A method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine

Abstract

The present invention relates to immunotherapy for hepatitis B and other infectious diseases, including immune cell therapy and immune factor therapy, and the application of vaccine development research. Specifically, it relates to the technical fields of induction templates, induction methods, and cell therapy for infectious diseases, tumors and other diseases, immunotherapy and vaccine development, and discloses a method for activating the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine, including the following steps: 1) Animal grouping and dosage: Experimental animal grouping method: Female and male animals are stratified and randomly grouped according to body weight, and are divided into 3 groups: 4 positive control groups, 4 low-dose cell therapy groups, and 2 high-dose cell therapy groups, half male and half female. The method for stimulating the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine does not use viral vector gene transcription or other vector gene editing technology methods, but has the effect of activating antibody-secreting B cells.

Description

Technical Field

[0001] The present invention relates to immunotherapy for hepatitis B and other infectious diseases, including immune cell therapy and immune factor therapy, and the application of vaccine development research. Specifically, it relates to the technical fields of induction templates, induction methods, cell therapy for infectious diseases, tumors and other diseases, immunotherapy and vaccine development, and specifically a method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine. Background Art

[0002] With the development of molecular biology and transgenic technology, the use of reasonable transgenic technology can direct the targeting of certain cells. For example, CART treatment of tumors, as long as the tumor antigen is specific, theoretically, a good therapeutic effect can be obtained. The same technology can also be used to develop treatments for infectious diseases. The development of biotechnology has opened up broad hopes for the treatment of hepatitis B. Since the 1990s, various biological agents have been tried to treat hepatitis B at home and abroad, such as subunit therapeutic vaccines, gene therapy, cell immunotherapy, etc. However, no breakthroughs have been made. Until 2008, four therapeutic biological agents that had undergone phase III clinical trials in my country did not achieve the expected therapeutic effect. Cellular immunotherapy, mostly in vitro, induces DC cells through lymphokines to try to break the immune tolerance mechanism of hepatitis B patients, but the effect is not certain.

[0003] Recently, gene editing technology has enabled the patient's own cytotoxic T cells to have the ability to specifically bind to and eliminate the hepatitis B virus; iRNA technology, which aims to inhibit the transcription process of the hepatitis B virus and thus completely kill the virus, has entered the clinical trial stage; however, to date, the above therapies are far from the natural immune clearance after hepatitis B infection.

[0004] Most hepatitis B infections are naturally cleared by their own immune system. The most critical sign is the ability to produce anti-HBV surface antibodies. However, to date, there has been no breakthrough in this regard. In the past, the idea of ​​activating DCs in vitro in cellular immunotherapy did not achieve the desired effect. This is because we do not have a thorough understanding of the pathogenesis of hepatitis B, or have not studied the key points. So far, there is no ideal animal model for hepatitis B, and there are different understandings.

[0005] In theory, immunotherapy should be the most effective. The current main idea is to activate antiviral-related immune cells, such as TC, NK, LAK and B cells. Among them, the activation therapy of TC, NK and LAK has a 20-year history of application, but the effect is uncertain. However, the activation of B cells to produce protective antibodies against hepatitis B virus is still a blank. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the shortcomings of the prior art, the present invention provides a method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine, which has the advantages of more effectively activating B cells and producing protective antibodies against hepatitis B virus, and solves the problems mentioned in the above background technology.

[0008] (II) Technical solution

[0009] To achieve the above object, the present invention provides the following technical solution: a method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine, comprising the following steps:

[0010] 1) Animal grouping and dosage: Experimental animal grouping method: Male and female animals were stratified and randomly divided into 3 groups according to body weight: a positive control group of 4 animals, a low-dose cell therapy group of 4 animals, and a high-dose cell therapy group of 2 animals, half of which were males and half were females.

[0011] 2) Administration-related information and dosage: Administration route and method: Cells were reinfused by intravenous injection; Reasons for selecting the administration route: consistent with the clinical administration route; Administration frequency and duration: Each animal in the cell therapy group received a single cell infusion, and all animals were injected with hepatitis B vaccine 4 times.

[0012] 3) Dosage design: Because there is no relevant reference for this cell therapy method, based on the dosage recommended by the client, the human treatment dose for a person weighing 60 kg is 1x108 cells per dose. It is estimated that the dosage for crab-eating macaques in this experiment should be: 1.6x106cells / kg. Since this is a preliminary experiment for toxicology research, a small-dose group is equivalent to a single human treatment dose, and a large-dose group is 5 times the small-dose group.

[0013] 4) Method for clearing HBsAg by in vitro culture of gene-induced lymphocytes:

[0014] Experimental method: in vitro cell culture method; cell source: animals in the cell therapy group; cell treatment: non-viral vector gene induction; positive control: derived from PBMC in the positive control group; negative control: derived from PBMC in the cell therapy group without gene induction.

[0015] 5) Serum HBsAb production after autologous gene-induced lymphocyte infusion:

[0016] Blood collection animals: all animals; Blood collection time: before administration, 2nd, 4th and 8th week after the first administration; Blood collection site: animal forelimb or hindlimb vein; Blood collection volume and anticoagulant: about 1 mL of blood was collected using an inert separation gel coagulation tube (combined with the determination of blood biochemical indicators in long-term toxicity tests, not collected separately).

[0017] 6) Peripheral blood T and B lymphocyte phenotype determination: Blood collection animals: all animals; Blood collection time: before administration, D15 and D24 after the first immunization; Blood collection site: animal forelimb or hindlimb vein; Blood collection volume and anticoagulant: about 1 mL of blood was collected using EDTA·K2 vacuum blood collection tubes; Sample identification: each sample was marked with the subject number, animal number, test day, sample type and collection date; Sample storage and transportation: blood samples were placed at room temperature after collection and sent to the Immunology Department for testing within 2 hours.

[0018] Preferably, on the fifth day of culturing in a medium containing 2 ng / ml of hepatitis B vaccine, the HBsAg titer in the supernatant was detected, and the gene-induced lymphocytes of animal No. 1001 showed a negative result after dilution of 1 / 16, and the non-gene-induced autologous lymphocytes turned negative only at 1 / 64, as shown in Table 3. On the tenth day of culturing in a medium containing 4 ng / ml of hepatitis B vaccine, the HBsAg titer in the supernatant was detected, and the gene-induced lymphocytes of animal No. 2003 showed negative holes at 1 / 256 dilution, and completely turned negative at 1 / 1024, while the non-gene-induced autologous lymphocytes had no negative holes.

[0019] Preferably, after the in vitro gene-induced lymphocytes are autologously infused, the initial inhibitory effect is manifested as a delay in antibody production time, and the serum HBsAb level after 1 and 2 immunizations is lower than that of the positive gene template donor animals; by 3 and 4 immunizations, the HBsAb level of the gene-induced lymphocyte autologous infusion animals increases in a leap-like manner, which is significantly higher than that of the positive gene template donor animals. The inhibitory effect stage of the high-dose group is slightly stronger than that of the low-dose group, and there is no significant difference between the high-dose and low-dose groups in the enhancement of HBsAb jump-like antibody secretion after 3 immunizations.

[0020] Preferably, the phenotype of T / B lymphocytes (CD3 / CD4, CD3 / CD8 and CD20) was compared before administration (D0) and at various time points after cell reinfusion, and compared with itself and with each group. Except for a slight decrease in CD3 in the low-dose group, there was no similar change in the high-dose group. The total analysis of the T / B lymphocyte phenotype test results showed no significant changes in the animals of each group.

[0021] Preferably, the administration-related information and dosage recovery period: none; administration time: 09:03-15:49; administration volume: 10 mL / animal; administration rate: 3 mL / min.

[0022] Preferably, the culture medium is a lymphocyte-specific culture medium containing 2 ng / ml and 4 ng / ml of hepatitis B vaccine; culture conditions: 50,000 cells / well, duplicate wells, inoculation of 96-well plates, 37° C., 5% CO2, and total culture for 10 days.

