WO2023125749A1 - 一种评价个体是否完成疫苗接种或个体免疫变化的方法 - Google Patents

一种评价个体是否完成疫苗接种或个体免疫变化的方法 Download PDF

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WO2023125749A1
WO2023125749A1 PCT/CN2022/143141 CN2022143141W WO2023125749A1 WO 2023125749 A1 WO2023125749 A1 WO 2023125749A1 CN 2022143141 W CN2022143141 W CN 2022143141W WO 2023125749 A1 WO2023125749 A1 WO 2023125749A1
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vaccine
characteristic
peak
peaks
virus
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French (fr)
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马庆伟
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吕倩
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北京毅新博创生物科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks

Definitions

  • the invention belongs to the field of biotechnology, relates to a method for evaluating vaccine quality and vaccine protection validity period based on immune response fingerprints by using time-of-flight mass spectrometry technology, and belongs to the field of vaccine detection.
  • Immune effect monitoring refers to judging an individual's immune protection by detecting whether the level of relevant antibodies in the body reaches the minimum level required for disease prevention.
  • IgG antibodies will be produced in the body after successful and effective vaccination or natural infection. However, the internal conditions of each body, the body's response to the vaccine, and the body's metabolism are different, so the level of antibodies produced by different bodies after vaccination, the speed at which antibodies decay in the body, and the retention time are also different. protection will also vary.
  • the traditional technology for evaluating the immune effect is to detect the level of relevant antibodies or related cytokines and pathogens in the body, that is, it is necessary to detect whether the relevant antibodies or cytokines in the body reach a certain level, or whether the pathogens drop to the expected level.
  • Peng Yi (“China School Health", Volume 21, Issue 1, 2000) reported a method for monitoring the level of tuberculosis immunity. This method uses BCG pure protein derivatives (PPD) as an indicator, and the positive conversion rate of the PPD test The card mark reaction is used to detect the tuberculosis immunity level of the vaccinators, and good results have been achieved.
  • PPD BCG pure protein derivatives
  • Geng Lina (China School Doctor", Volume 31, Issue 10, 2017) reported that the enzyme-linked immunosorbent assay was used to detect the concentration of rubella antibody in serum, and the overall antibody level of the population was analyzed by software, the immunity and age of the population were evaluated, and the effect of vaccination on antibody The level of influence also achieves good results.
  • enzyme-linked immunosorbent assay uses the sensitivity and specificity of the virus to obtain test results, and particle agglutination assay is obtained by culturing The virus is used to observe the changing process of the virus.
  • the rapid diagnostic reagent detection method uses the chemical reaction between the virus and the reagent to obtain the experimental results.
  • the antibody confirmation test method uses radioimmunoprecipitation and Western blotting to obtain the antibody detection results. Therefore, traditional vaccine effect evaluation techniques can only evaluate the immune effect of a single index, which is not universal.
  • This method does not target specific markers related to antibodies or their fragments, but monitors the changes in molecular immunity in body fluids after immune responses to various vaccines, which is highly versatile. sex.
  • the first principle of the present invention is that usually within 2-4 weeks of inoculation with DNA or RNA virus vaccines, effective titer antibodies can be produced.
  • the inventors found through mass spectrometry that some of the characteristic protein fragments or characteristic polypeptide fragments of the immune response produced by the body after vaccination are not exclusive to the antibodies or antibody fragments produced by the body.
  • By optimizing the evaluation system obtained by screening and screening the non-antibody characteristic polypeptide fragments related to the immune effect of the vaccine according to each known time point of DNA or RNA virus type antibody production, and establishing a characteristic mass spectrometry model it can be established A method for evaluating the immune effect of the vaccine. Because the method is based on the difference of characteristic polypeptides in vivo before and after vaccination rather than characteristic antibody fragments, it can quickly and accurately evaluate the changes in the body's immune response.
  • the second principle of the present invention is to construct a mass spectrometry model for detecting or evaluating the immune effect of the vaccine by analyzing the protein sequence of the characteristic protein or polypeptide fragment composition screened from different vaccines through the above principles and methods.
  • the first object of the present invention is to provide a method for constructing mass spectrometry models for evaluating the immune effects of various vaccines, including:
  • body fluid samples from multiple volunteers at multiple time points before and after vaccination, and freeze them for later use, wherein the body fluid samples are selected from serum, saliva, plasma, whole blood, saliva, urine, tissue fluid, and joint fluid , Nasopharyngeal swab extract or cerebral effusion.
  • the software is the BE-V 2.0 software researched and developed by the inventor himself;
  • step (1) when the vaccine needs to be vaccinated for 1 shot, the time point is 1 day before inoculation and the 3rd week after inoculation;
  • the time points are 1 day before the first injection, 3 weeks after the first injection, 1 week after the second injection, and 3 weeks after the second injection. At least 21 days between needles;
  • the time points are 1 day before the first dose, 3 weeks after the first dose, 1 week after the second dose, 3 weeks after the second dose, and the third week.
  • Week 1 after the first dose and week 3 after the third dose with at least 21 days between doses; and,
  • a group of characteristic peaks inherent in a certain type of body fluid sample is selected as a quality control internal standard peak, wherein the internal standard peak m/z is as follows: 6426m/z, 6623m/z, 8753m/z, 8785m/z, 8904m/z, 9118m/z, 9409m/z, 9700m/z, and the spectrum quality control conditions are: in the spectrum of a single sample, the quality control When the number of internal standard peaks is not less than 70% of the total number of quality control internal standard peaks and the deviation of the molecular weight of the internal standard peak is less than 0.002 or the deviation range does not exceed 2 ⁇ , the quality control is considered qualified.
  • the vaccine that needs to be inoculated for one shot is an adenovirus vector vaccine
  • the vaccine that needs to be inoculated for two shots is an inactivated vaccine
  • the vaccine that needs to be inoculated for three shots is a recombinant protein vaccine.
  • the pretreatment method in step (2) includes diluting the protein or polypeptide in the stable sample with a sample treatment solution (Beijing Yixin Bochuang Biotechnology Co., Ltd.).
  • the step (2) uses the immunoreaction fingerprinting kit (Beijing Yixin Bochuang Biotechnology Co., Ltd., article number: 1010307) to dilute and read the protein or polypeptide in the body fluid sample to obtain Peptide Fingerprints.
  • the immunoreaction fingerprinting kit Beijing Yixin Bochuang Biotechnology Co., Ltd., article number: 1010307
  • the same mass spectrometry parameters are used to detect the crystallization point of the blank matrix, and if an obvious mass spectrum peak appears, the quality of the matrix solution is considered unqualified.
  • the detection instrument used in the time-of-flight mass spectrometry is MALDI-TOF MS.
  • the vaccine that needs to be inoculated for one dose is an adenovirus vector vaccine
  • the vaccine that needs to be inoculated for two doses is an inactivated vaccine
  • the vaccine that needs to be inoculated for three doses is a recombinant protein vaccine.
  • the vaccine is a vaccine against a DNA viral disease or a vaccine against an RNA viral disease.
  • the DNA virus includes hepatitis B virus, herpes virus, rubella virus, human papilloma virus.
  • the RNA virus includes AIDS virus/hepatitis C virus, Japanese encephalitis virus/all influenza viruses, SARS virus, MERS virus, and novel coronavirus.
  • the vaccine is a vaccine against novel coronavirus disease
  • the body fluid sample is human serum
  • the characteristic polypeptides with specific mass-to-charge ratios include the characteristic peaks 3025m/z and 13761m/z of down-regulated expression , 13882m/z, 13939m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peaks of up-regulated expression are: 3198m/z, 3213m/z, 6609m/ z.
  • the characteristic polypeptides of specific mass-to-charge ratios include, the characteristic peaks 3025m/z, 13882m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z, and the polypeptide sequence is selected from:
  • the body fluid sample is human serum
  • the characteristic polypeptides of the specific mass-to-charge ratio include the characteristic peaks 3025m/z and 13882m/z of down-regulated expression , 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z.
  • the second object of the present invention is to construct a mass spectrometry model based on immune response fingerprints to characterize human immune changes through the above method.
  • the time point is 1 day before vaccination and 3 weeks after vaccination;
  • the time points are 1 day before the first injection, 3 weeks after the first injection, 1 week after the second injection, and 3 weeks after the second injection. At least 21 days between needles;
  • the time points are 1 day before the first dose, 3 weeks after the first dose, 1 week after the second dose, 3 weeks after the second dose, and the third week.
  • Week 1 after the first dose and week 3 after the third dose with at least 21 days between doses; and,
  • a group of characteristic peaks inherent in a certain type of body fluid sample is selected as a quality control internal standard peak, wherein the internal standard peak m/z is as follows: 6426m/z, 6623m/z, 8753m/z, 8785m/z, 8904m/z, 9118m/z, 9409m/z, 9700m/z, and the spectrum quality control conditions are: in the spectrum of a single sample, the quality control When the number of internal standard peaks is not less than 70% of the total number of quality control internal standard peaks and the deviation of the molecular weight of the internal standard peak is less than 0.002 or the deviation range does not exceed 2 ⁇ , the quality control is considered qualified.
  • the detection method of the model is time-of-flight mass spectrometry
  • the detection instrument is MALDI-TOF MS.
  • said model is prepared from immune response fingerprinting.
  • the model includes mass-to-charge ratio, molecular mass, mass spectrum peak area, mass spectrum peak intensity or relative intensity data of 5-200 characteristic peptide/protein peaks.
  • the vaccine is a vaccine against a DNA viral disease or a vaccine against an RNA viral disease.
  • the DNA virus includes hepatitis B virus, herpes virus, rubella virus, human papilloma virus.
  • the RNA virus includes AIDS virus/hepatitis C virus, Japanese encephalitis virus/all influenza viruses, SARS virus, MERS virus, and novel coronavirus.
  • the body fluid sample is human serum
  • the characteristic polypeptides of the specific mass-to-charge ratio include the characteristic peaks 3025m/z and 13761m/z of down-regulated expression , 13882m/z, 13939m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peaks of up-regulated expression are: 3198m/z, 3213m/z, 6609m/ z.
  • the characteristic polypeptide fragments include, the characteristic peaks 3025m/z, 13882m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z, and the polypeptide sequence is selected from:
  • the body fluid sample is human serum
  • the characteristic polypeptides of the specific mass-to-charge ratio include the characteristic peaks 3025m/z and 13882m/z of down-regulated expression , 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z.
  • the third object of the present invention is to obtain the characteristic polypeptide composition for evaluating the immune effect of vaccines obtained by the above-mentioned method for constructing a mass spectrometry model.
  • the vaccine is a vaccine against novel coronavirus disease
  • the body fluid sample is human serum
  • the characteristic polypeptides with specific mass-to-charge ratios include the characteristic peaks 3025m/z, 13882m/z, 14044m /z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z.
  • the characteristic polypeptide of the specific mass-to-charge ratio includes the characteristic peak 3484m/z of down-regulated expression; The characteristic peaks are: 3799m/z, 3878m/z, 4176m/z, 4186m/z, 4609m/z.
  • the characteristic polypeptide composition used to evaluate the immune effect of the vaccine is obtained.
  • the vaccine is a vaccine against novel coronavirus disease
  • the body fluid sample is human serum
  • the characteristic polypeptides with specific mass-to-charge ratios include the characteristic peaks 3025m/z, 13882m/z, 14044m /z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z.
  • the characteristic polypeptide of the specific mass-to-charge ratio includes the characteristic peak 3484m/z of down-regulated expression; The characteristic peaks are: 3799m/z, 3878m/z, 4176m/z, 4186m/z, 4609m/z.
  • the kit also includes a standard mass spectrometry sample tube to ensure the accuracy of the molecular weight measured by the mass spectrometer.
  • the sample tube can be multiple sample tubes containing a single characteristic polypeptide, or one sample tube containing multiple characteristic polypeptides.
  • the sample in the standard sample tube is used to perform a parallel mass spectrometry test with the sample to be tested to determine whether the molecular weight information of the sample to be tested is accurate and reliable.
  • the kit may contain software or a chip for a standard database of immune response fingerprints as described above. It can be used to provide comparison of standard data or curves when mass spectrometry is performed on samples to be tested, so as to judge the expression status of characteristic polypeptides in samples to be tested.
  • the fifth object of the present invention is to use the above-mentioned mass spectrometry model, characteristic polypeptide composition or kit to evaluate whether an individual has completed vaccination or the effect of individualized vaccination.
  • the sixth object of the present invention is to provide a method for evaluating vaccine quality, and/or, analyzing vaccine protective effect time series or vaccine protective efficacy period, and/or, vaccine protective time.
