A universal detection technology for antigen-specific thymus-dependent lymphocytes in a wide range of populations in East Asia

Abstract

The present invention discloses a technical scheme for detecting antigen-specific thymus-dependent lymphocytes, which can universally detect the specific thymus-dependent lymphocyte function of specific antigens in a wide range of people in Northeast Asia and Southeast Asia; the broad-spectrum T cell epitope peptide library of specific antigens used in the technical scheme comprises specific antigen T cell epitope peptides presented by 13 HLA-A molecules, 25 HLA-B molecules and 14 HLA-C molecules with allele frequencies greater than 1% in the Northeast Asian population; the total gene frequencies of these dominant HLA-A, B and C molecules account for about 95.5%, 94% and 78% of the Chinese population, respectively, and the total gene frequencies in the Northeast Asian and Southeast Asian populations are similar to those in the Chinese population; therefore, the technical scheme can be applied to the detection of the vast majority of individuals in the region, can meet the needs of clinical laboratories, and will provide accurate immunological indicators for clinical efficacy evaluation, prognosis and recurrence judgment, guidance of subsequent treatment plans, and research on the immune pathogenesis of virus-infected patients or cancer patients.

Description

Technical Field

[0001] The present invention belongs to the field of medical immunology and immunological testing technology, and specifically relates to a universal technical solution and application thereof for detecting antigen-specific thymus-dependent lymphocytes in a wide range of people in Northeast Asia and Southeast Asia including China. Background Art

[0002] 1. Antiviral infection and anti-tumor effects of antigen-specific T cell immunity and the clinical value of its detection:

[0003] 1.1 Antigen-specific T cell responses affect the disease progression of viral infection and tumors:

[0004] Modern immunology believes that antigen-specific T cells are the core cells that mediate adaptive immune responses. They play a major role in anti-viral infection and anti-tumor, and work synergistically with antibody-mediated humoral immunity to form the body's main anti-viral and anti-tumor immune mechanisms. The T cell receptor (TCR) on the T cell membrane can specifically recognize and bind to the complex of HLA molecules and antigen peptides on the surface of antigen-presenting cells or target cells, that is, the antigen peptide / HLA complex molecule. Virus-infected cells or tumor cells process intracellular viral antigens or tumor antigens into T cell epitope peptides, which are presented to the membrane surface after binding to HLA molecules. CD8 + After activation, T cells differentiate into cytotoxic T cells (CTLs), which specifically bind to pHLA on the surface of virus-infected cells or tumor cells and are the main effector cells for targeted killing of tumor cells; CD4 + After activation, T cells differentiate into effector Th cells, secrete a large number of pro-inflammatory cytokines, and assist CD8 + T cell or B cell activation. For example, in hepatitis B virus infection, the strength of the body's T cell immune response to hepatitis B virus antigens determines the outcome of HBV infection. Patients with strong T cell immune responses can clear the virus, while those with weak immune responses lead to chronic infection, and HBV-specific T cells gradually decrease and become functionally exhausted, eventually developing into cirrhosis and liver cancer. In acute infection with the new coronavirus, CD4 + T cells and CD8 + The number of T cells decreased significantly, but they were highly activated and cytotoxic, and the number of regulatory T cells decreased. In the acute phase of infection, specific T cells showed a highly activated cytotoxic phenotype, while in the recovery phase, mild cases and asymptomatic cases were multifunctional and had a stem cell-like memory phenotype. The detection rate of specific T cells was higher than that of antibodies. In hepatocellular carcinoma, the patient's spontaneous tumor antigen-specific T cell immune response, especially CD8 +T cell responses change dynamically at different stages of the disease and have important clinical pathological and prognostic significance. For example, it has been recently reported that in the process from early to late stages of liver cancer, the responses of specific T cells such as MAGE-A1, MAGE-A3, NY-ESO-1, SSX2 and SALL4 gradually decrease, which may play an immune protective role in the early stages of liver cancer; while the AFP-specific T cell response is weak in the early stages, but gradually increases in the late stages, which may be a sign of the malignant state of late liver cancer. Therefore, detecting the functional changes of tumor antigen-specific T cells can directly observe the treatment and judge the prognosis in real time.

[0005] 1.2 Antigen-specific T cells affect the effect of clinical treatment, prognosis and recurrence:

[0006] Many clinical treatments for viral infections and tumors can affect and change the intensity of antigen-specific T cell responses in individuals, affecting the final efficacy, especially immunotherapy. The dynamic changes in antigen-specific T cell responses or immune function are significantly correlated with prognosis and recurrence. For example, in hepatitis B patients, nucleoside drugs can only temporarily enhance the HBV antigen-specific CD4 + T cells and CD8 + T cell response. HBV-specific T cells play a vital role in the clearance of residual HBV in the body after drug withdrawal, and are a key influencing factor and candidate predictive indicator for relapse after antiviral treatment withdrawal; PEG-IFN-a has the dual effects of antiviral and immunomodulatory, and can partially correct the functional exhaustion of specific T cells in chronic patients; and the treatment of co-stimulatory molecules OX40 and CD28 and immune checkpoint inhibitors Anti-PD-1\CTLA-4 can significantly increase the reactivity of hepatitis B virus-specific T cells. In hepatocellular carcinoma, after many immunotherapies such as immune checkpoint inhibitors such as Anti-PD-1 / PD-L1 / CTLA-4, peptide vaccines such as MRP3\GPC3\AFP, DC modified with antigen peptides or adoptive T cells, tumor antigen-specific CD8 + The degree of enhancement of T cell activity is significantly positively correlated with the patient's recurrence-free, progression-free survival, and even the extension of overall survival and the reduction of recurrence rate. After traditional treatments such as surgical resection, radiofrequency ablation or microwave ablation, transcatheter arterial chemoembolization or tyrosine kinase inhibitors, tumor tissues and cells are destroyed, and tumor antigens are continuously released to stimulate specific T cell responses, which are enhanced in stages or newly occur. The degree of activity is also positively correlated with the patient's survival and the reduction of recurrence rate. In addition, the spontaneous T cell immune response specific to tumor antigens in liver cancer patients, especially CD8 +T cell responses change dynamically at different stages of the disease and have important clinical pathological and prognostic significance. For example, it has been recently reported that the responses of specific T cells such as MAGE-A1, MAGE-A3, NY-ESO-1, SSX2 and SALL4 gradually decrease from early to late stages of liver cancer, which may play an immune protective role in the early stages of liver cancer; while the response of AFP-specific T cells is weak in the early stages but gradually increases in the late stages, which may be a sign of the malignant state of liver cancer in the late stages.

[0007] 1.3 Antigen-specific T cell detection has very important clinical significance:

[0008] In summary, the detection of antigen-specific T cells has important clinical value in the immediate and direct evaluation of the immunotherapy effects of viral infection and tumors, predicting the prognosis and recurrence of other traditional therapies, and studying the mechanisms of virus-body and tumor-body immune interactions.

[0009] 2. Difficulties, current status and shortcomings of antigen-specific T cell detection technology:

[0010] Although some laboratories can detect changes in the frequency of functional subtypes such as activated / proliferating T cells, regulatory T cells, and functionally exhausted T cells in the patient's overall T cell pool, they cannot monitor changes in the individualized antigen-specific T cell immune function of a broad patient population, because there are no universal detection technologies and reagents suitable for a broad patient population at home and abroad, and there are no reagents and reference values ​​that conform to the HLA polymorphism of the Chinese population. This seriously hinders clinical physicians' immediate and direct evaluation of the effectiveness of immunotherapy, as well as the early prediction of the prognosis and recurrence of traditional treatments and the study of the immune interaction mechanisms between virus-human and tumor-human.

