CN115774102A

CN115774102A - Method for determining the state of a neocorona vaccine-mediated protective immune response

Info

Publication number: CN115774102A
Application number: CN202111050925.3A
Authority: CN
Inventors: 钱峰; 高健; 罗雅丽; 韩静轩
Original assignee: Shanghai International Human Phenotype Group Research Institute; Fudan University
Current assignee: Shanghai International Human Phenotype Group Research Institute; Fudan University
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2023-03-10

Abstract

The invention provides a method for determining a neocorona vaccine-mediated protective immune response state, and particularly provides a method for detecting a vaccine-mediated protective immune response state, which comprises the following steps: (a) Providing a sample to be tested, wherein the sample to be tested is a blood sample, and the sample to be tested is a sample after vaccination; (b) Determining the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the sample to be tested; and (c) adding an antigen stimulus, thereby obtaining an antigen-stimulated test sample; (d) Detecting the content of the cell factor and the content of the specific antibody in the antigen-stimulated sample to be detected; (e) According to the percentage of immune cell subgroup or the expression intensity of immune cell surface function marker, the content of cell factor and the content of specific antibody, the vaccine-mediated protective immune response state is obtained. The method can evaluate the protective immune response state mediated by the vaccine with high accuracy and comprehensiveness.

Description

Method for determining the state of a neocorona vaccine-mediated protective immune response

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a method for determining a protective immune response state mediated by a neocorona vaccine.

Background

The new coronavirus pneumonia caused by severe acute respiratory syndrome coronavirus 2 has become a new emergent infectious disease facing all over the world. The disease has the characteristics of high transmission speed, high lethality, long latent period, infectivity and the like.

Vaccines comprise attenuated or inactivated portions (antigens) of a particular organism that elicit an immune response in vivo. This weakened version does not cause disease in the person receiving the vaccine, but rather causes the immune system to respond to a degree comparable to the first response to the actual pathogen. For some COVID-19 vaccines, two doses are required for vaccination. The first dose is the first time to provide antigen (protein that stimulates antibody production) to the immune system, initiating an immune response. The second agent acts as an enhancer, ensuring that the immune system produces long-lasting antibodies and develops memory cells. In this way, the body can be trained against specific pathogenic organisms, building up memory against the pathogen, so that it can be quickly challenged by future exposure.

The titer of the vaccine needs to be evaluated, and the vaccine cannot protect a vaccinee by one hundred percent due to the limitation of the vaccine, so that the evaluation and determination are necessary for the response state of the protective immunity formed after the vaccination.

When the vaccine is injected into muscle, dendritic cells are activated by recognizing protein antigens through Pattern Recognition Receptors (PRRs), and then transported to draining lymph nodes. Here, MHC molecules on dendritic cells present polypeptides of vaccine protein antigens that activate T cells via the T Cell Receptor (TCR), and soluble vaccine protein antigens may also transmit immune response signals in conjunction with the B Cell Receptor (BCR). T cells help to activate B cells, promote the formation of the developmental center and class switching of immunoglobulins, and actively secrete high-affinity specific antibodies against vaccine proteins. CD8+ memory T cells can proliferate rapidly when they encounter pathogens, while CD8+ effector T cells are important for the clearance of infected cells.

At present, the immunoprotection-related factors against SARS-CoV-2 have not been completely determined.

The immune cells are basic components of an immune system, and under the influence of genetic and environmental factors, various immune cells in a body can be differentiated in different quantities due to individual differences to form immune cell subsets with different proportions, and the different immune cell subsets are definite in division and have strong functions. The ratio of the number of subpopulations of immune cells also changes during the course of disease development when the organism is stimulated by a vaccine. Cellular immunity is also important in controlling SARS-CoV-2 infection.

Cytokines have a wide range of biological activities and are capable of modulating cell growth, differentiation and effects by binding to the corresponding receptors and modulating immune responses.

The neutralizing antibody of humoral immunity can bind to new coronavirus surface protein and block the binding of virus protein and cell surface specific receptor ACE-2, thereby preventing the virus from invading cells (the non-neutralizing antibody does not have the function).

However, the existing method for evaluating the response state of the protective immunity formed after vaccination only detects the content of the current specific antibody, and has the advantages of narrow coverage, incomplete coverage, incapability of evaluating other important components of an immune system and incapability of evaluating the potential of vaccine-mediated protective immunity.

Therefore, there is an urgent need in the art to develop a method of response status with high accuracy and capable of full-scale development of protective immunity after vaccination.

Disclosure of Invention

The object of the present invention is to provide a method with high accuracy and capable of rapidly assessing the state of the response of the protective immunity developed after vaccination.

In a first aspect of the invention, there is provided a method of detecting a vaccine-mediated protective immune response status, comprising:

(a) Providing a sample to be tested, wherein the sample to be tested is a blood sample, and the sample to be tested is a sample after vaccination;

(b) Determining the percentage of the subpopulation of immune cells or the expression intensity of the markers of immune cell surface functions in the sample to be tested; and

(c) Adding an antigen stimulant to obtain an antigen-stimulated sample to be tested;

(d) Detecting the content of the cell factor and the content of the specific antibody in the antigen-stimulated sample to be detected;

(e) According to the percentage of immune cell subgroup or the expression intensity of immune cell surface function marker, the content of cell factor and the content of specific antibody, the vaccine-mediated protective immune response state is obtained.

In another preferred embodiment, the sample to be tested is from a person after vaccination.

In another preferred embodiment, the vaccine comprises inactivated vaccine, RNA vaccine, recombinant protein vaccine.

In another preferred embodiment, the blood sample is a whole blood sample.

In another preferred example, the blood sample comprises peripheral blood.

In another preferred embodiment, the vaccine comprises a coronavirus vaccine.

In another preferred embodiment, the coronavirus comprises a novel coronavirus.

In another preferred example, the percentage of the subpopulation of immune cells includes the percentage of the subpopulation of immune cells in total leukocytes of peripheral blood (CD 45+ total leukocytes) or cells of a parental population of immune cells (e.g., CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, etc.).

In another preferred embodiment, the immune cells comprise innate immune cells and/or adaptive immune cells.