  • the method comprises:
  • body fluid samples from multiple known volunteers at multiple time points before and after vaccination, and perform mass spectrometry pretreatment on the body fluid samples, wherein the body fluid samples are selected from serum, saliva, plasma, whole blood, saliva, and urine , interstitial fluid, joint fluid, nasopharyngeal swab extract or cerebral effusion;
  • Steps (1)-(3) are used to process the patients to be vaccinated, and then perform quality control processing on the obtained data to screen out characteristic polypeptides with specific mass-to-charge ratios;
  • step (6) Using the characteristic polypeptide with a specific mass-to-charge ratio obtained in step (5) and the standard fingerprint model described in step (4), use computer software to evaluate the quality of the vaccine, and/or analyze the time of vaccine protective effect The sequence or duration of vaccine protection, and/or, the duration of vaccine protection;
  • step (1) when the vaccine needs to be vaccinated for 1 shot, the time point is 1 day before inoculation and the 3rd week after inoculation;
  • the time points are 1 day before the first injection, 3 weeks after the first injection, 1 week after the second injection, and 3 weeks after the second injection. At least 21 days between needles;
  • the time points are 1 day before the first dose, 3 weeks after the first dose, 1 week after the second dose, 3 weeks after the second dose, and the third week.
  • Week 1 after the first dose and week 3 after the third dose with at least 21 days between doses; and,
  • a group of characteristic peaks inherent in a certain class of body fluid samples are selected as quality control internal standard peaks, wherein the internal standard peaks
  • the m/z are as follows: 6426m/z, 6623m/z, 8753m/z, 8785m/z, 8904m/z, 9118m/z, 9409m/z, 9700m/z
  • the spectral quality control conditions are: in the spectrum of a single sample
  • the software is the BE-V 2.0 software researched and developed by the inventor himself.
  • the vaccine that needs to be inoculated for one shot is an adenovirus vector vaccine
  • the vaccine that needs to be inoculated for two shots is an inactivated vaccine
  • the vaccine that needs to be inoculated for three shots is a recombinant protein vaccine.
  • the pretreatment method in step (2) includes diluting the protein or polypeptide in the stable sample with a sample treatment solution (Beijing Yixin Biotechnology Co., Ltd., Beijing Yixin Biotechnology Co., Ltd.).
  • the step (2) uses the immunoreaction fingerprinting kit (Beijing Yixin Bochuang Biotechnology Co., Ltd., article number: 1010307) to dilute and read the protein or polypeptide in the body fluid sample to obtain Peptide Fingerprints.
  • the immunoreaction fingerprinting kit Beijing Yixin Bochuang Biotechnology Co., Ltd., article number: 1010307
  • the quality control process described in step (4) and step (5), for the blank matrix use the same mass spectrometry parameters to detect the crystallization point of the blank matrix, if there is an obvious mass spectrum peak, it is considered that the quality of the matrix solution is not good. qualified.
  • the detection instrument used in the time-of-flight mass spectrometry is MALDI-TOF MS.
  • the vaccine that needs to be inoculated for one dose is an adenovirus vector vaccine
  • the vaccine that needs to be inoculated for two doses is an inactivated vaccine
  • the vaccine that needs to be inoculated for three doses is a recombinant protein vaccine.
  • the vaccine is a vaccine against a DNA viral disease or a vaccine against an RNA viral disease.
  • the DNA virus includes hepatitis B virus, herpes virus, rubella virus, human papilloma virus.
  • the RNA virus includes AIDS virus/hepatitis C virus, Japanese encephalitis virus/all influenza viruses, SARS virus, MERS virus, and novel coronavirus.
  • the vaccine is a vaccine against novel coronavirus disease
  • the body fluid sample is human serum
  • the characteristic polypeptides with specific mass-to-charge ratios include the characteristic peaks 3025m/z and 13761m/z of down-regulated expression , 13882m/z, 13939m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peaks of up-regulated expression are: 3198m/z, 3213m/z, 6609m/ z.
  • the characteristic polypeptides of specific mass-to-charge ratios include, the characteristic peaks 3025m/z, 13882m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z, and the polypeptide sequence is selected from:
  • the body fluid sample is human serum
  • the characteristic polypeptides of the specific mass-to-charge ratio include the characteristic peaks 3025m/z and 13882m/z of down-regulated expression , 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; the characteristic peak of up-regulated expression is: 6609m/z.
  • the invention breaks through the technical limitation that the prior art is only limited to a single index such as existing antibodies or cytokines to evaluate vaccine immune response.
  • the immune response fingerprint method is adopted to realize the joint monitoring of multiple characteristic peaks, which improves the accuracy of immune effect detection.