[0011] Table 1 Difficulties, current status and shortcomings of antigen-specific T cell detection technology

[0012]

[0013]

[0014] As shown in the table above, compared with the quantitative detection of antigens and antibodies, the detection of antigen-specific T cells is much more difficult, mainly limited by two factors. Currently, there are two types of antigen-specific T cell detection technologies that are widely used: one is pMHC polymer fluorescence staining, which is the most specific; the other is using CD8 + T cell epitope peptide (9-10aa) or CD4 +T cell epitope peptides (16-19aa) are co-cultured with patient lymphocytes, and then the cytokine secretion is detected by enzyme-linked immunosorbent assay (ELISpot / FluoroSpot) or intracellular cytokine fluorescence staining (ICS), which are the most widely used methods. The latest methods include single-cell transcriptome sequencing and TCR gene sequencing. However, these evaluation methods have obvious shortcomings:

[0015] 2.1 The high polymorphism of HLA in the population seriously restricts the universality of the detection method:

[0016] The MHC gene system encodes human major histocompatibility antigens (HLA), which are responsible for presenting antigenic peptides (T cell epitope peptides) to T cells and initiating the body's adaptive immune response. Among them, HLA-A, B, C and other class I molecules present antigenic peptides to activate CD8 + T cells differentiate into cytotoxic T cells (CTL), which are the main effector cells for targeted killing of tumor cells; HLA-DR, DQ, DP and other class II molecules present antigenic peptides to activate CD4 + T cells differentiate into effector Th cells, secrete a large number of pro-inflammatory cytokines, and assist CD8 + T cell or B cell activation. However, the HLA (human leukocyte antigen) gene system is a group of tightly linked genes. Each HLA gene locus has a large number of alleles in the population. These alleles are not exactly the same between different individuals and are highly polymorphic in the population. Due to differences in molecular structure, there are many differences in the antigen peptide sequences that each HLA molecule can bind to and present, and the T cell immune response caused is also different in strength. This is also one of the main reasons for the individual differences in the body's anti-infection and anti-tumor abilities, that is, the individual differences in HLA genes. The distribution frequency of dominant HLA genes varies more between different ethnic groups, so there are ethnic differences in the characteristics of immune responses to specific antigens.

[0017] Although there are many studies on viral antigens and tumor antigens, there are few studies on T cell epitopes (antigenic peptides) presented by various HLA molecules in these antigens. The reported epitope peptides cannot cover the HLA polymorphism of people in a specific region, so it is impossible to prepare universal detection reagents for methods such as ELISpot, FluoroSpot or ICS. For example, in the past 33 years, only 205 CD8 T cell epitopes of hepatitis B virus antigen have been confirmed by research. + T cell epitopes and 79 CD4 +T cell epitopes are mainly presented by a few HLA molecules (HLA-A0201, A2402, B0702, DR04 and DR12). Although there are many tumor antigens related to hepatocellular carcinoma, such as tumor-associated antigens AFP, GPC3, GP73, hTERT, MPR3, SART2, SART3, SALL4, etc.; cancer-testis antigens MAGE-A1, MAGE-A3, NY-ESO-1, SSX2, etc. and tumor neoantigens, there are very few experimentally verified liver cancer antigen T cell epitope peptides, such as CD8 + Among T cell epitopes, AFP has only 22, hTERT has 16, GPC3 has 11, and the others have even fewer, and are mainly presented by a few HLA-A molecules (HLA-A0201, A2402 or A1101).

[0018] 2.2 The abundance of T cell epitope peptides in antigens seriously restricts the standardization of detection methods:

[0019] There are many types of epitope peptides that stimulate T cell responses in viral antigens and tumor antigens, and the number cannot be determined. Therefore, it is impossible to establish a completely standard antigen peptide library to detect the patient's individualized specific T cell clone library. At present, most researchers can only use a few known epitope peptides to detect a small number of patients carrying specific HLA molecules, which cannot fully reflect the functional status of the patient's tumor antigen-specific T cell clone library. Some researchers use overlapping peptide libraries covering the full length of tumor antigens or computer-simulated predicted peptide libraries to detect a wide range of liver cancer patients. Overlapping peptides are generally designed as overlapping long peptides (15-19aa / peptide) with an interval of 5-8 amino acids, and most of the predicted peptides are CD8 + Short peptides (9-10aa / peptide) of T cell epitopes. However, overlapping peptide libraries or predicted peptide libraries have serious defects: ① They are not true epitope peptides verified by experiments, and most of them are false epitopes. Recently, it was reported that among the 499 predicted epitope peptides of liver cancer antigens, only 29 (5.81%) predicted peptides detected positive T cell responses in 46 HCC patients; among the 5,600 predicted epitope peptides of the new coronavirus, only 523 (9.34%) were positive CD8 in 99 patients. + T cell response; of the 1925 overlapping peptides (15aa / peptide) covering the entire genome of the new coronavirus, only 280 (14.55%) overlapping peptides were positive for CD4 in 99 patients +T cell response: Among the 82 overlapping peptides (15aa / peptide) covering the full length of the N protein of the new coronavirus, only 11 overlapping peptides (13.41%) showed positive T cell response in 7 infected people. Therefore, the number of pseudoepitope peptides in the overlapping peptide library and the predicted peptide library should be far greater than the number of true epitopes. ② The HLA restriction of these overlapping peptides or predicted peptides is unclear, and it is unknown which HLA molecules can be cross-bound. Therefore, it is blind and helpless to use overlapping peptide libraries or predicted peptide libraries to test a wide range of patients.

[0020] 2.3pMHC polymer staining and new genomics technologies are difficult to promote clinically:

[0021] The pMHC polymer staining method is the most specific, because the specificity of T cells is reflected in the specific binding of TCR and pMHC complex, but the preparation technology is difficult, the cost is high, and the stability period is short. It is difficult to prepare hundreds of pMHC polymer libraries covering many different HLA molecules and many T cell epitope peptides to detect a wide range of clinical patients. Single-cell expression profiling sequencing and TCR gene sequencing have been used to analyze the clonal characteristics of antigen-specific T cell libraries in vivo, but they are difficult to popularize in terms of both cost and technology, and the analysis of the results and the interpretation of clinical significance are difficult to be specific and simplified.

[0022] Due to the above limitations, the current detection system is neither applicable to a wide range of patients nor able to detect many specific T cell clones, which has long been a technical bottleneck and problem. Summary of the invention

[0023] To solve the above problems, the present invention discloses a universal detection technology for viral antigen or tumor antigen-specific T cells applicable to a wide range of people in East Asia.

[0024] To achieve the above object, the technical solution of the present invention is as follows:

[0025] One object of the present invention is to provide a universal detection technology scheme for antigen-specific thymus-dependent lymphocytes applicable to a wide range of people in East Asia, which specifically comprises the following steps:

[0026] (1) Screening and verification of a series of antigenic peptides presented by all HLA-A, B, and C molecules with an allele frequency greater than 1% in the East Asian population against specific viral antigens or tumor antigens: using multiple online epitope prediction databases to virtually predict candidate epitope peptides presented by 13 HLA-A molecules, 25 HLA-B molecules, and 14 HLA-C molecules with an allele frequency greater than 1% against hepatitis B virus antigens (HBsAg, HBeAg, HBpol, HBx), novel coronavirus antigens (S, E, N, M, and RdRp), or liver cancer-related tumor antigens (AFP, GPC3, SART2 / 3, SSX2, GP73, and hTERT, etc.). Then, lymphocyte functional experiments, peptide-cell competition binding experiments on HLA molecules on cell lines, molecular docking, and molecular dynamics simulations were performed using the patient's peripheral blood mononuclear cells to verify the immunogenicity of the candidate epitope peptides one by one, and to establish a positive epitope peptide library presented by each dominant HLA molecule;

[0027] The 13 HLA-A molecules (whose total gene frequency in the Chinese population is greater than 95%) are as follows: HLA-A1101, A2402, A0201, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601, etc.

[0028] The 25 HLA-B molecules (whose total gene frequency in the Chinese population is greater than 94%) are as follows: HLA-B4001, B4601, B5801, B5101, B1302, B1501, B1301, B4006, B1502, B3802, B3501, B5401, B5502, B3901, B5201, B4002, B4403, B4801, B0702, B1511, B5102, B2704, B5701, B1527, B3701, etc.

[0029] The 14 HLA-C molecules (whose total gene frequency in the Chinese population is greater than 78%) are as follows: HLA-Cw0702, Cw0102, Cw0304, Cw0901, Cw0602, Cw0303, Cw0302, Cw0401, Cw1402, Cw1502, Cw0701, Cw1202, Cw1403, Cw1203, etc. The w is added after C to distinguish it from complement.

[0030] (2) Establish a broad-spectrum T cell epitope peptide library to achieve universal detection. Dominant T cell epitope peptides have multiple meanings: strong immunogenicity, can be cross-bound by multiple dominant HLA molecules, and its specific T cell clones can be detected in most patients. For each viral antigen or tumor antigen, 100-200 epitope peptides are screened according to the length of the sequence to form a broad-spectrum T cell epitope peptide library. These broad-spectrum T cell epitope peptides are epitope peptides presented by the above 52 HLA molecules, and each HLA-A / B / C molecule is cross-bound to 10-20 epitope peptides. Then, each patient's 6 HLA-A / B / C molecules can cross-bind to 60-120 epitope peptides (an average of about 90). For 4 hepatitis B viruses, 5 new coronavirus antigens or 6 tumor antigens, an average of 240-480, 300-600 or 360-720 specific CD8 + T cell clones. This broad-spectrum epitope peptide library not only conforms to the HLA polymorphism of the Chinese and East Asian populations, but also fully reflects the richness of T cell epitopes in antigen molecules. Therefore, it can achieve universal detection and fully reflect the immune function status of the antigen-specific T cell clone library in individual patients.