In another preferred example, the innate immune cells include granulocytes and subsets thereof, monocytes and subsets thereof, dendritic Cells (DCs) and subsets thereof, natural killer cells (NK cells) and subsets thereof.

In another preferred embodiment, the adaptive immune cells comprise T cells and subpopulations thereof, B cells and subpopulations thereof, preferably CD4+ T cells, CD4+ memory T cells, naive CD4+ T, CD8+ T cells, CD8+ effector T cells, memory B cells, plasmablasts.

In another preferred embodiment, the marker of immune cell surface function is selected from the group consisting of: CD25, CD69, CD28, CD38, HLADR, CCR7, CD64, CD57, ICOS, CD39, CD86, CD24, NKp46, NKG2D, or a combination thereof.

In another preferred embodiment, the percentage of said subpopulation of immune cells is detected by one or more of the following methods: flow cytometry.

In another preferred embodiment, the expression intensity of the immune cell surface function marker is detected by one or more of the following methods: flow cytometry, western immunoblotting (Western Blot).

In another preferred embodiment, the antigen is selected from the group consisting of: a severe acute respiratory syndrome coronavirus 2 Spike Receptor Binding Domain (RBD), a severe acute respiratory syndrome coronavirus 2 Spike S1 subunit (Spike S1), or a combination thereof.

In another preferred embodiment, the antigen has the sequence shown in SEQ ID No. 1 or 2.

In another preferred embodiment, the cytokine is selected from the group consisting of: IFN-g, TNF-a, IL-2, IL-6, IL-10, or a combination thereof.

In another preferred embodiment, the cytokine content includes the concentration of the cytokine in the antigen-stimulated test sample.

In another preferred embodiment, the cytokine content is detected by one or more of the following methods: enzyme linked immunosorbent assay (ELISA), microsphere-based multi-factor assay (Cytokine Bead Assays), luminex platform-based multi-factor assay, and MSD (Meso Scale Discovery) platform-based electrochemiluminescence hypersensitivity multi-factor assay.

In another preferred embodiment, the specific antibody is selected from the group consisting of: anti-RBD IgG, anti-Spike S1 IgG, or a combination thereof.

In another preferred example, the content of the specific antibody includes the concentration of the specific antibody in the antigen-stimulated sample to be tested.

In another preferred embodiment, the content of said specific antibody is detected by one or more of the following methods: enzyme linked immunosorbent assay (ELISA), microsphere-based multi-factor assay (Cytokine Bead Assays), luminex platform-based multi-factor assay, and MSD (Meso Scale Discovery) platform-based electrochemiluminescence hypersensitivity multi-factor assay.

In another preferred embodiment, the vaccine-mediated protective immune response state comprises a low response state, a medium response state, and a high response state.

In another preferred example, when the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the test sample is in the low response range as shown in table A1 or A2, the vaccine-mediated protective immune response state is in the low response state.

In another preferred embodiment, when the percentage of the subpopulation of immune cells or the expression intensity of the markers of surface functions of immune cells in the test sample is within the medium response range as shown in table A1 or A2, the vaccine-mediated protective immune response status is the medium response status.

In another preferred example, when the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the test sample is in the high response range as shown in table A1 or A2, the vaccine-mediated protective immune response state is in the high response state.

In another preferred embodiment, when the content of the cytokine in the antigen-stimulated sample to be tested is in the low response range as shown in table A1, the vaccine-mediated protective immune response state is in the low response state.

In another preferred example, when the content of the cytokine in the antigen-stimulated test sample is in the middle response range as shown in table A1, the vaccine-mediated protective immune response state is in the middle response state.

In another preferred example, when the content of the cytokine in the antigen-stimulated test sample is in the high response range as shown in table A1, the vaccine-mediated protective immune response state is the high response state.

In another preferred example, when the content of the specific antibody in the antigen-stimulated sample to be tested is in the low response range as shown in table A1, the vaccine-mediated protective immune response state is in the low response state.

In another preferred example, when the content of the specific antibody in the antigen-stimulated sample to be tested is in the middle response range as shown in table A1, the vaccine-mediated protective immune response state is in the middle response state.

In another preferred example, when the content of the specific antibody in the antigen-stimulated sample to be tested is in the high response range as shown in table A1, the vaccine-mediated protective immune response state is in the high response state.

In another preferred example, when the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the test sample, the content of the cytokine in the antigen-stimulated test sample, and the content of the specific antibody in the antigen-stimulated test sample simultaneously satisfy the following conditions: and if at least two of the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker, the content of the cytokine in the antigen-stimulated test sample and the content of the specific antibody in the antigen-stimulated test sample are ranges corresponding to the low response state, indicating that the vaccine-mediated protective immune response state is the low response state.

In another preferred example, when the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the test sample, the content of the cytokine in the antigen-stimulated test sample, and the content of the specific antibody in the antigen-stimulated test sample simultaneously satisfy the following conditions: and if at least two of the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker, the content of the cytokine in the antigen-stimulated sample to be detected and the content of the specific antibody in the antigen-stimulated sample to be detected are ranges corresponding to the medium response state, indicating that the vaccine-mediated protective immune response state is the medium response state.

In another preferred example, when the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the test sample, the content of the cytokine in the antigen-stimulated test sample, and the content of the specific antibody in the antigen-stimulated test sample simultaneously satisfy the following conditions:

and if at least two of the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker, the content of the cytokine in the antigen-stimulated sample to be detected and the content of the specific antibody in the antigen-stimulated sample to be detected are ranges corresponding to the high response state, indicating that the vaccine-mediated protective immune response state is the high response state.

In a second aspect, the invention provides a method of determining the state of a vaccine-mediated protective immune response comprising:

(a) Providing a data set for the analysis, the data set comprising: a1 st data set from the percentage of immune cell subpopulations or the expression intensity of immune cell surface functional markers, a2 nd data set from the amount of cytokines and a3 rd data set from the amount of specific antibodies, each data set from a post-vaccination sample, and the 2 nd and 3 rd data sets also from antigen-stimulated test samples;

(b) Performing statistical analysis on each data set to obtain data of a low response range, a medium response range and a high response range corresponding to each data set;

(c) And determining the vaccine-mediated protective immune response state based on the data of the low response range, the medium response range and the high response range corresponding to each data set.

