Thymus dependent lymphocyte antigen epitope peptide of hepatitis B virus and application of thymus dependent lymphocyte antigen epitope peptide

Abstract

The invention discloses thymus dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application, and belongs to the field of medical immunology and infectious pathology. The thymus dependent lymphocyte antigen epitope peptide of the hepatitis B virus antigen is characterized in that the amino acid sequence of the epitope peptide comprises any one or more of LTFGRETVL, LTFGRETVL, LTFGRETVL, LTFGRETVL and LTFGRETVL; aYRPPNAPIL, AYRPPNAPIL ASRELVVSY, ASRELVVSY; an MMWYWGPSL is used as a core; the amino acid sequence is an amino acid sequence obtained by removing or replacing a single amino acid of the amino acid sequence. The epitope peptide disclosed by the invention can be specifically combined with cytotoxic thymus dependent lymphocytes to stimulate the cytotoxic thymus dependent lymphocytes to activate, proliferate and differentiate, so that the immune effect of resisting the hepatitis B virus is exerted; the antigen peptides can be used for preparing therapeutic and prophylactic vaccines for hepatitis B virus infection, can also be used for preparing detection reagents for detecting hepatitis B virus specific cytotoxic thymus dependent lymphocytes, and have potential application value in prevention, treatment and diagnosis of hepatitis B.

Description

Technical Field

[0001] The present invention belongs to the fields of medical immunology and infectious diseases, and in particular relates to a thymus-dependent lymphocyte antigen epitope peptide of a hepatitis B virus antigen and an application thereof. Background Art

[0002] Hepatitis B virus (HBV) infection is one of the most common infectious diseases, posing a serious threat to human health. Despite over 30 years of HBV vaccination, infection continues to spread worldwide, affecting approximately 10% of the global population, with over 257 million people suffering from chronic, lifelong infection. Currently, approximately 70 million people in my country are infected with HBV, with approximately 20-30 million suffering from chronic HBV. Approximately 77% of cases of liver cirrhosis and 84% of cases of hepatocellular carcinoma are caused by chronic HBV. Antiviral therapy, primarily consisting of nucleoside (acid) analogs and interferon, is the most effective treatment for HBV and is highly effective in most patients. However, because HBV persists in the nucleus of infected cells through the formation of covalently closed circular DNA (cccDNA) and integrated sequences, current standard therapies can only suppress viral replication but cannot completely eliminate HBV. HBsAg levels often fail to resolve, and HBV is prone to reactivation after drug discontinuation. Chronic transformation of HBV and relapse after drug discontinuation are serious problems we face.

[0003] The HBV genome consists of incomplete double-stranded circular DNA. The long negative strand (3020-3320 bases) and the short positive strand (1700-2800 bases) comprise four open reading frames (ORFs), namely the C, X, P, and S coding regions. The C region consists of the pre-C and C genes, each regulated by different start codons and terminating at the same stop codon. The pre-C and C genes together encode the pre-C protein, which is then cleaved and processed to form the e antigen (HBeAg). The C gene encodes the viral capsid protein, HBcAg. The S region consists of the S gene, PreS1, and PreS2 genes. Translation is initiated by three different start codons and terminates at the same stop codon. These genes encode the surface antigen (HBsAg), PreS1, and PreS2 proteins, respectively. The P region encodes the HBV polymerase protein (HBpol). The gene in region X encodes X protein (HBx), which contains 154 amino acids and is the smallest open reading frame.

[0004] Cytotoxic T lymphocytes (CTLs) are core cells that mediate adaptive immune responses and play a crucial role in anti-infection, anti-tumor, hypersensitivity reactions, and autoimmune diseases. The T cell receptor (TCR) on their cell membrane can specifically recognize and bind to the complex of MHC class I molecules and antigenic peptides on the surface of antigen-presenting cells, known as the MHC / antigen-peptide complex. CTL epitopes are antigenic peptides bound to MHC class I molecules. They are linear segments or spatial conformational structures within the antigen molecule that are specifically recognized by the TCR. They are the basic antigenic units that trigger immune responses and play a key role in CTL activation.