[0023] Preferably, the storage and transportation of the sample: the blood is placed in a sample transport box after collection and sent to the clinical inspection department for testing within 2 hours; the determination method: a finished ELISA kit is used, and an OD value > 0.1 is positive.

[0024] Preferably, the sample is collected by centrifuging at D5 and D10 respectively and collecting the supernatant; the detection index is HBsAg titer; the detection method is using a finished ELISA kit.

[0025] Preferably, the detection indicators are: peripheral blood T cells (CD3+), T cell subsets (CD3+CD4+, CD3+CD8+), B cells (CD3-CD20+); detection method: flow cytometer.

[0026] (III) Beneficial effects

[0027] Compared with the prior art, the present invention provides a method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine, which has the following beneficial effects:

[0028] The method for testing the antibody secretion effect of hepatitis B antibody positive B cells on hepatitis B vaccine is to test that after intravenous injection of autologous gene-induced lymphocytes into crab-eating macaques, the response to conventional hepatitis B vaccine is weakened. First, there is an inhibitory effect, which is manifested as a delay in antibody production time and a weakened titer compared with the positive control group. The possible reasons are as follows: ① Gene-induced lymphocytes have the effect of clearing the corresponding antigens of the hepatitis B vaccine, resulting in a decrease in the effective immune dose and a weakened antibody production response. ② Gene induction and the natural corresponding gene effects inhibit each other. As time and the number of immunizations increase, after 3 immunizations (about 1 month after reinfusion), the antibody production response of the lymphocyte autologous reinfusion animals increases in a leap-like manner, and is even significantly higher than the positive control group. The present invention does not have viral vector gene transcription or other vector gene editing methods, but has the effect of activating antibody-secreting B cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the structure of 1106 and 1002 of the present invention in comparison;

[0030] Figure 2 This is a schematic diagram of the comparative structure of the present invention 2018 and 2004;

[0031] Figure 3 It is a schematic diagram of the structure of 1005 and 9 of the present invention;

[0032] Figure 4 This is a schematic diagram of the structure of 2018 and 10 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] according to Figure 1-4 As shown in Table 2-6, a method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine comprises the following steps:

[0035] 1) Animal grouping and dosage: Experimental animal grouping method: Male and female animals were stratified and randomly divided into 3 groups according to body weight: a positive control group of 4 animals, a low-dose cell therapy group of 4 animals, and a high-dose cell therapy group of 2 animals, half of which were males and half were females.

[0036] 2) Administration-related information and dosage: Administration route and method: Cells were reinfused by intravenous injection; Reasons for selecting the administration route: consistent with the clinical administration route; Administration frequency and duration: Each animal in the cell therapy group received a single cell infusion, and all animals were injected with hepatitis B vaccine 4 times.

[0037] 3) Dosage design: Because there is no relevant reference for this cell therapy method, based on the dosage recommended by the client, the human treatment dose for a person weighing 60 kg is 1x108 cells per dose. It is estimated that the dosage for crab-eating macaques in this experiment should be: 1.6x106cells / kg. Since this is a preliminary experiment for toxicology research, a small-dose group is equivalent to a single human treatment dose, and a large-dose group is 5 times the small-dose group.

[0038] 4) Method for clearing HBsAg by in vitro culture of gene-induced lymphocytes:

[0039] Experimental method: in vitro cell culture method; cell source: animals in the cell therapy group; cell treatment: non-viral vector gene induction; positive control: derived from PBMC in the positive control group; negative control: derived from PBMC in the cell therapy group without gene induction.

[0040] 5) Serum HBsAb production after autologous gene-induced lymphocyte infusion:

[0041] Blood collection animals: all animals; Blood collection time: before administration, 2nd, 4th and 8th week after the first administration; Blood collection site: animal forelimb or hindlimb vein; Blood collection volume and anticoagulant: about 1 mL of blood was collected using an inert separation gel coagulation tube (combined with the determination of blood biochemical indicators in long-term toxicity tests, not collected separately).

[0042] 6) Peripheral blood T and B lymphocyte phenotype determination: Blood collection animals: all animals; Blood collection time: before administration, D15 and D24 after the first immunization; Blood collection site: animal forelimb or hindlimb vein; Blood collection volume and anticoagulant: about 1 mL of blood was collected using EDTA·K2 vacuum blood collection tubes; Sample identification: each sample was marked with the subject number, animal number, test day, sample type and collection date; Sample storage and transportation: blood samples were placed at room temperature after collection and sent to the Immunology Department for testing within 2 hours.

[0043] On the fifth day of culture in the medium containing 2ng / ml of hepatitis B vaccine, the HBsAg titer in the supernatant was detected. The gene-induced lymphocytes of animal No. 1001 showed negative results after dilution of 1 / 16, while the non-gene-induced autologous lymphocytes turned negative only at 1 / 64, as shown in Table 3. On the tenth day of culture in the medium containing 4ng / ml of hepatitis B vaccine, the HBsAg titer in the supernatant was detected. The gene-induced lymphocytes of animal No. 2003 showed negative holes at 1 / 256 dilution, and turned completely negative at 1 / 1024, while the non-gene-induced autologous lymphocytes had no negative holes.

[0044] After the in vitro gene-induced lymphocytes were autologously infused, the initial inhibitory effect was manifested as a delay in antibody production. The serum HBsAb levels after the first and second immunizations were lower than those of the positive gene template donor animals. By the third and fourth immunizations, the HBsAb levels of the gene-induced lymphocyte autologous infusion animals increased in a leap-like manner, significantly higher than those of the positive gene template donor animals. The inhibitory effect of the high-dose group was slightly stronger than that of the low-dose group, and there was no significant difference between the high-dose and low-dose groups in the enhancement of HBsAb secretion after the third immunization.

[0045] The phenotypes of T / B lymphocytes (CD3 / CD4, CD3 / CD8 and CD20) were compared before administration (D0) and at various time points after cell transfusion. The comparisons with each other and with each group showed that, except for a slight decrease in CD3 in the low-dose group, there were no similar changes in the high-dose group. The total analysis of the T / B lymphocyte phenotype test results showed no significant changes in the animals of each group.

[0046] Administration-related information and dosage recovery period: None; administration time: 09:03-15:49; administration volume: 10 mL / mouse; administration rate: 3 mL / min.

[0047] Culture medium: lymphocyte-specific culture medium, containing hepatitis B vaccine 2ng / ml and 4ng / ml; culture conditions: 50,000 cells / well, duplicate wells, inoculated in 96-well plates, 37°C, 5% CO2, and cultured for 10 days.

[0048] Sample storage and transportation: After blood is collected, it is placed in a sample transport box and sent to the clinical inspection department for testing within 2 hours; Determination method: Use a finished ELISA kit, and an OD value >0.1 is positive.

[0049] Specimen collection: centrifugation at D5 and D10, and collection of supernatant; detection index: HBsAg titer; detection method: using a finished ELISA kit.

[0050] Detection indicators: peripheral blood T cells (CD3+), T cell subsets (CD3+CD4+, CD3+CD8+), B cells (CD3-CD20+); detection method: flow cytometer.

[0051] The production of serum HBsAb after autologous gene-induced lymphocyte transfusion: After autologous transfusion of lymphocytes induced by in vitro gene, the initial inhibitory effect was manifested as a delay in antibody production. The serum HBsAb levels after 1 and 2 immunizations were lower than those of animals with positive gene template donors. By the 3rd and 4th immunizations, the HBsAb levels of animals with autologous gene-induced lymphocyte transfusion increased in a leap-like manner, which was significantly higher than that of animals with positive gene template donors. The inhibitory effect of the high-dose group was slightly stronger than that of the low-dose group. There was no significant difference in the enhancement of HBsAb secretion after 3 immunizations between the high-dose group and the low-dose group. Figure 1-4 , see attached picture)

[0052] Peripheral blood T and B lymphocyte phenotype: T / B lymphocyte (CD3 / CD4, CD3 / CD8 and CD20) phenotypes were compared before administration (D0) and at various time points after cell transfusion. The comparison between the two groups and between each group showed that, except for a slight decrease in CD3 in the low-dose group, there was no similar change in the high-dose group. The total analysis of T / B lymphocyte phenotype test results showed no significant changes in the animals of each group. The detailed results are shown in Tables 5-6.

[0053] Table 2. Groups and measurements

[0054]

[0055]

[0056] Table 3. HBsAg titer in supernatant on the fifth day of culture containing 2 ng / ml of hepatitis B vaccine

[0057]

[0058] Table 4. HBsAg titer in supernatant containing 4 ng / ml hepatitis B vaccine on day 10

[0059]

[0060]

[0061] Table 5. Changes of CD3 and CD20 cells after autologous gene-induced lymphocyte transfusion

[0062]

[0063] Table 6. Changes of CD4 and CD8 cells after autologous gene-induced lymphocyte transfusion

[0064]

[0065]

[0066] Conclusion: This experiment found that gene-induced lymphocytes cultured in vitro have the effect of clearing hepatitis B virus surface antigen, suggesting that they have the ability to specifically clear hepatitis B virus in clinical application.

[0067] Under the conditions of this experiment, the response of crab-eating macaques to conventional hepatitis B vaccine was weakened after intravenous injection of autologous gene-induced lymphocytes. The first was an inhibitory effect, which was manifested as a delay in antibody production and a decrease in titer compared with the positive control group. The possible reasons are as follows: ① Gene-induced lymphocytes have the function of clearing the corresponding antigens of the hepatitis B vaccine, resulting in a decrease in the effective immunization dose and a weakened antibody production response. ② Gene induction and the corresponding natural genes inhibit each other. As time and the number of immunizations increase, after the third immunization (about 1 month after reinfusion), the antibody production response of the animals with autologous lymphocyte reinfusion increased in a leap-like manner, and was even significantly higher than that of the positive control group. There is currently a lack of literature support for the explanation of this phenomenon. However, from the analysis of existing data, the reinfusion of gene-induced autologous lymphocytes did not cause obvious changes in T / B lymphocyte subsets, but only enhanced the antibody secretion function after immunization with hepatitis B vaccine, which means a breakthrough in clinical treatment. So far, no similar research has been reported.

[0068] This study used healthy subjects with positive anti-hepatitis B antibodies as induction templates. Unlike other gene editing technologies, this study did not involve viral vector gene transcription or other vector gene editing methods, but it has the effect of activating antibody-secreting B cells. The technical details are core secrets and will not be disclosed for the time being.

[0069] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine, characterized in that: The following steps are involved: 1) Animal grouping and dosage: Experimental animal grouping method: Male and female animals were randomly divided into 3 groups according to body weight: 4 animals in the positive control group, 4 animals in the low-dose cell therapy group, and 2 animals in the high-dose cell therapy group, with half males and half females; 2) Administration-related information and dosage: Administration route and method: Cells were reinfused by intravenous injection; Reasons for the selection of the administration route: consistent with the clinical administration route; Administration frequency and duration: Each animal in the cell therapy group received a single cell reinfusion, and all animals were injected with hepatitis B vaccine 4 times; 3) Dosage design: Assuming that the human therapeutic dose for a person weighing 60 kg is 1×108 cells per dose, the animal dose should be calculated to be 1.6×106 cells / kg. Since this is a preliminary experiment for toxicology research, the small dose group is equivalent to the single therapeutic dose for humans, and the large dose group is 5 times the small dose group; 4) Method for clearing HBsAg by in vitro culture of gene-induced lymphocytes: Experimental method: in vitro cell culture method; cell source: animals in the cell therapy group; cell treatment: non-viral vector gene induction; positive control: derived from PBMC in the positive control group; negative control: derived from PBMC in the cell therapy group without gene induction; 5) Serum HBsAb production after autologous gene-induced lymphocyte infusion: Blood collection animals: all animals; Blood collection time: before administration, 2, 4, and 8 weeks after the first administration; Blood collection site: animal forelimb or hindlimb vein; Blood collection volume and anticoagulant: about 1 mL of blood was collected using an inert separation gel coagulation tube; 6) Peripheral blood T and B lymphocyte phenotype determination: Blood collection animals: all animals; Blood collection time: before administration, D15 and D24 after the first immunization; Blood collection site: animal forelimb or hindlimb vein; Blood collection volume and anticoagulant: about 1 mL of blood was collected using EDTA·K2 vacuum blood collection tubes; Sample identification: each sample was marked with the subject number, animal number, test day, sample type and collection date; Sample storage and transportation: blood samples were placed at room temperature after collection and sent to the Immunology Department for testing within 2 hours; Detection indicators: peripheral blood T cell CD3+, T cell subpopulation CD3+CD4+CD3+CD8+, B cell CD3-CD20+; detection method: flow cytometer.

2. The method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine according to claim 1, characterized in that: On the fifth day of culture in a medium containing 2 ng / ml of hepatitis B vaccine, the HBsAg titer in the supernatant was detected. The gene-induced lymphocytes of animal No. 1001 turned negative after dilution of 1 / 16, while the non-gene-induced autologous lymphocytes turned negative only at 1 / 64; On the 10th day of culture in the medium containing 4 ng / ml of hepatitis B vaccine, the HBsAg titer in the supernatant was detected. The gene-induced lymphocytes of animal No. 2003 showed negative holes at a dilution of 1 / 256 and completely turned negative at 1 / 1024, while the non-genetically induced autologous lymphocytes showed no negative holes.

3. The method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine according to claim 1, characterized in that: After the in vitro gene-induced lymphocytes were autologously infused, the initial inhibitory effect was manifested as a delay in antibody production. The serum HBsAb levels after the first and second immunizations were lower than those of the animals with positive gene template donors. By the third and fourth immunizations, the HBsAb levels of the animals with gene-induced lymphocytes autologously infused increased in a leap-like manner, and were significantly higher than those of the animals with positive gene template donors. The inhibitory effect of the high-dose group was slightly stronger than that of the low-dose group. There was no significant difference in the enhancement of HBsAb jump antibody secretion between the high-dose and low-dose groups after 3 immunizations.

4. The method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine according to claim 1, characterized in that: The T / B lymphocyte CD3 / CD4, CD3 / CD8 and CD20 phenotypes were compared before administration and at various time points after cell reinfusion, and compared with each other and with each group. Except for a slight decrease in CD3 in the low-dose group, there was no similar change in the high-dose group. The total analysis of the T / B lymphocyte phenotype test results showed no significant changes in the animals of each group.

5. The method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine according to claim 1, characterized in that: The administration-related information and dosage recovery period: None; administration time: 09:03-15:49; administration volume: 10 mL / mouse; administration rate: 3 mL / min.

6. The method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine according to claim 1, characterized in that: Culture medium: lymphocyte-specific culture medium, containing hepatitis B vaccine 2ng / ml and 4ng / ml; culture conditions: 50,000 cells / well, duplicate wells, inoculated in 96-well plates, 37°C, 5% CO2, and cultured for 10 days.

7. The method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine according to claim 1, characterized in that: Sample storage and transportation: After blood is collected, it is placed in a sample transport box and sent to the clinical inspection department for testing within 2 hours; Determination method: Use a finished ELISA kit, and an OD value >0.1 is positive.

8. The method for testing the antibody secretion effect of hepatitis B antibody-positive B cells on hepatitis B vaccine according to claim 1, characterized in that: Specimen collection: centrifugation at D5 and D10, and collection of supernatant; detection index: HBsAg titer; detection method: using a finished ELISA kit.

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