Quantitative Detection Kit，For Hepatitis B Virus Specific T Cells

DESCRIPTION:

HLA-A molecules are main HLA class I molecules which present viral or tumor antigenic peptides to CD8+ T cells and induce cytotoxic effects in vivo. HBV has four main protein antigens: surface antigen (HBsAg), E antigen (HBeAg, including core antigen HBcAg), DNA polymerase (HBpol) and X protein (HBx). According to their amino acid sequences of these HBV antigens and bioinformatics analyses, we predict hundreds of antigenic peptides presented by 13 kinds of predominant HLA-A molecules which cover vast majority of populations, in Eastern Asian, Southeast Asian, American and European populations (see Additional Notes). Then flesh peripheral blood specimens are collected from more than 700 hepatitis B patients, PBMCs are co-cultured for 20 hours with each peptide followed by detection of IFN-γ-secreting T cells using the enzyme-linked immune spot (ELISPOT) assay and flow cytometry. Positive result means that there have been the epitope-specific active or memory T cells in the patients’ peripheral blood before the co-culture, implying the real immunogenicity of the indicated peptide in HBV-infected patients. Consequently, 105 kinds of peptides (9-10 aa per peptide) have been verified to function as antigenic T cell epitopes that can stimulate T cells to activate and produce IFN-γ. Furthermore, molecular structure bioinformatics analyses has been carried out. The epitope peptide and the HLA protein crystal structure were docked using the Glide 5.7 module in the Schrodinger Suite, and the Desmond module was used to complete the molecular dynamics simulation. Finally, the affinity of these positive peptides to related HLA-A molecules was determined according to the RMSD results. Then the corss-restriction of HLA-A molecules with indicated peptide was defined. Among these 105 kinds of antigenic peptides, 31, 25, 29 and 20 kinds derive from HBsAg, HBeAg (containing HBcAg), HBpol and HBx proteins, respectively. They almost cover the full length of each HBV protein antigen, and can be cross-presented by the 13 dominant HLA-A molecules. The 105 kinds of antigenic peptides are finally arranged into seven peptide pools to prepare the pool-array ELISPOT kit for the quantitative analyses of active/memory HBV-specific T cells in patients. It is suitable for the vast majority of hepatitis B patients. The HBV-specific T cells activated by these antigenic peptides are mainly CD8+ T cells, partly CD4+ T cells, because about 30% of antigenic peptides can also activate CD4+ T cells simultaneously in this co-culture system. According to the results of blood samples from more than 300 hepatitis B patients, the preliminary reference range of hepatitis B population in China was obtained.

DETAILS

Immunogen: ic peptide pool 7260 μL - 80 ℃
Background: 1. Current status of hepatitis B virus infection and treatment Hepatitis B virus (HBV) infection is a serious public health problem with about 10% of the global population infected with HBV, of which more than 350 million suffer from chronic lifelong infection[1]. Chronic HBV infection in China accounts for about 8% of the total population[2], and is a major risk factor for cirrhosis, liver failure and liver cancer. Currently, the main clinical treatment is the antiviral therapy with nucleotide analogues and/or interferon to inhibit viral replication. However, HBV can form covalent closed circular DNA (cccDNA) in the nucleus of infected cell, integrate into host genome, and persist in host cell for a long time. The current standard treatment cannot effectively eliminate HBV, and usually cannot turn HBsAg into negative[3]. Thus chronic transformation of hepatitis B and relapse after drug withdrawal have become serious problems that must be faced[1]. 2. Personalized differences of HLA genes and anti-infection ability HLA gene system encodes major histocompatibility antigens of human, which are responsible for presenting antigenic peptides to T cells and initiating specific immune responses of the body. The HLA molecules are distinctive from individual to individual, and each HLA molecule binds to and presents distinctive antigenic peptides[4], thus leading to different specific immune responses in different individuals against the same pathogen. This is one of the main reasons for personalized differences in the body's ability to resist infection, namely, personal differences in HLA genes[5]. 