[0031] (3) Purchase commercial reagents, use the peptide pool array of the broad-spectrum T cell epitope peptide library of the above-mentioned specific antigens, co-culture with patient PBMCs in a microwell reaction plate, establish an enzyme-linked immunosorbent spot (ELISpot) or fluorescent immunospot (FluroSpot) method, and quantitatively detect the number of cells secreting IFN-γ.

[0032] Furthermore, in step (3) of the present invention, the enzyme-linked immunosorbent assay or fluorescent immunospot assay can also be replaced by a magnetic microsphere chemiluminescence assay: purchasing streptavidin-labeled magnetic microspheres, biotinylated IFN-γ capture, enzyme-labeled IFN-γ detection antibody, chemiluminescence substrate, IFN-γ standard and other reagent materials, using the peptide pool array of the broad-spectrum T cell epitope peptide library of the specific antigen in step (2) above, co-culture with the patient's PBMCs in a 96-well cell culture plate, and collecting the culture supernatant of each well as the sample to be tested. A magnetic microsphere chemiluminescence assay is established in another microwell reaction plate to detect the level of IFN-γ in the culture supernatant.

[0033] Furthermore, in step (3) of the present invention, the enzyme-linked immunosorbent assay or fluorescent immunospot assay can also be replaced by an enzyme-linked immunosorbent assay (ELISA): purchasing reagent materials such as IFN-γ capture antibody, biotinylated IFN-γ detection antibody, streptavidin-labeled horseradish peroxidase or alkaline phosphatase, enzyme substrate and IFN-γ standard, and using the peptide pool array of the broad-spectrum T cell epitope peptide library of the specific antigen in step (2) above, co-culture with the patient's PBMCs in a 96-well cell culture plate, and collect the culture supernatant of each well as the sample to be tested. Establish an ELISA method in another microwell reaction plate to detect the level of IFN-γ in the culture supernatant.

[0034] Furthermore, in step (3) of the present invention, the enzyme-linked immunosorbent assay can be replaced by a cytometric beads array (CBA) combined with T cell multicolor fluorescent staining technology to analyze the functional subtypes of antigen-specific T cells: a set of reagents for detecting multiple molecules is assembled using commercial CBA single reagents. The peptide pool array of the specific antigen in step (2) above is co-cultured with the patient's PBMCs in a 96-well cell culture plate for 20 hours, and then the supernatant is taken, and the secretion levels of multiple soluble molecules secreted by the patient's PBMCs are detected using CBA reagents and flow cytometric analysis technology; at the same time, cells are taken for multi-combination multicolor fluorescent monoclonal antibody staining and flow analysis to quantitatively analyze the frequencies of multiple functional subpopulations of antigen-specific T cells.

[0035] Further, the soluble molecules secreted by the patient's PBMCs are any one or more of human interferon-γ, human tumor necrosis factor, human interleukin-17, granzyme A, granzyme B, perforin, tumor necrosis factor-α and tumor necrosis factor-β.

[0036] The technical solution of the invention can be used to universally detect the immune function status of antigen-specific T cells in patients infected with hepatitis B virus, patients infected with new coronavirus or patients with hepatocellular carcinoma. The test results will provide accurate immunological indicators for clinical efficacy evaluation, prognosis and recurrence judgment, guidance of subsequent treatment plans, and research on the body's immune pathogenesis, providing a powerful technical support for precision immunotherapy.

[0037] Another object of the present invention is to provide an application of the above-mentioned antigen-specific thymus-dependent lymphocyte detection technology scheme suitable for a wide range of people in East Asia in the preparation of specific T cell detection reagents or kits for detecting hepatitis B virus antigens, new coronavirus antigens or liver cancer-related tumor antigens.

[0038] The technical solution of the invention can be used to prepare specific T cell detection reagents or kits for detecting hepatitis B virus antigens, new coronavirus antigens or liver cancer-related tumor antigens.

[0039] Preferably, the reagent or kit is an enzyme-linked immunosorbent assay reagent or kit, a fluorescent immunospot assay reagent or kit, an enzyme-linked immunosorbent assay (ELISA) reagent or kit, a chemiluminescence assay reagent or kit, a Cytometric beads array reagent or kit, or a T cell membrane fluorescent staining flow cytometry reagent or kit.

[0040] The beneficial effects of the present invention are:

[0041] 1. Applicable to the vast majority of patients in the region, and universal testing is available:

[0042] The broad-spectrum T cell epitope peptide library used in this technical solution is suitable for detecting patients carrying the above 52 HLA alleles. The total gene frequencies of these 13 HLA-A molecules, 25 HLA-B molecules, and 14 HLA-C molecules with gene frequencies greater than 1% in the Chinese population are greater than 95%, 94%, and 78%, respectively. The total gene frequencies in the Northeast Asian population are also similar to those in the Chinese population, and are about 75-90% in the Southeast Asian population. That is, these 52 dominant HLA molecules can cover the vast majority of Northeast Asian and Southeast Asian populations, including China. The 2 A, 2 B, and 2 C molecules inherited from each patient's parents are very likely to belong to the above 52 HLA molecules. Therefore, the T cell epitope peptide library presented by these 52 dominant HLA molecules is universal and can be used for detection between different individuals in a wide range of populations in the region, without the need to screen patients with specific HLA molecules through HLA allele typing.

[0043] 2. The broad-spectrum T cell epitope peptide library can detect numerous specific T cell clones, which can fully reflect the immune function status of the specific T cell clone library for a specific antigen in the individual patient:

[0044] For each antigen, 100-200 epitope peptides are screened according to the length of the sequence to form a spectrum of T cell epitope peptide library. These broad-spectrum T cell epitope peptides are presented by the above 52 HLA molecules, and each HLA-A / B / C molecule is cross-bound to 10-20 epitope peptides. The 6 HLA-A / B / C molecules of each patient can cross-bind to 60-120 epitope peptides (an average of about 90). For 4 hepatitis B viruses, 5 new coronavirus antigens or 6 tumor antigens, each patient can detect an average of 240-480, 300-600 or 360-720 specific CD8 + T cell clones. This broad-spectrum epitope peptide library not only matches the polymorphism of HLA in East Asian populations, but also fully reflects the richness of T cell epitopes in antigen molecules. Therefore, it can achieve universal detection and fully reflect the immune function status of the antigen-specific T cell clone library in individual patients.

[0045] 3. Use a broad-spectrum epitope peptide library composed of experimentally verified T cell epitope peptides for personalized testing, rather than an overlapping peptide library or a predicted peptide library. Currently, there is no universal detection technology or similar kit on the market.

[0046] At present, there are no universal antigen-specific T cell detection kits for viral or tumor antigens in the domestic and foreign markets, and there are no antigen-specific T cell detection kits and reference values ​​that meet the HLA polymorphism of the Chinese population. This technical solution enables each patient to personalize the immune function status of the antigen-specific T cell clone library in his or her body based on his or her specific HLA-A, B, C molecules and the T cell epitope peptides presented by these molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 ELSPOT spot image of a representative sample of a patient with chronic hepatitis B detected by the universal detection method for hepatitis B virus antigen-specific T cells prepared in Example 1 of the present invention;

[0048] Figure 2 ELSPOT spot image of representative specimens of healthy blood donors tested by the universal detection method for novel coronavirus antigen-specific T cells prepared in Example 2 of the present invention;

[0049] Figure 3 Representative ELISPOT spot diagram (A) and spot statistical chart (B) of liver cancer patients and healthy subjects detected by the universal detection method for liver cancer-related tumor antigen-specific T cells prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0050] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0051] Embodiment 1:

[0052] This technical solution is used to prepare a broad-spectrum T cell epitope peptide library of HBV antigens presented by HLA-A molecules for universal detection of HBV-specific T cells in a wide range of HBV patients in China.

[0053] 1) Virtual prediction of candidate T cell epitope libraries of HBV antigens presented by 13 dominant HLA-A molecules:

[0054] ① The full-length amino acid sequences of HBsAg, HBeAg (covering HBcAg), HBpol and HBx proteins of hepatitis B virus were obtained through databases such as UniProt / NCBI and COSMIC (as follows), and multiple sequence alignment was performed using the ClustalW method in the Molecular Evolutionary Genetics Analysis (MEGA7, 1.0.0.0 version) software, and then the alignment results were analyzed using the multiple sequence alignment and analysis software GeneDoc (2.7.0 version) to obtain the conserved regions of the sequences. The conservative nature of each amino acid was judged using 100%, 95%, and 80% as critical points.

