A detection method for immunological indicators predicting HBsAg clearance in CHB patients

Abstract

The present invention discloses a detection method for immunological indicators predicting HBsAg clearance in CHB patients, comprising the following steps: screening of molecules, initially screening out immunological indicators for evaluating the function of the body's T cells, and collecting blood samples of HBsAg-positive and HBsAg-cleared CHB patients as well as healthy controls; detection method, collecting blood samples of patients, performing cell resuspension and seeding, and preparing antibodies; performing detection by flow cytometry, saving data, and performing data analysis using the software Flow Jo software V10.0.7; evaluation of prediction efficacy. The present invention has successfully discovered the T cell immunological indicator HLA-DR that can predict HBsAg seroconversion within 48 weeks in CHB patients. In the present invention, for the first time, the expression levels of HLA-DR of CD4 and CD8 T cells are used to predict HBsAg seroconversion within 48 weeks in CHB patients, and its sensitivity and specificity are as high as 83.33% and 72.34% respectively, which has important clinical significance for evaluating the antiviral efficacy of CHB patients.

Description

Technical Field

[0001] The present invention relates to a method for detecting immunological indexes, and particularly to a method for detecting immunological indexes for predicting the clearance of HBsAg in CHB patients, belonging to the technical field of medical immunology. Background Art

[0002] Hepatitis B virus (HBV) remains a major public health problem, with over 400 million people infected with HBV globally. The World Health Organization's global strategy on viral hepatitis proposes that the elimination of hepatitis B virus (HBV) is an achievable goal. Currently, most clinical practice guidelines recommend that the most ideal endpoint of chronic hepatitis B (CHB) treatment is to achieve functional cure, which is defined as the serological clearance of hepatitis B surface antigen (HBsAg) with or without serological conversion of HBsAg. The above goal is proposed because achieving the clearance of HBsAg can halt the progression of the disease and reduce the risk of primary liver cancer.

[0003] The incidence of HBsAg clearance in CHB patients who have not received antiviral treatment or have received nucleoside (nucleotide) analogs is extremely low. In recent years, with the application of interferon-α combination or sequential therapy, the clearance rate of HBsAg in CHB patients has increased significantly. Interferon-α mainly inhibits the production of RNA and related proteins in infected cells directly, and inhibits virus replication by blocking the assembly of nucleocapsid proteins. Moreover, interferon-α also has immunomodulatory effects, such as regulating the immune activation of T cells, NK cells, etc. Therefore, CHB patients who have received long-term effective antiviral treatment with nucleoside (nucleotide) analogs and then receive interferon-α treatment mainly achieve better antiviral effects, and even the clearance of HBsAg, by regulating the immune function of the body.

[0004] Interferon-α therapy mainly achieves a rapid decline and clearance of HBsAg by regulating the body's immune function. Currently, there are still a lack of immunologically relevant monitoring indicators in clinical practice to comprehensively evaluate the antiviral efficacy of patients. At present, there are many studies on the immunological mechanism of interferon-α in the antiviral effect of CHB patients, but there are few studies on the mechanism of interferon-α in achieving HBsAg clearance in CHB patients, and no effective immunological indicators for evaluating or predicting the antiviral efficacy of CHB patients have been found. For patients treated with combined or sequential interferon-α, currently, the antiviral efficacy and prognosis are mainly evaluated by detecting HBV-related virological indicators and biochemical indicators in clinical practice. The predictive effects of the above indicators are poor and lagging. Developing a method for detecting a simple and practical immunological indicator will be beneficial to more comprehensively evaluate the immunological function of patients, combine with the existing clinical virus and biochemical related indicators to evaluate the antiviral status and efficacy of patients, so as to assist in discovering potential advantageous populations that are expected to achieve HBsAg clearance, further optimize the antiviral treatment plan, and thus enable more patients to achieve HBsAg clearance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a detection method for immunological indicators predicting HBsAg clearance in CHB patients.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A detection method for immunological indicators predicting HBsAg clearance in CHB patients, comprising the following steps:

[0008] Step 1: Screening of molecules:

[0009] a. Initially screen out immunological indicators for evaluating the function of the body's T cells;

[0010] b. Collect blood samples from HBsAg-positive and HBsAg-cleared CHB patients and healthy controls;

[0011] Step 2: Detection method:

[0012] a. Collect blood samples from patients and perform cell resuspension and seeding;

[0013] b. Prepare antibodies;

[0014] c. Perform flow cytometry detection, save the data, and perform data analysis using the software Flow Jo software V10.0.7;

[0015] Step 3: Evaluation of predictive efficacy: Using the ROC curve, evaluate the predictive efficacy of the expression levels of HLA-DR on CD4 and CD8 T cells for HBsAg seroconversion within 48 weeks in 54 CHB patients.