3. Antiviral effects of T cells HBV-specific T cells play a more important role than HBV-specific antibodies in host antiviral responses, especially HBV-specific CD8+ T cells which mediate cytotoxic effects to clear virus and play the core role in host antiviral adaptive immune responses [6,7]. Clinical studies have shown that the strength of host T-cell response to viral antigens determines the outcome of HBV infection[8]. Patients with a strong T-cell immune response can clear the virus, while a weak immune response leads to chronic infection, in which HBV-specific T cells gradually reduce and eventually leading to cirrhosis and liver cancer [9-11]. Numerous studies have also shown a high risk of recurrence after withdrawal of antiviral therapy, where host specific T cells play a critical role in clearing the residual HBV[12-14]. 4. Clinical implications of HBV-specific T cell detection HBV DNA, antigen and antibody detection has been widely carried out in clinical practice, but these indicators can only reflect the degree of HBV replication and proliferation in vivo and the strength of specific humoral immune response, but cannot display the strength of specific T cell immune response that play a more important role in antiviral responses. The number of active and memory HBV-specific T cells in peripheral blood can directly reflect the strength of patient’s cellular immune function specific for HBV, so the quantitative detection of HBV-specific T cells and dynamic analyses have important clinical reference value, as a new laboratory indicator, in monitoring disease progression, predicting prognosis, guiding treatment regimen, evaluating the curative effects of antiviral therapy or immunotherapy, and predicting relapse after drug withdrawal. 5. Status of HBV-specific T cell detection By now, no commercial detection kit of HBV-specific T cells is available for research or clinical uses, because detecting HBV-specific T cells is much more difficult than detecting antigens, antibodies, and DNA. Two factors must be considered simultaneously when preparing the T cell detection kit: patients' distinct HLA molecules; a wide variety of antigenic peptides derived from the various HBV antigens and presented by these HLA molecules. But only a small number of HBV antigenic peptides have already been determined to be presented by various HLA molecules and can induce T cell responses[15-17]。The lack of commercial kits limits the specific T cell detection for hepatitis B patients who carrying different HLA alleles, thus obstruct the elucidation of the role of HBV specific T cells in the development of hepatitis B, and also limits the precision immunotherapy based on the personalized HLA genes and the HBV antigenic peptides presented by the individual HLA molecules.
References: [1] Guangyan Zhou. Principles of Immunology, 4th edition, Science Press, 2018 Chinese version). [2] ELAHI S, HORTON H. Association of Hlaw-Alleles with the immune regulation of chronic viral diseases [J]. Int J Biochem Cell Biol, 2012, 44(8): 1361-5. [3] AYALEW M B, HORSSA B A, GETACHEW N, et al. The Knowledge and attitude of the health care professionals regarding hepatitis B virus get infection and its vaccination, University of Gondar Hospital, Ethiopia [J]. Hepat Med, 2016, 8:135-42. [4] Lanjuan Li, Yuming Wang. Epidemiology, 3rd edition, People's Medical Publishing House, 2015 (Chinese version). [5] HAYES C N, CHAYAMA K. HBV Culture and Infectious Systems [J]. Hepatology International, 2016, 10(4): 559-66. [6] TSENG T C, HUANG, l. r. Immunopathogenesis of Hepatitis B Virus get [J]. J Infect Dis, 2017, 216 (suppl_8) : S765 - S770. [7] WANG Q, PAN W, LIU Y, et al. Hepatitis B Virus -specific CD8+ T Cells Maintain Functional after Antigen Reexposure in an Acute Activation Immune Environment [J]. Front Immunol, 2018, 9:219. [8] WANG F S, ZHANG Z. Host Immunity Influences Disease progression and passage selection in humans infected with hepatitis B virus [J]. Expert Rev Gastroenterol Hepatol, 2009, 3(5): 499-512. [9] LIU Q, ZHENG Y, YU Y, et al. Identification of HLA-a *0201-restricted CD8+ T-cell Epitope C64-72 From hepatitis B virus core protein [J]. Int Immunopharmacol, 2012, 13(2): 141-7. [10] LI J, HAN Y, JIN K, et al. Dynamic Changes of Cytotoxic T lymphocytes (CTLs), Natural Killer (NK) cells,And natural killer T (NKT) cells in patients with acute hepatitis B infection [J]. Virol J, 2011, 8:199. [11] CHISARI F V, ISOGAWA M, WIELAND s. f. Pathogenesis of hepatitis B virus get infection [J]. Am J Pathol Biol (Paris), 2010, 58 (4) : 258-66. [12] REIJNDERS J G, PERQUIN M J, ZHANG N, et al. Nucleos(T) IDE analogues only induce temporary hepatitis