[0055] ②Using the epitope prediction algorithms and tools listed in Table 2, virtually predict the epitope peptides presented by all 13 HLA-A with gene frequencies greater than 1% in the Chinese population. The epitope peptide length is set to 9-mer and 10-mer, and at least meets the evaluation criteria of more than 3 prediction algorithms. The top 5-10 peptides with the highest scores are selected as candidate epitope peptides to form a candidate epitope peptide library.

[0056] The amino acid sequences of the four HBV antigens are as follows:

[0057] Scientifically named biological genus and species: Virales family Hepaciviridae genus Hepacivirus species Hepatitis B virus type C

[0058] HBsAg (Protein_ID:P31868-1)

[0059]

[0060] HBeAg (Protein_ID: P0C6H5-1)

[0061]

[0062] HBpol (Protein_ID: P0C688-1)

[0063]

[0064]

[0065] HBxAg (Protein_ID: P0C686-1)

[0066] MAARVCCQLDPARDVLCLRPVGAESRGRPVSGPFGPLPSPSSSAVPADHGARLSLLRGLPV 60

[0067] CAFSSAGPCALRFTSARRMETTVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDW 120

[0068] EELGEEIRLMVFVLGGCRHKLVCSPAPCNFFTSA 154

[0069] 2) Using PBMCs from hepatitis B patients, the immunogenicity of candidate epitope peptides was verified by peptide-PBMCs co-stimulation experiment:

[0070] ①Peripheral blood of hepatitis B patients was collected from the Department of Infectious Diseases of Nanjing Second Hospital. PBMCs were routinely separated and frozen for future use, and some cells were taken for HLA-A allele typing.

[0071] Fresh anticoagulated whole blood stored at room temperature was taken and appropriately diluted with sterile PBS; 1 times the volume of the original blood was added to a 15 mL centrifuge tube with human lymphocyte separation solution (Dakoway Biotechnology, Shenzhen); the diluted blood was slowly spread on the separation solution and centrifuged at room temperature for 20 min at 2500 rpm; the mononuclear cell (PBMCs) layer was aspirated and washed twice by centrifugation; the cells were resuspended with serum-free medium (Dakoway Biotechnology, Shenzhen), and the cells were counted to adjust the cell concentration to 4×10 6 / mL for future use.

[0072] ②HLA-A allele typing

[0073] 200 μL of anticoagulated blood was taken, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Bio, Beijing); PCR was performed using HLA-A locus-specific primers A1 and A3 (Table 3) to amplify the DNA sequences of exon 2, intron 2, exon 3, and part of introns 1 and 3 of the A locus, with a product size of 985 bp. The amplification conditions were: 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; 72°C extension for 5 min. The amplified product was identified by 1% agarose gel electrophoresis and sent to Shanghai Sunny Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0074] Table 2 Epitope peptide prediction database website

[0075]

[0076]

[0077] Table 3 HLA-A locus-specific PCR amplification primers

[0078]

[0079] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA-A overlapping sequence (Contig) by using the Seqman software of the Lasergene program. The bases of the bidirectional sequencing were carefully checked to see if they were complete and consistent. The bases of the heterozygotes were found and replaced with annexed bases, such as M for A and C, R for A and G, W for A and T, S for C and G, Y for C and T, and K for G and T. Finally, the sequence fragments of the amplified HLA-A alleles were determined. The Nucleotide BLAST tool was used to compare the assembled HLA-A base sequence with the exon 2 and exon 3 sequences of all HLA-A alleles in the database until a completely matched gene combination was obtained, thereby determining the HLA-A alleles.

[0080] ③Identification of immunogenicity of HBV antigen T cell epitope peptides by IFN-γELISPOT method

[0081] First, HBV chronic infected persons who have a positive reaction to the mixed peptide group were screened: the epitope peptides to be identified for the four HBV proteins restricted by each HLA-A molecule were mixed into a group, with 8-9 epitope peptides in each group; the ELISPOT plate pre-coated with anti-human IFN-γ antibody (Dakoway Biotechnology, Shenzhen) was activated with serum-free culture medium (200 μL / well) for 8 minutes, and the PBMC suspension of HBV chronic infected persons was added to each well (100 μL / well); then, the mixed peptides restricted by each HLA-A molecule were added to the detection wells, with a single polypeptide in the mixed peptides at 1.5 μg / well, PHA (0.25 μg / well) was added to the positive control wells, and the negative control wells were added with the same peptide as the detection wells. The same concentration of DMSO peptide solution; incubate at 37°C, 5% CO2 incubator for 16-20h; lyse and wash cells according to the instructions of human IFN-γ ELISPOT kit; add biotin-labeled anti-human IFN-γ antibody working solution (100μL / well) to each well and incubate at 37°C for 1h; add enzyme-linked avidin working solution (100μL / well) after washing the plate, and continue to incubate at 37°C for 1h; add the freshly prepared AEC color development solution (100μL / well) after washing the plate, and color at room temperature in the dark for 20min; wash the plate with deionized water 4-5 times to stop color development; place the ELISPOT plate in the dark, dry it, and scan and count the spots with an enzyme-linked immunospot analyzer. If the number of spots in the negative well is 0-5, the number of spots in the test well-negative control well is ≧5, which is considered a positive T cell reaction; if the number of spots in the negative control well is ≧5, the number of spots in the test well ≧2 times the number of spots in the negative well is considered a positive T cell reaction.

[0082] Identify the immunogenicity of a single epitope peptide: recollect the peripheral blood PBMC of chronic HBV infected patients with positive CTL reaction to mixed peptides, add them to the above ELISPOT plate, add a single HBV peptide (3μg / well) in the positive mixed peptide to each test well, set up positive control wells and negative control wells as above, incubate in a 37℃, 5% CO2 incubator for 16-20h, and perform ELISPOT test as above. The reaction of the positive well indicates that the epitope peptide to be identified in the well is immunogenic and can stimulate the patient's T cell activation. Perform HLA-A allele typing on the HBV infected person, and combine the virtual affinity of the epitope peptide and the CTL response of the HLA-A gene homozygous patient to clarify which HLA-A molecule it is restricted and presented by.

[0083] 3) Use molecular docking and molecular dynamics simulation experiments to verify the cross-binding force between positive epitope peptides and various related HLA-A molecules:

[0084] The positive epitope peptides verified by the peptide-PBMCs co-stimulation experiment were classified. According to the predicted HLA molecule restriction and the actual HLA molecules of the patients in the verification experiment, the HLA molecules that each positive epitope peptide might bind to were listed and classified. Then, the positive epitope peptides were molecularly docked with the protein crystal structures of the relevant HLA molecules through the Glide 5.7 module in the Schrodinger Suite. The kinetic simulation of each pair of molecules was completed using the Desmond module. Finally, the binding free energy (kal / mol) was calculated based on the RMSD results to determine the affinity of each positive epitope peptide with the relevant HLA molecules and to clarify the cross-restriction of each epitope peptide with multiple HLA molecules (i.e., one epitope peptide can be bound and presented by multiple HLA molecules).

[0085] 4) Using the HMy2.CIR cell line that stably expresses specific HLA-A molecules, the peptide competition binding experiment of HLA molecules was carried out to verify the cross-binding ability of positive epitope peptides with various related HLA-A molecules:

[0086] ①HMy2.CIR is a human B lymphoblastoid cell line lacking HLA class I antigens. It does not express HLA-A and B molecules, but only expresses trace amounts of HLA-Cw4 molecules. This laboratory constructed 12 HMy2.CIR cell lines that stably express 12 dominant HLA-A molecules. The main steps are: extracting mRNA from PBMCs of healthy individuals, amplifying each HLA-A and cDNA, routinely constructing pcDNATM3.1 / myc-His(-)A recombinant plasmids, electroporating HMy2.CIR cell lines, selecting stable cell lines with G418, and then staining with HLA-ABC fluorescent monoclonal antibody W6 / 32. CIR cell lines that highly express the HLA molecule are sorted by flow cytometry for pure culture, and then sequenced and identified.

[0087] ② Classify the positive epitope peptides verified by the peptide-PBMCs costimulation experiment, and list and classify the positive epitope peptides that each HLA molecule may bind to as above. The HMy2.CIR cell line that stably expresses a specific HLA-A molecule was co-incubated with each positive epitope peptide associated with the HLA molecule and a reference peptide labeled with fluorescein to perform a competitive binding experiment: first, the peptides bound to the HLA molecules on the CIR cell membrane were eluted with an acid solution (0.131 mol / L citric acid and 0.061 mol / L disodium hydrogen phosphate, PH3.0), then neutralized with cell culture medium, and inoculated in a 96-well cell culture plate (1×10 5 cells / well / 100μL), added β2-microglobulin (1μg / mL), then added fluorescein-labeled reference peptide (200nM), and then added unlabeled positive epitope peptides (5μM, 15μM), respectively. After culturing for 24h, flow cytometry analysis was performed to determine the affinity of each positive epitope peptide to a specific HLA molecule through IC50 value and competitive inhibition percentage.