  • immunoreaction fingerprinting has the technical advantages of simple operation, low cost, high speed and high throughput.
  • the invention is applicable to the characterization of the immune response fingerprints of different body fluids, and provides multiple references for evaluating the immune effect of vaccines.
  • the invention characterizes the immune changes of the vaccinated human body, monitors the protective effect of the vaccine in different time series in real time, and provides powerful evidence for precise epidemic prevention.
  • the invention is also applicable to the characterization of immune response fingerprints of animal body fluids, can be used for the evaluation of preclinical vaccine effects, and accelerates the pace of vaccine research and development.
  • the present invention is more suitable for evaluating the immune effect of individual vaccines, not limited to the analysis of group immune responses, and can realize precise epidemic prevention.
  • the invention can continuously monitor the immune response changes at different time points after vaccination, continuously analyze the immune response changes of the body after vaccination, and evaluate whether the individual has completed vaccination or the effect of individualized vaccination.
  • the evaluation standard can be determined by the existing known method of the vaccine on the basis of the fingerprint comparison result obtained in the present invention.
  • the method of the present invention can be used to analyze the time series of vaccine protection effect or the duration of vaccine protection, and/or, the application in vaccine protection time.
  • the evaluation standard can be determined by the existing known method of the vaccine on the basis of the fingerprint comparison result obtained in the present invention.
  • Figure 1 Comparison of mass spectra of different groups of samples.
  • Figure a is the full spectrum of immune response fingerprints before and after vaccination
  • Figure b is a partial enlarged view of the main characteristic peaks.
  • the abscissa in the figure represents the m/z information of each characteristic peak, and the ordinate represents the normalized spectral peak intensity.
  • the characteristic peaks of 13761m/z, 13882m/z, 13939m/z, 14044m/z, 14091m/z, 14150m/z, and 28195m/z showed significant changes before and after vaccination.
  • Figure a is the principal component analysis diagram of the immune response fingerprint before inoculation with the new crown inactivated vaccine and 3 weeks after the first dose of vaccine;
  • Figure b is the principal component analysis diagram of the immune response fingerprint before inoculation with the new crown inactivated vaccine and 1 week after the second dose of vaccine;
  • Figure c is the principal component analysis diagram of the immune response fingerprint before inoculation with the new crown inactivated vaccine and 3 weeks after the second dose of vaccine;
  • Figure d is the principal component analysis diagram of the immune response fingerprint 3 weeks after the first injection of the vaccine and 1 week after the second injection of the vaccine;
  • Figure e is the principal component analysis diagram of the immune response fingerprint 3 weeks after the first injection of the vaccine and 3 weeks after the second injection of the vaccine;
  • Figure f is the principal component analysis diagram of the immune response fingerprint 1 week after the second dose of vaccine and 3 weeks after the second dose of vaccine.
  • FIG. 1 Partial least squares regression analysis (PLS-DA) diagram of neutralizing antibody positive samples and neutralizing antibody negative samples in the immune response fingerprint model. "P” in the figure means neutralizing antibody positive; “N” means neutralizing antibody negative.
  • Figure 5 The up- and down-regulation of each characteristic peak of the neutralizing antibody-related immune response fingerprint.
  • the figure shows the relative intensity comparison of 7 characteristic peaks in neutralizing antibody negative samples and positive samples. p-values were calculated by Mann-Whitney test. Asterisks indicate statistically significant differences between groups (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001).
  • N in the figure means neutralizing antibody negative;
  • P means neutralizing antibody positive.
  • the curve a in the figure is the saliva immune polypeptide fingerprint spectrum of people who have not been vaccinated with the new crown inactivated vaccine; the b curve is the saliva immune polypeptide fingerprint spectrum of people who have been vaccinated with 2 doses of the new crown inactivated vaccine.
  • Fig. 7 The up and down regulation of each characteristic peak of the saliva immune response fingerprint.
  • FIG. 8 The principal component analysis (PCA) diagram of the salivary immune response fingerprint model.
  • the green points in the figure represent the samples that have completed 2 injections of vaccine, and the red points represent the samples that have not completed the complete injection.
  • Figure 9. Peptide fingerprinting spectrum of influenza nasal swab.
  • the curve a in the figure is the polypeptide fingerprint spectrum of nasal swabs from people who have not been vaccinated against influenza;
  • the curve b in the figure is the polypeptide fingerprint spectrum of nasal swabs from people who have been vaccinated against influenza.
