CN114324889A - Vaccine quality control method, vaccine quality control reagent and application thereof - Google Patents

Vaccine quality control method, vaccine quality control reagent and application thereof Download PDF

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CN114324889A
CN114324889A CN202111453163.1A CN202111453163A CN114324889A CN 114324889 A CN114324889 A CN 114324889A CN 202111453163 A CN202111453163 A CN 202111453163A CN 114324889 A CN114324889 A CN 114324889A
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pathogenic bacteria
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张敬仁
王娟娟
安浩然
丁铭
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Tsinghua University
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Abstract

The invention relates to a vaccine quality control method, a vaccine quality control reagent and application thereof. The vaccine quality control method comprises the following steps: detecting the vaccine to be detected and determining the detection result, wherein the detection comprises antibody titer detection, pathogenic bacteria elimination half-life detection in blood, pathogenic bacteria capture rate in liver, pathogenic bacteria capture quantity detection in liver sinus cells and animal survival rate, and when the preliminary detection results of all the detection modes are the quality standard of the vaccine to be detected, the preliminary detection results are the indication that the final quality of the vaccine to be detected reaches the standard. The quality control method can track and quantify the immune protection effect of the vaccine on the host organism-organ-cell level, can evaluate the protection level of the vaccine quantitatively, quickly, accurately and comprehensively in real time, and has the advantage of high accuracy.

Description

Vaccine quality control method, vaccine quality control reagent and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a vaccine quality control method, a vaccine quality control reagent and application thereof, and more particularly relates to application of rapid and accurate evaluation of vaccine protection level by evaluating efficiency of pathogenic bacteria intercepted by liver in a blood stream infection experimental animal model.
Background
Preventive vaccination is one of the most important ten public health-affecting achievements of the last century, and has effectively controlled the spread of various infectious diseases worldwide, significantly reducing the mortality rate after infection. According to the statistics of the world health organization, the global mortality rate of children under 5 years of age decreased from 93/1000 people in 1990 to 39 in 2018, which is indistinguishable from the worldwide promotion of vaccination of children. As an effective strategy for controlling infectious diseases, preventive vaccination enables people to completely get rid of the trouble of the difficult problem of smallpox in the century in 1980, and diseases such as diphtheria, tetanus, pertussis, hepatitis B, poliomyelitis and the like are effectively prevented and controlled, so that two to three million people can be saved each year.
Vaccines are the most effective way to prevent and control infectious diseases. The accurate evaluation of the vaccine immune protection effect is a key link in the research and development and popularization and use processes of novel vaccines. However, the current vaccine evaluation index cannot accurately reflect the specific process of vaccine immunoprotection and the deep mechanism of the difference of the immune effect. Therefore, there is a need to develop a method for rapidly and precisely evaluating the protection level of a vaccine.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a vaccine quality control method, a vaccine quality control reagent and application thereof, and the method can quantitatively, quickly and accurately evaluate the vaccine protection level in real time.
It should be noted that the present invention is completed based on the following work of the inventors:
vaccines are the most effective way to prevent and control infectious diseases. The accurate evaluation of the vaccine immune protection effect is a key link in the research and development and popularization and use processes of novel vaccines, and the current method for evaluating the vaccine protection effect mainly depends on: 1) in vitro antibody titer and in vitro neutrophil opsonophagocytosis (HL60 sterilization test); 2) animal infection protection experiment. Although these judgment tools have played an important driving role in the development of streptococcus pneumoniae vaccines, they all have serious drawbacks. Among them, antibody titer detection only reflects the total antibody level generated by vaccination, and cannot be directed against antibody subpopulations with actual protective effects. The HL60 sterilization is an antibody functional detection method based on a human-derived neutrophil cell line HL60, which can reflect the antibody which can help the neutrophil phagocytosis sterilization in the serum, and has the defects that: 1) the HL60 cell line has long induced differentiation period, 2) low sensitivity, 3) can not reflect the function of other immune cells except the neutrophil; moreover, the reports that the survival rate of the infected animals can be effectively improved by the antibody without obvious effect in-vitro HL60 sterilization experiments are frequently available, which shows that the method can not truly reflect the protective efficacy of the vaccine. In addition, animal infection protection can demonstrate vaccine protection levels, but the reason (mechanism) for the development of immune protection is not revealed. Therefore, the current vaccine evaluation index cannot accurately reflect the specific process of vaccine immunoprotection and the deep mechanism of the difference of the immune effect.
Based on the above, the inventor carries out intensive research on a vaccine quality control method, and finds that the vaccine-induced immune protection sterilization process is mainly completed in the liver for the first time by taking gram-positive human pathogenic bacteria streptococcus pneumoniae and gram-negative human pathogenic bacteria klebsiella pneumoniae whole-bacterium inactivated vaccine and streptococcus pneumoniae polysaccharide combined vaccine as research objects; therefore, based on the findings, the inventor establishes a vaccine quality control method for accurately evaluating the protection level of the vaccine, and the method can track and quantify the immune protection effect of the vaccine on the host body-organ-cell level, so that the protection level of the vaccine can be quantitatively evaluated in real time, quickly and accurately.
In one aspect of the invention, the invention provides a vaccine quality control method. According to the embodiment of the invention, the vaccine quality control method comprises the following steps: step 1: the vaccine to be detected is detected in the following modes: mode 1: administering the vaccine to be detected to a subject, collecting the serum of the subject after the vaccine to be detected is administered, measuring the antibody titer corresponding to the vaccine to be detected in the serum, and preliminarily determining whether the quality of the vaccine to be detected reaches the standard or not based on the measurement result; mode 2: administering the vaccine to be tested for a predetermined time and then administering the pathogenic bacteria again, measuring the capture rate of the pathogenic bacteria in the liver of the subject after a period of time, and preliminarily determining whether the quality of the vaccine to be tested reaches the standard or not based on the measurement result; mode 3: administering the vaccine to be tested to the subject again with lethal dose of the pathogenic bacteria of the subject after a predetermined time, measuring the survival rate of the subject, and preliminarily determining whether the quality of the vaccine reaches the standard or not based on the measurement result; and step 2: determining whether the final quality of the vaccine to be detected reaches the standard or not based on the primary detection results of all detection modes in the step 1; and the preliminary detection results of all the detection modes are that the quality of the vaccine to be detected reaches the standard, and the preliminary detection results are an indication that the final quality of the vaccine to be detected reaches the standard.
The inventor finds out through a large number of experiments that the vaccine-induced immunoprotective bactericidal process is mainly completed in the liver. Therefore, the inventor can track and quantify the immune protection effect of the vaccine on the host organism-organ-cell level by detecting the capture rate of pathogenic bacteria in the liver of a subject (immune animal) to which the vaccine to be detected is applied and detecting the serum antibody titer and the infection protection index of the immune animal in combination, based on the detection results of the three modes, so that the protection level of the vaccine can be evaluated quantitatively, rapidly and accurately in real time, and the method has the advantage of high accuracy.
According to the embodiment of the invention, the vaccine quality control method can also have the following additional technical characteristics:
according to an embodiment of the present invention, step 1 further comprises at least one of the following detection modes: mode 4: administering the pathogenic bacteria again after the subject is administered with the vaccine to be detected for a preset time, recording the change of the loading capacity of the pathogenic bacteria in the blood of the subject within the preset time after the subject is administered with the pathogenic bacteria, determining the elimination half-life of the pathogenic bacteria, and preliminarily determining whether the quality of the vaccine to be detected reaches the standard or not based on the determination result; mode 5: and administering the vaccine to be tested again after a preset time, measuring the capture quantity of the pathogenic bacteria in the hepatic sinus cells of the tested person after a period of time, and preliminarily determining whether the quality of the vaccine to be tested reaches the standard or not based on the measurement result.
According to an embodiment of the invention, the sinusoidal hepatocytes are selected from kupffer cells and sinusoidal hepatocytes.
According to an embodiment of the present invention, the mode 1 includes the following steps: experimental groups: administering the vaccine to be tested to a subject, collecting the serum of the subject after the vaccine to be tested is administered, and determining the titer of the antibody generated by the vaccine to be tested in the serum and directed to a specific antigen; control group: administering an adjuvant equal in volume to the vaccine to be tested to a subject, collecting the serum of the subject after the adjuvant is given, and determining the titer of the antibody generated by the vaccine to be tested in the serum and directed to a specific antigen; and when the antibody titer of the experimental group is significantly different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard.
According to an embodiment of the present invention, the mode 2 includes the following steps: experimental groups: administering the vaccine to be tested for a predetermined time and then again administering the pathogenic bacteria to the subject, collecting the liver of the subject after administering the vaccine to be tested for a period of time, and measuring the capture rate of the pathogenic bacteria in the liver of the subject; control group: administering an adjuvant with the same volume as the vaccine to be tested to a subject, administering pathogenic bacteria again after a preset time, collecting the liver of the subject after the vaccine to be tested is administered after a period of time, and measuring the capture rate of the pathogenic bacteria in the liver of the subject; and when the capture rate of the experimental group is significantly different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard.
According to an embodiment of the present invention, the mode 3 includes the following steps: experimental groups: administering a second lethal dose of said pathogenic bacteria to the subject a predetermined time after administering said test vaccine to the subject, and determining the survival of the subject; control group: administering to the subject a lethal dose of said pathogenic bacteria to the subject a predetermined time after administering an equal volume of adjuvant to the subject to be tested, and determining the survival rate of the subject; and when the survival rate of the experimental group is remarkably different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard.
According to an embodiment of the present invention, the mode 4 includes the following steps: experimental groups: administering the pathogenic bacteria again after the subject is administered with the vaccine to be detected for a preset time, recording the change of the loading capacity of the pathogenic bacteria in the blood of the subject within the preset time after the administration of the pathogenic bacteria, and counting the elimination half-life of the pathogenic bacteria in the blood; control group: administering the pathogen again after the subject is administered with the adjuvant with the same volume as the vaccine to be detected for a preset time, recording the change of the loading capacity of the pathogen in the blood of the subject in the preset time after the pathogen is administered, and counting the elimination half-life of the pathogen in the blood; and when the elimination half-life period of the experimental group is obviously different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard.
According to an embodiment of the present invention, the mode 5 includes the following steps: experimental groups: administering the vaccine to the subject a second time after a predetermined period of time, and determining the number of antral cells in the liver of the subject capturing the pathogen after a period of time; control group: administering the pathogen again to the subject after a predetermined time with an adjuvant of the same volume as the vaccine to be tested, and measuring the capture amount of the pathogen by the antral cells in the liver of the subject after a period of time; and when the capturing quantity of the liver sinus cells in the liver of the experimental group to the pathogenic bacteria is obviously different from the capturing quantity of the liver sinus cells in the liver of the control group to the pathogenic bacteria, preliminarily determining that the quality of the vaccine to be detected reaches the standard.
According to an embodiment of the present invention, the period of time in the mode 2 is 20 to 40 minutes.
According to an embodiment of the invention, the predetermined time for recording the change in the pathogen load in mode 4 is 20-45 minutes.
According to an embodiment of the invention, the vaccine comprises a gram positive bacterial vaccine and/or a gram negative bacterial vaccine.
According to an embodiment of the invention, the vaccine includes, but is not limited to, a streptococcus pneumoniae vaccine and/or a klebsiella pneumoniae vaccine.
According to embodiments of the present invention, the vaccine includes, but is not limited to, whole-cell inactivated vaccines and/or polysaccharide conjugate vaccines.
In another aspect of the invention, the invention provides the use of an agent in the preparation of a kit for vaccine quality control. According to an embodiment of the invention, the agent is selected from the group consisting of an agent capable of detecting the number of pathogenic bacteria in the antral cells of the subject after a predetermined time of administration of the vaccine to the subject and a period of time after which the pathogenic bacteria are administered again. Therefore, the reagent in the kit can detect the number of the pathogenic bacteria in the hepatic sinus cells of the immunized animal, and can accurately evaluate the immune protection effect of the vaccine to be detected. Therefore, the kit can be used for quality control of vaccines and has the advantage of high quality control accuracy.
According to an embodiment of the invention, the agent is selected from the group consisting of an agent capable of detecting the number of pathogenic bacteria in sinusoidal endothelial cells and kupffer cells of the subject after a predetermined time of administration of the vaccine to the subject and a subsequent administration of the pathogen after a period of time.
According to an embodiment of the invention, the vaccine comprises a gram positive bacterial vaccine and/or a gram negative bacterial vaccine.
According to an embodiment of the invention, the vaccine comprises a streptococcus pneumoniae vaccine and/or a klebsiella pneumoniae vaccine.
According to embodiments of the present invention, the vaccine includes, but is not limited to, whole-cell inactivated vaccines and/or polysaccharide conjugate vaccines.
In yet another aspect of the invention, the invention provides the use of an agent that increases the activity of sinusoidal endothelial cells and kupffer cells in increasing the protective effect of a vaccine. The inventor finds that the reagent is used for improving the activity of hepatic sinus endothelial cells and kupffer cells in a body of a user through experiments, the utilization rate of the liver of the user to the vaccine can be improved, and the protective effect of the vaccine is further improved.
According to an embodiment of the invention, the vaccine comprises a gram positive bacterial vaccine and/or a gram negative bacterial vaccine.
According to an embodiment of the invention, the vaccine comprises a streptococcus pneumoniae vaccine and/or a klebsiella pneumoniae vaccine.