[0088] 5) Establish a broad-spectrum T cell epitope peptide library of four hepatitis B virus antigens and group the antigen peptides:

[0089] ① 215 patients with CTL positive response to HBV mixed peptides were screened out from 540 patients with chronic HBV infection by IFN-γELISPOT method. PBMCs of these patients were collected again, and the immunogenicity of single epitope peptides was verified by ELISPOT method. Then, the epitope peptides cross-bound by each dominant HLA-A molecule were comprehensively analyzed by combining the results of molecular docking, molecular dynamics simulation and HLA-peptide competition binding experiment. Finally, 103 dominant epitope peptides presented by 13 dominant HLA-A molecules were screened to form a broad-spectrum T cell peptide library of hepatitis B virus antigens. The number of epitope peptides that can cross-bind to each dominant HLA-A molecule is shown in Table 4.

[0090] ② Antigen peptide grouping: The above 103 synthetic hepatitis B virus antigens were dissolved in DMSO and divided into 8 groups (i.e. 8 antigen peptide libraries) according to the different charge and source antigen types. Each peptide library includes about 10 antigen peptides and is stored at -80°C for standby use to avoid repeated freezing and thawing. When testing each blood sample, each peptide library is added to a reaction well, 20μL, and each peptide is 2μg.

[0091] Table 4 HLA-restricted distribution of the broad-spectrum T cell epitope peptide library of HBV antigen

[0092]

[0093] 6) Prepare ELISPOT microwell reaction plate: Coat anti-human interferon-γ antibody in polyvinylidene fluoride PVDF membrane microwell reaction plate. The specific steps are all sterile in the clean bench:

[0094] ① Activate the strips: Take the required amount of PDVF membrane detachable strips (BD Bioscience), add 15 μL of 35% ethanol to each well, wet the wells for no more than 1 minute, then add 200 μL of sterile washing solution to each well, let it stand for 1 minute, shake off the liquid, repeat this washing process 5 times, and then place in a ventilated place to allow the ethanol to evaporate completely.

[0095] ② Plate coating: Add 100 μL of coating antibody (human IFN-γ coating antibody working solution; BD Bioscience) to each well, cover, and incubate in a humidified box at 4°C overnight (at least 12 hours).

[0096] ③ Wash the plate: On the second day, take out the strip plate from the wet box, shake off the coating solution in the wells, add 200 μL of sterile washing solution to each well, let it stand for 1 minute, shake off the liquid, repeat the washing process 5 times, dry it, put it in a pure aluminum foil vacuum bag (containing desiccant), evacuate it, seal the bag, and store it at 4°C for later use.

[0097] 7) Universal detection of HBV-specific T cells in a wide range of HBV patients:

[0098] ① Isolation of PBMC: peripheral anticoagulated blood samples were collected from the patients to be tested, and mononuclear cells (PBMCs) were isolated as above. The cells were counted and the cell concentration was adjusted to 2×10 using serum-free cell culture medium (Dakoway Biotechnology, Shenzhen). 6 / ml.

[0099] ② Add cell suspension: add PBMC from each patient's blood sample to 10 wells, 100 μL / well (i.e.: 4×10 5 cells / well), of which 8 wells were set as experimental wells, 1 well was set as negative well, and 1 well was set as positive control for PHA stimulation.

[0100] ③ Add antigen peptide: 10 wells were tested for each blood sample. No antigen peptide was added to the first well, and only 20 μL of negative well supplement solution (containing the same concentration of DMSO as other wells) was added to serve as the negative control well. Antigen peptide library 1, peptide library 2, peptide library 3, peptide library 4, peptide library 5, peptide library 6, peptide library 7, and peptide library 8 were added to wells 2 to 9, 20 μL / well, respectively, to serve as the test wells. PHA was added to the tenth well, which served as the positive control well.

[0101] ④ Incubation: Cover the strips and place in a 37°C, 5% CO2 cell culture incubator for 20 hours.

[0102] ⑤ Wash the plate: Take out the ELISPOT strip plate from the cell culture incubator, shake off the cell suspension, add 200 μL of deionized water to each well, place at 4°C for 10 minutes to lyse the cells, and discard the deionized water. Add 200 μL of washing solution (1×PBS) to each well, let it stand for 1 minute, shake off the liquid, and repeat the washing process 5 times. After shaking off the liquid for the last time, pat dry on absorbent paper.

[0103] ⑥ Add detection antibody: add 100 μL of biotin-IFN-γ detection antibody working solution (BD Bioscience) to each well and incubate at room temperature for 2 hours. Wash the plate as above: add 200 μL of washing solution (1×PBS) to each well, let it stand for 1 minute, shake off the liquid, repeat this 5 times, shake off the liquid for the last time and pat dry on absorbent paper.

[0104] ⑦ Add HRP-streptavidin: Add 100 μL HRP-streptavidin working solution (BD Bioscience) to each well and incubate at room temperature for 1 hour in the dark. Wash the plate 5 times as above, and pat dry on absorbent paper after shaking off the liquid for the last time.

[0105] ⑧ Color development: Add 100 μL AEC color development solution (prepared immediately before use) to each well and leave it at room temperature and away from light for 20 minutes. Then shake off the liquid, remove the plate base, and wash the front and back of the PVDF membrane and the base with deionized water 3-5 times to stop the color development. Place the strip plate in a dark place at room temperature and install the plate base after it is naturally dried.

[0106] ⑨ Spot counting: After drying, the strips are automatically photographed and the spots are counted using an ELISA spot analyzer. If the number of spots is small, the experimenter can also count the spots by himself under an upright optical microscope.

[0107] ⑩Result judgment:

[0108] The number of spots in each sample = (the sum of the number of spots in 8 experimental wells) - (the number of background spots in 8 experimental wells)

[0109] Note: When the actual number of spots in the experimental well is less than that in the negative control well, the actual number of spots is taken as the background number of spots in the well; when the actual number of spots in the well is equal to or more than that in the negative control well, the number of spots in the negative control well is taken as the background number of spots in the well.

[0110] 8) Analysis of personalized test results:

[0111] ①The number of spots obtained from each specimen represents 4×10 5 The number of reactive HBV-specific T cells in each PBMC population.

[0112] ② Single test results: When the number of spots is large, it indicates that the patient's HBV-specific T cell immune function is strong, indicating that the patient's HBV-specific T cells are activated, proliferated and have a strong ability to kill the virus and its infected liver cells after being stimulated by HBV. When the number of spots is small, it indicates that the patient's specific T cells have a weak ability to kill the virus and its infected liver cells.

[0113] ③ Dynamic test results: Dynamic analysis results are more meaningful. Repeat the test every 2-3 months to observe the dynamic changes in the number of spots in patients during treatment or at different stages of the disease. If the number of spots increases or decreases significantly, even within the mid-range of the reference value, it can still indicate that the patient's HBV-specific T cell immune function has increased or decreased significantly, and the clinic should analyze the possible causes and treatment measures.

[0114] The representative ELSPOT spot image of the specimens of chronic hepatitis B patients detected by the universal detection method of hepatitis B virus antigen-specific T cells prepared by the technical solution of the present invention is as follows: Figure 1 This figure is the result of step (7). Quantitative detection of HBV-specific T cells in PBMCs of chronic hepatitis B patients using a broad spectrum T cell epitope peptide library of 103 HBV antigens (IFN-γ ELISpot method). Negative wells and experimental wells: 4×10 5 PBMCs / well, PHA positive well: 2×10 5 PBMCs / well. The number of spots secreting IFN-γ was detected after 20 h of co-culture. The number of spots in each negative well and experimental well represents 4×10 5 The number of reactive specific T cells in PBMCs after being stimulated by HBV T cell epitope peptides. This result diagram shows that the PBMC specimens of different individuals with hepatitis B can be quantitatively detected by this technical solution to detect reactive hepatitis B virus antigen-specific T cells.