In another preferred example, in the step (b), the statistical analysis is performed on each of the data sets in the following calculation manner:

5) The following objects are defined and calculated: (1) a first tertile equal to the maximum + (max-min) x 33% value in the dataset; (2) a second quartile equal to the value of maximum + (max-min) x 67% in the data set;

6) Calculating the average value x of sample detection values in the data set, which detection values are lower than the first tertile ₁ And standard deviation s ₁ The "low response range" is: x is less than or equal to ₁ +s ₁ 。

7) Calculating an average value x of sample detection values in the dataset having detection values higher than the second quartile ₂ And standard deviation s ₂ The "high response range" is: not less than x ₂ -s ₂ 。

8) The "low response range" and the "high response range" are in between the "medium response range".

In another preferred example, in step (b), the data of the low response range, the medium response range and the high response range corresponding to each data set is shown in table A1 or A2.

In another preferred example, in step (c), when at least 2 data sets among the data of the low response range, the medium response range and the high response range corresponding to each data set correspond to the low response range, the vaccine-mediated protective immune response state is indicated as the low response state.

In another preferred example, in step (c), when at least 2 data sets among the data of low response range, medium response range and high response range corresponding to each data set correspond to medium response range, the vaccine-mediated protective immune response state is indicated as medium response state.

In another preferred example, in step (c), when at least 2 data sets among the data of low response range, medium response range and high response range corresponding to each data set correspond to high response range, the vaccine-mediated protective immune response state is indicated as high response state.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred example, the 1 st data set is the detection result from the following technology: flow cytometry, western immunoblotting (Western Blot), or a combination thereof. In another preferred example, the 2 nd data set is the detection result from the following technology: enzyme linked immunosorbent assay (ELISA), microsphere-based multi-factor assay (Cytokine Bead Assays), luminex platform-based multi-factor assay, MSD (Meso Scale Discovery) platform-based electrochemiluminescence hypersensitivity multi-factor assay, or a combination thereof.

In another preferred example, the 3 rd data set is the detection result from the following technology: enzyme linked immunosorbent assay (ELISA), microsphere-based multi-factor assay (Cytokine Bead Assays), luminex platform-based multi-factor assay, MSD (Meso Scale Discovery) platform-based electrochemiluminescence hypersensitivity multi-factor assay, or a combination thereof.

In another preferred example, the data sets are obtained by a method comprising the following steps:

(a) Providing a sample to be detected, wherein the sample to be detected is a blood sample;

(b) Determining the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the sample to be tested, and obtaining the data of the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the sample to be tested, thereby obtaining a1 st data set; and

(d) And detecting the content of the cell factor and the content of the specific antibody in the antigen-stimulated sample to be detected, and obtaining data of the cell factor and the specific antibody in the sample to be detected, thereby obtaining a2 nd data set and a3 rd data set.

In a third aspect the invention provides an apparatus for determining the state of a vaccine-mediated protective immune response comprising:

(a) A data input unit for inputting 3 data sets for the analysis, wherein the data sets comprise: a1 st data set from the percentage of immune cell subpopulations or the expression intensity of immune cell surface functional markers, a2 nd data set from the amount of cytokines and a3 rd data set from the amount of specific antibodies, each data set being from a post-vaccination sample, and the 2 nd and 3 rd data sets also being from an antigen stimulated test sample;

(b) The statistical analysis unit is used for carrying out statistical analysis on each data set so as to obtain data of a low response range, a medium response range and a high response range corresponding to each data set; the statistical analysis unit is configured to perform the following operations:

(i) The following objects are defined and calculated: (1) a first tertile equal to the maximum + (max-min) x 33% value in the dataset; (2) a second tertile equal to the maximum + (max-min) x 67% value in the dataset;

(ii) Calculating an average value x of sample detection values in the data set having detection values below a first quartile ₁ And standard deviation s ₁ The "low response range" is: x is less than or equal to ₁ +s ₁ 。

(iii) Calculating the average value x of sample detection values in the data set, wherein the detection values are higher than the second third quantile ₂ And standard deviation s ₂ The "high response range" is: not less than x ₂ -s ₂ 。

(iv) The "medium response range" is between the "low response range" and the "high response range";

(c) A data analysis unit for further analyzing and processing the data obtained by the statistical analysis unit so as to determine the vaccine-mediated protective immunity response state; the data analysis unit is configured to perform the following operations:

(1) When at least 2 data sets in the data of the low response range, the medium response range and the high response range corresponding to each data set correspond to the low response range, the vaccine-mediated protective immune response state is indicated to be a low response state;

(2) When at least 2 data sets in the data of the low response range, the medium response range and the high response range corresponding to each data set correspond to the medium response range, the vaccine-mediated protective immune response state is indicated to be the medium response state;

(3) When at least 2 data sets in the data of the low response range, the medium response range and the high response range corresponding to each data set correspond to the high response range, indicating that the vaccine-mediated protective immune response state is the high response state;

(d) And the output unit is used for outputting the analysis result of the data analysis unit.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

Fig. 1 is a flow chart of the method for determining the protective immune response state mediated by the new corona vaccine provided by the invention.

FIG. 2 is a graph showing the analysis results of the percentage of CD45+ cells in naive CD4+ T cells in "low response state", "medium response state" and "high response state" provided in example two.

FIG. 3 is the analysis chart of the results of detecting the concentration of IFN-g in the supernatant after peripheral blood is stimulated by specific antigen in "low response state", "medium response state" and "high response state" provided in example two.

FIG. 4 is an analysis chart showing the results of detecting the concentration of IgG antibody specific to RBD in the supernatant after peripheral blood is stimulated with specific antigen, in the "low response state", "medium response state" and "high response state" provided in example two.

FIG. 5 is an analysis chart of the results of the detection of the expression intensity of "low response status", "medium response status" and "high response status" HLADR on pDC provided in example three.

