCAUG Coding sequence 6 of NE1 (NE1V6) SEQ ID NO: 23: AUGCACCGGGACGGCGUGAGAAGACCACAGAAGAGGCCCAGCUGCAUUUUUCACCCCGUGGGCGAUGCCGACUAUUUCGAGUAUCUGCAGGAGGGAGGCCCUGACGGCGAACCAGAUGUGCCACCUGGAGCCAUUGAACAGGGACCUACUGAUGAUCCUGGAGAAGGACCAUCAACAGGACCUAGAGGACAGGGAGACGGAGGAAGAAGAAAGAAAGGCGGCUGGUUCGGCAAACACAGGGGACAGGGCGGAAGCAAUCCCAAGUUUGAGAAUAUCGCUGAGGGCCUGAGAGUGCUGCUGGCCAGGUCCCACGUGGAAAGAACAACCGAGGAAGGCAAUUGGGUGGCCGGUGUGUUUGUGUAUGGAGGAAGCAAGACCAGCCUGUACAACCUGAGAAGGGGCAUUGCUCUGGCUGUGCCACAGUGUAGAAUUACCCCCCUGUCUAGGCUGCCCUUUGGAAUGGCCCCUGGACCUGGACCACAGCCAGGCCCCCUGAGAGAGAGCAUUGUGUGUUACUUCAUGGUGUUCCUGCAGACACACAUUUUCGCCGAGGUGCUGAAAGACGCCAUCAAGGACCUGGUGAUGACCAAGCCUGCCCCUACAUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGAUGGCGUGGACCUGCCUCCUUGGUUCCCUCCCAUG Coding sequence 7 of NE1 (NE1V7) SEQ ID NO: 24: AUGCACAGAGACGGCGUGAGAAGACCCCAGAAGAGGCCCAGCUGCAUCUUCCACCCAGUGGGCGACGCCGAUUACUUCGAGUACCUGCAGGAGGGCGGCCCAGAUGGCGAGCCUGACGUGCCACCAGGAGCUAUCGAACAGGGCCCUACAGAUGAUCCUGGAGAAGGCCCAUCUACAGGACCUAGAGGACAGGGAGAUGGAGGCAGAAGAAAGAAGGGCGGAUGGUUUGGCAAGCACAGAGGCCAGGGCGGCAGCAACCCCAAAUUCGAGAACAUCGCCGAGGGCCUGAGGGUGCUGCUGGCCCGGAGCCAUGUGGAGAGAACCACAGAGGAAGGAAAUUGGGUGGCCGGCGUGUUUGUGUACGGAGGCAGCAAGACCAGCCUGUACAACCUGAGAAGAGGCAUUGCUCUGGCUGUGCCUCAGUGUCGGAUUACCCCACUGAGCAGGCUGCCAUUUGGCAUGGCUCCUGGCCCUGGACCUCAGCCUGGCCCCCUGCGGGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAGGAUGCCAUCAAGGACCUGGUGAUGACCAAGCCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGAUGGCGUGGACCUGCCUCCCUGGUUCCCACCCAUG Coding sequence 8 of NE1 (NE1V8) SEQ ID NO: 25: AUGCACAGAGACGGCGUGAGAAGACCCCAGAAGAGGCCCAGCUGCAUCUUCCACCCAGUGGGCGAUGCCGAUUACUUCGAGUACCUGCAGGAGGGCGGCCCAGAUGGCGAGCCUGACGUGCCACCAGGAGCUAUCGAACAGGGCCCUACAGAUGAUCCUGGAGAAGGACCAUCUACAGGCCCUAGAGGACAGGGAGAUGGAGGCAGAAGAAAGAAGGGCGGAUGGUUUGGCAAGCACAGAGGCCAGGGCGGCAGCAACCCCAAAUUCGAGAACAUCGCCGAAGGCCUGAGGGUGCUGCUGGCCCGGAGCCACGUGGAAAGGACCACAGAGGAGGGAAAUUGGGUGGCCGGCGUGUUUGUGUACGGAGGAAGCAAAACCAGCCUGUACAACCUGAGGAGAGGCAUUGCUCUGGCUGUGCCUCAGUGUCGGAUUACCCCACUGAGCAGGCUGCCAUUUGGCAUGGCUCCUGGCCCUGGACCUCAGCCUGGCCCCCUGCGGGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAGGAUGCCAUCAAGGACCUGGUGAUGACCAAGCCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGACGGCGUGGACCUGCCUCCCUGGUUCCCACCCAUG Coding sequence 9 of NE1 (NE1V9) SEQ ID NO: 26: AUGCACAGAGACGGCGUGAGAAGACCUCAGAAGAGGCCCAGCUGCAUCUUCCACCCAGUGGGCGACGCCGAUUACUUUGAGUACCUGCAGGAGGGCGGCCCAGAUGGCGAGCCCGAUGUGCCACCAGGAGCUAUCGAACAGGGCCCUACAGAUGAUCCUGGAGAAGGACCAUCUACAGGCCCUAGAGGACAGGGAGAUGGAGGCAGAAGAAAGAAGGGCGGCUGGUUUGGCAAGCACAGAGGCCAGGGCGGCAGCAACCCCAAAUUCGAGAAUAUCGCCGAAGGCCUGAGGGUGCUGCUGGCCCGGAGCCAUGUGGAACGGACCACAGAGGAGGGAAAUUGGGUGGCCGGAGUGUUUGUGUACGGCGGAAGCAAAACCAGCCUGUACAAUCUGCGGAGAGGCAUUGCUCUGGCUGUGCCUCAGUGUCGGAUUACCCCACUGAGCAGGCUGCCAUUUGGCAUGGCUCCUGGCCCUGGACCUCAGCCUGGCCCCCUGCGCGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAGGAUGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGACGGCGUGGACCUGCCUCCCUGGUUCCCACCCAUG Coding sequence 10 of NE1 (NE1V10) SEQ ID NO: 27: AUGCACAGAGACGGCGUGAGAAGACCUCAGAAGCGGCCCAGUUGCAUCUUCCACCCAGUGGGCGACGCCGAUUACUUCGAAUACCUGCAGGAGGGCGGUCCUGACGGAGAGCCCGAUGUGCCACCAGGAGCUAUCGAACAGGGACCCACAGAUGACCCUGGAGAAGGACCAUCUACAGGCCCUAGAGGACAGGGAGAUGGAGGCAGAAGAAAGAAGGGCGGCUGGUUUGGCAAACACAGAGGCCAGGGCGGCAGCAACCCCAAAUUCGAGAAUAUCGCCGAAGGCCUGAGGGUGCUGCUGGCCCGCAGCCAUGUGGAGCGCACCACAGAGGAAGGAAACUGGGUGGCUGGCGUGUUUGUGUACGGCGGAAGCAAAACCUCCCUGUACAAUCUGCGGAGAGGCAUUGCUCUGGCUGUGCCUCAGUGUCGGAUUACCCCACUGAGCAGGCUGCCAUUUGGCAUGGCACCUGGCCCUGGACCUCAGCCUGGCCCCCUGCGGGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAGGAUGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCUACCUGCAACAUCAAGGUGACCGUGUGUAGCUUCGACGACGGCGUGGAUCUGCCUCCCUGGUUCCCACCCAUG Coding sequence 1 of NE2 (NE2V1) SEQ ID NO: 28: AUGGCCGCCCCCGGCAGCGCCAGAAGGCCUCUGCUGCUGCUCCUGCUGCUGUUGCUGCUGGGCCUGAUGCACUGUGCCUCUGCCCAUAGAGAUGGCGUGAGAAGACCUCAGAAAAGACCCUCUUGUAUUUUCCACCCCGUGGGCGAUGCCGAUUACUUUGAGUACCUGCAGGAGGGAGGACCUGAUGGAGAGCCUGAUGUGCCACCUGGAGCUAUUGAACAGGGACCUACCGAUGACCCAGGAGAGGGACCUUCUACAGGCCCUAGAGGACAGGGCGAUGGAGGCAGAAGAAAAAAGGGCGGCUGGUUUGGAAAACACAGAGGCCAGGGAGGCAGCAAUCCUAAGUUUGAGAAUAUCGCCGAAGGCCUGAGAGUGCUGCUGGCUAGAAGCCACGUGGAAAGGACCACAGAGGAAGGCAAUUGGGUGGCCGGCGUGUUUGUGUAUGGCGGCAGCAAAACCAGCCUGUACAACCUGAGAAGAGGCAUUGCCCUGGCCGUGCCUCAGUGUAGAAUUACCCCACUGUCUAGGCUGCCAUUUGGAAUGGCUCCAGGACCUGGACCUCAGCCAGGACCACUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAGGAUGCCAUCAAGGAUCUGGUGAUGACCAAGCCUGCCCCCACAUGCAACAUCAAGGUGACCGUGUGCAGCUUCGAUGAUGGCGUGGAUCUGCCUCCUUGGUUUCCUCCAAUGAGCAUGCUGAUCCCUAUCGCUGUGGGCGGAGCUCUGGCUGGAUUGGUGCUGAUCGUGCUGAUUGCCUAUCUGGUGGGCAGAAAGAGAAGCCAUGCCGGAUAUCAGACAAUC NE2 coding sequence 2 (NE2V2) SEQ ID NO: 29: AUGGCCGCCCCCGGCAGCGCCAGAAGGCCUCUGCUGCUGCUCCUGCUGCUGUUGCUGCUGGGCCUGAUGCACUGUGCCUCUGCCCAUAGAGAUGGCGUGAGAAGACCUCAGAAAAGACCCUCUUGUAUUUUCCACCCCGUGGGCGAUGCCGAUUACUUUGAGUACCUGCAGGAGGGAGGACCUGAUGGAGAGCCUGAUGUGCCACCUGGAGCUAUUGAACAGGGACCUACCGAUGACCCAGGAGAGGGACCUUCUACAGGCCCUAGAGGACAGGGCGAUGGAGGCAGAAGAAAAAAAGGCGGCUGGUUUGGCAAACACAGAGGCCAGGGAGGCAGCAAUCCUAAGUUUGAGAAUAUCGCCGAAGGCCUGAGAGUGCUGCUGGCUAGAAGCCACGUGGAAAGGACCACAGAGGAAGGCAAUUGGGUGGCCGGCGUGUUUGUGUAUGGCGGCAGCAAAACCAGCCUGUACAACCUGAGAAGAGGCAUUGCCCUGGCCGUGCCUCAGUGUAGAAUUACCCCACUGUCUAGGCUGCCAUUUGGAAUGGCUCCAGGACCUGGACCUCAGCCAGGACCACUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAAGUGCUGAAGGACGCCAUCAAGGAUCUGGUGAUGACCAAGCCUGCCCCCACAUGCAACAUCAAGGUGACCGUGUGCAGCUUCGAUGAUGGCGUGGAUCUGCCUCCUUGGUUUCCUCCAAUGAGCAUGCUGAUCCCUAUCGCUGUGGGCGGAGCUCUGGCUGGAUUGGUGCUGAUCGUGCUGAUUGCCUAUCUGGUGGGCAGAAAGAGAAGCCAUGCCGGAUAUCAGACAAUC