[0016] Step 4: Discover the T cell immunological index HLA-DR that can predict the negative conversion of HBsAg within 48 weeks in CHB patients.

[0017] A further improvement of the technical solution of the present invention lies in that: the immunological indexes in the a process of step 1 include those representing cell activation (CD69, HLA-DR, CD95, CD25, CD40L), exhaustion (PD-1, TIM-3, CTLA-4), and effector functions (Granzyme B, CD107a).

[0018] A further improvement of the technical solution of the present invention lies in that: the cell resuspension and planting in the a process of step 2 include the following steps:

[0019] I. Take fresh anticoagulated blood and blood sample and invert and suspend them to obtain an anticoagulated blood sample to prevent the blood sample from coagulating and failing to achieve the accuracy of the detection result;

[0020] II. Slowly pour the anticoagulated blood sample obtained in step I above into a lymphocyte separation tube for separation; centrifuge at 20°C, 800g, for 20 min, and adjust the acceleration and deceleration to the third gear (if there are ten gears in total) to obtain a milky white mononuclear cell layer;

[0021] III. Aspirate the milky white mononuclear cell layer obtained in step II above into a centrifuge tube, add 3 - 5 ml of red blood cell lysate, mix well and centrifuge, centrifuge at 20°C, 300g, for 10 min, discard the supernatant, add an appropriate amount of fetal bovine serum, pipette and resuspend and mix well, centrifuge at 20°C, 250g, for 10 min, discard the supernatant, and add 3 - 5 ml of phosphate buffer to resuspend the cells for standby;

[0022] IV. Aspirate 10ul of trypan blue and mix it with 10ul of cell suspension, count under a microscope, wash the cells, resuspend them, and plant 1*10 5 ~1*10 6 cells in a 96-well U-bottom plate.

[0023] A further improvement of the technical solution of the present invention lies in that: the preparation of antibodies in the b process of step 2 includes the following steps:

[0024] V. Mix 1.5ul of CD4 antibody, 1.5ul of CD8 antibody, 1.5ul of CD3 antibody, 2ul of HLA-DR antibody, 0.5ul of FVD antibody and 43ul of sheath fluid;

[0025] Ⅵ. Centrifuge the 96-well U-bottom plate seeded with cells in step Ⅳ at 1500 rpm for 5 min. After discarding the supernatant, add 50 μl of the prepared antibody mixture to the corresponding cells. Resuspend by gently scraping the bottom and incubate in a 4°C refrigerator for 30 minutes.

[0026] Ⅶ. After incubating for 30 minutes, take out the 96-well U-bottom plate, add 150 μl of sheath fluid for washing, centrifuge at 1500 rpm for 5 min, discard the supernatant, add 100 μl of sheath fluid, resuspend and transfer to a flow tube containing 100 μl of sheath fluid for flow cytometry detection.

[0027] A further improvement of the technical solution of the present invention lies in: according to the results of step 3 above, the expression of HLA-DR on CD4 or CD8 T cells has a certain predictive efficacy for the negative conversion of HBsAg within 48 weeks.

[0028] Compared with the prior art, the present invention provides a detection method for immunological indicators predicting HBsAg clearance in CHB patients, and successfully discovers the T cell immunological indicator HLA-DR that can predict the negative conversion of HBsAg within 48 weeks in CHB patients. In the present invention, for the first time, the expression levels of HLA-DR on CD4 and CD8 T cells are used to predict the negative conversion of HBsAg within 48 weeks in CHB patients, and its sensitivity and specificity are as high as 83.33% and 72.34% respectively, which has important clinical significance for evaluating the antiviral efficacy of CHB patients. Furthermore, it helps to discover potential advantageous populations that are expected to achieve HBsAg clearance, further optimize the antiviral treatment plan, so that more patients can achieve HBsAg clearance. Description of the Drawings

[0029] Figure 1 It is a schematic diagram of the gating method for flow cytometry detection and analysis in the embodiment of the present invention;

[0030] Figure 2 It is a schematic diagram of the area under the ROC curve of HLA-DR on CD4 T cells in the embodiment of the present invention;

[0031] Figure 3 It is a schematic diagram of the area under the ROC curve of HLA-DR on CD8 T cells in the embodiment of the present invention. Detailed Embodiments

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The following will further elaborate on the present invention in detail with specific embodiments:

[0033] As Figures 1 to 3 shown, the present invention provides a detection method for immunological indicators predicting HBsAg clearance in CHB patients, including the following steps:

[0034] Step 1. Screening of molecules:

[0035] a. Initially screen out immunological indicators for evaluating the function of the body's T cells;

[0036] b. Collect blood samples from HBsAg-positive and HBsAg-cleared CHB patients as well as healthy controls.