[0005] The MHC system refers to the major histocompatibility complex (MHC), which is a group of tightly linked genes in the vertebrate genome that encode and express MHC class I and class II protein molecules. HLA (human leukocyte antigen) is the human MHC system and the most complex gene group in the human body, with high polymorphism in the human population. HLA plays an important role in the body's immune processes such as antigen recognition and antigen presentation, and is the main factor affecting the human immune response. HLA class I molecules are mainly responsible for presenting endogenous HBV antigens to CD8 + CTLs, activated CTLs secrete perforin and granzyme to induce apoptosis of virus-infected hepatocytes, and secrete specific cytokines to inhibit HBV replication. + The number and function of T cells can accurately reflect the specific immune function status of hepatitis B infected patients against HBV. Due to the different HLA molecular types of different people, their processing, handling and presentation capabilities of different HBV antigens are also different, thus causing different degrees of HBV antigen-specific immune response. According to the different HLA molecular types of hepatitis B patients, the HBV specific antigen peptides presented by them are selected and the CD8 specific HBV antigen peptides are dynamically monitored. + The number and function of T cells are crucial for monitoring the disease progression, developing diagnostic and treatment plans, observing efficacy, and determining prognosis in HBV-infected patients. They are a crucial technical tool for achieving precision medicine in the treatment of hepatitis B. Furthermore, utilizing these HBV-specific antigenic peptides that bind with high affinity to HLA-C molecules can also be used to develop peptide or gene vaccines for the prevention and treatment of HBV infection.

[0006] However, there are still few HBV antigen peptides that have been clearly presented by various HLA molecules and can stimulate the body to induce T cell responses. This limits the specific T cell detection of hepatitis B patients carrying different HLA alleles, the role of HBV-specific T cells in the occurrence and development of hepatitis B, and personalized testing and precision immunotherapy based on individual differences in HLA genes and the differences in the HBV antigen peptides they present. Summary of the Invention

[0007] In view of the deficiencies in the prior art, the present invention proposes a thymus-dependent lymphocyte antigen epitope peptide of a hepatitis B virus antigen and its application.

[0008] The technical solutions of the present invention are as follows:

[0009] The first aspect of the present invention relates to a thymus-dependent lymphocyte antigen epitope peptide of a hepatitis B virus antigen, wherein the amino acid sequence of the antigen epitope peptide is any one or more of the following sequences:

[0010] LTFGRETVL;

[0011] AYRPPNAPIL;

[0012] ASRELVVSY;

[0013] MMWYWGPSL;

[0014] The amino acid sequence above is obtained by removing or replacing a single amino acid.

[0015] The second aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen in the preparation of hepatitis B polypeptide vaccine or gene vaccine.

[0016] The third aspect of the present invention relates to the nucleic acid molecule of the thymus-dependent lymphocyte antigen epitope peptide of the above-mentioned hepatitis B virus antigen.

[0017] The fourth aspect of the present invention relates to a nucleic acid molecule vector.

[0018] The fifth aspect of the present invention relates to a vaccine for treating or preventing hepatitis B virus, comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0019] The sixth aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen in the preparation of a drug for treating and preventing hepatitis B.

[0020] The seventh aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen in the preparation of a kit for detecting hepatitis B virus antigen-specific T cells.

[0021] Optionally, the kit includes: enzyme-linked immunospot reagents, intracellular cytokine fluorescent staining reagents, enzyme-linked immunosorbent assay reagents, human leukocyte antigen multimer fluorescent staining or flow cytometry reagents.

[0022] The eighth aspect of the present invention relates to a monoclonal antibody capable of specifically binding to the above-mentioned epitope peptide.

[0023] The ninth aspect of the present invention relates to a drug for treating hepatitis B, comprising the above-mentioned monoclonal antibody.

[0024] More specifically, the present invention provides a novel thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen, the amino acid sequence of which is any one of the epitope peptide sequences shown below:

[0025]

[0026] As can be seen from the above table, the above sequences are the antigen peptide sequences of hepatitis B virus e antigen HBeAg and surface antigen HBsAg, which bind to HLA-C0701, C0702, C0303, C1202, C0602 and other molecules with high affinity, form MHC / antigen peptide complex molecules on the surface of antigen presenting cells, and bind to antigen peptide specific CD8 + T cell clones bind to stimulate their activation, proliferation and differentiation, and exert immune effect against hepatitis B virus.