FIG. 6 is an analysis chart of the results of measuring the concentration of TNF-a in the supernatant after stimulation with specific antigen in peripheral blood in "low response state", "medium response state" and "high response state" provided in example three.

FIG. 7 is the analysis chart of the results of the detection of anti-Spike S1 IgG antibody concentration in the supernatant after the stimulation of specific antigen in peripheral blood in "Low response State", "Medium response State" and "high response State" provided in example three

Fig. 8 exemplarily shows "low response state", "medium response state", and "high response state" of the 4 subjects provided in example four, based on the percentage of peripheral blood immune cell subpopulations (naive CD4+ T cells), the cytokine IFN-g concentration in supernatant after peripheral blood stimulation with specific antigen, and the RBD-specific antibody IgG concentration, which determine the protective immune response mediated by the corona vaccine.

Detailed Description

The present inventors have made extensive and intensive studies and have for the first time unexpectedly found that a vaccine-mediated protective immune response state can be obtained depending on the percentage of immune cell subsets or the expression intensity of immune cell surface functional markers, the content of cytokines and the content of specific antibodies. The method has the advantages of high accuracy and wide coverage. On this basis, the present inventors have completed the present invention.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of or" consisting of 823030A ".

As used herein, the term "subject" refers to a human, particularly a subject vaccinated with a new corona vaccine.

The immune cells in the peripheral blood sample of the subject are selected from innate immune cells and/or adaptive immune cells.

Wherein said innate immune cell is preferably selected from the group consisting of: granulocytes and subsets thereof, monocytes and subsets thereof, dendritic cells (DC cells) and subsets thereof, natural killer cells (NK cells) and subsets thereof.

Wherein said adaptive immune cell is preferably selected from the group consisting of: t cells and subsets thereof, B cells and subsets thereof, in particular CD4+ T cells, CD4+ memory T cells, naive CD4+ T, CD8+ T cells, CD8+ effector T cells, memory B cells, plasmablasts.

Wherein the at least one immune cell preferably comprises one or more immune cells selected from the group described above. The immune cell parameters refer to the percentage of each immune cell subgroup in peripheral blood leukocytes or immune cell father group cells and/or the expression strength of immune cell surface functional molecules.

The immune cell surface functional molecule is preferably selected from the group consisting of: CD25, CD69, CD28, CD38, HLADR, CCR7, CD64, CD57, ICOS, CD39, CD86, CD24, NKp46, NKG2D.

Peripheral blood samples of the subject are stimulated by at least one specific antigen, preferably selected from the group consisting of the severe acute respiratory syndrome coronavirus 2-Spike Receptor Binding Domain (RBD) and/or the severe acute respiratory syndrome coronavirus 2-Spike S1 subunit (Spike S1), wherein

The sequence of a spinous process protein Receptor Binding Domain (RBD) of the severe acute respiratory syndrome coronavirus 2 is as follows:

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP KKSTNLVKNKCVNF(SEQ ID NO.1)

the sequence of the S1 subunit (Spike S1) of the spinous process protein of the severe acute respiratory syndrome coronavirus 2 is as follows:

SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRF DNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFS ALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA DYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY QPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAV RDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRA GCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR(SEQ ID NO.2)

a cytokine in a peripheral blood sample of the subject following stimulation by a specific antigen selected from the group consisting of: IFN-g, TNF-a, IL-2, IL-6, IL-10.

Wherein the at least one cytokine preferably comprises one or more cytokines selected from the group described above. The cytokine parameter refers to the concentration of the cytokine in the peripheral blood supernatant after stimulation.

Specific antibodies in a peripheral blood sample of the subject, preferably anti-RBD IgG andor anti-Spike S1 IgG. Wherein the at least one specific antibody parameter preferably comprises one or more specific antibodies selected from the group mentioned above, the specific antibody parameter being the concentration of specific antibodies in the peripheral blood supernatant after stimulation.

For the measured immune cell parameters, cytokine parameters and specific antibody parameters, the protective immune response status range corresponding to each parameter is determined according to tables A1 and A2.

TABLE A1

TABLE A2

In a more preferred embodiment, the "low response status" range, "medium response status" range, and "high response status" range for the percentage of CD45+ cells, IFN-g concentration, and anti-RBD IgG antibody concentration for naive CD4+ T cells are shown in Table A3:

TABLE A3

In a preferred embodiment, the "low response status" range, "medium response status" range, and "high response status" range for the percentage of CD45+ cells, IFN-g concentration, and anti-RBD IgG antibody concentration for naive CD4+ T cells are shown in Table A4:

TABLE A4

In a more preferred embodiment, the "low response" status range, "medium response" status range, and "high response" status range for the expression intensity of HLADR on pDC (HLADR MFI in pDC), TNF-a concentration, and anti-Spike S1 IgG antibody concentration are shown in Table A5:

TABLE A5

At least two parameters of three parameters of immune cell parameters, cytokine parameters and specific antibody parameters of the subject are in a low response state, and the immune response state mediated by the subject neocorona vaccine is considered to be in the low response state.

At least two parameters of three parameters of immune cell parameters, cytokine parameters and specific antibody parameters of the subject are in a medium response state, and the immune response state mediated by the subject neocorona vaccine is considered to be in the medium response state.

At least two parameters of three parameters of immune cell parameters, cytokine parameters and specific antibody parameters of the subject are in a high response state, and the immune response state mediated by the subject neocorona vaccine is considered to be in the high response state.

Immune cell

The immune cells are basic components of an immune system, and under the influence of genetic and environmental factors, various immune cells in a body can be differentiated in different quantities due to individual differences to form immune cell subsets with different proportions, and the different immune cell subsets are definite in division and have strong functions. The ratio of the number of subpopulations of immune cells also changes during the course of disease development when the organism is stimulated by a vaccine. Cellular immunity is also important in controlling SARS-CoV-2 infection. Studies have shown that not all patients develop a protective humoral immune response, whereas asymptomatic or mild patients often develop a strong T cell response.

The number of SARS-CoV specific CD8+ T cells correlates with virus clearance and increased survival in a mouse model.