B e seroseroconversion in most patients with chronic hepatitis B [J]. Gastroenterology,2010, 139 (2) : 491-8. [13]PAPATHEODORIDIS G, VLACHOGIANNAKOS I, CHOLONGITAS E, et al. Discontinuation of oral cnet in chronic hepatitis B: A systematic Review [J]. Hepatology, 2016, 63(5): 1481-92. [14] EUROPEAN ASSOCIATION FOR THE STUDY OF THE liver.Electronic ADDRESS E E E,EUROPEAN ASSOCIATION FOR THE STUDY OF THE L. EASL 2017 Clinical Practice Guidelines on THE Management OF hepatitis B virus infection [J]. J Hepatol, 2017, 67(2): 370-98. [15] Brinck-Jensen NS, Vorup-Jensen T, LEUTSCHER P D,Et al. Immunogenicity of twenty peptides epitopes of the hepatitis B core and surface antigens by IFN- Gamma Response in chronic and Resolved HBV [J]. BMC Immunol, 2015, 16:65 5. [16] YAMAMIYA D, MIZUKOSHI E, KAJI K, et al. Immune Responses of Human T lymphocytes to novel hepatitis B virus-derived Peptides [J]. PLoS One, 2018, 13(6): E0198264. [17] ZHENG J, OU Z, LIN X, et al. Analysis of epitopo-based vaccine candidates against the E antigen of the hepatitis B virus based on the B Genotype sequence:Silico and in silico approach [J]. Cell Immunol, 2018, 329:56-65. Examples of clinical specimen detection case diagram
Positive Control: well: spot number > 180 for 95% of patientsExperimental wells:For 15% of patients, spot number > 238, implying strong immune reaction of HBV-specific T cells;For 70% patients, spot number 16- 238, implying medium immune reaction of HBV-specific T cellsFor 15% patients, spot number 0-16, implying weak immune reaction of HBV-specific T cellsNote: the reference value will be varied in different countries and regions, even in different laboratories due to the individual operational variation. Thus the local laboratories should determine their own reference value which fit to the regional patients.Results analysesSingle test result: When the spot number (>238) indicates the strong HBV-specific T cell reaction, that means the HBV-specific T cells in the patient have a strong ability to activate, proliferate, secrete inflammatory factors and kill HBV-infect liver cells after being stimulated by the virus, suggesting better disease progression and prognosis. However, this result is not necessarily consistent with the test results of liver function, HBV antigen, antibody and DNA copy number at that time. When the spot number is in the low range, it is suggested that the ability of killing virus and infected hepatocytes of patients with HBV-specific T cells is weak, so it is difficult to deplete thoroughly HBV in patient, and the risk of chronic outcome and recurrence should be considered in clinic.2. Dynamic analyses is more significant: since the HLA genes of each patient are distinct, the HBV antigenic peptide presented by each HLA molecule is also different, so the immune response of HBV-specific T cells from different individuals should be significantly varied. The above reference values do not necessarily apply to every patient. A better approach is to repeat the tests every 2-3 months to observe the dynamic changes in the spot number for patient’s blood samples during treatment or at different stages of the disease. If the spot number is significantly increased or decreased, even if in the mid-range of reference values, it can still indicate that the immune reaction of patients' HBV-specific T cells is significantly increased or decreased. Clinicians should think about the possible causes and resulting treatment changes, in combination with the results of other detection indicators, such as HBV DNA copy number, antigens and antibodies.3. Negative control: for a small number of patients, their blood samples show strong positive reactions (spot number >53) in the negative wells, which may imply that the patients are recently in the acute infection stage of HBV or other pathogens, or other stress conditions, and the patient's immune cells are in a highly activated state, worthy of clinical attention and observation.4. A small number of patients: the actual spot number in each experimental well may be less than or close to the spot number in the negative control well, suggesting that the immune activity of HBV-specific T cells in these wells is low, or there are many activated immune cells in the negative well, but may not be HBV-specific T cells.5. Positive control: PHA is a non-specific polyclonal mitogen for T lymphocytes. The spot number in the positive control well should be more than 180, indicating the good or normal reactivity of overall T cell repertoire in the patient. When the spot