[0027] The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used to prepare a hepatitis B polypeptide vaccine or a gene vaccine: Preparation of polypeptide vaccine: one or more antigenic peptides are artificially synthesized according to the antigenic epitope peptide sequence of the present invention, mixed with an adjuvant to form a soluble preparation, or loaded with biological nanomaterials to form a nanopeptide vaccine, which is injected into the body of a hepatitis B virus infected person or hepatitis B patient to stimulate the activation and proliferation of the patient's hepatitis B virus antigen-specific T cells and enhance their tumor cell-killing activity, thereby preparing a hepatitis B polypeptide vaccine. Preparation of gene vaccine: according to the polypeptide sequence of the present invention, a recombinant DNA gene fragment, recombinant plasmid or recombinant viral vector of one or more polypeptides is constructed and injected into the body of a hepatitis B virus infected person or hepatitis B patient to allow the recombinant gene to express one or more polypeptides in vivo, stimulate the activation and proliferation of hepatitis B virus antigen-specific T cells and enhance their activity in killing virus-infected cells, thereby preparing a hepatitis B gene vaccine.

[0028] The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used to prepare a detection preparation or kit for detecting hepatitis B virus antigen-specific T cells: according to the antigen epitope peptide sequence of the present invention, one or more antigen peptides are artificially synthesized, and in the enzyme-linked immunosorbent assay, intracellular cytokine fluorescence staining method and enzyme-linked immunosorbent assay, as an antigen preparation, mixed with the patient's peripheral blood mononuclear cells (PBMC) to stimulate the activation, proliferation and secretion of cytokines by hepatitis B virus antigen-specific T cells, and then the synthesis amount of the cytokine is detected by other combination reagents, thereby reflecting the number and reactivity of specific T cells; these polypeptides can also be used to prepare human leukocyte antigen and polypeptide complexes (peptide-HLA complex) and their polymers through genetic engineering technology and protein engineering technology, and then further prepared into fluorescently labeled preparations, and the number of hepatitis B virus antigen-specific T cells in the patient's peripheral blood PBMC cell population is detected by flow cytometry. The related kit refers to a hepatitis B virus antigen-specific T cell detection kit assembled by the above-mentioned preparation and other reagents commonly used in different detection methods.

[0029] The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used to prepare drugs for treating hepatitis B: the above-mentioned polypeptide vaccine or gene vaccine based on the antigen epitope peptide sequence of the present invention is combined with other immunotherapy preparations or chemotherapy preparations to prepare clinical drugs for treating hepatitis B.

[0030] The present invention utilizes six online epitope prediction databases to virtually predict HLA-C molecule-restricted HBV-specific antigen epitope peptide sequences, thereby obtaining a group of HBV-specific antigen epitope peptide sequences that can respectively bind to HLA-C0102, C0602, C0702, C0801, C0304, C0302, C0303, C0401, C1402, C1202, C1502, C1403, C1203, and C0701 molecules with high affinity. The immunogenicity of the peptides is then verified by intracellular cell staining experiments, thereby providing specific antigen peptides for the preparation of therapeutic and preventive vaccines for HBV infection and the development of HBV antigen-specific T cell detection reagents.

[0031] 1. Select the amino acid sequence of hepatitis B virus e antigen (HBeAg, including the sequence of HBcAg) and surface antigen (HBsAg) as the targeting sequence;

[0032] 2. Six commonly used epitope prediction databases with high accuracy and recognized by researchers were selected: SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, and EPIJEN to predict the HBV-specific antigen epitope peptide sequences restricted by the above 14 HLA-C molecules;

[0033] 3. According to certain prediction criteria, the prediction results of six online epitope prediction websites were integrated and analyzed to obtain candidate antigen peptide sequences with relatively consistent prediction results from the six websites.

[0034] 4. The immunogenicity of HBV antigen epitope peptide was verified through IFN-γICS cell functional experiment.

[0035] 5. HLA-C peptide competition binding experiments were performed using the HMy2.CIR cell line expressing specific HLA-C molecules to analyze the binding affinity of positive epitope peptides to specific HLA-C molecules.

[0036] 6. The immunogenicity of the positive epitope peptide in stimulating specific T cell activation and proliferation in vivo was verified by inoculating C57BL / 6J mice.

[0037] The present invention relates to antigenic peptide sequences in hepatitis B virus e antigen (HBeAg, including HBcAg sequences) and surface antigen (HBsAg) that can bind to HLA-C0701, C0702, C0303, C1202, and C0602 molecules with high affinity and are immunogenic; it also relates to hepatitis B polypeptide vaccines, gene vaccines, and methods for treating and preventing hepatitis B based on the above-mentioned antigenic peptides, as well as reagents and methods for detecting hepatitis B virus antigen-specific T cells based on the above-mentioned antigenic peptides.

[0038] Compared with the prior art, the advantages of the present invention are as follows:

[0039] The HBV-specific epitope peptides restricted to HLA-C0701, C0702, C0303, C1202, and C0602 molecules obtained through online virtual prediction and functional experimental validation have not been previously reported. These HLA-C molecules have also not been previously reported to have restricted HBV antigenic peptides. Therefore, these new epitope peptide sequences will provide key antigenic components, namely, epitope peptide sequences, required for the development of therapeutic and preventive peptide and gene vaccines for hepatitis B, as well as for the design of reagents and methods for detecting hepatitis B virus antigen-specific T cells. Furthermore, these epitope peptides provide key antigenic components for personalized testing and precision medicine for hepatitis B patients with these specific HLA-C alleles. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 PBMCs from hepatitis B patients were seeded into 96-well plates and incubated with candidate epitope peptides corresponding to the patient's HLA-C genotype for 6 hours. Then, IFN-γICS was used to detect CD3 + / CD8 + The proportion of IFN-γ secreting cells in the T cell population. (A) CD3+ / CD8 + Flow cytometric analysis of IFN-γ secretion in T cell populations after stimulation with specific epitope peptides; (B) CD3 + / CD8 + Statistical graph of the proportion of T cells that secrete IFN-γ after stimulation with specific epitope peptides. Figure 1: Epitope peptide 1 (SEQ ID NO. 1); Figure 2: Epitope peptide 2 (SEQ ID NO. 2); Figure 3: Epitope peptide 3 (SEQ ID NO. 3); Figure 4: Epitope peptide 4 (SEQ ID NO. 4).

[0041] Figure 2 Peptide competition binding assays were performed to analyze the binding affinities of four hepatitis B T cell epitopes to specific HLA-C molecules. NC, negative control; Peptide 1: Epitope 1 peptide (SEQ ID NO. 1); Peptide 2: Epitope 2 peptide (SEQ ID NO. 2); Peptide 3: Epitope 3 peptide (SEQ ID NO. 3); Peptide 4: Epitope 4 peptide (SEQ ID NO. 4). The blue and red solid lines represent the peak fluorescence intensity of cells at 5 μM and 15 μM concentrations of the test peptide, respectively, while the gray filled line represents the maximum fluorescence intensity of cells in the presence of the GFP-reference peptide alone. The dark blue solid line in the NC plot represents the negative control background fluorescence of cells in the no-peptide well.

[0042] Figure 3 Provides a technical roadmap for epitope peptide prediction and screening, immunogenicity validation, and peptide competition binding experiments.

[0043] Figure 4 Epitope peptides induced specific CD8 in C57BL / 6J mice + T cell response

[0044] A mixed polypeptide vaccine was prepared with four positive epitope peptides and used in combination with the adjuvant poly(I:C). C57BL / 6J mice were immunized on days 0, 7, and 21, respectively. Seven days after the last immunization, spleen cells were obtained from the mice and restimulated with PBS and each positive epitope peptide for 10 h. BFA / Monesion (to block cytokine release) was then added and cultured for another 6 h. IFN-γ intracellular staining and flow cytometry analysis were then performed to detect IFN-γ. + / CD3 + / CD8 + T cell frequency. Figure 4 A is a representative flow cytometry analysis; B is the IFN-γ + / CD3 + / CD8 + T cell frequency statistics. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0046] This example discloses HLA-C0701, C0702, C0303, C1202, and C0602 molecule-restricted HBV-specific antigen peptides, the sequences of which were screened and identified by the following steps:

[0047] 1. Online virtual prediction of dominant T cell epitope peptides of 2 HBV antigens restricted by 14 HLA-C molecules

[0048] Two HBV proteins were selected: surface antigen (HBsAg, sequence is SEQ ID NO.7) and e antigen (HBeAg, sequence is SEQ ID NO.8, including the sequence of HBcAg). Their amino acid sequences were obtained by searching the UniProt global protein resource database, and the HBV C type HBV protein sequence with the most infection in China was selected. Then, six commonly used epitope peptide prediction databases, including SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, and EPIJEN, were used to virtually predict the T cell epitope peptides for each of the above HBV proteins restricted by HLA-C0102, C0602, C0702, C0801, C0304, C0302, C0303, C0401, C1402, C1202, C1502, C1403, C1203, and C0701 molecules. The SVMHC database includes MHCPEP The IEDB database includes two prediction algorithms: ANN, SMM, and ARB. In this example, the ANN and SMM methods with better statistical significance were selected. The websites of these epitope peptide prediction databases are shown in Table 1.

[0049] The antigen-binding groove of HLA class I molecules is closed at both ends and accepts antigenic peptides with a length of 8-11 amino acid residues, with 9 and 10 amino acids being the most common. Therefore, in this example, peptides of 9 and 10 amino acids in length were selected as the research objects. The amino acid sequence of each HBV protein was entered into the corresponding amino acid sequence input box of the prediction database website. Then, epitope peptides of 9 and 10 amino acids in length were selected, respectively. Specific HLA-C molecules were then selected to perform online virtual prediction of T cell epitope peptides of HBV antigens.

[0050] Table 1 Epitope peptide prediction database website

[0051]

[0052] For each HLA-C molecule and each HBV protein, the nine and ten peptides predicted from different databases were ranked from high to low by score. Epitope peptides that met the scoring criteria of at least two prediction methods were selected as candidate epitope peptides. For each HLA-C molecule and each HBV protein, 1-4 peptides with the highest scores (highest affinity) were selected from the candidate epitope peptides as epitope peptides to be identified. A total of 38 dominant T cell epitope peptides were screened and predicted for identification.

[0053] 2. Isolation of PBMC from peripheral blood of patients with chronic HBV infection

[0054] Fresh anticoagulated whole blood stored at room temperature was diluted appropriately with sterile PBS. One volume of human lymphocyte separation medium (Dakoway Biotech, Shenzhen) was added to a 15 mL centrifuge tube. The diluted blood was slowly layered on the separation medium and centrifuged at 2500 rpm for 20 min at room temperature. The mononuclear cell (PBMC) layer was aspirated and washed twice by centrifugation. The cells were resuspended in serum-free medium (Dakoway Biotech, Shenzhen) and counted.

[0055] 3. Identification of immunogenicity of HBV antigen T cell epitope peptides using intracellular cytokine staining Figure 3 As shown:

[0056] 200 μL of peripheral blood was taken from fresh anticoagulated blood samples of hepatitis B patients for HLA-C allele typing, and the remaining blood samples were used to isolate and prepare PBMCs. The cells were seeded in 96-well U-shaped cell culture plates at 5×10 5 cells, 200 μL / well; according to the HLA-C typing results, the candidate epitope peptides predicted to bind to the HLA-C molecule were added to a 96-well plate (2 μg / well, final concentration 80 μg / mL) and co-cultured with PBMCs. At the same time, PHA control wells (PHA final concentration 10 μg / mL) and peptide-free wells were set up, and 10% FBS-RPMI 1640 medium was added to 200 μL / well. The cells were placed in a cell culture incubator and incubated for 6 hours. Brefeldin ASolution / Monensin Solution (1000×) blocking agent was added for co-culture, and then IFN-γ intracellular fluorescence staining was performed, and flow cytometry analysis was performed to calculate CD3 + / CD8 + IFN-γ in T cell populations + Compared with the wells without peptide, the frequency of IFN-γ + / CD8 +The candidate epitope peptides whose T cell frequency increased by more than 2 times were defined as positive epitope peptides, that is, the epitope peptides could stimulate the memory CD8 + T cells are activated and secrete cytokines.