Cytokine

The cell factor has wide biological activity, can regulate cell growth, differentiation and effect by combining with corresponding receptors, regulates immune response, and detects the level of the cell factor after vaccination, which is very important for determining the protective immune response state mediated by the new corona vaccine.

Neutralizing antibodies for humoral immunity

The neutralizing antibody of humoral immunity can bind to the new coronavirus surface protein and block the binding of the virus protein and a cell surface specific receptor ACE-2, thereby preventing the virus from invading cells (the non-neutralizing antibody does not have the function), so that the serological detection of the neutralizing antibody is an important method for evaluating the protective efficacy of the new coronavirus vaccine at present. But antibody serological tests exclude the evaluation of immune cells and cytokines. In humoral immunity, the titer of neutralizing antibodies can be used as an important biomarker in the clinical research of evaluating SARS-CoV-2 vaccine. Neutralizing antibodies isolated from convalescent corona patients in convalescent phase have been shown to control SARS-CoV-2 infection in both prevention and treatment.

Method for detecting vaccine-mediated protective immune response status

The invention provides a method for detecting vaccine-mediated protective immune response state, which comprises the following steps:

(b) Determining the percentage of the immune cell subpopulation or the expression intensity of the immune cell surface function marker in the sample to be tested; and

(d) Detecting the content of the cytokine and the content of the specific antibody in the antigen-stimulated sample to be detected;

Specifically, the method detects the percentage of the peripheral blood immune cell subpopulation of the target object or the expression intensity parameter of the functional molecules on the surface of the immune cells through multicolor flow cytometry: labeling a peripheral blood sample of the subject with an agent capable of detecting the expression of a subpopulation of immune cells or of a functional marker on the surface of immune cells, such as: anti-human CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti-human CD19 antibody, anti-human CD56 antibody, anti-human CD16 antibody, anti-human CD14 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human HLADR antibody, anti-human CD25 antibody, anti-human CD69 antibody, anti-human CCR7 antibody, anti-human CD64 antibody, anti-human CD57 antibody, anti-human ICOS antibody, anti-human CD39 antibody, anti-human CD86 antibody, anti-human CD24 antibody, anti-human NKp46 antibody, anti-human NKG2D antibody, etc., the used markers are detected by using a flow cytometry, and the percentage of each immune cell sub-population in peripheral blood leukocytes or immune cell parent population cells and the expression intensity of immune cell surface function markers are analyzed and calculated.

A peripheral blood sample of a subject is stimulated with at least one specific antigen to express a purified antigenic protein, a severe acute respiratory syndrome coronavirus 2-spike receptor binding domain or a severe acute respiratory syndrome coronavirus 2-spike protein S1 subunit, added to a peripheral blood sample of the subject to be tested, and stimulated at 37 ℃ and 5% CO2 for 24 hours.

The supernatant after the antigen stimulation is collected, and microspheres coated with specific detection antibodies are used, such as: the method comprises the following steps of detecting the concentration of a cytokine and the concentration of at least one specific antibody in supernatant fluid of a target peripheral blood sample after stimulation of at least one antigen by a microsphere-based multi-factor detection method, wherein the microspheres coated with the specific antibody are marked with fluorescein APC (immunoglobulin C), but the sizes or the fluorescence intensities are different, and the microspheres coated with the IFN-g antibody, the microspheres coated with the RBD IgG antibody and the like can be distinguished in flow cytometry detection.

As used herein, "low response state" means having fewer memory cells, a lower degree of immune cell activation, less cytokine and antibody secretion when the new corona vaccine is vaccinated. By "high response state" is meant more memory cells, higher immune cell activation levels, more cytokines and antibody secretion when new corona vaccination is followed. The "medium response state" is between the "low response state" and the "high response state".

Respectively collecting 63 peripheral blood samples, and determining response ranges of various parameters according to the following principles:

(1) Performing statistical analysis on the detection value after the new corona vaccine is inoculated, and defining and calculating the following objects: (1) a first tertile equal to the maximum + (max-min) x 33% value in the dataset; (2) the second tertile, which is equal to the maximum + (max-min) x 67% value in the dataset.

(2) Calculating the average value x of the samples with the detection value lower than the first three-place number ₁ And standard deviation s ₁ The "low response range" is: x is less than or equal to ₁ +s ₁ 。

(3) Calculating the average value x of the samples with the detection value higher than the second three-thirds number ₂ And standard deviation s ₂ The "high response range" is: not less than x ₂ -s ₂ 。

(4) The "low response range" and the "high response range" are in between the "medium response range".

TABLE A3

In a more preferred embodiment, the "low response status" range, the "medium response status" range, and the "high response status" range for the percentage of CD45+ cells, IFN-g concentration, and anti-RBD IgG antibody concentration for naive CD4+ T cells are shown in Table A4 below:

TABLE A4

In a more preferred embodiment, the "low response" status range, "medium response" status range and "high response" status range for the expression intensity of HLADR on pDC (HLADR MFI in pDC), TNF-a concentration and anti-Spike S1 IgG antibody concentration are shown in Table A5:

TABLE A5

In the present invention, the method for detecting the percentage of the subpopulation of immune cells or the expression intensity of the markers of immune cell surface functions in the sample to be tested: detecting the percentage of the target subject's peripheral blood immune cell subpopulation or the expression intensity parameter of the immune cell surface functional molecule by multicolor flow cytometry, and labeling the target subject's peripheral blood sample with an antibody capable of detecting the expression of the immune cell subpopulation or the immune cell surface functional marker, such as: the antibody can be selected from anti-human CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti-human CD19 antibody, anti-human CD56 antibody, anti-human CD16 antibody, anti-human CD14 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human HLADR antibody, anti-human CD25 antibody, anti-human CD69 antibody, anti-human CCR7 antibody, anti-human CD64 antibody, anti-human CD57 antibody, anti-human ICOS antibody, anti-human CD39 antibody, anti-human CD86 antibody, anti-human CD24 antibody, anti-human NKp46 antibody, anti-human NKG2D antibody, etc., and the percentage of each immune cell subset in peripheral blood leukocytes or immune cell parent population cells and the expression intensity of immune cell surface functional marker can be analyzed and calculated by using a flow cytometry.