  • Fig. 10 Partial least squares discriminant (PLS-DA) analysis chart of influenza nasal swab immune response fingerprint model.
  • the left area (n) represents the sample distribution of those who have not been vaccinated against influenza
  • the right area (p) represents the sample distribution of those who have been vaccinated against influenza.
  • the immune response fingerprint analysis was carried out on the serum of volunteers who had not been vaccinated with the new crown inactivated vaccine and who had been vaccinated at different time points. Through the immune response fingerprint, it is hoped that the serum changes during the immunization process can be evaluated, and the immune effect of the vaccine can be evaluated.
  • Mass spectrometry pretreatment of serum samples Before performing mass spectrometry detection experiments, take out the aliquoted serum samples from the low-temperature refrigerator and place them on wet ice. Thaw for 60-90 minutes.
  • Clin-TOF mass spectrometer (Beijing Yixin Bochuang Biotechnology Co., Ltd.) was used. Set the appropriate laser energy to collect a certain point of the crystallization point of the sample. 50 laser bombardment positions were selected for each sample point, and each position was bombarded 10 times, that is, 500 laser bombardments were performed on each sample crystallization point to collect spectra. Laser frequency: 30Hz. Data collection range: 3 ⁇ 30KDa. Before the crystallization point of each sample is collected, external standard calibration is performed with a standard, and the average molecular weight deviation is less than 500ppm.
  • the internal standard peak m/z is as follows: 6426m/z, 6623m/z, 8753m/z, 8785m/z, 8904m/z, 9118m/z, 9409m/z, 9700m/z.
  • the raw data of MALDI-TOF was recalibrated with internal standard by BE-V 2.0 software (Beijing Yixin Bochuang Biotechnology Co., Ltd.).
  • the internal standard peak m/z used is: 6426m/z, 6623m/z, 8753m/z, 8785m/z, 8904m/z, 9118m/z, 9409m/z, 9700m/z.
  • BE-V 2.0 software (Beijing Yixin Bochuang Biotechnology Co., Ltd.) processed the spectrum. Spectrum processing includes variance stabilization, smoothing and noise reduction, baseline removal, peak intensity normalization, spectral averaging, spectral alignment, spectral peak identification, spectral peak binning, etc. Keep the peaks whose peak frequency is not lower than 25% in the group.
  • the resulting matrix is used in the following analysis. After log2 transformation, the peak intensity matrix was quantile normalized. In all samples, missing values are filled with the minimum value.
  • the peaks of the training set were analyzed using the following three machine learning methods: Partial Least Squares Regression Analysis (PLS-DA), Volcano Plot Analysis (Volcano Plot) and Pairwise t test method.
  • PLS-DA Partial Least Squares Regression Analysis
  • Volcano Plot Volcano Plot
  • Pairwise t test method select the peak with vip greater than 1.8; in the volcano map analysis method, set Fc greater than 1.6 and P equal to 0.05; in the paired t test method, select the peak with P value less than 0.05 and the proportion greater than 1.2 .
  • the common difference peaks were selected as the characteristic peaks to establish the model.
  • characteristic peaks of down-regulated expression are: 3025m/z, 13761m/z, 13882m/z, 13939m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, 15868m/z, 28195m/z; up-regulated expression
  • the characteristic peaks are: 3198m/z, 3213m/z, 6609m/z.
  • the comparison chart of the immune response fingerprints of different groups of samples is shown in Figure 1, in which from bottom to top are the serum profiles of healthy people who have not been vaccinated with the new crown vaccine, the serum profiles of volunteers 3 weeks after receiving 1 injection of inactivated vaccine, and the serum profiles of volunteers who received 2 doses of inactivated vaccine. Serum profiles of volunteers 1 week after receiving inactivated vaccines, serum profiles of volunteers 3 weeks after receiving 2 injections of inactivated vaccines).
  • the serum inoculated with 1 to 2 injections was at 3025m/z, 13761m/z, 13882m/z, 13939m/z, 14044m/z, 14092m/z, 14150m/z, 15124m/z, At 15868m/z and 28195m/z, the peak intensity decreased obviously; while at 3198m/z, 3213m/z, and 6609m/z, the peak intensity increased obviously.
  • the details of the up and down regulation of the 13 characteristic peaks are shown in Figure 2.