Coding sequence 3 of NE2 (NE2V3) SEQ ID NO: 30: AUGGCCGCCCCCGGCAGCGCCAGAAGGCCUCUGCUGCUGCUCCUGCUGCUGUUGCUGCUGGGCCUGAUGCACUGUGCCUCUGCCCAUAGAGAUGGCGUGAGAAGACCUCAGAAAAGACCCUCUUGUAUUUUCCACCCCGUGGGCGAUGCCGAUUACUUUGAGUACCUGCAGGAGGGAGGACCUGAUGGAGAGCCUGAUGUGCCACCUGGAGCUAUUGAACAGGGACCUACCGAUGACCCAGGAGAGGGACCUUCUACAGGCCCUAGAGGACAGGGCGAUGGAGGCAGAAGAAAAAAAGGCGGCUGGUUUGGCAAACACAGAGGCCAGGGAGGCAGCAAUCCUAAGUUUGAGAAUAUCGCCGAAGGCCUGAGAGUGCUGCUGGCUAGAAGCCACGUGGAAAGGACCACAGAGGAAGGCAAUUGGGUGGCCGGCGUGUUUGUGUAUGGCGGCAGCAAAACCAGCCUGUACAACCUGAGAAGAGGAAUUGCCCUGGCCGUGCCUCAGUGUAGAAUCACCCCACUGUCUAGGCUGCCAUUUGGAAUGGCUCCAGGACCUGGACCUCAGCCAGGACCACUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAAGUGCUGAAGGACGCCAUCAAGGAUCUGGUGAUGACCAAGCCUGCCCCCACAUGCAACAUCAAGGUGACCGUGUGCAGCUUCGAUGAUGGCGUGGAUCUGCCUCCUUGGUUUCCUCCAAUGAGCAUGCUGAUCCCUAUCGCUGUGGGCGGAGCUCUGGCUGGAUUGGUGCUGAUCGUGCUGAUUGCCUAUCUGGUGGGCAGAAAGAGAAGCCAUGCCGGAUAUCAGACAAUC Coding sequence 4 of NE2 (NE2V4) SEQ ID NO: 31: AUGGCCGCCCCCGGCAGCGCCAGAAGGCCUCUGCUGCUGCUCCUGCUGCUGUUGCUGCUGGGCCUGAUGCACUGUGCCUCUGCCCAUAGAGAUGGCGUGAGAAGACCUCAGAAAAGACCCUCUUGUAUUUUCCACCCCGUGGGCGAUGCCGAUUACUUUGAGUACCUGCAGGAGGGAGGACCUGAUGGAGAGCCUGAUGUGCCACCUGGAGCUAUUGAACAGGGACCUACCGAUGACCCAGGAGAGGGACCUUCUACAGGCCCUAGAGGACAGGGCGAUGGAGGAAGAAGAAAGAAGGGCGGAUGGUUUGGCAAACAUAGGGGCCAGGGAGGCAGCAAUCCUAAGUUUGAGAACAUCGCCGAAGGCCUGAGAGUGCUGCUGGCUAGAAGCCACGUGGAAAGGACCACAGAAGAAGGCAAUUGGGUGGCCGGCGUGUUUGUGUAUGGCGGCAGCAAAACCAGCCUGUACAACCUGAGAAGAGGAAUUGCCCUGGCCGUGCCUCAGUGUAGAAUCACCCCACUGUCUAGGCUGCCAUUUGGAAUGGCUCCAGGACCUGGACCUCAGCCAGGACCACUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAAGACGCCAUUAAGGACCUGGUGAUGACCAAGCCUGCCCCCACAUGCAACAUCAAGGUGACCGUGUGCAGCUUCGAUGAUGGCGUGGAUCUGCCUCCUUGGUUUCCUCCAAUGAGCAUGCUGAUCCCCAUUGCUGUGGGCGGAGCUCUGGCUGGAUUGGUGCUGAUCGUGCUGAUUGCCUAUCUGGUGGGCAGAAAGAGAAGCCAUGCCGGAUAUCAGACAAUC Coding Sequence 5 of NE2 (NE2V5) SEQ ID NO: 32: AUGGCCGCCCCCGGCAGCGCCAGAAGGCCUCUGCUGCUGCUCCUGCUGCUGUUGCUGCUGGGCCUGAUGCACUGUGCUUCUGCUCACAGAGAUGGCGUGAGAAGACCUCAGAAAAGACCCUCUUGUAUUUUCCACCCCGUGGGCGAUGCCGACUACUUUGAGUAUCUGCAGGAGGGAGGCCCUGAUGGAGAGCCUGAUGUGCCACCUGGAGCUAUUGAACAGGGACCUACAGAUGACCCAGGAGAGGGACCUUCUACAGGCCCUAGAGGACAGGGCGAUGGAGGAAGAAGAAAGAAGGGCGGCUGGUUUGGAAAACAUAGGGGCCAGGGAGGCAGCAAUCCUAAGUUUGAGAACAUCGCCGAAGGCCUGAGAGUGCUGCUGGCUAGAAGCCACGUGGAAAGGACCACAGAAGAAGGCAAUUGGGUGGCCGGCGUGUUUGUGUAUGGCGGCAGCAAAACCAGCCUGUACAACCUGAGAAGAGGAAUUGCCCUGGCCGUGCCUCAGUGUAGAAUCACCCCACUGUCUAGGCUGCCAUUUGGAAUGGCACCAGGACCUGGACCUCAGCCAGGACCACUGAGAGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAAGACGCCAUUAAGGACCUGGUGAUGACCAAGCCUGCCCCCACAUGCAACAUCAAGGUGACCGUGUGCAGCUUCGAUGAUGGCGUGGAUCUGCCUCCUUGGUUUCCUCCAAUGAGCAUGCUGAUCCCCAUUGCUGUGGGCGGAGCUCUGGCUGGAUUGGUGCUGAUCGUGCUGAUUGCCUAUCUGGUGGGCAGAAAGAGAAGCCAUGCCGGAUAUCAGACAAUC Coding sequence 6 of NE2 (NE2V6) SEQ ID NO: 33: AUGGCCGCCCCUGGCAGCGCCAGAAGGCCACUGCUGCUGCUCCUGCUGCUGUUGCUGCUGGGCCUGAUGCACUGCGCAUCUGCUCACAGGGAUGGCGUUAGAAGACCACAGAAAAGACCAUCCUGUAUUUUCCACCCUGUCGGCGAUGCCGACUACUUCGAGUAUCUGCAGGAGGGAGGCCCAGAUGGAGAGCCUGAUGUGCCACCUGGAGCCAUUGAACAGGGACCUACAGAUGAUCCAGGAGAGGGACCUUCUACAGGCCCUAGAGGACAGGGGGAUGGAGGAAGAAGAAAGAAGGGAGGCUGGUUCGGAAAACACAGGGGACAGGGAGGCAGCAAUCCUAAGUUUGAGAACAUCGCCGAAGGCCUGAGAGUGCUGCUGGCUAGAUCCCACGUGGAACGCACCACUGAAGAGGGCAAUUGGGUGGCUGGAGUGUUUGUGUAUGGCGGGAGCAAGACAUCCCUGUACAACCUGAGAAGAGGAAUUGCCCUGGCCGUGCCUCAGUGUCGGAUUACCCCACUGUCUAGGCUGCCAUUUGGCAUGGCACCAGGACCUGGACCUCAGCCAGGACCACUGAGAGAGAGCAUUGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCCGAGGUGCUGAAAGACGCCAUUAAGGACCUGGUGAUGACCAAGCCUGCCCCCACAUGCAACAUCAAGGUGACAGUGUGCAGCUUCGACGAUGGCGUGGAUCUGCCUCCAUGGUUUCCACCAAUGUCCAUGCUGAUUCCCAUUGCUGUGGGCGGAGCUCUGGCCGGAUUGGUGCUGAUCGUGCUGAUUGCUUAUCUGGUGGGCAGAAAGAGAAGCCAUGCCGGAUAUCAGACCAUC Coding sequence 7 of NE2 (NE2V7) SEQ ID NO: 34: AUGGCCGCCCCCGGCAGCGCCAGAAGACCACUGCUGCUGCUCCUGCUGCUUCUGCUGCUGGGCCUGAUGCACUGUGCCAGCGCUCACAGAGACGGCGUGAGAAGGCCUCAGAAGAGGCCAAGCUGCAUCUUUCACCCUGUGGGCGAUGCUGAUUACUUCGAGUACCUGCAGGAGGGCGGCCCUGAUGGAGAACCUGAUGUGCCUCCUGGAGCCAUUGAGCAGGGCCCAACCGAUGAUCCUGGAGAGGGACCUUCUACAGGACCUAGAGGACAGGGCGAUGGCGGCAGAAGGAAGAAGGGCGGCUGGUUUGGCAAGCAUAGAGGCCAGGGCGGCAGCAAUCCCAAGUUUGAGAACAUCGCUGAGGGCCUGAGAGUGCUGCUGGCUAGGAGCCACGUGGAGAGAACAACCGAGGAGGGCAAUUGGGUGGCUGGAGUGUUCGUGUACGGAGGCAGCAAGACAAGCCUGUACAACCUGAGACGGGGCAUUGCCCUGGCUGUGCCCCAGUGUAGGAUCACACCUCUGAGCAGGCUGCCAUUUGGAAUGGCCCCAGGACCCGGACCACAGCCUGGCCCACUGAGGGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUUGCCGAGGUGCUGAAGGACGCCAUCAAGGACCUGGUGAUGACCAAGCCUGCCCCCACCUGCAACAUCAAGGUGACCGUGUGCAGCUUCGACGAUGGCGUGGAUCUGCCUCCUUGGUUUCCACCCAUGAGCAUGCUGAUCCCUAUCGCCGUGGGCGGAGCCCUGGCCGGCCUGGUGCUGAUCGUGCUGAUUGCUUAUCUGGUGGGAAGAAAGAGAAGCCACGCUGGCUACCAGACCAUC Coding sequence 8 of NE2 (NE2V8) SEQ ID NO: 35: AUGGCCGCCCCUGGGUCCGCCAGAAGGCCACUGCUGCUGCUCCUGCUGUUGCUGCUGUUGGGCCUGAUGCACUGCGCCUCUGCCCACAGAGACGGAGUGAGACGGCCUCAGAAGAGGCCAUCUUGCAUCUUCCACCCUGUGGGCGACGCUGAUUAUUUUGAGUAUCUGCAGGAGGGCGGCCCAGAUGGAGAGCCAGAUGUGCCUCCAGGAGCCAUUGAGCAGGGCCCUACCGAUGAUCCAGGAGAGGGACCUUCUACAGGACCAAGAGGACAGGGCGAUGGCGGCAGACGGAAGAAGGGGGGCUGGUUCGGCAAACACAGAGGCCAGGGCGGAAGCAAUCCAAAGUUUGAGAACAUCGCUGAGGGCCUGAGGGUGCUGCUGGCUAGGAGCCACGUGGAGAGAACAACCGAGGAGGGCAAUUGGGUGGCUGGAGUGUUUGUGUACGGAGGCUCCAAGACAAGCCUGUACAACCUGCGGCGCGGCAUUGCACUGGCUGUGCCCCAGUGUAGGAUUACACCUCUGAGCAGGCUGCCAUUUGGAAUGGCCCCAGGCCCCGGACCACAGCCUGGCCCACUGAGGGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUUGCCGAGGUGCUGAAGGACGCCAUCAAGGACCUGGUGAUGACCAAGCCUGCCCCCACCUGCAACAUCAAGGUGACCGUGUGCAGCUUCGAUGACGGCGUGGAUCUGCCUCCCUGGUUUCCACCAAUGAGCAUGCUGAUCCCUAUCGCCGUGGGCGGAGCCCUUGCCGGCCUGGUGCUGAUCGUGCUGAUUGCUUAUCUGGUGGGCAGGAAAAGAAGCCACGCCGGCUACCAGACCAUU Coding sequence 9 of NE2 (NE2V9) SEQ ID NO: 36: AUGGCCGCCCCCGGCAGCGCCAGAAGACCUCUGCUGCUCCUGCUGUUGCUGCUGCUGCUGUUGGGCCUGAUGCACUGUGCCUCUGCCCACAGAGAUGGCGUGAGAAGACCACAGAAGAGGCCCAGCUGCAUCUUCCACCCAGUGGGAGACGCCGACUACUUUGAGUACCUGCAGGAGGGCGGACCAGAUGGAGAACCAGAUGUGCCUCCUGGAGCUAUCGAGCAGGGACCUACCGAUGAUCCUGGAGAAGGACCAUCUACAGGCCCUAGAGGACAGGGCGAUGGCGGCAGAAGAAAGAAGGGCGGAUGGUUUGGCAAACACAGAGGCCAGGGGGGCAGCAAUCCCAAGUUUGAGAACAUCGCCGAGGGCCUGAGAGUGCUGCUGGCCAGGAGCCACGUGGAAAGAACCACAGAGGAGGGAAAUUGGGUGGCCGGCGUGUUUGUGUACGGCGGCAGCAAAACAAGCCUGUACAACCUGAGGAGAGGCAUUGCCCUGGCCGUGCCACAGUGUAGAAUUACACCCCUGAGCAGGCUGCCUUUUGGAAUGGCUCCAGGCCCUGGCCCUCAGCCAGGCCCUCUGAGGGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUUGCCGAGGUGCUGAAGGACGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCCACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGACGGCGUGGACCUGCCUCCUUGGUUUCCCCCUAUGAGCAUGCUGAUUCCCAUCGCCGUGGGAGGCGCCCUGGCUGGCCUGGUGCUGAUUGUUCUGAUUGCCUAUCUGGUGGGCAGAAAGAGAAGCCAUGCCGGCUACCAGACCAUU NE2 coding sequence 10 (NE2V10) SEQ ID NO: 37: AUGGCCGCCCCAGGGAGUGCCAGAAGGCCUCUGCUGCUCCUGCUGUUGCUGCUGCUGCUGUUGGGCCUCAUGCACUGCGCCUCCGCCCAUAGAGAUGGCGUGAGAAGACCACAGAAGAGGCCCAGCUGCAUCUUCCACCCAGUGGGAGACGCCGACUACUUUGAGUACCUGCAGGAGGGAGGACCAGAUGGAGAGCCAGACGUGCCUCCUGGAGCUAUUGAACAGGGACCUACCGAUGAUCCUGGAGAAGGACCAUCUACUGGCCCUAGAGGCCAGGGCGAUGGCGGCAGAAGAAAGAAAGGCGGAUGGUUUGGCAAGCACAGGGGCCAGGGAGGCAGCAAUCCCAAGUUUGAGAACAUCGCCGAGGGCCUGAGAGUGCUGCUGGCCAGGUCCCACGUGGAAAGAACAACCGAGGAGGGAAAUUGGGUGGCUGGCGUGUUCGUGUACGGCGGAAGCAAAACAAGCCUGUACAACCUGAGGAGAGGAAUUGCCCUGGCCGUGCCACAGUGUAGAAUUACACCCCUGUCCCGGCUGCCUUUUGGAAUGGCUCCAGGCCCUGGCCCUCAGCCAGGCCCUCUGAGGGAGAGCAUCGUGUGCUACUUCAUGGUGUUCCUGCAGACCCACAUCUUCGCUGAGGUGCUGAAGGACGCCAUCAAGGACCUGGUGAUGACCAAACCUGCCCCCACCUGCAACAUCAAGGUGACCGUGUGUAGCUUUGACGACGGCGUGGACCUGCCUCCUUGGUUUCCCCCUAUGAGCAUGCUGAUUCCCAUCGCCGUGGGAGGCGCCCUGGCUGGCCUGGUGCUGAUCGUUCUGAUUGCCUAUCUGGUGGGCAGAAAGAGAAGCCACGCCGGAUAUCAGACCAUU Coding sequence 1 of NE3 (NE3V1) SEQ ID NO: 38: The coding sequence of NE3, 2 (NE3V2), SEQ ID NO: 39: The coding sequence 3 of NE3 (NE3V3) SEQ ID NO: 40: The coding sequence of NE3, 4 (NE3V4), is SEQ ID NO: 41. The coding sequence of NE3, 5 (NE3V5), SEQ ID NO: 42: The coding sequence of NE3, 6 (NE3V6), SEQ ID NO: 43: The coding sequence of NE3, 7 (NE3V7), is SEQ ID NO: 44. The coding sequence of NE3, 8 (NE3V8), is SEQ ID NO: 45. The coding sequence of NE3, 9 (NE3V9), SEQ ID NO: 46: The coding sequence 10 of NE3 (NE3V10) SEQ ID NO: 47: NE4 coding sequence 2 (NE4V2) SEQ ID NO: 49: NE4 coding sequence 3 (NE4V3) SEQ ID NO: 50: The coding sequence of NE4, 4 (NE4V4), is SEQ ID NO: 51. The coding sequence of NE4, 5 (NE4V5), is SEQ ID NO: 52. The coding sequence of NE4, 6 (NE4V6), is SEQ ID NO: 53. The coding sequence of NE4, SEQ ID NO: 54 (NE4V7): The coding sequence of NE4, 8 (NE4V8), is SEQ ID NO: 55. The coding sequence of NE4, 9 (NE4V9), is SEQ ID NO: 56. The coding sequence 10 of NE4 (NE4V10) SEQ ID NO: 57: Coding sequence 1 of NE5 (NE5V1) SEQ ID NO: 58: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGACCCUUACCAGGUGCCCUUUGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCAGAACCACUGCCUCAGGGACAGCUGACAGCUUAUCAUGUGUCUGCAGCUCCUACAGGAUCUUGGUUUCCAGCACCUCAGCCUGCCCCAGAGAAUGCCUAUCAGGCCUAUGCCGCUCCUCAGCUGUUUCCUGUGUCUGAUAUCACCCAGAACCAGCUGACAAAUCAGGCUGGAGGAGAAGCCCCUCAGCCAGGCGACAAUUCUACAGUGCAGCCAGCUGCUGCUGUGGUGCUGGCUUGUCCUGGAGCUAAUCAGGAACAGCAACUGGCUGAUAUUGGAGCCCCACAGCCUGCUCCUGCUGCUGCUCCAGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAAUCUCUGGAGGAGUGUGAUAGCGAGCACCUGCUGCAGCAUUACAGAGAGGUGGCCUCUGCCAAGAGCAGCGAGAACGAUAGGCUGAGGCUGCUCCUGAAGCAGAUGUGUCCUAGCCUGGAUGUGGAUUCUAUCAUCCCUAGGACCCCCGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 2 of NE5 (NE5V2) SEQ ID NO: 59: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGACCCUUACCAGGUGCCCUUUGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCAGAACCACUGCCUCAGGGACAGCUGACAGCUUAUCAUGUGUCUGCAGCUCCUACAGGAUCUUGGUUUCCAGCACCUCAGCCUGCCCCAGAGAAUGCCUAUCAGGCCUAUGCCGCUCCUCAGCUGUUUCCUGUGUCUGAUAUCACCCAGAACCAGCUGACAAAUCAGGCUGGAGGAGAAGCCCCUCAGCCAGGCGACAAUUCUACAGUGCAGCCAGCUGCUGCUGUGGUGCUGGCUUGUCCUGGAGCUAAUCAGGAACAGCAACUGGCUGAUAUUGGAGCCCCACAGCCUGCUCCUGCUGCUGCUCCAGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAAUCUCUGGAGGAGUGUGACAGCGAACACCUGCUGCAGCACUACAGAGAGGUGGCCUCUGCCAAAAGCAGCGAGAACGAUAGGCUGAGGCUGCUCCUGAAGCAGAUGUGUCCUAGCCUGGAUGUGGAUUCUAUCAUCCCUAGGACCCCCGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 3 of NE5 (NE5V3) SEQ ID NO: 60: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGACCCUUACCAGGUGCCCUUUGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCAGAACCACUGCCUCAGGGACAGCUGACAGCUUAUCAUGUGUCUGCAGCUCCUACAGGAUCUUGGUUUCCAGCACCUCAGCCUGCCCCAGAGAAUGCCUAUCAGGCCUAUGCCGCUCCUCAGCUGUUUCCUGUGUCUGAUAUCACCCAGAACCAGCUGACAAAUCAGGCUGGAGGAGAAGCCCCUCAGCCAGGCGACAAUUCUACAGUGCAGCCAGCUGCUGCUGUGGUGCUGGCUUGUCCUGGAGCUAAUCAGGAACAGCAACUGGCUGAUAUUGGAGCCCCACAGCCUGCUCCUGCUGCUGCUCCAGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAAUCUCUGGAGGAGUGUGACAGCGAACACCUGCUGCAGCACUACAGAGAGGUGGCCUCUGCCAAAAGCAGCGAGAACGAUAGGCUGAGGCUGCUCCUGAAGCAGAUGUGUCCUAGCCUGGAUGUGGAUUCUAUCAUCCCUAGGACCCCCGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 4 of NE5 (NE5V4) SEQ ID NO: 61: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCAGACCCUUACCAGGUGCCCUUUGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCAGAACCACUGCCUCAGGGACAGCUGACAGCUUAUCAUGUGUCUGCAGCUCCUACAGGAUCUUGGUUUCCAGCACCUCAGCCUGCCCCAGAGAAUGCCUAUCAGGCCUAUGCCGCUCCUCAGCUGUUUCCUGUGAGCGAUAUUACCCAGAACCAGCUGACAAAUCAGGCUGGCGGAGAAGCCCCUCAGCCAGGCGAUAAUUCUACAGUGCAGCCAGCUGCUGCUGUGGUGCUGGCUUGUCCUGGAGCUAAUCAGGAACAGCAACUGGCUGAUAUUGGAGCCCCACAGCCUGCUCCUGCUGCUGCUCCAGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAAUCUCUGGAGGAGUGUGACAGCGAACAUCUGCUGCAGCACUACAGAGAGGUGGCCUCUGCCAAAAGCAGCGAGAACGAUAGGCUGCGGCUGCUCCUGAAGCAGAUGUGUCCUAGCCUGGAUGUGGAUUCUAUCAUCCCUAGGACCCCCGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 5 of NE5 (NE5V5) SEQ ID NO: 62: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCAGACCCUUACCAGGUGCCCUUUGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCAGAACCACUGCCUCAGGGACAGCUGACAGCAUAUCAUGUGUCUGCAGCUCCUACAGGAUCUUGGUUUCCAGCACCUCAGCCUGCCCCAGAGAAUGCCUAUCAGGCCUAUGCCGCUCCUCAGCUGUUUCCUGUGAGCGAUAUUACCCAGAACCAGCUGACAAAUCAGGCUGGCGGAGAAGCCCCUCAGCCAGGCGAUAAUUCUACAGUGCAGCCAGCUGCUGCUGUGGUGCUGGCUUGUCCUGGAGCUAAUCAGGAACAGCAACUGGCUGAUAUUGGAGCCCCACAGCCUGCUCCUGCUGCUGCUCCAGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAAUCUCUGGAGGAGUGUGACAGCGAACACCUGCUGCAGCACUACAGAGAGGUGGCCUCUGCUAAAAGCAGCGAGAACGACAGGCUGCGGCUGCUCCUGAAACAGAUGUGUCCUAGCCUGGAUGUGGAUUCUAUCAUCCCUCGGACCCCCGAUGUGCUGCACGAGGACCUGCUGAAUUUC The coding sequence 6 of NE5 (NE5V6) SEQ ID NO: 63: AUGAUGGACCCCAACAGCACCAGUGAGGACGUGAAGUUUACCCCAGACCCCUACCAGGUGCCCUUUGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCAGAACCACUGCCACAGGGACAGUUAACAGCCUAUCAUGUGUCUGCAGCUCCUACAGGAUCUUGGUUUCCAGCACCUCAGCCUGCCCCAGAGAAUGCCUAUCAGGCCUAUGCCGCUCCUCAGCUGUUUCCUGUGAGCGAUAUUACCCAGAACCAGCUGACAAAUCAGGCUGGCGGAGAAGCCCCUCAGCCAGGCGAUAAUUCUACAGUGCAGCCAGCUGCUGCUGUGGUGCUGGCUUGUCCUGGAGCUAAUCAGGAACAGCAACUGGCUGAUAUUGGAGCUCCCCAGCCUGCUCCUGCUGCUGCUCCAGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAAUCUCUGGAGGAGUGUGACAGCGAACACCUGCUGCAGCACUACAGAGAGGUGGCCUCUGCUAAAAGCAGCGAGAAUGACAGGCUGCGGCUGCUCCUGAAACAGAUGUGCCCUAGCCUGGAUGUGGAUUCUAUCAUCCCUCGGACCCCCGAUGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 7 of NE5 (NE5V7) SEQ ID NO: 64: AUGAUGGACCCCAACAGCACCAGCGAGGAUGUGAAGUUUACCCCCGACCCCUACCAGGUGCCCUUCGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCCCCACUGCCCUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCACAGGGACAGCUGACCGCCUAUCACGUGUCUGCUGCACCAACAGGCUCUUGGUUUCCUGCUCCUCAGCCUGCCCCUGAGAAUGCCUAUCAGGCCUAUGCCGCUCCCCAGCUGUUUCCAGUGAGCGAUAUCACCCAGAAUCAGCUGACCAAUCAGGCCGGCGGCGAAGCUCCACAGCCUGGCGACAACUCUACAGUGCAGCCAGCUGCUGCCGUGGUGCUGGCUUGUCCAGGAGCUAAUCAGGAGCAGCAGCUUGCUGAUAUUGGAGCCCCUCAGCCAGCCCCUGCUGCUGCCCCUGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAAAGCCUGGAAGAGUGCGACAGCGAGCACCUGCUGCAGCACUACAGGGAGGUGGCCUCUGCCAAAAGCAGCGAGAACGACCGGCUGAGGCUGCUGCUGAAGCAGAUGUGCCCUAGCCUGGAUGUGGAUAGCAUCAUCCCCAGGACCCCUGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding Sequence 8 of NE5 (NE5V8) SEQ ID NO: 65: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCCGACCCCUACCAGGUGCCCUUCGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCCCCACUGCCCUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCACAGGGACAGCUGACCGCCUAUCACGUGUCUGCUGCACCAACAGGCUCUUGGUUUCCUGCUCCUCAGCCUGCCCCUGAGAAUGCCUAUCAGGCCUAUGCCGCUCCCCAGCUGUUUCCAGUGAGCGAUAUCACCCAGAAUCAGCUGACCAAUCAGGCCGGCGGCGAAGCUCCACAGCCUGGCGACAACUCUACAGUGCAGCCAGCUGCUGCCGUGGUGCUGGCUUGUCCAGGAGCUAAUCAGGAGCAGCAGCUUGCUGAUAUUGGAGCCCCUCAGCCAGCCCCUGCUGCUGCCCCUGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAGAGCCUGGAAGAGUGUGACAGCGAGCACCUGCUGCAGCACUACAGGGAGGUGGCCUCUGCCAAAAGCAGCGAGAACGACCGGCUGAGGCUGCUGCUGAAGCAGAUGUGCCCUAGCCUGGAUGUGGAUAGCAUCAUCCCCAGGACCCCUGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 9 of NE5 (NE5V9) SEQ ID NO: 66: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCCGACCCCUACCAGGUGCCCUUCGUGCAGGCCUUUGAUCAGGCCACUAGAGUGUAUCAGGAUCUGGGAGGACCAUCUCAGGCCCCACUGCCCUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCACAGGGACAGCUGACCGCCUAUCAUGUGUCUGCUGCACCAACAGGCUCUUGGUUUCCUGCUCCUCAGCCUGCCCCUGAGAAUGCCUACCAGGCCUACGCUGCUCCCCAGCUGUUUCCAGUGAGCGAUAUCACCCAGAAUCAGCUGACCAAUCAGGCCGGCGGCGAAGCUCCACAGCCUGGCGACAACUCUACAGUGCAGCCAGCUGCUGCCGUGGUGCUGGCUUGUCCAGGAGCUAAUCAGGAGCAGCAGCUUGCUGAUAUUGGAGCCCCUCAGCCAGCUCCUGCUGCUGCCCCUGCUAGAAGAACCAGAAAACCUCUGCAGCCUGAGAGCCUGGAAGAGUGUGACAGCGAGCACCUGCUGCAGCACUACCGGGAGGUGGCUAGCGCCAAAAGCAGCGAGAACGACCGGCUGAGGCUGCUGCUGAAGCAGAUGUGCCCUAGCCUGGAUGUGGAUAGCAUCAUCCCCCGGACACCUGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 10 of NE5 (NE5V10) SEQ ID NO: 67: AUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGAUCCCUACCAGGUGCCCUUCGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUACCAGGAUCUGGGAGGACCAUCUCAGGCCCCACUGCCCUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCACAGGGACAGCUGACCGCCUAUCAUGUGUCUGCUGCACCAACAGGCUCUUGGUUUCCUGCUCCUCAGCCUGCCCCUGAGAAUGCCUACCAGGCCUACGCUGCUCCCCAGCUGUUCCCAGUGUCCGAUAUUACCCAGAAUCAGCUGACCAAUCAGGCCGGCGGCGAAGCUCCACAGCCUGGUGACAAUUCUACAGUGCAGCCCGCUGCAGCCGUGGUGCUGGCUUGUCCAGGAGCAAAUCAGGAGCAGCAGUUGGCUGACAUUGGAGCCCCUCAGCCAGCUCCUGCUGCCGCUCCUGCUAGAAGAACCAGAAAGCCUCUGCAGCCUGAAUCCCUGGAGGAGUGUGACAGCGAGCAUCUGCUGCAGCACUACCGGGAAGUGGCAAGCGCCAAAAGCUCCGAGAACGACCGGCUGAGGCUGCUGCUGAAGCAGAUGUGCCCUAGCCUGGAUGUGGAUAGCAUCAUCCCCCGGACCCCUGACGUGCUGCACGAGGACCUGCUGAAUUUC Coding sequence 1 of NE6 (NE6V1) SEQ ID NO: 68: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUGUUACUGCUGCUCCUCCUGGGCCUGAUGCAUUGUGCCUCUGCCAUGGACCCUAACUCUACCUCUGAGGACGUGAAGUUUACCCCCGAUCCUUACCAGGUGCCUUUUGUGCAGGCCUUUGAUCAGGCUACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCUCAGGGACAGCUGACAGCCUAUCAUGUGAGCGCUGCUCCUACAGGAUCUUGGUUUCCUGCUCCACAGCCUGCUCCAGAGAAUGCCUAUCAGGCUUAUGCUGCCCCACAGCUGUUUCCUGUGAGCGAUAUUACACAGAACCAGCUGACAAAUCAGGCCGGAGGCGAAGCACCUCAGCCUGGCGAUAAUUCUACAGUGCAGCCAGCUGCCGCUGUGGUGCUGGCUUGUCCUGGAGCCAAUCAGGAACAGCAGCUGGCAGAUAUUGGAGCUCCUCAGCCAGCUCCAGCAGCUGCUCCAGCUAGAAGAACAAGAAAACCUCUGCAGCCAGAAUCUCUGGAAGAGUGUGACAGCGAGCACCUGCUGCAGCACUAUAGAGAGGUGGCCAGCGCCAAAAGCAGCGAGAAUGAUCGGCUGAGGCUGCUCCUGAAGCAGAUGUGCCCAUCUCUGGACGUGGACAGCAUCAUCCCCAGAACACCAGACGUGCUGCACGAGGAUCUGCUGAAUUUCUCUAUGCUGAUCCCUAUUGCUGUGGGAGGAGCUCUGGCUGGCCUGGUGCUGAUUGUGCUGAUCGCCUACCUGGUGGGCAGAAAGAGAUCUCACGCCGGAUAUCAGACCAUC Coding sequence 2 of NE6 (NE6V2) SEQ ID NO: 69: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUGUUACUGCUGCUCCUCCUGGGCCUGAUGCAUUGUGCCUCUGCCAUGGACCCUAACUCUACCUCUGAGGACGUGAAGUUUACCCCCGAUCCUUACCAGGUGCCUUUUGUGCAGGCCUUUGAUCAGGCUACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCUCAGGGACAGCUGACAGCCUAUCAUGUGAGCGCUGCUCCAACAGGAUCUUGGUUUCCUGCUCCACAGCCUGCUCCAGAGAAUGCCUAUCAGGCUUAUGCUGCCCCACAGCUGUUUCCUGUGAGCGAUAUUACACAGAACCAGCUGACAAAUCAGGCCGGAGGCGAAGCACCUCAGCCUGGCGAUAAUUCUACAGUGCAGCCAGCUGCCGCUGUGGUGCUGGCUUGUCCUGGAGCCAAUCAGGAACAGCAGCUGGCAGAUAUUGGAGCUCCUCAGCCAGCUCCAGCAGCUGCUCCAGCUAGAAGAACAAGAAAACCUCUGCAGCCAGAAUCUCUGGAGGAGUGUGACAGCGAGCACCUGCUGCAGCACUAUAGAGAGGUGGCCAGCGCCAAAAGCAGCGAAAAUGAUCGGCUGAGGCUGCUCCUGAAGCAGAUGUGCCCAUCUCUGGACGUGGACAGCAUCAUCCCCAGAACACCAGACGUGCUGCACGAGGAUCUGCUGAAUUUCUCUAUGCUGAUCCCUAUCGCUGUGGGAGGAGCACUGGCUGGCCUGGUGCUGAUUGUGCUGAUCGCCUAUCUGGUGGGCAGAAAGAGAUCUCACGCCGGAUAUCAGACCAUC Coding sequence 3 of NE6 (NE6V3) SEQ ID NO: 70: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUGUUACUGCUGCUCCUCCUGGGCCUGAUGCAUUGUGCCUCUGCCAUGGACCCUAACUCUACCUCUGAGGACGUGAAGUUUACCCCUGAUCCUUACCAGGUGCCUUUUGUGCAGGCCUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCUCAGGGACAGCUGACAGCCUAUCAUGUGAGCGCUGCUCCAACAGGAUCUUGGUUUCCUGCUCCACAGCCUGCUCCAGAGAAUGCCUAUCAGGCUUAUGCUGCCCCACAGCUGUUUCCUGUGAGCGACAUUACACAGAACCAGCUGACAAAUCAGGCCGGAGGCGAAGCACCUCAGCCUGGCGAUAAUUCUACAGUGCAGCCAGCUGCCGCUGUGGUUCUGGCUUGUCCUGGAGCCAAUCAGGAACAGCAGCUGGCAGAUAUUGGAGCUCCUCAGCCAGCUCCUGCAGCUGCUCCAGCUAGAAGAACAAGAAAACCUCUGCAGCCAGAAUCUCUGGAGGAGUGUGACAGCGAGCACCUGCUGCAGCACUAUAGAGAGGUGGCCAGCGCCAAAAGCAGCGAGAAUGAUCGGCUGAGGCUGCUCCUGAAGCAGAUGUGCCCAUCUCUGGACGUGGAUAGCAUCAUCCCCAGAACACCAGACGUGCUGCACGAGGAUCUGCUGAAUUUCUCUAUGCUGAUCCCUAUCGCUGUGGGAGGAGCCCUGGCUGGCCUGGUGCUGAUUGUGCUGAUCGCCUAUCUGGUGGGCAGAAAGAGAUCUCACGCCGGAUAUCAGACCAUC Coding sequence 4 of NE6 (NE6V4) SEQ ID NO: 71: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUGUUACUGCUGCUCUUGCUGGGCCUGAUGCAUUGUGCCUCUGCCAUGGACCCUAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGAUCCUUACCAGGUGCCUUUUGUGCAGGCUUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCUCAGGGACAGCUGACAGCCUAUCAUGUGAGCGCUGCUCCAACAGGAUCUUGGUUUCCUGCUCCACAGCCUGCUCCAGAGAAUGCCUAUCAGGCUUAUGCUGCCCCACAGCUGUUUCCUGUGAGCGACAUUACACAGAACCAGCUGACAAAUCAGGCCGGAGGCGAAGCACCUCAGCCUGGCGAUAAUUCUACAGUGCAGCCAGCUGCCGCUGUGGUUCUGGCUUGUCCUGGAGCCAAUCAGGAACAGCAGCUGGCAGAUAUUGGAGCUCCUCAGCCAGCUCCUGCAGCUGCUCCAGCUAGAAGAACAAGAAAACCUCUGCAGCCAGAAUCUCUGGAGGAGUGUGACAGCGAGCACCUGCUGCAGCACUACAGAGAGGUGGCCAGCGCCAAAAGCAGCGAAAAUGAUCGGCUGAGGCUGCUCCUGAAGCAGAUGUGCCCAUCUCUGGACGUGGAUAGCAUCAUCCCCAGAACACCAGACGUGCUGCACGAGGAUCUGCUGAAUUUCUCUAUGCUGAUUCCUAUCGCCGUGGGAGGAGCCCUGGCUGGCCUGGUGCUGAUUGUGCUGAUCGCCUAUCUGGUGGGCAGAAAGAGAUCUCACGCCGGAUAUCAGACCAUC Coding sequence 5 of NE6 (NE6V5) SEQ ID NO: 72: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUGUUACUGCUGCUCUUGCUGGGCCUGAUGCAUUGUGCCUCUGCCAUGGACCCUAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGAUCCUUACCAGGUGCCUUUUGUGCAGGCUUUUGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCUCAGGGACAGCUGACAGCCUAUCAUGUGUCUGCUGCUCCAACAGGCUCUUGGUUUCCUGCUCCACAGCCUGCUCCAGAGAAUGCCUAUCAGGCUUAUGCUGCCCCACAGCUGUUUCCUGUGAGCGACAUUACACAGAACCAGCUGACAAAUCAGGCCGGAGGCGAAGCACCUCAGCCUGGCGAUAAUUCUACAGUGCAGCCAGCUGCCGCUGUGGUUCUGGCUUGUCCUGGAGCCAAUCAGGAACAGCAGCUGGCAGAUAUUGGAGCUCCUCAGCCAGCUCCUGCAGCUGCUCCAGCUAGAAGAACAAGAAAACCUCUGCAGCCAGAAUCUCUGGAGGAGUGUGACAGCGAGCACCUGCUGCAGCACUACAGAGAGGUGGCCAGCGCCAAAAGCAGCGAAAAUGAUCGGCUGAGGCUGCUCCUGAAGCAGAUGUGCCCAUCUCUGGACGUGGAUAGCAUCAUCCCCAGAACACCAGACGUGCUGCACGAGGAUCUGCUGAAUUUCUCUAUGCUGAUUCCUAUCGCCGUGGGAGGAGCCCUGGCUGGCCUGGUGCUGAUUGUGCUGAUCGCCUAUCUGGUGGGCAGAAAGAGAUCUCACGCCGGAUAUCAGACCAUC Coding sequence 6 of NE6 (NE6V6) SEQ ID NO: 73: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUGUUACUGCUGCUCUUGCUGGGCCUGAUGCAUUGUGCCUCUGCCAUGGACCCUAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGAUCCUUACCAGGUGCCUUUUGUGCAGGCUUUCGAUCAGGCUACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCUCCACUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCUCUGCCUCAGGGACAGCUGACAGCCUAUCAUGUGUCUGCUGCUCCAACAGGCUCUUGGUUUCCUGCUCCACAGCCUGCACCAGAGAAUGCCUAUCAGGCUUAUGCUGCCCCACAGCUGUUUCCUGUGAGCGACAUUACACAGAACCAGCUGACAAAUCAGGCCGGAGGCGAAGCACCUCAGCCUGGCGAUAAUUCUACAGUGCAGCCAGCUGCCGCUGUGGUUCUGGCUUGUCCUGGAGCCAAUCAGGAACAGCAGCUGGCAGAUAUUGGAGCUCCUCAGCCAGCUCCUGCAGCUGCUCCAGCUAGAAGAACAAGAAAACCUCUGCAGCCAGAAUCUCUGGAGGAGUGUGACAGCGAGCACCUGCUGCAGCACUACAGAGAGGUGGCCAGCGCCAAAAGCAGCGAAAAUGAUCGGCUGAGGCUGCUCCUGAAGCAGAUGUGCCCAUCUCUGGACGUGGAUAGCAUCAUCCCCAGAACACCAGACGUGCUGCACGAGGAUCUGCUGAAUUUCUCUAUGCUCAUUCCUAUCGCCGUGGGAGGAGCCCUGGCUGGCCUGGUGCUGAUUGUGCUGAUCGCCUAUCUGGUGGGCAGAAAGAGAUCUCACGCCGGAUAUCAGACCAUC The coding sequence 7 of NE6 (NE6V7) SEQ ID NO: 74: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUCCUGCUGCUUCUGCUGCUGGGCCUGAUGCACUGUGCCAGCGCCAUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGACCCCUACCAGGUGCCUUUUGUGCAGGCCUUCGAUCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCCCCUCUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCACUGCCACAGGGCCAGCUGACAGCCUACCAUGUGUCUGCUGCCCCAACAGGCUCUUGGUUUCCUGCUCCCCAGCCAGCCCCUGAGAAUGCCUAUCAGGCCUAUGCCGCCCCUCAGCUGUUUCCUGUGUCUGACAUUACCCAGAAUCAGCUGACCAACCAGGCUGGCGGAGAAGCCCCACAGCCAGGCGACAAUUCUACAGUGCAGCCUGCCGCUGCUGUGGUGCUGGCUUGUCCAGGCGCCAAUCAGGAACAGCAGCUUGCUGAUAUUGGAGCUCCUCAGCCAGCUCCAGCAGCUGCCCCUGCCCGGAGAACCAGAAAACCACUGCAGCCUGAAAGCCUGGAGGAGUGCGACAGCGAGCAUCUGCUGCAGCACUAUAGGGAGGUGGCCAGCGCUAAGAGCUCUGAGAACGAUCGGCUGAGGCUGCUGCUGAAGCAGAUGUGUCCCAGCCUGGACGUGGAUUCUAUCAUCCCCAGAACCCCCGAUGUGCUGCACGAGGACCUGCUGAAUUUCAGCAUGCUCAUCCCUAUCGCUGUGGGCGGAGCCCUGGCCGGCCUGGUGCUGAUCGUGCUGAUUGCCUACCUGGUGGGCAGAAAGAGGAGCCACGCCGGCUAUCAGACCAUC Coding sequence 8 of NE6 (NE6V8) SEQ ID NO: 75: AUGGCCGCCCCCGGCAGCGCCAGAAGACCCCUGCUGCUGCUCCUGCUGCUUCUGCUGCUGGGCCUGAUGCACUGUGCCAGCGCCAUGAUGGACCCCAACAGCACCUCCGAGGACGUGAAGUUUACCCCUGACCCUUACCAGGUGCCAUUUGUGCAGGCCUUUGACCAGGCCACAAGAGUGUAUCAGGAUCUGGGAGGACCUUCUCAGGCCCCACUGCCUUGUGUGCUGUGGCCUGUGCUGCCUGAACCACUGCCACAGGGCCAGCUGACAGCCUACCAUGUGUCUGCUGCCCCAACAGGCUCUUGGUUUCCUGCUCCCCAGCCAGCCCCUGAGAAUGCCUAUCAGGCCUAUGCCGCCCCUCAGCUGUUUCCUGUGUCUGACAUUACCCAGAAUCAGCUGACCAACCAGGCUGGCGGAGAAGCCCCACAGCCAGGCGACAAUUCUACAGUGCAGCCUGCCGCUGCUGUGGUGCUGGCUUGUCCAGGCGCCAAUCAGGAACAGCAGUUGGCUGAUAUUGGAGCUCCUCAGCCAGCUCCAGCAGCUGCCCCUGCCCGGAGAACCAGAAAACCCUUACAGCCUGAAAGCCUGGAGGAGUGCGAUAGCGAGCACCUGCUGCAGCACUACAGGGAGGUGGCCAGCGCUAAGAGCUCUGAGAACGAUCGGCUGAGGCUGCUGCUGAAGCAGAUGUGCCCCAGCCUGGACGUGGAUUCUAUCAUCCCUAGAACCCCCGAUGUGCUGCACGAGGACCUGCUGAAUUUCAGCAUGCUCAUCCCUAUCGCUGUGGGCGGAGCCCUGGCCGGCCUGGUGCUGAUCGUGCUGAUUGCCUACCUGGUGGGCAGAAAGAGGAGCCACGCCGGCUAUCAGACCAUC Coding sequence 9 of NE6 (NE6V9) SEQ ID NO: 76: AUGGCCGCCCCAGGCAGCGCCAGAAGACCUCUGCUCCUCCUCCUGCUGCUUCUGUUGCUGGGCCUGAUGCACUGCGCCAGCGCCAUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGACCCAUACCAGGUGCCUUUCGUGCAGGCCUUUGAUCAGGCUACAAGAGUUUAUCAGGACCUGGGAGGACCAAGCCAGGCCCCUCUGCCUUGUGUGCUGUGGCCAGUGCUGCCAGAACCACUGCCACAGGGCCAGCUGACAGCCUACCAUGUGUCCGCUGCCCCAACUGGCUCUUGGUUUCCUGCUCCCCAGCCAGCCCCUGAAAAUGCCUAUCAGGCCUACGCCGCCCCUCAGCUGUUCCCUGUGUCUGAUAUUACCCAGAAUCAGCUGACAAACCAGGCUGGCGGAGAGGCCCCACAGCCAGGCGAUAACUCUACAGUGCAGCCUGCCGCUGCAGUGGUGCUGGCAUGUCCAGGCGCCAAUCAGGAACAGCAGUUAGCUGAUAUCGGAGCUCCUCAGCCAGCUCCAGCAGCUGCCCCUGCCCGGAGAACCAGAAAACCCUUACAGCCUGAAUCCCUGGAGGAGUGCGAUAGCGAGCACCUGCUGCAGCACUACAGGGAGGUGGCCAGCGCUAAGAGCUCUGAGAACGAUCGCCUGAGGCUGCUGCUGAAGCAGAUGUGCCCCAGCCUGGACGUGGAUUCUAUCAUCCCUAGAACCCCCGAUGUGCUGCACGAGGACCUGCUGAAUUUCAGCAUGCUCAUCCCUAUCGCAGUGGGCGGAGCCCUGGCCGGCCUGGUGCUGAUCGUGCUCAUCGCCUAUCUGGUGGGCAGAAAGAGGAGCCACGCUGGCUAUCAGACCAUC Coding sequence 10 of NE6 (NE6V10) SEQ ID NO: 77: AUGGCCGCCCCAGGCAGCGCCAGAAGACCUCUGCUCCUCCUCCUGCUGCUUCUGUUGCUGGGCCUGAUGCACUGCGCCAGCGCCAUGAUGGACCCCAACAGCACCAGCGAGGACGUGAAGUUUACCCCUGACCCAUACCAGGUGCCUUUCGUGCAGGCCUUUGAUCAGGCUACAAGAGUUUAUCAGGACCUGGGAGGACCAAGCCAGGCCCCUCUGCCUUGUGUGCUGUGGCCAGUGCUGCCAGAACCACUGCCACAGGGCCAGCUGACAGCCUACCAUGUGUCCGCUGCCCCAACUGGCUCUUGGUUUCCUGCUCCCCAGCCAGCCCCUGAAAAUGCCUAUCAGGCCUACGCCGCCCCUCAGCUGUUCCCUGUGUCUGAUAUUACCCAGAAUCAGCUGACAAACCAGGCUGGCGGAGAGGCCCCACAGCCAGGCGAUAACUCUACAGUGCAGCCUGCCGCUGCAGUGGUGCUGGCAUGUCCAGGCGCCAAUCAGGAACAGCAGUUAGCUGAUAUCGGAGCUCCUCAGCCAGCUCCAGCAGCUGCCCCUGCCCGGAGAACCAGAAAACCCUUACAGCCUGAAUCCCUGGAGGAGUGCGAUAGCGAGCACCUGCUGCAGCACUACAGGGAGGUGGCCAGCGCUAAGAGCUCUGAGAACGAUCGCCUGAGGCUGCUGCUGAAGCAGAUGUGCCCCAGCCUGGACGUGGAUUCUAUCAUCCCUAGAACCCCCGAUGUGCUGCACGAGGACCUGCUGAAUUUCAGCAUGCUCAUCCCUAUCGCAGUGGGCGGAGCCCUGGCCGGCCUGGUGCUGAUCGUGCUCAUCGCCUAUCUGGUGGGCAGAAAGAGGAGCCACGCUGGCUAUCAGACCAUC Coding sequence 1 of NE7 (NE7V1) SEQ ID NO: 78: NE7 coding sequence 2 (NE7V2) SEQ ID NO: 79: The coding sequence 3 of NE7 (NE7V3) SEQ ID NO: 80: The coding sequence of NE7, 4 (NE7V4), is SEQ ID NO: 81. The coding sequence 5 of NE7 (NE7V5) SEQ ID NO: 82: The coding sequence of NE7, 6 (NE7V6), is SEQ ID NO: 83. The coding sequence 7 of NE7 (NE7V7) is SEQ ID NO: 84. The coding sequence of NE7, SEQ ID NO: 85 (NE7V8): The coding sequence of NE7, 9 (NE7V9), SEQ ID NO: 86: The coding sequence 10 of NE7 (NE7V10) SEQ ID NO: 87: NE8 coding sequence 1 (NE8V1) SEQ ID NO: 88: NE8 coding sequence 2 (NE8V2) SEQ ID NO: 89: NE8 coding sequence 3 (NE8V3) SEQ ID NO: 90: NE8 coding sequence 4 (NE8V4) SEQ ID NO: 91: The coding sequence 5 of NE8 (NE8V5) SEQ ID NO: 92: The coding sequence of NE8, 6 (NE8V6), is SEQ ID NO: 93. The coding sequence of NE8, 7 (NE8V7), is SEQ ID NO: 94. The coding sequence of NE8 (NE8V8) is SEQ ID NO: 95. The coding sequence of NE8, SEQ ID NO: 96 (NE8V9): The coding sequence of NE8, SEQ ID NO: 97 (NE8V10): NE9 coding sequence 1 (NE9V1) SEQ ID NO: 98: NE9 coding sequence 2 (NE9V2) SEQ ID NO: 99: NE9 coding sequence 3 (NE9V3) SEQ ID NO: 100: The coding sequence of NE9, 4 (NE9V4), is SEQ ID NO: 101. The coding sequence 5 of NE9 (NE9V5) SEQ ID NO: 102: The coding sequence of NE9, 6 (NE9V6), SEQ ID NO: 103: The coding sequence 7 of NE9 (NE9V7) SEQ ID NO: 104: The coding sequence 8 of NE9 (NE9V8) SEQ ID NO: 105: The coding sequence of NE9, 9 (NE9V9), is SEQ ID NO: 106. The coding sequence of NE9, SEQ ID NO: 107 (NE9V10): The coding sequence 1 of NE10 (NE10V1) SEQ ID NO: 108: NE10 coding sequence 2 (NE10V2) SEQ ID NO: 109: The coding sequence 3 of NE10 (NE10V3) SEQ ID NO: 110: The coding sequence of NE10, 4 (NE10V4), SEQ ID NO: 111: The coding sequence 5 of NE10 (NE10V5) SEQ ID NO: 112: The coding sequence of NE10, 6 (NE10V6), SEQ ID NO: 113: The coding sequence 7 of NE10 (NE10V7) SEQ ID NO: 114: The coding sequence 8 of NE10 (NE10V8) SEQ ID NO: 115: The coding sequence of NE10, 9 (NE10V9), SEQ ID NO: 116: The coding sequence 10 of NE10 (NE10V10) SEQ ID NO: 117: The coding sequence 1 of NE15 (NE15V1) SEQ ID NO: 118: The coding sequence 2 of NE15 (NE15V2) SEQ ID NO: 119: The coding sequence 3 of NE15 (NE15V3) SEQ ID NO: 120: The coding sequence 4 of NE15 (NE15V4) SEQ ID NO: 121: The coding sequence 5 of NE15 (NE15V5) SEQ ID NO: 122: The coding sequence 6 of NE15 (NE15V6) SEQ ID NO: 123: The coding sequence 7 of NE15 (NE15V7) SEQ ID NO: 124: The coding sequence 8 of NE15 (NE15V8) SEQ ID NO: 125: The coding sequence of NE15, 9 (NE15V9), SEQ ID NO: 126: The coding sequence 10 of NE15 (NE15V10) SEQ ID NO: 127: The coding sequence 1 of NE16 (NE16V1) SEQ ID NO: 128: NE16 coding sequence 2 (NE16V2) SEQ ID NO: 129: The coding sequence 3 of NE16 (NE16V3) SEQ ID NO: 130: The coding sequence of NE16, 4 (NE16V4), SEQ ID NO: 131: The coding sequence 5 of NE16 (NE16V5) SEQ ID NO: 132: The coding sequence 6 of NE16 (NE16V6) SEQ ID NO: 133: The coding sequence 7 of NE16 (NE16V7) SEQ ID NO: 134: The coding sequence 8 of NE16 (NE16V8) SEQ ID NO: 135: The coding sequence of NE16, 9 (NE16V9), SEQ ID NO: 136: The coding sequence 10 of NE16 (NE16V10) SEQ ID NO: 137: The coding sequence 1 of NE17 (NE17V1) SEQ ID NO: 138: The coding sequence 2 of NE17 (NE17V2) SEQ ID NO: 139: The coding sequence 3 of NE17 (NE17V3) SEQ ID NO: 140: The coding sequence of NE17, 4 (NE17V4), is SEQ ID NO: 141. The coding sequence 5 of NE17 (NE17V5) SEQ ID NO: 142: The coding sequence 6 of NE17 (NE17V6) SEQ ID NO: 143: The coding sequence 7 of NE17 (NE17V7) SEQ ID NO: 144: The coding sequence 8 of NE17 (NE17V8) SEQ ID NO: 145: The coding sequence of NE17, 9 (NE17V9), SEQ ID NO: 146: The coding sequence 10 of NE17 (NE17V10) SEQ ID NO: 147: The coding sequence 1 of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 153: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 154: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 155: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 156: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 157: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 158: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 159: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 160: The coding sequence of LMP2A (SEQ ID NO: 14) is SEQ ID NO: 161: The coding sequence 1 of LMP2A (SEQ ID NO: 10) is SEQ ID NO: 162: The coding sequence 1 of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 163. The coding sequence 2 of the LMP2A truncated variant (the antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 164. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 165. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 166. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 167. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 168. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 169. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 170. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 171. The coding sequence of the LMP2A truncated variant (antigen of NE15, SEQ ID NO: 149) is SEQ ID NO: 172.

Claims

1. An antigenic protein derived from Epstein-Barr virus, characterized in that, The antigen protein is selected from (1) or (2): (1) The antigen protein comprises or has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 149, SEQ ID NO: 13, SEQ ID NO: 5, SEQ ID NO: 8 or SEQ ID NO: 10; (2) The antigen protein comprises an amino acid sequence that has at least 90% sequence identity with the antigen protein defined in (1) and has comparable or better immunogenicity.

2. An antigen protein composition, characterized in that, The antigen protein composition comprises one or more of the antigen proteins as described in claim 1, preferably two; Preferably, the antigen protein composition comprises antigen proteins with amino acid sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 149, respectively.

3. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antigen protein as described in claim 1; Preferably, the nucleic acid is RNA or DNA, and more preferably mRNA; More preferably, the mRNA comprises a nucleotide sequence selected from any of SEQ ID NO: 18-27, SEQ ID NO: 38-47, SEQ ID NO: 58-67, SEQ ID NO: 88-97, SEQ ID NO: 108-117, SEQ ID NO: 138-147, SEQ ID NO: 153-162 and SEQ ID NO: 163-172.

4. The nucleic acid as described in claim 3, characterized in that, The mRNA also contains nucleotide sequences encoding signal peptides, membrane anchoring segments, and / or lysosomal localization signals; Preferably, the signal peptide is the signal peptide of LAMP1, and the amino acid sequence of the signal peptide is preferably as shown in SEQ ID NO:16; and / or, The membrane anchoring segment originates from the transmembrane region of LAMP1, and the preferred amino acid sequence of the membrane anchoring segment is as shown in SEQ ID NO:15; and / or, The lysosomal localization signal originates from the cytoplasmic tail of LAMP1, and the preferred amino acid sequence of the lysosomal localization signal is shown in SEQ ID NO:

148.

5. The nucleic acid as described in claim 3 or 4, characterized in that, The mRNA comprises a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 12; Preferably, the mRNA comprises the nucleotide sequence shown in any of SEQ ID NO: 28-37, SEQ ID NO: 48-57, SEQ ID NO: 68-77, SEQ ID NO: 78-87, SEQ ID NO: 98-107, SEQ ID NO: 118-127 and SEQ ID NO: 128-137.

6. The nucleic acid according to any one of claims 3-5, characterized in that, The mRNA further comprises a 5'-cap structure, a 5'UTR, a 3'UTR, and / or Poly(A); preferably, the 5'-cap structure is CAP O, CAP I, or CAP II; and / or, the Poly(A) comprises 25-400 adenosine nucleotides; Optionally, the mRNA further comprises a nucleotide sequence encoding a protein tag; Preferably, the mRNA includes modifications at one or more of the following locations: a 5' cap structure, a 5' UTR, an open reading frame, a 3' UTR, and Poly(A); More preferably, the modification is selected from one or more of the following: pseudouridine modification, N1-methyl-pseudouridine modification, 5-methoxy-uridine modification, N6-methyl-adenosine modification, and 5-methyl-cytidine modification.

7. A nucleic acid composition, characterized in that, The nucleic acid composition comprises the nucleic acid encoding the antigen protein composition as described in claim 2; Preferably, the encoding nucleic acid is mRNA as defined in any one of claims 2-6; More preferably, the nucleic acid composition comprises two mRNAs, each mRNA comprising a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 7 and SEQ ID NO: 11; or, the mRNAs comprising a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 8 and SEQ ID NO:

149. More preferably, the nucleic acid composition comprises two mRNAs, each mRNA comprising a nucleotide sequence shown in any one of SEQ ID NO: 78-87 and / or any one of SEQ ID NO: 118-127, preferably the nucleic acid composition comprising nucleotide sequences such as SEQ ID NO: 82 and SEQ ID NO: 118; or, the mRNAs comprising nucleotide sequences shown in any one of SEQ ID NO: 88-97 and / or any one of SEQ ID NO: 163-172.

8. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in any one of claims 3-6 or the nucleic acid composition as described in claim 7.

9. A transformant, characterized in that, The transformant comprises the nucleic acid as described in any one of claims 3-6 or the recombinant expression vector as described in claim 8.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antigen protein as claimed in claim 1, the antigen protein composition as claimed in claim 2, the nucleic acid as claimed in any one of claims 3-6, the nucleic acid composition as claimed in claim 7, the recombinant expression vector as claimed in claim 8, or the transformant as claimed in claim 9; preferably, it further comprises a pharmaceutically acceptable carrier and / or excipients; Preferably, the pharmaceutical composition is a vaccine, the vaccine comprising (i) a nucleic acid as described in any one of claims 3-6 or a nucleic acid composition as described in claim 7; and (2) a delivery vector; More preferably, when the vaccine comprises the nucleic acid composition, the mass ratio of the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 11 is 40-50% : 50-60%; or, the mass ratio of the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 149 is 40-50% : 50-60%; and / or, The delivery carrier is a lipid nanoparticle, an ionic lipid complex, a lipid polymer complex, a polymer nanoparticle, an inorganic nanoparticle, a cationic nanoemulsion, or an exosome; the lipid nanoparticle is preferably composed of cationic lipids, cholesterol, phospholipids, and lipid conjugates, for example, composed of RL151, cholesterol, DSPC, and DMG50-PEG2000. More preferably, the molar ratio of RL151:cholesterol:DSPC:DMG50-PEG2000 is (30~60): (20~40): (10~30): (1~5), for example, 50: 38.5: 10:1.

5.

11. A medicine kit, characterized in that, The drug kit includes the antigen protein as described in claim 1, the antigen protein composition as described in claim 2, the nucleic acid as described in any one of claims 3-6, the nucleic acid composition as described in claim 7, the recombinant expression vector as described in claim 8, or the transformant as described in claim 9, or the drug composition as described in claim 10, and other drugs for the prevention, relief and / or treatment of tumors, such as immunosuppressants, cytotoxic drugs, tumor vaccines, antibodies, peptides and / or chemotherapeutic agents. Preferably, the chemotherapeutic agent is paclitaxel, cisplatin, vinorelbine, docetaxel, gemcitabine, temozolomide, irinotecan and / or carboplatin; and the antibody is a PD-1 antibody.

12. A method for preparing the antigen protein as described in claim 1 or the antigen protein composition as described in claim 2, characterized in that, The method includes culturing the transformant as described in claim 9 under conditions suitable for the expression of antigen proteins derived from EB virus.

13. The use of the antigen protein of claim 1, the antigen protein composition of claim 2, the nucleic acid of any one of claims 3-6, the nucleic acid composition of claim 7, the recombinant expression vector of claim 8, or the transformant of claim 9 in the preparation of a medicament for the prevention, mitigation, and / or treatment of diseases caused by EB virus and / or EB virus-positive diseases; Preferably, the disease is a tumor that highly expresses EBNA1, Zta and / or LMP2A; More preferably, the tumor is nasopharyngeal carcinoma, Burkitt lymphoma, gastric cancer, or Hodgkin lymphoma.

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