The detection method of the content of the cytokine and the content of the specific antibody in the antigen-stimulated sample to be detected comprises the following steps: expressing the purified antigenic protein Severe acute respiratory syndrome coronavirus 2-spike protein receptor binding domain or Severe acute respiratory syndrome coronavirus 2-spike protein S1 subunit, adding into a peripheral blood sample to be tested of a target subject, and stimulating at 37 ℃ and 5% by weight of CO2 for 24 hours. The supernatant after the antigen stimulation is collected, and microspheres coated with specific detection antibodies are used, such as: the method comprises the following steps of detecting the concentration of a cytokine and the concentration of at least one specific antibody in supernatant fluid of a target peripheral blood sample after stimulation of at least one antigen by a microsphere-based multi-factor detection method, wherein the microspheres coated with the specific antibody are marked with fluorescein APC (immunoglobulin C), but the sizes or the fluorescence intensities are different, and the microspheres coated with the IFN-g antibody, the microspheres coated with the RBD IgG antibody and the like can be distinguished in flow cytometry detection. .

The main advantages of the present invention include:

(1) The invention discovers for the first time that the protective immune response state mediated by the vaccine can be comprehensively obtained with high accuracy according to the percentage of the immune cell subgroup or the expression strength of the immune cell surface functional marker, the content of the cell factor and the content of the specific antibody.

(2) The present invention for the first time has found a method for determining the state of a protective immune response mediated by a neocorona vaccine in a peripheral blood sample of a subject, based on the determination of the state of the at least one protective immune response after stimulation by at least one antigen, a cytokine parameter and at least one immune cell parameter as well as at least one specific antibody parameter in a peripheral blood sample of a subject.

(3) The invention discloses a kit for simultaneously detecting a cytokine and a specific antibody for the first time.

The invention is further described with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless otherwise specified, reagents and materials used in examples of the present invention are commercially available products.

The microspheres and antibody reagent materials used in the examples

1) Coating a microsphere mixture of a biotin-labeled antibody: consists of a plurality of microspheres coated with different antibodies (the amount of substances of each microsphere is equal), and the microspheres are respectively (1) the microspheres coated with the IFN-g antibody marked by biotin, and the number of the microspheres is A6; (2) a microsphere coated with a biotin-labeled TNF-a antibody, wherein the microsphere is numbered A4; (3) coating a microsphere of an IL-2 antibody marked by biotin, wherein the microsphere is numbered A10; (4) coating a biotin-labeled microsphere of the anti-RBD IgG antibody, wherein the microsphere is numbered B5; (5) and the microsphere coated with the biotin-labeled anti-Spike S1 IgG antibody is numbered B2. Microspheres were purchased from BioLegend and product numbers are listed in table 1. The microspheres are all labeled with fluorescein APC, but the sizes and fluorescence intensities are different, and the A4, A6 and A10 microspheres are smaller than the B2 and B5 microspheres, so that the microspheres can be distinguished in flow cytometry detection.

TABLE 1 cargo number of microspheres coated with different antibodies

2) PE-labeled streptavidin detection antibody: the PE-labeled streptavidin detection antibody can be combined with the microspheres coated with the biotin-labeled antibody to amplify a detection signal and improve sensitivity, and is purchased from BioLegend (Cat. No. 77743).

3) Fluorescent-labeled anti-immune cell surface functional molecule antibody: the relevant antibody sources are shown in table 2.

TABLE 2 fluorescent-labeled anti-immunocyte surface-functional molecule antibodies, inc. and Cat # s

Name of antibody	Company (SA)	Goods number
			Anti-human CD45-BV510 antibody	BD	563204
Anti-human CD3-PerCP/Cy5.5 antibody	BioLegend	300430
			Anti-human CD4-APC/Fire750 antibody	BioLegend	300560
Anti-human CCR7-FITC antibodies	BioLegend	353216
			Anti-human CD45RA-AF700 antibody	BD	560673
Anti-human CD3-APC/Fire750 antibody	BioLegend	300470
			Anti-human CD19-APC/Fire750 antibody	BioLegend	302258
Anti-human CD56-APC/Fire750 antibody	BioLegend	392408
			Anti-human HLADR-AF700 antibodies	BioLegend	307626
Anti-human CD123-PE/Cy7 antibody	BioLegend	306010
			Anti-human CD11c-PE/CF594 antibody	BD	562393

Example 1: preparation of RBD protein standard solution

1) Plasmid pET-32a (+) (vector purchased from Sigma-Aldrich, cat # 69015) containing RBD gene was transformed into competent DH5a (purchased from Digital organisms, cat # DL 1001), and shake-cultured to OD using LB medium ₆₀₀ ＝1。

2) RBD expression was induced using 0.1mM IPTG for 4-6 hours at 37 ℃.

3) The cell suspension was removed, centrifuged at 4000rpm for 30 minutes at 4 ℃ and the supernatant was discarded.

4) The cells were collected, washed 2 times with PBS buffer, and then resuspended in 4 ℃ precooled PBS buffer.

5) The cells were disrupted under high pressure, centrifuged at 10000rpm for 1 hour at 4 ℃ and the supernatant was discarded.

6) The pellet was resuspended with 8mol/L urea and incubated at 4 ℃ for 24 hours with shaking.

7) The supernatant was collected by centrifugation at 13900 Xg for 40 minutes at 4 ℃.

8) The supernatant was purified by passing through a nickel column using an AKTA protein purification system (available from Cytiva, inc.).

9) Protein renaturation was performed using dialysis bags.

10 The proteins after the completion of the dialytic renaturation were concentrated using an ultrafiltration tube and filtered using a sterile filter to obtain a sterile RBD protein solution.

11 Carrying out protein concentration quantification on a sterile RBD protein solution, diluting the protein concentration to 0.25mg/mL by using a sterile PBS buffer solution to obtain an RBD protein standard solution, subpackaging and storing at-20 ℃.

Example 2 determination of immune cell parameters (naive CD4+ T cells as a percentage of CD45+ cells), cytokine parameters after antigen stimulation (IFN-g concentration) and parameters of the protective immune response status for specific antibody parameters (anti-RBD IgG antibody concentration).