[0037] Step 2. Detection method:

[0038] a. Collect blood samples from patients and perform cell resuspension and seeding;

[0039] b. Prepare antibodies;

[0040] c. Perform flow cytometry detection, save the data, and perform data analysis using the software Flow Jo software V10.0.7;

[0041] Step 3. Evaluation of prediction efficacy: Use the ROC curve to evaluate the prediction efficacy of the HLA-DR expression levels on CD4 and CD8 T cells for HBsAg seroconversion within 48 weeks in 54 CHB patients;

[0042] Step 4. Successfully discovered the T cell immunological indicator HLA-DR that can predict HBsAg seroconversion within 48 weeks in CHB patients.

[0043] Example 1

[0044] As Figures 1 to 3As shown in the figure, the present invention further provides an improved technical solution: Preferably, the immunological indicators in the a process of step 1 include those representing cell activation (CD69, HLA-DR, CD95, CD25, CD40L), exhaustion (PD-1, TIM-3, CTLA-4), and effector functions (Granzyme B, CD107a). Blood samples of HBsAg-positive and HBsAg-cleared CHB patients and healthy controls were collected. In this example, the expression differences of the above T cell indicators were detected. The results showed that the HLA-DR expression levels of CD4 and CD8 T cells in HBsAg-cleared patients were significantly higher than those in HBsAg-positive patients and healthy individuals, and were positively correlated with the decline of HBsAg and the response intensity of HBV-specific T cells, suggesting that the HLA-DR expression levels of CD4 and CD8 T cells may have a predictive effect on the clearance of HBsAg.

[0045] Example 2

[0046] As Figure 1 shown, on the basis of Example 1, the present invention provides an improved technical solution: Preferably, for the cell resuspension and planting in the a process of step 2, first, take fresh anticoagulated blood and blood samples and invert and suspend them to obtain anticoagulated blood samples to prevent the blood samples from coagulating and failing to achieve the accuracy of the detection results; secondly, slowly pour the obtained anticoagulated blood samples into a lymphocyte separation tube for separation; control the centrifugation at 20 °C, 800 g, for 20 min, and adjust the acceleration and deceleration to the third gear (if there are a total of ten gears) to obtain a milky white mononuclear cell layer. Again, carefully aspirate the obtained milky white mononuclear cell layer into a centrifuge tube, add 3 - 5 ml of red blood cell lysate, mix well and centrifuge, control the centrifugation at 20 °C, 300 g, for 10 min, discard the supernatant, add an appropriate amount of fetal bovine serum, resuspend and mix well by pipetting, control the centrifugation at 20 °C, 250 g, for 10 min, discard the supernatant, and add 3 - 5 ml of phosphate buffer to resuspend the cells for standby; furthermore, aspirate 10 ul of trypan blue and mix it with 10 ul of cell suspension, count under a microscope, wash the cells, resuspend them, and plant 1*10 5 ~1*10 6 cells in a 96-well U-bottom plate.

[0047] Example 3

[0048] As Figure 1As shown, on the basis of Example 2, the present invention provides an improved technical solution: in the b process of step 2 in this embodiment, antibodies are configured. First, 1.5 μl of CD4 antibody, 1.5 μl of CD8 antibody, 1.5 μl of CD3 antibody, 2 μl of HLA-DR antibody, 0.5 μl of FVD antibody and 43 μl of sheath fluid are mixed evenly. Secondly, the 96-well U-bottom plate with planted cells in Example 2 is centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant, 50 μl of the configured antibody mixture is added to the corresponding cells. After gently scraping the bottom to resuspend, it is placed in a refrigerator at 4°C for incubation for 30 minutes. Thirdly, after incubating for 30 minutes, the 96-well U-bottom plate is taken out, 150 μl of sheath fluid is added for washing, and after centrifuging at 1500 rpm for 5 minutes, the supernatant is discarded. 100 μl of sheath fluid is added, and it is resuspended and transferred to a flow tube containing 100 μl of sheath fluid for flow cytometry detection, and the data is saved. Furthermore, software Flow Jo software V10.0.7 is used for data analysis.

[0049] Example 4

[0050] As Figures 2 to 3 shown, on the basis of Example 1, Example 2, and Example 3, the present invention provides an improved technical solution: according to the evaluation of the HLA-DR prediction efficacy of CD4 and CD8 T cells in the above step 3, the results show that the expression of HLA-DR on CD4 or CD8 T cells has a certain prediction efficacy for the negative conversion of HBsAg within 48 weeks. Among them, the area under the ROC curve (AUC) of HLA-DR on CD4 T cells is 0.773 (P = 0.031, 95% confidence interval is 0.607 - 0.939), and the optimal prediction cut-off value is 7.35% (sensitivity 100%, specificity 55.3%). In addition, the area under the ROC curve (AUC) of HLA-DR on CD8 T cells is 0.807 (P = 0.005, 95% confidence interval is 0.637 - 0.977), and the optimal prediction cut-off value is 12.10% (sensitivity 83.33%, specificity 72.34%).

[0051] Next, the working principle of the detection method for the immunological indexes predicting HBsAg clearance in CHB patients will be specifically described.

[0052] As Figures 1 to 3As shown, the present invention has successfully discovered the T cell immunological index HLA-DR that can predict the negative conversion of HBsAg within 48 weeks in CHB patients. In the present invention, for the first time, the expression levels of HLA-DR of CD4 and CD8 T cells are used to predict the negative conversion of HBsAg within 48 weeks in CHB patients, and its sensitivity and specificity are as high as 83.33% and 72.34% respectively, which has important clinical significance for evaluating the antiviral efficacy of CHB patients, and further helps to discover potential advantageous populations that are expected to achieve HBsAg clearance, further optimize the antiviral treatment plan, so that more patients can achieve HBsAg clearance.

[0053] The above has described in detail the detection method of the immunological index for predicting HBsAg clearance in CHB patients provided by the present invention. For those of ordinary skill in the art, any obvious changes made to it without departing from the essence of the present invention will constitute an infringement of the patent right of the present invention and will bear corresponding legal responsibilities.

Claims

1. A method for predicting immunological indicators of HBsAg clearance in CHB patients, characterized in that It includes the following steps: Step 1, screening of molecules: a. Initially screen out the immunological indexes for evaluating the function of the body's T cells; b. Collect blood samples of HBsAg-positive and CHB-cleared patients as well as healthy controls; Step 2, detection method: a. Collect blood samples of patients and perform cell resuspension and seeding; b. Prepare antibodies; c. Perform detection by flow cytometry, save the data, and perform data analysis using the software Flow Jo software V10.0.7; Step 3, evaluation of the predictive efficacy of HLA-DR on CD4 and CD8 T cells: Use the ROC curve to evaluate the predictive efficacy of the HLA-DR expression level on CD4 and CD8 T cells for HBsAg seroconversion within 48 weeks in 54 CHB patients; Step 4, discover the T cell immunological index HLA-DR that can predict HBsAg seroconversion in CHB patients within 48 weeks; In the process a of Step 1, the immunological indexes include those representing cell activation, exhaustion, and effector function; Among them, the immunological indexes of cell activation are CD69, HLA-DR, CD95, CD25, and CD40L; The immunological indexes of exhaustion are PD-1, TIM-3, and CTLA-4; The immunological indexes of effector function are Granzyme B and CD107a.

2. The method for predicting immunological indexes for HBsAg clearance in CHB patients as described in claim 1, wherein: The cell resuspension and seeding in the process a of Step 2 includes the following steps: I. Take fresh anticoagulated blood and the blood sample and invert and suspend them to obtain an anticoagulated blood sample; II. Slowly pour the anticoagulated blood sample obtained in the above Step I into a lymphocyte separation tube for separation to obtain a milky mononuclear cell layer; III. Use a pipette to aspirate the milky mononuclear cell layer obtained in the above Step II into a centrifuge tube, add 3 - 5 ml of red blood cell lysate, mix well and centrifuge, discard the supernatant, add an appropriate amount of fetal bovine serum, pipette and resuspend well, discard the supernatant, and add 3 - 5 ml of phosphate buffer to resuspend the cells for standby; IV. Mix 10 μl of trypan blue with 10 μl of cell suspension, count under a microscope, wash the cells, resuspend them, and seed 1×10 5 ~1×10 6 cells into a 96-well U-bottom plate.

3. The method for predicting immunological indexes for HBsAg clearance in CHB patients as described in claim 1, wherein: According to the results of the above Step 3, the expression of HLA-DR on CD4 or CD8 T cells has a predictive efficacy for HBsAg seroconversion within 48 weeks of HBsAg.

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