number is obviously less than 180, it indicates a weak immune reactivity of overall T cell repertoire. In this case, the results of experimental wells should be influenced by the overall weak immune responses of the patient. When the positive control wells for all samples show obvious less than 180 spots, the experiment should be repeated to confirm the reagent and operation is normal.Additional NotesPBMC separation diagram:Cell countingAfter the isolation of peripheral blood mononuclear cells (PBMC), cell concentration should be counted and adjusted. There are two counting methods: One is to use automatic cell counting instrument; the second is to use the traditional blood cell count plate. The following is a supplementary introduction to the blood cell count plate method.Reagents and materialsBlood cell count plate (figure below); 2 ml EP tubeWhite blood cell dilution solution: add 2 mL glacial acetic acid into 100 mL deionized water, mix well, store at room temperature for later use.Protocols :( aseptic conditions is needed for the first step)1. Mix the PBMC suspension with a straw, transfer 10 μL cell suspension into a EP tube, add 190μL white blood cell dilution solution (diluted 20 times), mix well.2. Suck the cell suspension with a pipette, and infuse the cells into cell count plate, full but not overflowing.3. Enumerate the cells in 4 large squares under microscope. One large square contains 16 medium squares. Be careful not to count remaining red blood cells, dead cells and impurities.4. Calculate the cell concentration in PBMC suspension using the formula below:Prepare cell suspension at density of 2×106 cells/ml: (aseptic operation in hood)For each sample, PBMCs need to be prepared to a density of 2×106 cells/mL with a total volume of 1mL. Therefore, 2×106 PBMCs ( x μL) are harvested from PBMC stock suspension, added into another sterile 2mL EP tube, then serum-free cell culture medium is added until 1.0 mL, store in the cell incubator until use.
Storagestability: The antigenic peptide pools in this kit must be stored at -80 0C. It is strongly recommended to divide the antigenic peptide pool into small aliquots for single use. These aliquots should be stored at -80 0C; Fetal bovine serum, PHA working solution, and negative well auxiliary buffer should be stored at -20 0C. All other reagents and materials should be stored at 4 0C and kept sterile. Under these conditions the reagents and materials in this kit are stable for minimal 5 months.
Recommended Dilutions: 1. The reference value range provided in this manual depends on the results obtained from a large number of Chinese hepatitis B patients, using daily fresh anticoagulant blood samples tested within 5 hours. Considering the ethnic and geographic differences of patients and the inter-laboratory errors such as cell counting, washing and spot judgment criteria, it is better for the local laboratory to determine their own reference range for the regional patients.2. The whole process from blood cell separation to cell culture should be strictly aseptic to prevent bacterial contamination. If there is slight bacterial contamination, experimental wells and negative control well will be strongly positive, thus leading to fail.3. The pipette tip should not touch the PVDF membrane in the well bottom. The indentation or notch caused by tip may lead to misjudgment of spots.4. The microplate is placed inside the protective plate base and can be removed only after the color developing is completed.5. Different pipettes, washing protocols, incubation time and temperature may affect the experimental results.6. Different batches of reagents should not be mixed.Warning:1. The blood samples detected are derived from the human body and should take into account the potential risk of infection. The treatment, use, storage and placement of all samples and their components shall comply with the relevant national regulations.2. The reagents contain biological preservatives. Avoid contact with skin. Wear appropriate gloves during operation.3. The chromogenic solution is irritating to eyes, respiratory system and skin. Respiratory inhalation, skin contact or oral intake are harmful to the human body. When using the chromogenic solution, avoid inhalation of steam and spray, avoid skin and eye contact, use appropriate protective clothing, glasses and take skin protection facilities.4. The test results of the kit are only used as the basis for clinical auxiliary diagnosis of the disease for research use only.

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