[0057] Through IFN-γICS experiments, PBMCs from 100 HBV-infected patients were collected and co-cultured with relevant peptides from 38 candidate epitope peptides. Among them, 4 candidate epitope peptides (sequences were LTFGRETVL; AYRPPNAPIL; ASRELVVSY; MMWYWGPSL) induced strong CD8 + T cell response, PBMCs of HBV-infected patients produce IFN-γ + / CD8 + T cell frequency increased more than 10-fold or IFN-γ was observed in two patients with HBV infection. + / CD8 + The T cell frequency increased by more than 3 times, so the four candidate epitope peptides were determined to be positive epitopes and dominant epitope peptides with strong immunogenicity ( Figure 1 ).

[0058] 4. HLA-C allele typing

[0059] Chronic HBV infection patients who demonstrated CTL-positive responses in epitope peptide identification were selected. 200 μL of anticoagulated blood was collected, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Biotechnology, Beijing). PCR was performed using HLA-C locus-specific primers CF and CR to amplify the DNA sequence of exon 2, intron 2, exon 3, and portions of introns 1 and 3 within the C locus. The product size was 985 bp. Amplification conditions were as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s; 62°C annealing for 15 s; 72°C extension for 90 s; 35 cycles; and 72°C extension for 5 min. The amplified product was size-confirmed by 1% agarose gel electrophoresis and sent to Shanghai Sangon Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novozymes Biotechnology Co., Ltd. (see Table 2).

[0060] Table 2 HLA-C locus-specific PCR amplification primers

[0061]

[0062] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA-C contig using the Lasergene Seqman software. The bidirectional sequencing was carefully checked for base integrity and consistency. Heterozygous bases were identified and replaced with degenerate bases (e.g., M represents A and C, R represents A and G, W represents A and T, S represents C and G, Y represents C and T, and K represents G and T). The resulting sequence fragments were then identified as the amplified HLA-C alleles. The Nucleotide BLAST tool was then used to align the assembled HLA-C base sequence with the exon 2 and exon 3 sequences of all HLA-C alleles in the database until a perfect match was obtained, thereby confirming the HLA-C allele.

[0063] 5. Peptide competition binding assay for HLA-C molecules

[0064] 38 candidate epitopes, including the four positive epitope peptides No. 1, No. 2, No. 3, and No. 4 screened in the previous step and verified by the peptide-PBMCs costimulation experiment, were assigned to the relevant epitope peptide queues of each HLA-C molecule based on the predicted HLA-C restriction and the HLA-C allele type of the patient with a positive T cell response. As test peptides, peptide competition binding experiments were performed with the fluorescent reference peptide of the HLA-C molecule. Finally, these four positive peptides all caused the cell fluorescence peak to shift to the left ( Figure 2 ), suggesting that these positive epitope peptides can compete with the fluorescent reference peptides for binding to the relevant HLA-C molecules on the HMy2.CIR cell membrane. These four positive epitope peptides all showed high or medium affinity binding to the corresponding HLA-C molecules (Table 3).

[0065] Table 3 Peptide competition binding assay analysis of the binding affinity of four HBV positive T cell epitopes to nine dominant HLA-C molecules

[0066]

[0067] 6. Immunization of C57BL / 6J mice with epitope peptides to verify their immunogenicity in inducing specific T cell responses in vivo

[0068] Four positive epitope peptides were prepared as a cocktail and then mixed with the adjuvant poly I:C. C57BL / 6J mice were randomly divided into two groups of three. The experimental group received the peptide cocktail / poly(I:C) (epitope peptide: 10 μg / single peptide, poly(I:C): 100 μg / mouse) on days 0, 7, and 21, respectively; the adjuvant group received the poly(I:C) (100 μg / mouse) on days 0, 7, and 21, respectively. Subcutaneous injections were administered at four sites (the nape of the neck, the base of the tail, and the bilateral groins).

[0069] On the 28th day, the mice were killed and spleen cell suspension was prepared routinely. The cell concentration was adjusted to 1×10 7 / mL, inoculated into 48-well cell culture plates (0.5mL / well), each mouse spleen cell inoculation well was divided into 5 groups, and PBS (negative control) and each positive epitope peptide (20μg / mL / peptide) were added respectively; after culturing for 10 hours, BFA and Monensin were added to each well and incubated for another 6 hours; then, cells in each well were collected, blocked with FcR Blocking Reagent Mouse for 15 minutes, and co-stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8a for 30 minutes; after washing, each tube was treated with a membrane permeabilization agent, and finally, APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30 minutes; after washing, CD3 was analyzed by flow cytometry. + / CD8 + / IFN-γ + T cell frequency, observing the specific CD8 induced by epitope peptide + The strength of the T cell response.

[0070] The results showed that the four positive epitope peptides in this example (peptide cocktail / polyI:C group) all induced strong epitope peptide-specific CD8 + T cell response. Compared with the adjuvant group, the IFN-γ induced by these four epitope peptides + / CD8 + The T cell frequency was 2-7 times that of the adjuvant group.

[0071] The above results show that the epitope peptides of this example can induce specific immune responses in mice and T cells are effectively activated. Therefore, the epitope peptides of this example can be used in the preparation of hepatitis B vaccine. + The activation of T cells reflects that the above-mentioned epitope peptides may also have certain preventive or therapeutic effects on tumors (such as those caused by hepatitis B).

[0072] It should also be noted that in practical applications, such as as hepatitis B drugs or vaccines, the epitope peptides of this embodiment may also be combined with other pharmaceutically acceptable excipients to further optimize the product's efficacy, such as excipients for maintaining drug or vaccine stability and for delivering specific targeting functions. These excipients can be directly obtained by those skilled in the art from existing technologies and will not be described in detail in this application.

[0073] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0074] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrative purposes. Without departing from the spirit and scope of the present invention, the present invention may be subject to various changes and improvements, which fall within the scope of the invention claimed.

[0075] The amino acid sequences of two hepatitis B virus antigens used to predict antigenic epitopes are as follows:

[0076] SEQ ID NO.7

[0077] HBsAg(Q913A6):

[0078] Molecular type of sequence: PRT

[0079] Scientifically named genus and species: Human hepatitis B virus

[0080]

[0081] SEQ ID NO.8

[0082] HBeAg (Q913A8):

[0083] Molecular type of sequence: PRT

[0084] Scientifically named genus and species: Human hepatitis B virus

[0085]

Claims

1. A thymus-dependent lymphocyte antigen epitope peptide of a hepatitis B virus antigen, characterized in that: The amino acid sequence of the antigen epitope peptide is any one or more of the following sequences: LTFGRETVL; AYRPPNAPIL; ASRELVVSY; MMWYWGPSL; The amino acid sequence above is an amino acid sequence after a single amino acid is removed or replaced.

2. Use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen according to claim 1 in the preparation of hepatitis B polypeptide vaccine or gene vaccine.

3. A nucleic acid molecule encoding the thymus-dependent lymphocyte antigen epitope peptide of the hepatitis B virus antigen according to claim 1. A vector comprising the nucleic acid molecule according to claim 3.

5. A vaccine for treating or preventing hepatitis B virus, characterized in that: Containing the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. Use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen according to claim 1 in the preparation of a drug for treating and preventing hepatitis B.

7. Use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen according to claim 1 in preparing a kit for detecting hepatitis B virus antigen-specific T cells.

8. The use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus according to claim 1 in a kit for preparing hepatitis B virus antigen-specific T cells according to claim 7, characterized in that: The kit comprises: an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, an enzyme-linked immunosorbent assay reagent, and a human leukocyte antigen polymer fluorescent staining or flow cytometric analysis reagent.

9. A monoclonal antibody, characterized in that Capable of specifically binding to the epitope peptide according to claim 1.

10. A drug for treating hepatitis B, characterized in that: Comprising the monoclonal antibody according to claim 9.

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