S1, detecting the percentage of the peripheral blood immune cell subpopulation of the target object or the expression intensity parameter of the immune cell surface functional molecules by multicolor flow cytometry. As shown in block a of fig. 1.

1) Collecting peripheral blood sample of target object inoculated by novel coronavirus SARS-CoV-2 inactivated vaccine, transporting at normal temperature, and completing sample analysis within 24 h.

2) Antibodies capable of detecting naive CD4+ T cells (CD 45+ CD3+ CD4+ CCR7+ CD45RA +) were added to peripheral blood samples: anti-human CD45-BV510 antibody, anti-human CD3-PerCP/Cy5.5 antibody, anti-human CD4-APC/Fire750 antibody, anti-human CCR7-FITC antibody and anti-human CD45RA-AF700 antibody, and incubating for 15 minutes at room temperature in the dark.

3) Add erythrocyte lysate, vortex and mix well, incubate 15 minutes in dark.

4) Centrifuge at 500 Xg for 5 min at room temperature, discard the supernatant, add PBS, vortex and mix well, centrifuge at 500 Xg for 5 min at room temperature.

5) Discard supernatant, add 1% PFA resuspension, detect fluorescence signal using flow cytometer

6) The percentage of each immune cell subset in peripheral blood and the median of fluorescence intensity of surface functional markers of each immune cell subset were calculated as their expression intensity using flow analysis software FlowJo circle gate analysis.

And S2, stimulating the peripheral blood sample of the target object by using the specific antigen. As shown in block B of fig. 1.

500ul of peripheral blood of the target subject (peripheral blood sample inoculated with inactivated vaccine against SARS-CoV-2, a novel coronavirus) was applied to a cell culture plate, 10ul of RBD protein standard solution was added thereto, mixed, and stimulated at 37 ℃ for 24 hours at 5% CO2.

And S3, detecting at least one antigen-stimulated cytokine parameter and at least one specific antibody parameter in a target peripheral blood sample (peripheral blood sample inoculated by the novel coronavirus SARS-CoV-2 inactivated vaccine) by a multi-factor detection method based on microspheres. As shown in block C of fig. 1.

1) Collecting a detection sample: and (3) transferring all the peripheral blood samples after the specific antigen stimulation to a new 1.5mL centrifuge tube, centrifuging the peripheral blood samples at the room temperature by 500 Xg for 10 minutes, and collecting supernate to be tested.

2) A96-well plate was taken, 25ul of the supernatant after stimulation with the specific antigen was added first, and 20ul of the microsphere mixture coated with the detection antibody was added. The entire well plate was wrapped with tinfoil and placed on a shaker and incubated for 2 hours at room temperature with shaking.

3) Add 1 × washing solution and repeat washing twice.

4) 25ul of PE-labeled streptavidin detection antibody was added, the entire well plate was wrapped with tinfoil paper and placed on a shaker, and incubated for 1 hour at room temperature with shaking.

5) Add 1 × Wash solution and repeat twice.

6) The samples were transferred from the well plate into a flow tube and detected using a flow cytometer.

7) Analyzing the data by using a LEGENDplex analysis tool, and calculating the concentration of the cytokine and the concentration of the specific antibody after antigen stimulation of the peripheral blood sample of the target object according to a standard concentration curve (detection concentration = stimulated group concentration-unstimulated group concentration).

According to the above-mentioned process, the percentage of CD45+ cells occupied by naive CD4+ T cells, IFN-g concentration, and the "low response" status range, "medium response" status range, and "high response" status range of anti-RBD IgG antibody concentration were obtained by detection and calculation, as shown in Table 3

TABLE 3 percentage of CD4+ T cells in CD45+ cells, IFN-g concentration, and "Low response" status range, "Medium response status" range, and "high response" status range for anti-RBD IgG antibody concentration

As shown in fig. 2, the difference in the percentage of naive CD4+ T cells to CD45+ cells in the low response state, the medium response state, and the high response state.

As shown in FIG. 3, the difference in IFN-g concentration between the low response state, the medium response state and the high response state.

As shown in FIG. 4, the difference in the anti-RBD IgG antibody concentration among the low response state, the medium response state and the high response state is shown.

Wherein, at least two parameters of the three parameters of the percentage of the juvenile CD4+ T cells to the CD4+ T cells, the IFN-g concentration and the anti-RBD IgG antibody concentration of the subject are determined to be in a low response state, and the immune response state mediated by the new corona vaccine of the subject is considered to be in the low response state.

Wherein, at least two parameters of the three parameters of the percentage of the CD4+ T cells in the naive CD4+ T cells, the IFN-g concentration and the anti-RBD IgG antibody concentration of the subject are determined to be in a middle response state, and the immune response state mediated by the new corona vaccine of the subject is considered to be in the middle response state.

Wherein, at least two parameters of the three parameters of the percentage of CD4+ T cells in the larvae of the subject to the CD4+ T cells, the IFN-g concentration and the anti-RBD IgG antibody concentration are determined to be in a high response state, and the immune response state mediated by the subject neocoronary vaccine is considered to be in the high response state.

Example 3 protective immune response status parameters were determined for immune cell parameters (intensity of expression of HLADR on pDC, HLADR MFI in pDC), cytokine parameters after antigen stimulation (TNF-a concentration) and specific antibody parameters (anti-Spike S1 IgG antibody concentration).

The procedure was carried out as in example 2.

The flow cytometry antibodies for detecting the immune cell subgroup pDC (CD 45+ CD3-CD19-CD56-HLADR + CD123+ CD11 c-) and the functional markers are as follows: anti-human CD45-BV510 antibody, anti-human CD3-APC/Fire750 antibody, anti-human CD19-APC/Fire750 antibody, anti-human CD56-APC/Fire750 antibody, anti-human HLADR-AF700 antibody, anti-human CD123-PE/Cy7 antibody, anti-human CD11c-PE/CF594 antibody.

According to the above-described procedure, the "low response" state range, "medium response state" range and "high response" state range for detecting and calculating the expression intensity of HLADR on pDC (HLADR MFI in pDC), TNF-a concentration and anti-Spike S1 IgG antibody concentration are shown in Table 4.