According to embodiments of the present invention, the vaccine includes, but is not limited to, whole-cell inactivated vaccines and/or polysaccharide conjugate vaccines.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
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The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a flow chart of a vaccine evaluation system in one embodiment of the present invention;
FIG. 2 is a flow chart of a vaccine evaluation system in one embodiment of the present invention;
FIG. 3 shows the result of detecting the titer of specific IgG antibody against an antigen of interest in the serum of a mouse according to example 2 of the present invention;
FIG. 4 shows the result of the test for clearance of Streptococcus pneumoniae from blood of mice according to example 3 of the present invention;
FIG. 5 shows the result of the detection of the clearance of Klebsiella pneumoniae from the blood of the mouse in example 3 of the present invention;
FIG. 6 shows the result of the test for clearance of Streptococcus pneumoniae from blood of mice according to example 3 of the present invention;
FIG. 7 shows the capture of Streptococcus pneumoniae by the liver of the mouse of example 4 of the present invention;
FIG. 8 shows the capture of Klebsiella pneumoniae by the liver of the mouse of example 4;
FIG. 9 shows the capture of Streptococcus pneumoniae by the liver of the mouse of example 4 of the present invention;
FIG. 10 shows the capture of Streptococcus pneumoniae by mouse liver sinus cells of example 5 of the present invention;
FIG. 11 shows the capture of Klebsiella pneumoniae by mouse liver sinusoidal cells of example 5 of the present invention;
FIG. 12 shows the capture of Streptococcus pneumoniae by mouse liver sinus cells of example 5 of the present invention;
FIG. 13 is a graph showing the survival rate of mice infected with Streptococcus pneumoniae of example 6 of the present invention;
FIG. 14 shows the survival rate of mice infected with Klebsiella pneumoniae according to example 6 of the present invention;
FIG. 15 is a graph showing the survival rate of mice infected with Streptococcus pneumoniae of example 6 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
The invention provides a vaccine quality control method, a vaccine quality control reagent and application thereof, which are respectively described in detail below.
Vaccine quality control method
In one aspect of the invention, the invention provides a vaccine quality control method. According to an embodiment of the present invention, as shown in fig. 1, the vaccine quality control method includes:
step 1: detecting the vaccine to be detected in the following way:
mode 1, antibody titer detection: administering a vaccine to be detected to a subject, collecting serum of the subject after the vaccine to be detected is administered, determining the antibody titer corresponding to the vaccine to be detected in the serum, and preliminarily determining whether the quality of the vaccine to be detected reaches the standard or not based on the determination result;
mode 2, detection of liver bacterial capture rate: administering the vaccine to be detected for a preset time and then administering the pathogenic bacteria again, measuring the capture rate of the pathogenic bacteria in the liver of the subject after a period of time, and preliminarily determining whether the quality of the vaccine to be detected reaches the standard or not based on the measurement result;
mode 3, detection of animal infection protection rate: and (3) administering the vaccine to be tested to the subject again after a preset time, determining the survival rate of the subject, and preliminarily determining whether the quality of the vaccine reaches the standard or not based on the determination result.
The inventor finds out through a large number of experiments that the vaccine-induced immunoprotective bactericidal process is mainly completed in the liver. Therefore, the inventor can track and quantify the immune protection effect of the vaccine on the host organism-organ-cell level by detecting the capture rate of pathogenic bacteria in the liver of a subject (immune animal) to which the vaccine to be detected is applied and combining the detection results of serum antibody titer and animal infection protection indexes in the immune animal.
It should be noted that, the process of administering the vaccine to be tested to the subject is to construct an immune animal model, and the specific operation steps are not limited, and the process of administering the vaccine to be tested to the subject is an immunization procedure known in the art.
In the present invention, all of the applied pathogens are active pathogens.
Step 2: determining a detection result: determining whether the final quality of the vaccine to be detected reaches the standard or not based on the primary detection results of all the detection modes in the step 1; the initial detection results of all detection modes are the quality standard of the vaccine to be detected, and are the indication of the final quality standard of the vaccine to be detected.
According to the embodiment of the invention, when all the detection results in the step 1 are the quality standards of the vaccine to be detected, the later protection effect of the vaccine to be detected can be ensured, so that the quality control method can comprehensively detect the vaccine, can evaluate the protection level of the vaccine quantitatively, quickly and accurately in real time, and has the advantage of high accuracy.
According to an embodiment of the present invention, step 1 further comprises at least one of the following detection modes:
mode 4, detection of the half-life of the blood stream bacteria clearance: administering the pathogenic bacteria again after the tested vaccine is administered to the subject for a preset time, recording the change of the pathogenic bacteria load in the blood of the subject within the preset time after the pathogenic bacteria are administered, determining the elimination half-life of the pathogenic bacteria, and preliminarily determining whether the quality of the tested vaccine reaches the standard or not based on the determination result;
mode 5, localization detection of bacteria on the surface of liver sinus cells: and (3) administering the vaccine to be tested again after a preset time, measuring the capture quantity of the pathogenic bacteria in the hepatic sinus cells of the tested person after a period of time, and preliminarily determining whether the quality of the vaccine to be tested reaches the standard or not based on the measurement result.
The inventor finds that, through a large number of experiments, when the elimination half-life period of pathogenic bacteria in the blood (also referred to as blood flow) of an immunized animal and/or the capture amount of pathogenic bacteria by liver antrum cells are detected, the accuracy of evaluating the protection effect of a vaccine to be tested can be further improved, so that the situation that the evaluation on the protection effect of the vaccine is too high and the immunity of a later-stage vaccinee is too low is avoided, the situation that the evaluation on the protection effect of the vaccine is too low and the protection effect of the vaccine cannot be truly reflected is also avoided, and the accuracy of quality control of the vaccine is further improved.
It should be noted that the standard for determining whether the final quality of the vaccine to be detected reaches the standard in the present invention is that all detection modes in step 1 reach the standard, that is, the subsequent mode 4 and/or mode 5 also reach the standard, and can be used as the result for determining that the final quality of the vaccine to be detected reaches the standard.
Illustratively, the vaccine quality control method of the present invention may be mode 1, mode 2, or mode 3.
Illustratively, the vaccine quality control method of the present invention may be a mode 1, a mode 2, a mode 3, or a mode 4.
Illustratively, the vaccine quality control method of the present invention may be a mode 1, a mode 2, a mode 3, or a mode 5.
Illustratively, as shown in fig. 2, the vaccine quality control method of the present invention may be a method 1, a method 2, a method 3, a method 4, or a method 5.
According to an embodiment of the present invention, in step 1, mode 2, mode 3, mode 4 and mode 5 detections are all performed after mode 1 detection. The detection sequences of the modes 2, 3, 4 and 5 are not limited to the front and the back, and may be performed simultaneously or in any sequence.
According to an embodiment of the present invention, the antral hepatocytes are selected from kupffer cells and antral endothelial cells (also referred to herein as "endothelial cells"). The inventors have found through extensive experiments that the pathogenic bacteria after immunization (especially "streptococcus pneumoniae" and "klebsiella pneumoniae") are mainly captured by the hepatic sinus cells in the liver. Therefore, the inventors have examined the number of trapped pathogenic bacteria in the antral cells of the liver to evaluate the protective effect of the vaccine more accurately.
It should be noted that the term "hepatic sinus cell" as used herein refers to a cell located in a hepatic sinus (also referred to as "hepatic sinus").
According to an embodiment of the present invention, mode 1 includes the steps of: experimental groups: administering a vaccine to be detected to a subject, collecting the serum of the subject after the vaccine to be detected is administered, and measuring the antibody titer corresponding to the vaccine to be detected in the serum; control group: administering an adjuvant with the same volume as the vaccine to be detected to a subject, collecting the serum of the subject given the adjuvant, and measuring the antibody titer corresponding to the vaccine to be detected in the serum; and when the antibody titer of the experimental group is obviously different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard. Thus, by the above steps, the antibody titer in the blood of the subject administered with the test vaccine can be detected, wherein the antibody is used as the cell secretion, and the cell level is used as one of the indexes for evaluating the vaccine protection effect.
It should be noted that the term "significant difference" as used herein refers to the evaluation of data difference on Statistics (staticiscs), and significant difference includes significant difference or extremely significant difference. Wherein, the experimental result reaches 0.05 level or 0.01 level, which means that the difference between the data is significant or the difference is extremely significant.
The phrase "the antibody titer of the experimental group is significantly different from the antibody titer of the control group" means that the antibody titer of the experimental group is significantly improved as compared with the antibody titer of the control group. Wherein, the higher the titer, the higher the level of antibodies generated in the organism against the target antigen, which indicates that the vaccine effect of the experimental group is better.
It is noted that the adjuvant of the present invention can be selected from the group consisting of vaccine and pathogen free solutions, including but not limited to water, sodium chloride buffer.
According to an embodiment of the present invention, mode 2 includes the following steps: experimental groups: administering the to-be-detected vaccine to the subject again after a preset time, collecting the liver of the subject after administration of the to-be-detected vaccine after a period of time, and measuring the capture rate of the pathogenic bacteria in the liver of the subject; control group: administering the adjuvant with the same volume as the vaccine to be detected for a preset time, then administering the pathogenic bacteria again, collecting the liver of the subject after the vaccine to be detected is administered after a period of time, and measuring the capture rate of the pathogenic bacteria in the liver of the subject; and when the capture rate of the experimental group is obviously different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard. Thus, by adopting the steps, the capture rate of pathogenic bacteria in the liver of a subject to be administered with the vaccine to be tested can be detected, wherein the liver is used as a body organ, and the organ level is used as one index for evaluating the protective effect of the vaccine.
The phrase "the capture rate of the experimental group is significantly different from the capture rate of the control group" means that the capture rate of the experimental group is significantly higher than the capture rate of the control group. Wherein, the higher the capture rate is, the higher the capture effect of the liver on pathogenic bacteria is, the better the vaccine effect of the experimental group is.
According to an embodiment of the present invention, mode 3 includes the steps of: experimental groups: administering the vaccine to the subject for a predetermined period of time and then administering a lethal dose of the pathogenic bacteria to the subject again to determine the survival rate of the subject; control group: administering the adjuvant with the same volume as the vaccine to be tested to the subject for a preset time, then administering the pathogenic bacteria with lethal dose to the subject again, and determining the survival rate of the subject; and when the survival rate of the experimental group is obviously different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard. By adopting the steps, the survival rate of the subject to be administered with the vaccine to be detected can be detected, and the organism level is used as one index of the evaluation of the vaccine protection effect.
The phrase "the survival rate of the experimental group is significantly different from that of the control group" means that the survival rate of the experimental group is significantly improved as compared with that of the control group. Wherein, the higher the survival rate, the better the protective effect of the vaccine.
According to an embodiment of the present invention, mode 4 includes the following steps: experimental groups: after the tested vaccine is applied to the testee for a preset time, the pathogenic bacteria are applied again, the change of the pathogenic bacteria load in the blood of the testee in the preset time after the pathogenic bacteria are applied is recorded, and the elimination half-life of the pathogenic bacteria in the blood is counted; control group: after the subject is administered with the adjuvant with the same volume as the vaccine to be detected for a preset time, the pathogenic bacteria are administered again, the change of the pathogenic bacteria load in the blood of the subject in the preset time after the pathogenic bacteria are administered is recorded, and the elimination half-life of the pathogenic bacteria in the blood is counted; and when the elimination half-life period of the experimental group is obviously different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard. Thus, the above steps can be used to detect the elimination half-life of pathogenic bacteria in the blood of a subject to whom a test vaccine is administered, as one of the indexes for evaluating the vaccine protection effect.
The phrase "the clearance half-life of the experimental group is significantly different from that of the control group" means that the clearance half-life of the experimental group is significantly reduced as compared with that of the control group. Wherein, the lower the elimination half-life, the faster the elimination of pathogens, indicating the better vaccine efficacy of the experimental group.
According to an embodiment of the present invention, mode 5 includes the following steps: experimental groups: administering the vaccine to be tested to the subject for a predetermined time, then administering the pathogenic bacteria again, and measuring the capture quantity of the liver sinusoidal cells in the liver of the subject to the pathogenic bacteria after a period of time; control group: administering the adjuvant with the same volume as the vaccine to be detected to the subject for a preset time, then administering the pathogenic bacteria again, and measuring the capture quantity of the hepatic sinus cells in the liver of the subject to the pathogenic bacteria after a period of time; and when the capturing quantity of the liver sinus cells in the liver of the experimental group to the pathogenic bacteria is obviously different from the capturing quantity of the liver sinus cells in the liver of the control group to the pathogenic bacteria, preliminarily determining that the quality of the vaccine to be detected reaches the standard. Thus, the number of trapped pathogenic bacteria in the antral cells of a subject administered a test vaccine can be detected by the above procedure, which is one of the indicators for evaluating the protective effect of the vaccine at the cellular level.
The phrase "the number of captured samples in the experimental group is significantly different from the number of captured samples in the control group" means that the number of captured samples in the experimental group is significantly increased as compared with the number of captured samples in the control group. Wherein, the larger the capture amount is, the better the capture effect of the antral cells on pathogenic bacteria is, which indicates that the vaccine effect of the experimental group is better.
According to an embodiment of the present invention, the period of time in mode 2 is 20 to 40 minutes (e.g., 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes). The inventors have obtained the above-mentioned excellent detection time by a large number of experiments, and thus have been able to accurately detect the capture rate of pathogenic bacteria in the liver of a subject to whom a vaccine to be tested is administered. If the detection time is too early (e.g., within 20 minutes), the vaccine may not be sufficiently effective in the liver; if the detection time is too late (e.g. after 40 minutes), the capture of pathogenic bacteria in the liver is substantially complete, and the capture efficiency does not significantly improve with increasing detection time.
According to an embodiment of the present invention, the predetermined time for recording the change in the pathogen load in mode 4 is 20 to 45 minutes (e.g., 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes). The inventor obtains the excellent detection time through a large number of tests, thereby accurately detecting the pathogenic bacteria elimination half-life in the blood of a subject to which the vaccine to be detected is applied.
According to an embodiment of the invention, the vaccine comprises a gram positive bacterial vaccine and/or a gram negative bacterial vaccine. Therefore, the vaccine quality control method can accurately evaluate the protection effect of the vaccine.
The vaccine quality control method of the present application is applicable to both gram-positive and gram-negative vaccines, and the types of pathogenic bacteria are not particularly limited.
Illustratively, vaccines in the present application include, but are not limited to, streptococcus pneumoniae vaccines and/or klebsiella pneumoniae vaccines. Therefore, the vaccine quality control method can accurately evaluate the protection effect of the vaccine.
According to embodiments of the present invention, the vaccine includes, but is not limited to, whole killed vaccines (also referred to herein as "killed vaccines" or "whole killed vaccines") and/or polysaccharide conjugate vaccines (also referred to herein as "conjugate vaccines" or "polysaccharide conjugate vaccines").
According to an embodiment of the present invention, when the vaccine is a whole-cell inactivated vaccine, the sinusoidal hepatocytes in mode 5 are selected from kupffer cells.
According to an embodiment of the present invention, when the vaccine is a polysaccharide conjugate vaccine, the sinusoidal hepatocytes in mode 5 are selected from sinusoidal endothelial cells.
Application of reagent in preparation of kit for vaccine quality control
In another aspect of the invention, the invention provides the use of an agent in the preparation of a kit for vaccine quality control. According to an embodiment of the invention, the agent is selected from the group consisting of an agent capable of detecting the number of pathogenic bacteria in the antral cells of the subject after a predetermined time of administration of the vaccine to the subject and a period of time after which the pathogenic bacteria are administered again. The inventor finds out through a large number of experiments that the number of pathogenic bacteria in the hepatic sinus cells of the immunized animals can be detected through the reagent in the kit, so that the immune protection effect of the vaccine to be detected can be accurately evaluated. Therefore, the kit can be used for quality control of vaccines and has the advantage of high quality control accuracy.
According to an embodiment of the invention, the agent is selected from the group consisting of an agent capable of detecting the number of pathogenic bacteria in sinusoidal endothelial cells and kupffer cells of a subject administered the vaccine to the subject a predetermined time after which the pathogenic bacteria are administered again. The inventor finds out through experiments that pathogenic bacteria (especially streptococcus pneumoniae and klebsiella pneumoniae) after immunization are mainly captured by hepatic sinus cells in the liver. Therefore, the reagent has higher detection accuracy for the capture quantity of pathogenic bacteria in the hepatic sinus cells.
The reagent contains a fluorescent-labeled antibody for labeling antral cells and pathogenic bacteria in a subject.
Illustratively, the reagent comprises at least one of FITC, AF594 anti-CD31, AF647 anti-F4/80 and PE anti-Ly6G, wherein FITC is used for labeling streptococcus pneumoniae or Klebsiella pneumoniae, AF594 anti-CD31 is used for labeling endothelial cells of liver sinuses, AF647 anti-F4/80 is used for labeling Kupffer cells, and PE anti-Ly6G is used for labeling neutrophils.
According to an embodiment of the invention, the vaccine comprises a gram positive bacterial vaccine and/or a gram negative bacterial vaccine. Thus, the protective effect of the vaccine can be accurately evaluated by using the kit of the present invention.
It should be noted that the kit of the present application is applicable to both gram-positive bacterial vaccines and gram-negative bacterial vaccines, and the types of pathogenic bacteria are not particularly limited.
Illustratively, vaccines in the present application include, but are not limited to, streptococcus pneumoniae vaccines and/or klebsiella pneumoniae vaccines. Thus, the protective effect of the vaccine can be accurately evaluated by using the kit of the present invention.
Use of agents to improve vaccine protection
In yet another aspect of the invention, the invention provides the use of an agent that increases the activity of sinusoidal endothelial cells and kupffer cells in increasing the protective effect of a vaccine. The inventor finds that the reagent is used for improving the activity of hepatic sinus endothelial cells and kupffer cells in a body of a user through experiments, the utilization rate of the liver of the user to the vaccine can be improved, and the protective effect of the vaccine is further improved.
According to an embodiment of the invention, the vaccine comprises a gram positive bacterial vaccine and/or a gram negative bacterial vaccine. Therefore, by adopting the reagent for improving the activity of the endothelial cells and kupffer cells of the liver sinuses, the pathogenic bacteria capturing capacity of the liver sinuses cells can be obviously improved, so that the protection effect of the vaccine is improved.
The reagent of the present application is suitable for use in both gram-positive and gram-negative bacterial vaccines, and the type of pathogenic bacteria is not particularly limited.
Illustratively, vaccines in the present application include, but are not limited to, streptococcus pneumoniae vaccines and/or klebsiella pneumoniae vaccines. Therefore, by adopting the reagent for improving the activity of the endothelial cells and kupffer cells of the liver sinuses, the pathogenic bacteria capturing capacity of the liver sinuses cells can be obviously improved, so that the protection effect of the vaccine is improved.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: streptococcus pneumoniae whole-bacterium inactivated vaccine, klebsiella pneumoniae whole-bacterium inactivated vaccine and streptococcus pneumoniae polysaccharide combined vaccine immunization program
Material sources are as follows: wild type CD1 mouse (provided by the animal center at the university of qinghua); streptococcus pneumoniae type 4 strain TIGR 4; klebsiella pneumoniae strain K2 TH 13157; THB (Oxoid Corp.); 13 valent streptococcus pneumoniae polysaccharide conjugate vaccine (beijing national sea biotechnology limited); yeast powder (Oxoid Corp.); tryptone (Oxoid Corp.); sodium chloride (west longa science ltd); 37% formaldehyde solution (Xiong chemical Co., Ltd.); PBS solution (Solarbio corporation); aluminum phosphate adjuvant (Thermo corporation).
The experimental method comprises the following steps: the streptococcus pneumoniae type 4 strain TIGR4 is inoculated into THY liquid culture medium (THB +0.5 percent yeast powder) and cultured to logarithmic growth phase in a 5 percent carbon dioxide incubator at 37 ℃; klebsiella pneumoniae strain K2, strain TH13157, was inoculated into LB liquid medium (1% tryptone, 1% sodium chloride, 0.5% yeast powder) and cultured in a shaker at 37 ℃ until logarithmic growth phase. According to 108The bacterial load of each mouse was collected by centrifugation, washed twice with PBS and resuspended. The resuspended suspension was incubated overnight at 4 ℃ with formaldehyde to a final concentration of 0.5% for inactivation. After the inactivated bacterial liquid is added with 0.5 percent of aluminum phosphate adjuvant and mixed, each 5-week-old female CD1 mouse is immunized twice in a subcutaneous injection mode, and 0.5 ml of 10 mixed with 0.5 percent of aluminum phosphate adjuvant is injected for each time8Inactivating the bacteria at intervals of 7 days; while the control group female CD1 mice were subjected to the same treatment each timeThe vaccine groups were treated with equal amounts of adjuvant injection. The streptococcus pneumoniae polysaccharide conjugate vaccine group is used for immunizing female CD1 mice of 5 weeks old three times by subcutaneous injection, and 0.05ml of 13-valent streptococcus pneumoniae polysaccharide conjugate vaccine is injected every time at intervals of 14 days; while control group female CD1 mice were treated with each injection of adjuvant in an amount equal to that of the vaccine group.
Example 2: determination of serum antibody titer of mice after vaccine immunization
Material sources are as follows: streptococcus pneumoniae type 4 strain TIGR 4; klebsiella pneumoniae strain K2 TH 13157; THB (Oxoid Corp.); type 4 streptococcus pneumoniae polysaccharide capsule (beijing national sea biotechnology limited); yeast powder (Oxoid Corp.); sodium chloride (west longa science ltd); tryptone (Oxoid Corp.); skim milk powder (BD corporation); alkaline phosphatase-labeled goat anti-mouse IgG (available from bosjie EASYBIO); soluble one-component TMB substrate solution (tiangen); phosphoric acid (Beijing chemical plant); capillary blood collection tubes (huakang corporation); a 96-well microplate (Biofil Corp.); synergy H1 microplate reader (BioTek).
The experimental method comprises the following steps: by subcutaneous injection 108A CD1 mouse is immunized by formaldehyde inactivated streptococcus pneumoniae type 4, K2 klebsiella pneumoniae and 0.05ml of 13-valent streptococcus pneumoniae polysaccharide conjugate vaccine respectively, and blood is taken from a retroorbital venous plexus of the mouse by a capillary blood collection tube and centrifuged at low speed 14 days after the last immunization. IgG antibody titers against both pathogens were assessed by enzyme-linked immunosorbent assay (ELISA). Culturing Streptococcus pneumoniae type 4 TIGR4 and K2 Klebsiella pneumoniae TH13157 in THY liquid culture medium and LB liquid culture medium respectively to OD600nmAfter standing at 0.1, resuspend in PBS. 100. mu.l of the bacterial suspension or 100. mu.l of type 4 capsular polysaccharide coating at a concentration of 5. mu.g/ml was added to each well of a 96-well microplate and incubated overnight at 4 ℃. After adding 200. mu.l of 5% skim milk powder per well and blocking at room temperature for 1 hour and washing three times with PBS, mouse serum was added in a gradient diluted in PBS and incubated at 37 ℃ for 2 hours. Three washes were performed with 200. mu.l PBS per well, alkaline phosphatase-labeled goat anti-mouse IgG (1:2000 dilution) was added and incubated at 37 ℃ for 1 hour. Add 200. mu.l PBS per well and wash threeNext, after developing by adding 100. mu.l of TMB substrate solution (5 minutes), 100. mu.l of 1M H concentration was added to each well3The PO4 solution stopped the color reaction. Finally, the absorbance was measured by a BioTek Synergy H1 microplate reader at a wavelength of 450 nm. Control sera were collected and tested in the same manner from CD1 mice immunized with equal amounts of adjuvant. Control sera were collected and tested in the same manner from CD1 mice immunized with equal amounts of adjuvant.
Referring to fig. 3, compared with the serum of a control group of mice, the titer of specific IgG antibodies in the serum of mice immunized with inactivated vaccines against corresponding streptococcus pneumoniae and klebsiella pneumoniae and combined vaccines against type 4 capsular polysaccharide is significantly improved.
Example 3: early clearance of bacteria in the bloodstream of mice following vaccine immunization
Material sources are as follows: streptococcus pneumoniae type 4 strain TIGR 4; klebsiella pneumoniae strain K2 TH 13157; tail vein syringe (jingxin corporation); sterile deionized water; capillary blood collection tubes (huakang corporation); blood agar plates; tribromoethanol anesthetic (provided by the animal center of the university of Qinghua); ringer's solution.
The experimental method comprises the following steps: injecting 300mg/kg tribromoethanol anesthetic into abdominal cavity of immunized group mouse and control group mouse, and infecting 10 via tail vein injection6Type 4 streptococcus pneumoniae or type K2 klebsiella pneumoniae, 10 μ l blood samples were collected at different time points post infection (2, 5, 10, 20, 30 and 45 minutes) through the retroorbital venous plexus using capillary blood collection tubes and mixed with 90 μ l sterile deionized water. The blood samples were diluted in ringer's solution in steps and inoculated onto blood agar plates, cultured overnight at 37 ℃ in a 5% carbon dioxide incubator to count the number of blood bacteria. The results of the early clearance of Streptococcus pneumoniae in blood demonstrated that the proportion of bacteria cleared 30 minutes after infection was not significantly different from the proportion 45 minutes after infection, and therefore 10 subsequent times6Klebsiella pneumoniae K2 (K) was infected in the same manner and blood was collected only within 30 minutes (2, 5, 10, 20 and 30 minutes) after infection for determination and analysis of the bacterial count. Clearance at a particular time point is the percentage of blood bacterial load at that point in relation to the initial bacterial infection. When mean max clearAll removal rate is>At 50%, a time-percent clearance Nonlinear Regression curve (Nonlinear Regression-exponentials-One phase association) is drawn by using GraphPad software; the regression equation is Y ═ Y0+(Plateau-Y0)·(1-e-K·x) Wherein Y is the percent clearance, Y0 Initial value 0, x time, Plateau upper percentage purge, and K purge rate constant. The time CT required to eliminate 50% of the bacteria (i.e. Y50) is deduced back according to the above equation50Ln (1-50/Plateau)/(-K). When the average maximum clearance rate is equal to<CT at 50%50Set to the maximum time detected.
As shown in FIG. 4, the blood flow clearing half-life (CT50) of Streptococcus pneumoniae was 2.1 min after immunization of inactivated vaccine, compared with CT in control mice50Greater than 45 minutes; the proportion of the extent to which Streptococcus pneumoniae was cleared in the bloodstream 30 minutes after infection (99.8%) was not significantly different from 45 minutes after infection (99.8%). As shown in FIG. 5, the blood flow clearing half-life (CT) of Klebsiella pneumoniae after immunization of inactivated vaccine50) 0.7 minutes; and CT in control mice50Then greater than 30 minutes. As shown in FIG. 6, blood flow clearing half-life (CT) of Streptococcus pneumoniae after combined vaccine immunization with inactivated vaccine after vaccine immunization50) 1.0 min, compared to CT in control mice50Then greater than 30 minutes. Thus, the immunization allows the bacteria to be rapidly cleared 20-45 minutes (e.g., 30 minutes) into the bloodstream.
Example 4: distribution of bacteria in various organs after vaccine immunization
Material sources are as follows: streptococcus pneumoniae type 4 strain TIGR 4; klebsiella pneumoniae strain K2 TH 13157; tail vein syringe (jingxin corporation); sterile deionized water; capillary blood collection tubes (huakang corporation); grinding the tube; grinders (OMNI corporation); blood agar plates; ringer's solution.
The experimental method comprises the following steps: after anesthetizing the mice of the immunization group and the mice of the control group according to the method, the mice are infected by a tail vein injection mode 106 Streptococcus pneumoniae type 4 or Klebsiella pneumoniae type K2, blood was collected 2 and 30 minutes after infection and mice were sacrificed by cervical dislocation to collect liver, spleen, kidney, lung and heartThe zang organs. Each organ was homogenized by grinding with a grinder, then diluted with a gradient of ringer's solution and plated on blood agar plates to count the number of bacteria. The proportion of bacteria in each organ was the bacterial load in the organ divided by the total bacterial load remaining at that time point (sum of bacterial loads in heart, liver, spleen, lung, kidney and blood). The results of the distribution of streptococcus pneumoniae in each organ and the ratio of total remaining bacteria after inactivated vaccine immunization demonstrated that the ratio of bacteria in the liver was the highest 30 minutes after infection compared to 2 minutes after infection. Thus, the following 106The K2 Klebsiella pneumoniae inactivated vaccine and the Streptococcus pneumoniae conjugate vaccine group were infected in the same manner and blood and major organs were collected 20-40 minutes (e.g., 30 minutes) after infection for assay analysis.
As shown in fig. 7, after immunization with inactivated vaccine streptococcus pneumoniae was mainly captured by the liver with capture rates of 41.5% and 77.0% at 2 and 30 minutes, respectively; while the bacteria in the control mice were mainly distributed in the blood, the liver capture rate was only 1.1% and 13.9%, respectively. As shown in fig. 8, after the inactivated vaccine is immunized, klebsiella pneumoniae is mainly captured by the liver, and the capture rate is 86.0%; while the bacteria in the control mice are mainly distributed in the blood, the liver capture rate is only 8.9%. As shown in fig. 9, after combined vaccine immunization, streptococcus pneumoniae was mainly captured by the liver, with a capture rate of 89.0%; while the bacteria in the control mice are mainly distributed in the blood, the liver capture rate is only 14.2%. Therefore, the results show that the immunization significantly improved the capacity of the liver to capture bacteria.
Example 5: real-time localization of bacteria in the liver following vaccine immunization
Material sources are as follows: streptococcus pneumoniae type 4 strain TIGR 4; klebsiella pneumoniae strain K2 TH 13157; AF594 anti-CD31 antibody (biolegend Co.), AF647 anti-F4/80 antibody (Invitrogen Co.), PE anti-Ly6G antibody (eBioscience Co.); FITC (Invitrogen corporation); leica TCS-SP8 fluorescence confocal microscope.
The experimental method comprises the following steps: after anesthetizing the mice in the above manner, the tail vein catheter was placed for intravenous injection. 2.5. mu.g of fluorescently labeled antibody (AF594 anti-CD31 labeled hepatic sinus endothelial cells, AF647 anti-F4/80 labeled Kupffer cells, PE anti-Ly6G labeled neutrophils). Bacteria were labeled by incubating with 0.2mg/ml FITC for 30 minutes at room temperature and washing twice with PBS for resuspension. Mice were abdominally exposed and placed on coverslips and injected 5X 10 through the tail vein7FITC-labeled bacteria. Living body imaging of liver microvessels was performed on an inverted Leica TCS-SP8 microscope using a 20X/0.80 NA HC PL APO objective to detect the status of antral cell and bacterial binding. A photomultiplier tube (PMT) and a hybrid photodetector (HyD) were used to detect signals of 1024X 1024 pixel size, with four wavelengths (488, 535, 594 and 647nm) excited by white laser light (1.5mw, laser suite WLL2, 470-670 nm). Five random fields of 1024 × 1024 pixel size were selected within 10-15 minutes after infection, and the number of bacteria captured by Kupffer Cell Capture (VEKC) and hepatic sinus endothelial Cell Capture (VEEC) in each field of view (FOV) was counted.
As shown in FIG. 10, after inactivated vaccine immunization, Streptococcus pneumoniae was mainly captured by liver macrophages-Kupffer cells and liver sinus endothelial cells in the liver, and the numbers of bacteria VEKC and VEEC captured by liver sinus cells per unit field of view were 84 and 2, respectively, whereas bacteria in control mice were not captured by both cells effectively. As shown in fig. 11, after inactivated vaccine immunization, klebsiella pneumoniae was mainly captured by liver macrophages, kupffer cells and liver sinus endothelial cells, in the liver, and the numbers of bacteria captured by liver sinus cells in unit field of view, VEKC and VEEC, were 57 and 7, respectively, whereas the bacteria in the control mice were not captured by both cells effectively. As shown in FIG. 12, after the combined vaccine immunization, Streptococcus pneumoniae was mainly captured by sinusoidal endothelial cells and Kupffer cells, which are liver macrophages, in the liver, and the numbers of bacteria VEKC and VEEC captured by sinusoidal cells per unit field were 34 and 123, respectively, whereas the bacteria in the control mice were not substantially captured by both cells efficiently. Therefore, the results show that the immune effect remarkably improves the bacterial capture capacity of liver macrophage kupffer cells and liver sinus endothelial cells.
Example 6: monitoring of the survival protection effect of mice after vaccine immunization
Material sources are as follows: streptococcus pneumoniae type 4 strain TIGR 4; klebsiella pneumoniae strain K2 TH 13157; sterile deionized water; capillary blood collection tubes (huakang corporation); blood agar plates.
The experimental method comprises the following steps: respectively adding 10 aiming at the whole bacteria-inactivated seedlings4、105Or 106The streptococcus pneumoniae or the klebsiella pneumoniae of the vaccine is used for infecting mice of an immune group and a control group in an intraperitoneal injection mode, and the infection dose is determined by titration on a blood agar plate immediately after infection; respectively combine 10 against the combined seedlings7Or 108The streptococcus pneumoniae of (2) by intraperitoneal injection, the mice of the immune group and the control group are infected, and the infection dose is determined by titration on a blood agar plate immediately after infection. Animal survival was recorded over 7 days, while the number of bacteria in the blood was measured every 12 hours by retroorbital bleeding and blood agar titration. The protection rate (survivval%) at each infectious dose is the percentage of the number of surviving mice in the total number of mice.
As shown in fig. 13, at 104After the mice are immunized by streptococcus pneumoniae infection, the survival protection rate can be 100 percent; whereas the control mice infected with the same dose all died. At higher doses 105And 106The survival rate of the mice after the streptococcus pneumoniae is infected and immunized respectively is reduced to 60 percent and 0 percent.
As shown in fig. 14, at 104After the mice are immunized by the Klebsiella pneumoniae infection, the survival protection rate can be 100 percent; whereas the control mice infected with the same dose all died. At higher doses 105And 106The survival rates of the mice infected with the Klebsiella pneumoniae are respectively reduced to 60% and 0%.
As shown in fig. 15, at 107After the mice are immunized by streptococcus pneumoniae infection, the survival protection rate can be 100 percent; whereas the control mice infected with the same dose all died. At higher doses 108The mice were all dead after the immunization with streptococcus pneumoniae infection.
As can be seen from FIGS. 13-14, the number of control mice was greater than 104None of the mice survived at the dose of infection, and the mice immunized with the whole-germ-free vaccine were at 104Infection with viral infectionThe survival rate under the dosage can reach 100 percent and is 10 percent5The survival rate under the infection dose can reach 60 percent, and the mouse in the combined vaccine immunization group is 10 percent7The survival rate under the infection dose can reach 100 percent. Therefore, the results show that vaccination can effectively improve the survival rate of the infected mice; also, the limit of the dose of streptococcus pneumoniae infection in combination vaccine-protected immunized mice is higher compared to the dose of streptococcus pneumoniae infection in inactivated vaccine-protected immunized mice.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (10)

1. A method for controlling the quality of a vaccine, comprising:
step 1: the vaccine to be detected is detected in the following modes:
mode 1: administering the vaccine to be detected to a subject, collecting the serum of the subject after the vaccine to be detected is administered, measuring the antibody titer corresponding to the vaccine to be detected in the serum, and preliminarily determining whether the quality of the vaccine to be detected reaches the standard or not based on the measurement result;
mode 2: administering the vaccine to be tested for a predetermined time and then administering the pathogenic bacteria again, measuring the capture rate of the pathogenic bacteria in the liver of the subject after a period of time, and preliminarily determining whether the quality of the vaccine to be tested reaches the standard or not based on the measurement result;
mode 3: administering the vaccine to be tested to the subject again with lethal dose of the pathogenic bacteria of the subject after a predetermined time, measuring the survival rate of the subject, and preliminarily determining whether the quality of the vaccine reaches the standard or not based on the measurement result;
step 2: determining whether the final quality of the vaccine to be detected reaches the standard or not based on the primary detection results of all detection modes in the step 1;
and the preliminary detection results of all the detection modes are that the quality of the vaccine to be detected reaches the standard, and the preliminary detection results are an indication that the final quality of the vaccine to be detected reaches the standard.
2. The method for controlling the quality of a vaccine according to claim 1, wherein the step 1 further comprises at least one of the following detection modes:
mode 4: administering the pathogenic bacteria again after the subject is administered with the vaccine to be detected for a preset time, recording the change of the loading capacity of the pathogenic bacteria in the blood of the subject within the preset time after the subject is administered with the pathogenic bacteria, determining the elimination half-life of the pathogenic bacteria, and preliminarily determining whether the quality of the vaccine to be detected reaches the standard or not based on the determination result;
mode 5: and administering the vaccine to be tested again after a preset time, measuring the capture quantity of the pathogenic bacteria in the hepatic sinus cells of the tested person after a period of time, and preliminarily determining whether the quality of the vaccine to be tested reaches the standard or not based on the measurement result.
3. The method of claim 2, wherein the sinusoidal hepatocytes are selected from kupffer cells and sinusoidal hepatocytes.
4. The method for controlling the quality of a vaccine according to claim 1 or 2, wherein the method 1 comprises the following steps:
experimental groups: administering the vaccine to be detected to a subject, collecting the serum of the subject after the vaccine to be detected is administered, and measuring the antibody titer corresponding to the vaccine to be detected in the serum;
control group: administering an adjuvant with the same volume as the vaccine to be tested to a subject, collecting the serum of the subject after the adjuvant is given, and measuring the antibody titer corresponding to the vaccine to be tested in the serum;
when the antibody titer of the experimental group is significantly different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard;
optionally, the mode 2 comprises the following steps:
experimental groups: administering the vaccine to be tested for a predetermined time and then again administering the pathogenic bacteria to the subject, collecting the liver of the subject after administering the vaccine to be tested for a period of time, and measuring the capture rate of the pathogenic bacteria in the liver of the subject;
control group: administering the adjuvant with the same volume as the vaccine to be tested to the subject for a preset time, then administering the pathogenic bacteria again, collecting the liver of the subject after the vaccine to be tested is administered after a period of time, and measuring the capture rate of the pathogenic bacteria in the liver of the subject;
when the capture rate of the experimental group is significantly different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard;
optionally, the mode 3 comprises the following steps:
experimental groups: administering a second lethal dose of said pathogenic bacteria to the subject a predetermined time after administering said test vaccine to the subject, and determining the survival of the subject;
control group: administering to the subject a lethal dose of said pathogenic bacteria to the subject a predetermined time after administering an equal volume of adjuvant to the subject to be tested, and determining the survival rate of the subject;
when the survival rate of the experimental group is significantly different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard;
optionally, the mode 4 comprises the following steps:
experimental groups: administering the pathogenic bacteria again after the subject is administered with the vaccine to be detected for a preset time, recording the change of the loading capacity of the pathogenic bacteria in the blood of the subject within the preset time after the administration of the pathogenic bacteria, and counting the elimination half-life of the pathogenic bacteria in the blood;
control group: administering the pathogen again after the subject is administered with the adjuvant with the same volume as the vaccine to be detected for a preset time, recording the change of the loading capacity of the pathogen in the blood of the subject in the preset time after the pathogen is administered, and counting the elimination half-life of the pathogen in the blood;
when the elimination half-life period of the experimental group is significantly different from that of the control group, preliminarily determining that the quality of the vaccine to be detected reaches the standard;
optionally, the means 5 comprises the steps of:
experimental groups: administering the vaccine to the subject a second time after a predetermined period of time, and determining the number of antral cells in the liver of the subject capturing the pathogen after a period of time;
control group: administering the pathogen again to the subject after a predetermined time with an adjuvant of the same volume as the vaccine to be tested, and measuring the capture amount of the pathogen by the antral cells in the liver of the subject after a period of time;
and when the capturing quantity of the hepatic sinus cells in the liver of the experimental group to the pathogenic bacteria is obviously different from the capturing quantity of the hepatic sinus cells in the liver of the control group to the pathogenic bacteria, preliminarily determining that the quality of the vaccine to be detected reaches the standard.
5. The method for controlling the quality of a vaccine according to claim 4, wherein the period of time in the mode 2 is 20 to 40 minutes;
optionally, the predetermined time for recording the change in pathogen load in mode 4 is 20 to 45 minutes.
6. The method for controlling the quality of the vaccine according to claim 1, wherein the vaccine comprises a gram-positive bacterial vaccine and/or a gram-negative bacterial vaccine;
optionally, the vaccine comprises a streptococcus pneumoniae vaccine and/or a klebsiella pneumoniae vaccine;
optionally, the vaccine comprises a whole-cell inactivated vaccine and/or a polysaccharide conjugate vaccine.
7. Use of an agent for the manufacture of a kit for the quality control of a vaccine, wherein said agent is selected from the group consisting of agents which detect the re-administration of a pathogen to a subject a predetermined time after administration of the vaccine to said subject, the number of said pathogen in the antral cells of the subject after said period of time.
8. The use according to claim 7, wherein the agent is selected from the group consisting of agents which detect the number of pathogenic bacteria in sinusoidal endothelial cells and kupffer cells in a subject after a predetermined time of administration of the vaccine to the subject;
optionally, the vaccine comprises a gram positive bacterial vaccine and/or a gram negative bacterial vaccine;
optionally, the vaccine comprises a streptococcus pneumoniae vaccine and/or a klebsiella pneumoniae vaccine;
optionally, the vaccine comprises a whole-cell inactivated vaccine and/or a polysaccharide conjugate vaccine.
9. Use of an agent that increases the activity of sinusoidal endothelial cells and kupffer cells in improving the protective effect of a vaccine.
10. Use according to claim 9, wherein the vaccine comprises a gram-positive bacterial vaccine and/or a gram-negative bacterial vaccine;
optionally, the vaccine comprises a streptococcus pneumoniae vaccine and/or a klebsiella pneumoniae vaccine;
optionally, the vaccine comprises a whole-cell inactivated vaccine and/or a polysaccharide conjugate vaccine.
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