[0115] Embodiment 2:

[0116] This technical solution is used to prepare a broad-spectrum T cell epitope peptide library of the new coronavirus (SARS-CoV-2) antigen presented by HLA-A molecules for universal detection of new coronavirus-specific T cells in a wide range of patients in China

[0117] 1) Virtual prediction of candidate T cell epitope spectra of the new coronavirus presented by 13 dominant HLA-A molecules:

[0118] ① Obtain the amino acid sequences of five proteins, including E, N, M, S and RdRp, of various representative virus strains and variants of SARS-CoV-2 through databases such as UniProt / NCBI (as follows), and identify the sequence difference sites between them and coronaviruses such as SARS-CoV and HCoV-OC43, HCoV-HKU1, HCoV-NL63 and HCoV-229E.

[0119] ② For the above five protein sequences and their variants and differential regions, the T cell epitope prediction algorithm in Table 1 was used to virtually predict the epitope peptides presented by all 13 HLA-A molecules with a gene frequency greater than 1% in the Chinese population.

[0120] ③The length of HLA-A molecule restricted epitope peptides was set to 9-mer and 10-mer, meeting the scoring criteria of at least three prediction methods, and then the top 5-10 peptides were selected as candidate epitope peptides.

[0121] The amino acid sequences of the five novel coronavirus (SARS-CoV-2) antigens are as follows:

[0122] Scientifically named biological genus and species: Virales, Coronaviridae, Genus Coronavirus, Novel Coronavirus

[0123] E protein (envelope protein) (Protein_ID=YP_009724392.1):

[0124] MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLLCAYCCNIVNVSLVKPSFYVYS 60

[0125] RVKNLNSSRVPDLLV 75

[0126] M protein (membrane protein) (Protein_ID=YP_009724393.1):

[0127]

[0128] N protein (nucleocapsid protein) (Protein_ID=YP_009724397.2):

[0129]

[0130] S protein (spike protein) (Protein_ID=YP_009724390.1):

[0131]

[0132] RdRp protein (RNA-dependent RNA polymerase) (Protein-ID=YP_009725307.1):

[0133]

[0134]

[0135] 2) Verify the immunogenicity of candidate epitope peptides through a modified DC-peptide-PBL co-stimulation assay:

[0136] ① Isolation of PBMC from blood donors:

[0137] Leukocyte filter disks (discarded leukocyte filter disks used to separate 400 mL of peripheral anticoagulant blood from red blood cells) were collected from peripheral blood donors at Jiangsu Provincial Blood Center. After disinfection with alcohol, the inlet and outlet tubes were cut open, and 300 mL of PBS was pushed inward from the outlet of the filter disk with a syringe. The cell suspension was collected from the inlet and centrifuged at room temperature. The cell precipitate was appropriately diluted with PBS, PBMCs were routinely separated with human lymphocyte separation medium, and the cells were resuspended in serum-free 1640 cell culture medium for later use.

[0138] ② Induce DC and cryopreserve PBL:

[0139] PBMC from blood donors were inoculated into cell culture flasks and incubated at 37°C for about 4 hours. Non-adherent cells were collected and cultured overnight with 10% fetal bovine serum. PBL were frozen the next day for later use. Meanwhile, adherent cells were cultured for 7 days in 1640 culture medium containing 10% Australian fetal serum. GM-CSF (1000 IU / mL) and rIL-4 (500 IU / mL) were added on days 1, 3, and 5. LPS (1 μg / mL) was added on day 5 to stimulate DC maturation. DC were harvested on day 7 and stained with 5 fluorescent monoclonal antibodies (CD83\CD80\CD86\DR\ABC). The percentage of mature DC was analyzed by flow cytometry.

[0140] ③ Improved DC-peptide-PBL co-stimulation culture:

[0141] That is, the conventional DC loaded with peptide and co-cultured with T cells was improved: PBMC was used to induce and culture DC for 7 days, and then PBL (peripheral blood lymphocyte group after removing adherent monocytes, including B cells) of the same blood donor resuscitated one day in advance and a single candidate epitope peptide corresponding to the HLA molecule of the blood donor were co-cultured in a 48-well plate for 14 days. During this period, the epitope peptide was first incubated with DC for 2 hours on the first day, and then the epitope peptide was not removed, and the epitope peptide was added again on the 7th day for co-stimulation culture; IL-2 (20IU / mL) was added on the 4th day, and half the amount of IL-2 was added on the 10th day. Each well has 1×10 PBL.6 cells / well (PBL:DC=20:1), single candidate epitope peptide 20μg / mL; about 20 candidate epitope peptides were verified in each blood sample, and three control wells were set up: DC-PBL negative control well (without epitope peptide), PBL positive well (with a small amount of PHA / PMA), PBL single well (without any stimulant).

[0142] ④Intracellular IFN-γ fluorescence staining:

[0143] On the evening of the 14th day, the cells were collected and transferred to a 48-well plate. The corresponding single candidate epitope peptide (20 μg / mL) was added to each well and cultured for 16 h in serum-free 1640 medium. Then, BFA / Monensin was added and cultured for 6 h. Anti-IFN-γ intracellular staining and anti-CD3 / anti-CD8 membrane staining were performed, and CD3 was analyzed by flow cytometry. + / CD8 + or CD3 + / CD8 - The proportion of IFN-γ+ cells in the cell population. Compared with the DC-PBL co-culture wells without peptide, the IFN-γ + The co-culture wells with a cell ratio increase of more than 100% were defined as positive wells, i.e., the epitope peptide in the wells could stimulate CD8 + T cells or CD4 + T cells are activated and secrete IFN-γ, which is immunogenic.

[0144] 3) Use molecular docking and molecular dynamics simulation experiments to verify the cross-binding force between positive epitope peptides and various related HLA-A molecules:

[0145] The positive epitope peptides verified by the DC-peptide-PBL co-stimulation experiment were classified. According to the predicted HLA-A molecule restriction and the actual HLA molecules of the blood donors in the verification experiment, the possible restrictive HLA-A molecules of each positive epitope peptide were listed and classified. Then, the positive epitope peptides were molecularly docked with the protein crystal structures of the relevant HLA molecules through the Glide 5.7 module in the Schrodinger Suite. The dynamics simulation of each pair of molecules was completed using the Desmond module. Finally, the binding free energy (kal / mol) was calculated based on the RMSD results to determine the affinity of each positive epitope peptide with the relevant HLA molecules and to clarify the cross-restriction of each epitope peptide with multiple HLA molecules (i.e., one epitope peptide can be presented by multiple HLA molecules).

[0146] 4) Using the HMy2.CIR cell line expressing a specific HLA-A molecule, a competitive binding experiment between HLA molecules and peptides was performed to verify the cross-binding ability of the positive epitope peptide with various related HLA-A molecules:

[0147] The positive epitope peptides verified by the DC-peptide-PBL costimulation experiment were classified, and the positive epitope peptides that each HLA molecule may present were listed and classified according to the predicted HLA molecule restriction and the actual HLA molecules of the blood donors in the verification experiment. Then, the peptide competition binding experiment of HLA molecules was carried out in the same way as in Example 1.

[0148] 5) Using HLA-A2 transgenic mice immunization experiments, the in vivo immunogenicity of 31 positive epitope peptides presented by HLA-A2 molecules was verified:

[0149] Using HLA-A0201 / DR1 transgenic mice, 31 positive epitope peptides restricted by the HLA-A0201 molecule were prepared into several mixed peptide pools, mixed with adjuvant Poly I:C (InvivoGen), and subcutaneously inoculated into mice at multiple points, with each peptide pool inoculated at one point, and 4 peptide pools / time / mouse. The inoculation amount was 10μg / epitope peptide / time / mouse + 25μg Poly I:C / point / time / mouse, immunized 3 times, with an interval of 2 weeks. One week after the last inoculation, the mice were killed and spleen cells were obtained. They were co-incubated with a single epitope peptide in a 96-well plate for 20 hours, and IFN-γ ELISPOT and intracellular IFN-γ staining plus flow cytometry were performed to detect the specific T cell response intensity induced by each epitope peptide, and the immunogenicity of some positive epitope peptides was verified in animals.

[0150] 6) Establish a broad-spectrum T cell epitope peptide library containing 215 positive epitope peptides and establish the ELISPOT method:

[0151] A broad-spectrum epitope peptide library was composed of 215 experimentally verified positive epitope peptides of five antigens of the new coronavirus presented by 13 dominant HLA-A molecules. 11 mixed peptide pools were prepared according to the different protein sources and acidity / alkalinity / neutrality. As PBMC stimulators, they were assembled into ELISPOT reagents with PVDF membrane 96-well strips (Merck & Millipore, M8IPS4510), IFN-γ capture antibody / detection antibody (BD, 551873), HRP-streptavidin (BD, 557630) and AEC display agent (BD, 551951).