TABLE 4 expression Strength of HLADR on pDC (HLADR MFI in pDC), TNF-a concentration and "Low response" State Range, "Medium response State" Range and "high response" State Range for anti-Spike S1 IgG antibody concentration

As shown in FIG. 5, the difference in expression level expression intensity of HLADR on pDC (HLADR MFI in pDC) in low response state, medium response state and high response state.

As shown in FIG. 6, the difference in TNF-a concentration among the low response state, the medium response state and the high response state was observed.

As shown in FIG. 7, the differences in the concentrations of anti-Spike S1 IgG antibodies were observed among the low response state, the medium response state and the high response state.

Example 4: use of neocorona vaccine mediated immune response status

The procedure was carried out as in example 2.

Three parameters, namely percentage of CD4+ T cells in the naive CD4+ T cells, IFN-g concentration and anti-RBD IgG antibody concentration of the four subjects A, B, C and D were measured, respectively, as shown in FIG. 8.

Wherein, the percentage of the juvenile CD4+ T cells in the CD4+ T cells of the A subject is in a low response range, the concentration of IFN-g is in a high response range, and the concentration of the anti-RBD IgG antibody is in a low response range, so that the immune response state mediated by the new corona vaccine of the A subject is judged to be a low response state.

Wherein, the percentage of the juvenile CD4+ T cells in the CD4+ T cells of the B subject is in a low response range, the concentration of IFN-g is in a low response range, and the concentration of the anti-RBD IgG antibody is in a high response range, so that the immune response state mediated by the new corona vaccine of the B subject is judged to be in a low response state.

Wherein, the percentage of the C object juvenile CD4+ T cells in the CD4+ T cells is in a high response range, the concentration of IFN-g is in a medium response range, and the concentration of the anti-RBD IgG antibody is in the medium response range, so that the immune response state mediated by the new corona vaccine of the object is judged to be in a medium response state.

Wherein, the percentage of the D object juvenile CD4+ T cells in the CD4+ T cells is in the middle response range, the IFN-g concentration is in the high response range, and the anti-RBD IgG antibody concentration is in the high response range, so that the immune response state mediated by the new crown vaccine of the object is judged to be the high response state.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Shanghai International institute for human phenotype groups

<120> method for determining protective immune response status mediated by neocorona vaccine

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Claims

1. A method of detecting a vaccine-mediated protective immune response status comprising:

(a) Providing a sample to be detected, wherein the sample to be detected is a blood sample, and the sample to be detected is a sample after vaccination;

(e) According to the percentage of the immune cell subgroup or the expression intensity of the immune cell surface functional marker, the content of the cell factor and the content of the specific antibody, the state of the protective immune response mediated by the vaccine is obtained.

2. The method of claim 1, wherein the vaccine comprises a coronavirus vaccine.

3. The method of claim 1, wherein the percentage of the subpopulation of immune cells is detected by one or more of the following: flow cytometry.

4. The method of claim 1, wherein the intensity of expression of the immune cell surface function marker is detected by one or more of the following: flow cytometry, western immunoblotting (Western Blot).

5. The method of claim 1, wherein the cytokine content is detected by one or more of the following: enzyme linked immunosorbent assay (ELISA), microsphere-based multi-factor assay (Cytokine Bead Assays), luminex platform-based multi-factor assay, and MSD (Meso Scale Discovery) platform-based electrochemiluminescence hypersensitivity multi-factor assay.

6. The method of claim 1, wherein the amount of specific antibody is detected by one or more of the following: enzyme linked immunosorbent assay (ELISA), microsphere-based multi-factor assay (Cytokine Bead Assays), luminex platform-based multi-factor assay, and MSD (Meso Scale Discovery) platform-based electrochemiluminescence hypersensitivity multi-factor assay.

7. The method of claim 1, wherein the vaccine-mediated protective immune response state comprises a low response state, a medium response state, and a high response state.

8. A method of determining the status of a vaccine-mediated protective immune response comprising:

(a) Providing a data set for the analysis, the data set comprising: a1 st data set from the percentage of immune cell subpopulations or the expression intensity of immune cell surface functional markers, a2 nd data set from the amount of cytokines and a3 rd data set from the amount of specific antibodies, each data set being from a post-vaccination sample, and the 2 nd and 3 rd data sets also being from an antigen stimulated test sample;

9. The method of claim 8, wherein in step (b), each of said data sets is statistically analyzed as follows:

1) The following objects are defined and calculated: (1) a first quartile equal to the value of maximum + (max-min) x 33% in the data set; (2) a second tertile equal to the maximum + (max-min) x 67% value in the dataset;

2) Calculating an average value x of sample detection values in the data set having detection values below a first quartile ₁ And standard deviation s ₁ The "low response range" is: x is less than or equal to ₁ +s ₁ 。

3) Calculating an average value x of sample detection values in the dataset having detection values higher than the second quartile ₂ And standard deviation s ₂ Said "high response range"is: not less than x ₂ -s ₂ 。

4) The "medium response range" is between the "low response range" and the "high response range".

10. An apparatus for determining the status of a vaccine-mediated protective immune response comprising:

(a) A data input unit for inputting 3 data sets for the analysis, wherein the data sets comprise: a1 st data set from the percentage of immune cell subpopulations or the expression intensity of immune cell surface functional markers, a2 nd data set from the amount of cytokines and a3 rd data set from the amount of specific antibodies, each data set from a post-vaccination sample, and the 2 nd and 3 rd data sets also from antigen-stimulated test samples;

(b) The statistical analysis unit is used for performing statistical analysis on each data set so as to obtain data of a low response range, a medium response range and a high response range corresponding to each data set; the statistical analysis unit is configured to perform the following operations:

(iii) Calculating an average value x of sample detection values in the dataset having detection values higher than the second quartile ₂ And standard deviation s ₂ The "high response range" is: not less than x ₂ -s ₂ 。

(c) The data analysis unit is used for further analyzing and processing the data obtained by the statistical analysis unit so as to determine the vaccine-mediated protective immune response state; the data analysis unit is configured to perform the following operations: