EB (Epstein-Barr) virus related antigen long peptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an EB virus related antigen long peptide and application thereof. The invention provides an EBV (Epstein-Barr Virus) related antigen long peptide. The sequence of the EBV related antigen long peptide is shown as any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.21. The invention further provides a preparation method of the EBV related antigen long peptide. According to the research, the residence time and the immunogenicity of the long peptide in vivo are further improved by modifying the long peptide with serum albumin through the screened long peptide capable of remarkably enhancing the immunogenicity. Besides, nanoparticles of the long peptide are also constructed through an antigen-adjuvant self-assembly strategy, and the nanoparticles not only optimize the solubility and stability of the long peptide, but also enhance the antigen presentation efficiency and induce stronger immune response. The nano-particles can be combined with an immune checkpoint inhibitor to further enhance the tumor treatment effect, and the nano-particles have good safety and good application prospects and value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a long peptide of EB virus-associated antigen and its applications. Background Technology

[0002] Epstein-Barr virus (EBV) has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). It was the first human oncogenic virus discovered and is highly associated with more than 10 types of malignant tumors, including nasopharyngeal carcinoma, lymphoma, and gastric cancer. Statistics show that over 90% of adults worldwide are infected with EBV, and approximately 200,000 new cases of EBV-related malignant tumors are diagnosed each year.

[0003] In the current field of innovative drug development, cancer therapeutic vaccines have become one of the breakthrough directions. However, directly using soluble proteins as vaccine antigens often fails to achieve ideal immune effects, mainly due to: 1) antigens are easily degraded in vivo; 2) low antigen presentation efficiency; and 3) insufficient immune response strength. Therefore, developing efficient cancer vaccine delivery systems is crucial for improving vaccine efficacy.

[0004] Nanoparticles, as novel drug delivery systems, have demonstrated unique advantages in the field of tumor vaccines. They can protect antigens from degradation, enhance antigen presentation, and target lymph nodes through surface modification, thereby inducing a stronger immune response. However, research on specific nanoparticle vaccines against EBV-related tumors is still in the exploratory stage. Summary of the Invention

[0005] The present invention aims to solve the technical problem that the immunization effect of EB virus-related tumor-specific vaccines in the prior art is not ideal, and provides a polypeptide vaccine for the prevention and treatment of EB virus-related tumors and its preparation method.

[0006] The first objective of this invention is to provide a long peptide of EBV virus-associated antigen.

[0007] A second objective of this invention is to provide a nanoparticle.

[0008] A third objective of this invention is to provide applications of the aforementioned long peptides and nanoparticles.

[0009] The fourth objective of this invention is to provide an EB virus vaccine.

[0010] The fifth object of the present invention is to provide a medicine for treating EB virus infection or diseases caused by EB virus.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] The present invention provides a long peptide of EBV virus-associated antigen, the sequence of which is shown in any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.21.

[0013] Preferably, the long peptide is modified with serum albumin.

[0014] This invention provides a nanoparticle containing the aforementioned long peptide.

[0015] As an alternative implementation, the nanoparticles also include an adjuvant.

[0016] As an alternative implementation, the adjuvant is imiquimod.

[0017] As an alternative implementation, the nanoparticles are prepared by adding an imiquimod solution to the above-mentioned long peptide solution.

[0018] As an alternative implementation, in the preparation method, imiquimod solution is added to the above-mentioned long peptide solution to obtain a mixed solution, wherein the mass ratio of imiquimod to long peptide in the mixed solution is 1:(1-2).

[0019] As an alternative implementation, in the preparation method, imiquimod solution is added to the above-mentioned long peptide solution to obtain a mixed solution, wherein the mass ratio of imiquimod to long peptide in the mixed solution is 1:1.

[0020] As an alternative implementation, in the preparation method, nanoparticles are prepared by dialysis of the mixed solution.

[0021] As an alternative implementation, the solvent for the long peptide solution is PBS buffer.

[0022] As an alternative implementation, the concentration of the PBS buffer is 4-8M (preferably 6M).

[0023] As an alternative implementation, the pH of the PBS buffer is 7-8 (preferably pH 7.4).

[0024] As an alternative implementation, the PBS buffer also includes urea.

[0025] As an alternative implementation, the solvent for the imiquimod solution is tetrahydrofuran.

[0026] As an alternative implementation method, the imiquimod solution is prepared by dissolving imiquimod in tetrahydrofuran and then subjecting it to ultrasonic treatment.

[0027] As an alternative implementation, the method for preparing the nanoparticles includes the following steps:

[0028] 1. The above-mentioned long peptide was dissolved in PBS buffer to obtain a long peptide solution;

[0029] 2. Imiquimod is dissolved in tetrahydrofuran to obtain an imiquimod solution;

[0030] 3. Imiquimod solution was added dropwise to long peptide solution to obtain a mixed solution with a mass ratio of long peptide to imiquimod of 1:1. Nanoparticles were prepared by dialysis.

[0031] This invention provides the use of the above-described long peptides or nanoparticles in the preparation of medicaments for the prevention or treatment of EB virus infection or diseases induced by it.

[0032] Specifically, the drug includes a vaccine for the prevention or treatment of EB virus infection or diseases it causes.

[0033] This invention provides an EB virus vaccine comprising the above-mentioned long peptide or the above-mentioned nanoparticles.

[0034] This invention provides a drug for treating EB virus infection or diseases caused by EB virus, including the above-mentioned EB virus vaccine and immune checkpoint inhibitor.

[0035] As an alternative implementation, the immune checkpoint inhibitor includes a PD-1 inhibitor.

[0036] The present invention has the following beneficial effects:

[0037] This invention studies long peptides with significantly enhanced immunogenicity, which can be further improved in vivo by modifying serum albumin to increase their retention time and immunogenicity. Furthermore, this invention constructs nanoparticles of these long peptides using an antigen-adjuvant self-assembly strategy. The preparation of nanoparticles not only optimizes the solubility and stability of the long peptides and protects the antigen from degradation, but also enhances antigen presentation efficiency and induces a stronger immune response, making them suitable as a vaccine for treating EBV-induced tumors. These nanoparticles can also be combined with immune checkpoint inhibitors (such as anti-PD-1 antibodies) to further enhance the therapeutic effect on tumors, and exhibit excellent safety, without causing significant weight loss or other adverse reactions during treatment. The long peptide vaccine provided by this invention can not only be used to prevent or treat EBV infection, but also to prevent and treat EBV-induced tumors (such as nasopharyngeal carcinoma and lymphoma), demonstrating excellent application prospects and value. Attached Figure Description

[0038] Figure 1The results show the specific immune response of hPBMCs to EB virus-related long peptides (Figure A shows the level of IFN-γ produced by hPBMCs under different long peptide stimulation detected by ELISPOT; Figure B shows the percentage of T cell immune responses induced by each long peptide group, n=15).

[0039] Figure 2 The results show the effect of MSA-LP on mouse T cell immune response (Figure A shows CD4). + Percentage of T cell-specific IFN-γ secretion; Figure B shows CD8. + (Percentage of T cell-specific IFN-γ secretion).

[0040] Figure 3 The results of particle size determination and electron micrographs of the nanoparticles are shown (electron micrograph scale bar is 100 nm).

[0041] Figure 4 This is a schematic diagram of an experiment using an EBV-CT26 tumor mouse model.

[0042] Figure 5 Tumor growth curves for different treatment groups.

[0043] Figure 6 To enhance the anti-EBV tumor effect of IMQ@MSA-LP NPs in combination with anti-PD-1 antibody (Figure A is the flowchart of the experimental protocol; Figure B is the mouse body weight change curve; Figure C is the mouse survival curve; Figure D is the mouse tumor volume growth curve; Figure E is the individual tumor growth curve of mice in each treatment group). Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] Mouse IFN-γ Precoated ELISPOT Kit, brand name: Dayou, item number: 2210004.

[0047] Imiquimod, CAS number 99011-02-6, structural formula is

[0048] The studies described below were approved by the Research and Ethics Committee of Sun Yat-sen University Cancer Center (SYSUCC). Experiments involving human participants were conducted in accordance with the recommendations of the Research and Ethics Committee. Full informed consent was obtained from all participants, and the studies were conducted in accordance with the Declaration of Helsinki (2013 edition).

[0049] Example 1

[0050] For systematic screening, we divided the EBV antigen proteins EBNA1, LMP-1, and LMP-2B into a series of overlapping peptides, each 50 amino acids long, with a 10-amino acid overlap between adjacent peptides to ensure complete coverage of potential antigenic epitopes. For the EBNA1 protein, due to its unique structural features, we excluded the repetitive sequence region composed of glycine-alanine (GAr) and started peptide design from amino acid position 330. The EBNA1 peptides were numbered according to the following nomenclature system: E1-1 (amino acids 330-379), E1-2 (amino acids 370-419), E1-3 (amino acids 410-459), and so on, synthesizing a total of 8 peptides (E1-1 to E1-8), completely covering the EBNA1 protein region from position 330 to 641. For LMP-1 and LMP-2B proteins, we designed full-length sequences and synthesized 10 LMP-1-related long peptides (L1-1 to L1-10) and 10 LMP-2B-related long peptides (L2-1 to L2-10), respectively.

[0051] All peptides were commercially synthesized by Shanghai Jier Biochemical Co., Ltd. using solid-phase peptide synthesis technology. Since highly hydrophobic (low hydrophilic) peptides or proteins typically exhibit weak antigenicity, the inward folding of hydrophobic amino acids reduces surface exposure, decreases solubility leading to aggregation, and creates a rigid structure unfavorable for antibody binding, while also interfering with MHC presentation. We rigorously screened all synthesized peptides, discarding long peptides with poor hydrophilicity and insufficient purity during the screening process. Only 16 long peptides with a purity greater than 90% confirmed by HPLC and mass spectrometry analysis were retained. The amino acid sequences of these long peptides are shown in Table 1, including E1-1, E1-3 to E1-8, L1-5 to L1-10, and L2-2, L2-3, and L2-10.

[0052] Table 1 Long peptide sequences

[0053]

[0054]

[0055]

[0056] Example 2: Screening and Validation of Advantageous Long Peptides of EB Virus

[0057] I. Experimental Methods

[0058] Patient-derived peripheral blood mononuclear cells (hPBMCs) were obtained from Sun Yat-sen University Cancer Center. 10 mL of anticoagulated whole blood was collected from each clinical patient and diluted with an equal volume of PBS buffer. An equal volume of human lymphocyte separation medium was added to a centrifuge tube, and the diluted blood sample was carefully spread evenly on top of the separation medium, maintaining a clear interface between the two liquids. The tube was centrifuged at 700-800 g for 30 minutes at room temperature using the third speed setting. After centrifugation, the bottom layer was a red blood cell layer, the middle layer was the separation medium layer, and the top layer was a plasma homogenate layer. Between the plasma layer and the separation medium layer was a thin, dense white membrane, i.e., the mononuclear cell layer. The white membrane layer was carefully transferred to another centrifuge tube, diluted with PBS buffer to 20 mL, and inverted to mix. The tube was centrifuged at 250 g for 10 minutes at room temperature, the supernatant was discarded, and the washing was repeated twice. Finally, the hPBMCs were resuspended in PBS buffer for later use.

[0059] Before the experiment, the pre-coated plate was activated with 200 μL / well of RPMI-1640 medium, incubated at room temperature for 10 minutes, and then aspirated. The isolated peripheral blood mononuclear cells (hPBMCs) were then cultured at 2 × 10⁻⁶ cells / well. 5 Cell / well concentration plating was performed. Subsequently, 16 long peptide stimulants synthesized in Example 1 (10 μL / well, final concentration 5 μg / mL) were added, and the secretion of the specific cytokine IFN-γ was measured using the Mouse IFN-γ Precoated ELISPOT Kit. Blank control wells and the stimulants provided with the kit were set up as positive control groups. The assay method was performed according to the kit instructions, specifically as follows:

[0060] (1) After gently mixing the cells in the plate, cover the plate and place it at 37°C and 5% humidity. Incubate in an incubator for 24 hours. After incubation, pour off the cells and culture medium from the wells, add pre-cooled deionized water (200 μL / well), and lyse the cells in a hypotonic manner at 4°C for 10 minutes. Then, shake off the liquid from the wells, add 1× Washing Buffer (260 μL / well), let stand for 1 minute, then discard the liquid from the wells. Repeat the washing process six times, patting dry on absorbent paper each time.

[0061] (2) After washing the plate, add 1×Biotinylated Antibody working solution (100 μL / well) to each well and incubate at 37°C for 1 hour. Repeat the washing steps. Then add 1×Streptavidin-HRP working solution (100 μL / well) and incubate at 37°C for 1 hour. After incubation, wash the plate thoroughly six times, patting it dry on absorbent paper each time. Then remove the plate base, rinse the bottom of the membrane and the base with deionized water, carefully blot dry any remaining water, close the base, add 1×WashingBuffer (260 μL / well) again, let stand for 1 minute, discard the liquid in the wells, and pat dry thoroughly.

[0062] (3) Finally, add freshly prepared AEC colorimetric solution (100 μL / well) to each experimental well, let it stand at room temperature in the dark for 30 minutes, then pour out the liquid in the well, remove the plate base, and thoroughly clean the front and back of the experimental well and the base with deionized water to terminate the colorimetric reaction. Place the plate in a cool place at room temperature to air dry naturally, then close the base, complete the ELISPOT plate spot counting and record various parameters for statistical analysis.

[0063] II. Experimental Results

[0064] The results of the specific immune response of hPBMCs to EB virus-associated long peptides are as follows: Figure 1 As shown, the results indicate that the E1-4 long peptide derived from EBNA1 protein significantly induced higher levels of the specific cytokine IFN-γ secretion compared to other groups. Other long peptides with better responses included E1-5, E1-6, L2-3, L1-6, and L1-5, suggesting that these regions may contain important T cell epitopes. Notably, the EBNA1 protein-related long peptides generally elicited stronger T cell responses than the long peptides of LMP1 and LMP2B proteins. This difference may be attributed to several factors: firstly, the membrane proteins LMP1 and LMP2B have poor hydrophilicity, leading to reduced antigen processing and presentation efficiency; secondly, EBNA1 protein is expressed at all stages of EBV infection, while LMP protein expression is phase-specific and tissue-specific, which may also affect the establishment and maintenance of the corresponding memory T cell pool.

[0065] Example 2: Modification and Optimization of Advantageous Long Peptides

[0066] I. Experimental Methods

[0067] To optimize the retention time of long peptides in vivo, E1-4, E1-5, E1-6, L2-3, L1-6, and L1-5 long peptides were sent to Shanghai Jier Biochemical Hospital for conjugation with mouse albumin to obtain long peptides conjugated with mouse albumin (MSA-LP), which were designated as MSA-E1-4, MSA-E1-5, MSA-E1-6, MSA-L1-5, MSA-L1-6, and MSA-L2-3, respectively.

[0068] BALB / c mice (female, 6-8 weeks old) were used as an animal model and randomly divided into 13 groups (n=5 per group), with one group receiving an equal volume of PBS as a control. The experimental groups received subcutaneous injections of either long peptides conjugated with different mouse albumin or free long peptides, at a dose of 5 μg per mouse. The mice were immunized twice, with a two-week interval. On the third day after the second immunization, the mice were sacrificed to measure cellular immunity.

[0069] The specific method for measuring cellular immunity was as follows: Mice were sacrificed and their spleens were isolated. After mechanical grinding, monocytes were obtained by density gradient centrifugation. The monocytes were plated and stimulated with long peptide antigens (5 μg / mL) for 12 hours. BFA was added to block cytokine secretion. After 4 hours, the cells were labeled with anti-CD3, CD8, CD4 and IFN-γ fluorescent antibodies, and analyzed by flow cytometry. The percentage of IFN-γ positive cells was calculated using FlowJo software.

[0070] II. Experimental Results

[0071] The effects of MSA-LP on mouse T-cell immune responses are as follows: Figure 2 As shown, the results indicate that conjugation with mouse albumin significantly enhances the immunogenicity of most long peptides in mice. Compared with the unconjugated control group, MSA-conjugated E1-4, E1-5, E1-6, L1-5, and L2-3 all induced significantly elevated CD4 levels. + and CD8 + T cell-specific IFN-γ secretion levels, including CD4 + T-cell response shows a clear advantage.

[0072] Example 3: Construction and Characterization of Nanoparticle Delivery System

[0073] I. Experimental Methods

[0074] This embodiment constructs a nanoparticle system for delivering modified, dominant long peptides;

[0075] 1) Using imiquimod as an adjuvant, carrier-free nanoparticles were prepared;

[0076] 2) The dominant long peptide of the modified mouse albumin was rapidly pipetted in the aqueous phase with Imiquimod in the oil phase at a volume ratio of 10:1.

[0077] 3) The physicochemical properties of nanoparticles were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0078] Based on the amphiphilic characteristics of MSA-LP, an antigen-adjuvant self-assembly strategy was used to construct a nanodelivery system. The specific preparation method is as follows:

[0079] 1. Preparation of MSA-LP aqueous phase: Dissolve the six MSA-LPs (MSA-E1-4, MSA-E1-5, MSA-E1-6, MSA-L1-5, MSA-L1-6, MSA-L2-3) in PBS buffer (pH 7.4) containing 6M urea, where urea is used as a denaturant to improve protein solubility;

[0080] 2. Preparation of the IMQ organic phase: Imiquimod (IMQ) is dissolved in tetrahydrofuran (THF) and sonicated (an appropriate amount of hydrochloric acid can be added to increase the solubility of IMQ);

[0081] 3. The organic phase of IMQ was added dropwise to the aqueous phase of MSA-LP (the mass concentration ratio of MSA-LP to IMQ was 1:10, and the volume ratio was 10:1). The final mass ratio of IMQ to MSA-LP was maintained at 1:1. Nanoparticles were prepared by dialysis, and the particle size distribution was monitored in real time by dynamic light scattering (DLS) technology.

[0082] II. Experimental Results

[0083] The particle size measurements of the various nanoparticles showed little difference; therefore, MSA-E1-4 nanoparticles were used as a representative for data presentation. The particle size measurements and electron micrographs of MSA-E1-4 nanoparticles are shown below. Figure 3 As shown, the results indicate that the average particle size of the prepared nanoparticles is 120±15 nm, and the PDI is 0.18±0.03. TEM observation shows that the particles have regular morphology and good dispersibility.

[0084] Example 4: Evaluation of the therapeutic effect of nanoparticle vaccines in EBV-related tumor models

[0085] This embodiment evaluates the therapeutic efficacy of nanoparticle vaccines in an EBV-related tumor model.

[0086] I. Experimental Methods

[0087] Following the method in Example 3, self-assembled nanoparticles of MSA conjugated with six dominant long peptides (E1-4, E1-5, E1-6, L1-5, L1-6, L2-3) were prepared.

[0088] Using an EBV-CT26 cell xenograft mouse model, 2×10 5EBV-CT26 cells were transplanted into mice to establish a tumor model. Following tumor inoculation, mice were injected with a vaccine (10 micrograms of each long peptide per mouse) at different time points (day 0, day 3, and day 8). A schematic diagram of the EBV-CT26 tumor mouse model experiment is shown below. Figure 4 As shown.

[0089] The specific groups were: IMQ@MSA-LP NPs group, MSA-LPs(IMQ) group, and PBS group. All groups were administered intramuscularly. The IMQ@MSA-LP NPs group and the MSA-LPs(IMQ) group had the same amount of antigen and adjuvant. The main difference was that the former was a nanoparticle group with an antigen content of 10 micrograms of 6 long peptides per mouse, while the latter was a free group. PBS served as the control group.

[0090] Tumor volume was measured every two days, and tumor growth was continuously monitored until the tumor volume reached 2000 mmHg. 3 Or on day 16. Measure the length and width of the tumor using calipers, and then use the formula V = (length × width) 2 The tumor volume is calculated as 1 / 2, and the unit is cubic millimeters (mm). 3 )

[0091] II. Experimental Results

[0092] Experimental results are as follows Figure 5 As shown, the results indicate significant differences among the three groups: the PBS control group exhibited the most rapid tumor growth, reaching a tumor volume of approximately 1750 mmHg by day 12. 3 The MSA-LPs (IMQ) group showed a slight inhibition of tumor growth, with the tumor volume reaching approximately 1500 mm² on day 14. 3 The IMQ@MSA-LP NPs treatment group showed the most significant tumor suppression effect, with the tumor volume reaching only about 900 mm² on day 15. 3 Compared to the control group, it decreased by nearly 50%.