[0152] 7) Detection of memory T cells that cross-react with the new coronavirus in PBMCs of 50 healthy blood donors:

[0153] ① Collect leukocyte filter disks from blood donors from Jiangsu Provincial Blood Center, separate PBMCs as above and freeze for future use. After overnight recovery and culture, use the above 215 epitope peptides for detection. Each specimen is divided into 13 wells for detection. Well 1 is a negative control well without peptide stimulation, wells 2-12 are experimental wells, and peptide libraries 1 to 11 are added respectively. Well 13 is a positive control well stimulated by PHA. The rest of the steps are the same as Example 1. Negative wells and experimental wells: 4×10 5 PBMCs / well, PHA positive well: 2×10 5 PBMCs / well. The number of spots secreting IFN-γ was detected after 20 h of co-culture.

[0154] ② Based on the actual number of spots in 50 healthy blood donors, the normal range of healthy people is preliminarily obtained as follows: 95% of the population reference range: 83-377 SFUs / 4×10 5 PBMCs; negative control: 0-73 SFUs / 4×10 5 PBMCs; PHA positive control: >180SFUs / 2×10 5 PBMCs

[0155] The representative ELSPOT spot diagram results of healthy blood donors tested using the universal detection method for novel coronavirus antigen-specific T cells prepared by the technical solution of the present invention are as follows: Figure 2 As shown. This figure is a representative result of step (7). A broad-spectrum T cell epitope peptide library of 215 SARS-CoV-2 antigens was used to quantitatively detect the specific T cell reactivity that cross-reacts with SARS-CoV-2 in PBMCs of healthy blood donors (IFN-γ ELISpot method), because about 10% of the 215 epitope peptides are T epitope peptides that are highly homologous to the four common coronavirus antigens. Negative wells and experimental wells: 4×10 5 PBMCs / well, PHA positive well: 2×10 5 PBMCs / well. The number of spots secreting IFN-γ was detected after 20 h of co-culture. The number of spots in each negative well and experimental well represents 4×10 5 The number of cross-reactive T cells in PBMCs that are reactive after being stimulated by the SARS-CoV-2 T cell epitope peptide. This result graph shows that individual PBMC specimens from different healthy individuals can be quantitatively detected using this technical solution to detect specific T cells that cross-react with the SARS-CoV-2.

[0156] Embodiment 3:

[0157] This technical solution is used to prepare a broad-spectrum T cell epitope peptide library of liver cancer-related tumor antigens presented by HLA-A molecules, which is used for universal detection of tumor antigen-specific T cells in a wide range of liver cancer patients in China

[0158] 1) Virtual prediction of candidate T cell epitopes of liver cancer-associated antigens presented by HLA-A molecules:

[0159] ① The full-length amino acid sequences of 6 tumor antigens (AFP, GPC3, SART2 / 3, SSX2, GP73 and hTERT) were obtained from databases such as UniProt / NCBI and COSMIC (as follows), and multiple sequence alignment was performed using the ClustalW method in the Molecular Evolutionary Genetics Analysis (MEGA7, 1.0.0.0 version) software. The alignment results were then analyzed using the multiple sequence alignment and analysis software GeneDoc (2.7.0 version) to obtain the conserved regions of the sequences. The conservative nature of each amino acid was judged using 100%, 95%, and 80% as critical points. The positive expression rates of AFP, GPC3, SART2 / 3, SSX2, GP73, and hTERT in the HCC patient group were approximately 50-80%, 65-80%, 100%, 75%, 63-72%, and 61-84%, respectively.

[0160] ②Using the epitope prediction algorithms and tools listed in Table 1, virtually predict the epitope peptides presented by all 13 HLA-A with gene frequencies greater than 1% in the Chinese population. The epitope peptide length is set to 9-mer and 10-mer, and at least meets the criteria of more than 3 prediction algorithms, and the peptides with the top 5-10 scores are selected as candidate epitope peptides.

[0161] The amino acid sequences of the seven hepatocellular carcinoma-associated tumor antigens are as follows:

[0162] Scientifically named genera and species: Vertebrata, Eutheria, Homo

[0163] AFP(Alpha fetoprotein)(ID:P02771-1)

[0164]

[0165] GPC3(Glypican 3)(ID:P51654-3)

[0166]

[0167] SART2(Squamous cell carcinoma antigen recognized by T-cells 2)(ID:Q9UL01-1)

[0168]

[0169] SART3(Squamous cell carcinoma antigen recognized by T-cells 3)(ID:Q15020-1)

[0170]

[0171]

[0172] SSX2(Synovial sarcoma,X breakpoint 2)(ID:Q16385-2)

[0173]

[0174] GP73(Golgi membrane protein 73)(ID:Q8NBJ4-1)

[0175]

[0176] hTERT(Telomerase reverse transcriptase)(ID:O94807-1)

[0177]

[0178] 2) Using PBMCs from patients with liver cancer, the immunogenicity of candidate epitope peptides was verified by peptide-PBMCs co-stimulation experiment:

[0179] ①Peripheral blood of liver cancer patients was collected from the Liver Tumor Treatment Center of Nanjing Second Hospital, PBMCs were routinely separated and frozen for future use, and some cells were taken for HLA-A allele typing in the same way as in Example 1.

[0180] ② Single peptide immunogenicity verification:

[0181] Based on the results of HLA-A\B\C allele identification, candidate epitope peptides corresponding to the specific HLA molecules of the patient were selected and co-cultured with PBMCs for 6 hours. Intracellular IFN-γ fluorescence staining was performed to detect CD8 + T cells and CD4 + T cell frequency, compared with the group without polypeptide culture. + Epitope peptides with significantly increased T cell frequency are immunogenic and are positive epitope peptides. Each candidate epitope peptide was tested with PBMCs of at least 5 patients, and then epitope peptides with strong immunogenicity and positive reactions in most patients (3 / 5) were selected and included in the dominant epitope peptide library.

[0182] 3) Using the HMy2.CIR cell line expressing a specific HLA-A molecule, a competitive binding experiment between HLA molecules and peptides was performed to verify the cross-binding ability of the positive epitope peptide with various related HLA-A molecules:

[0183] The positive epitope peptides verified by the peptide-PBMCs costimulation experiment were classified, and the positive epitope peptides that each HLA molecule may present were listed and classified according to the predicted HLA molecule restriction and the actual HLA molecules of the blood donors in the verification experiment. Then, the peptide competition binding experiment of HLA molecules was carried out in the same way as in Example 1.

[0184] 4) Establish a broad-spectrum T cell epitope peptide library of 6 liver cancer-related antigens to detect tumor antigen-specific T cells in PBMCs of a wide range of liver cancer patients:

[0185] Through the above-mentioned single peptide immunogenicity verification and HLA molecule and peptide competitive binding experiments, 106 dominant epitope peptides cross-bound by 13 dominant HLA-A molecules were screened out to form a broad-spectrum T cell epitope peptide library of tumor antigens. The number of epitope peptides cross-bound by each HLA-A molecule is 5. According to the different antigen sources and acidity and alkalinity, the 106 epitope peptides were divided into 8 mixed peptide libraries.

[0186] Table 5 Distribution of cross-restriction of 13 HLA-A molecules for 106 liver cancer-related antigen epitope peptides

[0187]

[0188] 5) Universal detection of liver cancer-related antigen-specific T cells:

[0189] ① Isolation of PBMC: peripheral anticoagulated blood samples were collected from the patients to be tested, and mononuclear cells (PBMCs) were isolated as above. The cells were counted and the cell concentration was adjusted to 2×10 using serum-free cell culture medium (Dakoway Biotechnology, Shenzhen). 6 / ml.

[0190] ② Add cell suspension: add PBMC from each patient's blood sample to 9 wells, 100 μL / well (i.e.: 2×10 5 cells / well), of which 8 wells were set as experimental wells and 1 well was set as negative well.

[0191] ③ Add antigen peptides: 9 wells were tested for each blood sample. Antigen peptide library A, peptide library B, peptide library C, peptide library D, peptide library E, peptide library F, peptide library G, and peptide library H were added to the 2nd to 9th wells, 20 μL / well, respectively, which were experimental wells; no antigen peptide was added to the 1st well, only 20 μL of sterile washing solution was added, which was the negative well.

[0192] ④ Incubation: Cover the strips and place in a 37°C, 5% CO2 cell culture incubator for 16-20 hours.

[0193] ⑤ Wash the plate: Take out the ELISPOT strip plate from the cell culture incubator, shake off the cell suspension, add 200 μL of deionized water to each well, place at 4°C for 10 minutes to lyse the cells, and discard the deionized water. Add 200 μL of washing solution (1×PBS) to each well, let it stand for 1 minute, shake off the liquid, and repeat the washing process 5 times. After shaking off the liquid for the last time, pat dry on absorbent paper.

[0194] ⑥ Add detection antibody: add 100 μL of biotin-IFN-γ detection antibody working solution (BD Bioscience) to each well and incubate at room temperature for 2 hours. Wash the plate as above: add 200 μL of washing solution (1×PBS) to each well, let it stand for 1 minute, shake off the liquid, repeat this 5 times, shake off the liquid for the last time and pat dry on absorbent paper.

[0195] ⑦ Add HRP-streptavidin: Add 100 μL HRP-streptavidin working solution (BD Bioscience) to each well and incubate at room temperature for 1 hour in the dark. Wash the plate 5 times as above, and pat dry on absorbent paper after shaking off the liquid for the last time.

[0196] ⑧ Color development: Add 100 μL AEC color development solution (prepared immediately before use) to each well and leave it at room temperature and away from light for 20 minutes. Then shake off the liquid, remove the plate base, and wash the front and back of the PVDF membrane and the base with deionized water 3-5 times to stop the color development. Place the strip plate in a dark place at room temperature and install the plate base after it is naturally dried.

[0197] ⑨ Spot counting: After drying, the strips are automatically photographed and the spots are counted using an ELISA spot analyzer. If the number of spots is small, the experimenter can also count the spots by himself under an upright optical microscope.

[0198] ⑩Result judgment:

[0199] The number of spots in each sample = (the sum of the number of spots in 8 experimental wells) - (the number of background spots in 8 experimental wells)

[0200] Note: When the actual number of spots in the experimental well is less than that in the negative control well, the actual number of spots is taken as the background number of spots in the well; when the actual number of spots in the well is equal to or more than that in the negative control well, the number of spots in the negative control well is taken as the background number of spots in the well.

[0201] 6) Analysis of personalized test results:

[0202] By testing 58 hepatocellular carcinoma patients and 16 healthy blood donors, the number of tumor antigen-specific T cells in hepatocellular carcinoma patients was significantly higher than that in healthy blood donors. The results are shown in the attached Figure 3 .

[0203] Figure 3 ELISPOT spot diagram A and spot statistical diagram B for detecting liver cancer patients and healthy subjects using the universal detection scheme for liver cancer-related tumor antigen-specific T cells prepared by the method of the present invention. + T cell epitope peptide library was used to detect the reactivity of liver cancer antigen-specific T cells in PBMCs of 58 hepatocellular carcinoma patients and 16 healthy subjects (IFN-γ ELISPOT method). Figure A is a representative result diagram of step (5), and Figure B is a statistical diagram of the test results of 58 hepatocellular carcinoma patients and 16 healthy blood donors. The results showed that the reactivity of tumor antigen-specific T cells in hepatocellular carcinoma patients was significantly higher than that in healthy blood donors. These results also show that PBMC specimens of different hepatocellular carcinoma patients can be quantitatively detected with this technical solution to detect reactive tumor antigen-specific T cells.

[0204] It should be noted that the above embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. The universal antigen-specific T cell detection method based on the HLA-B and HLA-C restricted broad-spectrum T cell epitope peptide library and the replacement or substitution thereof made on the above basis all fall within the scope of protection of the present invention.

[0205] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A non-diagnostic antigen-specific thymus-dependent lymphocyte detection scheme suitable for a wide range of people in East Asia, characterized in that: The following steps are involved: (1) Screening and verification of a series of antigenic peptides presented by HLA-A, B, and C molecules with an allele frequency greater than 1% in East Asian populations against specific viral antigens or tumor antigens: using multiple online epitope prediction databases to virtually predict candidate epitope peptides presented by 13 HLA-A molecules, 25 HLA-B molecules, and 14 HLA-C molecules with an allele frequency greater than 1% against hepatitis B virus antigens HBsAg, HBcAg, HBpol, and HBx, novel coronavirus antigens S, E, N, M, and RdRp, or liver cancer-related tumor antigens AFP, GPC3, SART2 / 3, SSX2, GP73, and hTERT; then using patients' peripheral blood mononuclear cells to conduct lymphocyte functional experiments, peptide-cell competition binding experiments with HLA-A, B, or C molecules on cell lines, molecular docking, and molecular dynamics simulation techniques, the immunogenicity of candidate epitope peptides was verified one by one, and a positive epitope peptide library presented by each dominant HLA molecule was established; (2) Establishment of a broad-spectrum T cell epitope peptide library: According to the length of each viral antigen or tumor antigen sequence, 100-200 epitope peptides are screened from the above positive epitope peptide library to form a broad-spectrum T cell epitope peptide library; these broad-spectrum T cell epitope peptides are epitope peptides presented by the above 52 HLA molecules, and each HLA-A / B / C molecule cross-binds to 10-20 epitope peptides; therefore, each patient's 6 HLA-A / B / C molecules can cross-bind to 60-120 epitope peptides in the broad-spectrum T cell epitope peptide library; for 4 hepatitis B virus, 5 new coronavirus antigens or 6 tumor antigens, each patient can detect 240-480, 300-600 or 360-720 specific CD8 + T cell clones; (3) Using the peptide pool array of the broad-spectrum T cell epitope peptide library of the above-mentioned specific antigen, co-culture with the patient's PBMCs in a microwell reaction plate, establish an enzyme-linked immunosorbent assay or fluorescent immunospot assay to quantitatively detect the number of cells secreting IFN-γ; In step (1) The 13 HLA-A molecules are as follows: HLA-A1101, A2402, A0201, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601; The 25 HLA-B molecules are as follows: HLA-B4001, B4601, B5801, B5101, B1302, B1501, B1301, B4006, B1502, B3802, B3501, B5401, B5502, B3901, B5201, B4002, B4403, B4801, B0702, B1511, B5102, B2704, B5701, B1527, B3701; The 14 HLA-C molecules are as follows: HLA-Cw0702, Cw0102, Cw0304, Cw0901, Cw0602, Cw0303, Cw0302, Cw0401, Cw1402, Cw1502, Cw0701, Cw1202, Cw1403, Cw1203.

2. The antigen-specific thymus-dependent lymphocyte detection scheme for non-diagnostic purposes applicable to a wide range of people in East Asia according to claim 1, characterized in that: In the step (3), the enzyme-linked immunosorbent assay or fluorescent immunospot assay can also be replaced by a magnetic microsphere chemiluminescence assay: a peptide pool array of a broad-spectrum T cell epitope peptide library of a specific antigen in step (2) is used to co-culture with the patient's PBMCs in a 96-well cell culture plate, and the culture supernatant of each well is collected as a sample to be tested; a magnetic microsphere chemiluminescence assay is established in another microwell reaction plate to detect the level of IFN-γ in the culture supernatant.

3. The antigen-specific thymus-dependent lymphocyte detection scheme for non-diagnostic purposes applicable to a wide range of people in East Asia according to claim 1, characterized in that: In the step (3), the enzyme-linked immunosorbent assay or fluorescent immunospot assay can also be replaced by an enzyme-linked immunosorbent assay: a peptide pool array of a broad-spectrum T cell epitope peptide library of a specific antigen in step (2) is co-cultured with the patient's PBMCs in a 96-well cell culture plate, and the culture supernatant of each well is collected as a sample to be tested; an enzyme-linked immunosorbent assay is established in another microwell reaction plate to detect the level of IFN-γ in the culture supernatant.

4. The antigen-specific thymus-dependent lymphocyte detection scheme for non-diagnostic purposes applicable to a wide range of people in East Asia according to claim 1, characterized in that: In the step (3), the enzyme-linked immunosorbent assay can be replaced by a cytometric beads array combined with T cell multicolor fluorescent staining technology to analyze the functional subtypes of antigen-specific T cells: the peptide pool array of the broad-spectrum T cell epitope peptide library of the specific antigen in step (2) is co-cultured with the patient's PBMCs in a 96-well cell culture plate for 20 hours, and then the supernatant is taken and the secretion levels of various soluble molecules secreted by the patient's PBMCs are detected using cytometric beads array reagents and flow cytometric analysis technology; at the same time, cells are taken for multi-combination multicolor fluorescent monoclonal antibody staining and flow cytometry analysis to quantitatively analyze the frequencies of various functional subpopulations of antigen-specific T cells.

5. The antigen-specific thymus-dependent lymphocyte detection scheme for non-diagnostic purposes applicable to a wide range of people in East Asia according to claim 4, characterized in that: The soluble molecules secreted by the patient's PBMCs are any one or more of human interferon-γ, human tumor necrosis factor, human interleukin-17, granzyme A, granzyme B, perforin, tumor necrosis factor-α and tumor necrosis factor-β.

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