[0093] Example 5: Antitumor effect enhanced by IMQ@MSA-LP NPs combined with PD-1

[0094] Although the aforementioned experiments demonstrated that IMQ@MSA-LP NPs can significantly activate cellular immunity and inhibit tumor growth, the immunosuppressive properties of the solid tumor microenvironment often result in limited immune response. To further improve therapeutic efficacy, we designed a treatment regimen combining nanoparticles with immune checkpoint inhibitors and evaluated its therapeutic effect on EBV-related solid tumors.

[0095] I. Experimental Methods

[0096] Using an EBV-CT26 cell xenograft mouse model, mice were inoculated with 4×10⁻⁶ cells on day 0. 5 A tumor model was established using EBV-CT26 cells. Mice were then vaccinated on days 0, 3, and 8, followed by a day of combination therapy with 100 μg of anti-PD-1 antibody.

[0097] The specific groupings were: PBS group, Ant-PD-1 group, IMQ@MSA-LP NPs group, and IMQ@MSA-LP NPs+Ant-PD-1 group. IMQ@MSA-LP NPs were administered intramuscularly, and Ant-PD-1 was administered intraperitoneally. During IMQ@MSA-LP NPs+Ant-PD-1 treatment, the same doses of antigen and Ant-PD-1 served as control groups, respectively. PBS was the non-intervention group.

[0098] II. Experimental Results

[0099] Experimental results are as follows Figure 6 As shown, the results indicated that the body weight of mice in the PBS control group gradually decreased, while the body weight of mice in the nanoparticle group and the combined treatment group remained relatively stable, indicating the safety of the treatment. Figure 6 Figure B). Survival curves showed that mice in the PBS group began to die around 10 days after inoculation, and all died within 15 days; while mice treated with MSA-LPs or anti-PD-1 antibodies alone had slightly longer survival; the IMQ@MSA-LP NPs alone group showed better survival benefit; the IMQ@MSA-LP NPs combined with anti-PD-1 group showed the best treatment effect, with almost all mice surviving during the observation period. Figure 6 (Figure C).

[0100] Tumor volume monitoring further confirmed the advantages of combination therapy. Tumors in the PBS group grew rapidly, reaching approximately 2000 mm² in volume at day 16. 3 Tumor growth was slowed in the groups treated with anti-PD-1 or MSA-LPs alone; the group treated with IMQ@MSA-LP NPs alone showed a significant tumor-suppressing effect; while the group treated with IMQ@MSA-LP NPs in combination with anti-PD-1 showed the strongest tumor-suppressing activity, with the tumor volume only about 300 mm at 16 days. 3 And it has a consistent and significant antitumor effect. Figure 6 (D diagram, E diagram).

[0101] In summary, these results indicate that although IMQ@MSA-LP NPs can effectively activate anti-tumor immunity, immunosuppressive factors (such as the PD-1 / PD-L1 pathway) present in the solid tumor microenvironment limit their efficacy. Combined use of anti-PD-1 antibodies can relieve T cell function inhibition, allowing the tumor-specific T cells activated by the nanoparticles to exert their anti-tumor effects more effectively. This synergistic effect not only improved tumor control rates but also significantly prolonged the survival of mice.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A long peptide of EBV virus-associated antigen, characterized in that, Its sequence is shown as any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.

21.

2. The long peptide according to claim 1, characterized in that, The long peptide was modified with serum albumin.

3. A nanoparticle, characterized in that, Includes the long peptide described in claim 1 or 2.

4. The nanoparticles according to claim 3, characterized in that, It also includes adjuvants.

5. The nanoparticles according to claim 4, characterized in that, The adjuvant is imiquimod.

6. The nanoparticles according to claim 5, characterized in that, The preparation method involves adding imiquimod solution to the long peptide solution described in claim 1 or 2 to prepare nanoparticles.

7. The use of the long peptide of claim 1 or 2 or the nanoparticle of any one of claims 3-6 in the preparation of a medicament for the prevention or treatment of EB virus infection or diseases induced by it.

8. An EB virus vaccine, characterized in that, It includes the long peptide of claim 1 or 2 or the nanoparticles of any one of claims 3-6.

9. A drug for treating EB virus infection or diseases caused by EB virus, characterized in that, Includes the EB virus vaccine and immune checkpoint inhibitor as described in claim 8.

10. The drug according to claim 9, characterized in that, The immune checkpoint inhibitors include PD-1 inhibitors.

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