  • Example 1 Three serum samples with higher characteristic peak intensities were selected, and the characteristic peaks determined in Example 1 were identified by secondary mass spectrometry. After the samples were reduced by DTT, they were separated by tricine-SDS-PAGE. Each band was identified by secondary mass spectrometry after in-gel digestion.
  • the nano-LC-MS/MS platform was used for peptide sequence identification, including nanoflow HPLC (Thermo Fisher Scientific, USA) and Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA).
  • the ion mode is positive ion mode, and the scanning range is 300-1400m/z.
  • the resolution of the primary mass spectrometer is 70,000, and the resolution of the secondary mass spectrometry is 17,500.
  • Liquid phase analysis column model: Exsil Pure 120 C18 (Dr.Maisch GmbH, USA); specification: 360 ⁇ m ⁇ 12cm; inner diameter: 150 ⁇ m; particle: 1.9um.
  • Elution mode mobile phase from 7% B solution (80% acetonitrile, 0.1% formic acid) to 45% B solution, linear elution. Flow rate: 600 nl/min; total time 38 minutes.
  • the peptide fingerprints verified in step (7) are processed by BE-V 2.0 software to construct a standard mass spectrometry model of the immune response of the new crown vaccine.
  • the 10 samples of the verification group were collected with the same method for immune response fingerprinting, and compared with the above-mentioned standard mass spectrometry model through the BE-V 2.0 software.
  • the results of the verification model sensitivity, specificity, accuracy, and precision are detailed in the table below .
  • the time series analysis of MALDI-TOF mass spectrometry was performed on the peptide fingerprints collected at three sampling time points before and after inoculation by principal component analysis (PCA), and the results are shown in Figure 3.
  • PCA principal component analysis
  • the immune peptide fingerprint on day 0 could be well distinguished from days 21, 28, and 42. It shows that after the first injection of the vaccine, the proteins and peptides in human serum will undergo significant changes.
  • a standard peptide fingerprint has been constructed, which indicates that the immune effect of the new crown vaccine can be characterized, and it can be used to evaluate the quality of the vaccine, and/or analyze the time series of vaccine protection effect or vaccine protection. The validity period of the vaccine, and/or, the duration of vaccine protection.
  • the immune effect of the vaccine can be evaluated by the level of antibodies (ie, binding antibodies, or immunoglobulins) produced by the human body after vaccination, for certain viral antigens before or in the human body, if the body exists Smaller proteins (neutralizing antibodies) that bind to particles inside the virus, then prevent the virus from infecting cells, destroy the virus particles. Therefore, not only the total antibody level should be measured in clinical trials, but more importantly, the neutralizing antibody level should be measured separately. Therefore, detecting the production of antibodies, especially neutralizing antibodies (NAbs), is of great significance for most COVID-19 vaccines to elicit the human body's protective mechanism against SARS-COV-2.
  • antibodies ie, binding antibodies, or immunoglobulins
  • the serum of volunteers 3 weeks after inoculation with 2 doses of the new crown inactivated vaccine was divided into two groups according to whether neutralizing antibodies were produced, and the immune response fingerprint analysis was performed in an attempt to confirm the immune response Fingerprinting (or fingerprinting) can be used to characterize whether a vaccinated person will develop neutralizing antibodies.
  • Mass spectrometry pretreatment of serum samples Before performing mass spectrometry detection experiments, take out the aliquoted serum samples from the low-temperature refrigerator and place them on wet ice. Thaw for 60-90 minutes.
  • Clin-TOF mass spectrometer (Beijing Yixin Bochuang Biotechnology Co., Ltd.) was used. Set the appropriate laser energy to collect a certain point of the crystallization point of the sample. For each sample point, 50 laser bombardment positions were selected, and each position was bombarded 10 times, that is, 500 laser bombardments were performed on each sample crystallization point to collect spectra. Laser frequency: 30Hz. Data collection range: 3 ⁇ 30KDa. Before the crystallization point of each sample is collected, external standard calibration is performed with a standard, and the average molecular weight deviation is less than 500ppm.
  • the internal standard peak m/z is as follows: 6426m/z, 6623m/z, 8753m/z, 8785m/z, 8904m/z, 9118m/z, 9409m/z, 9700m/z.
  • the raw data of MALDI-TOF was recalibrated with internal standard by BE-V 2.0 software (Beijing Yixin Bochuang Biotechnology Co., Ltd.).
  • the internal standard peak m/z used is: 6426m/z, 6623m/z, 8753m/z, 8785m/z, 8904m/z, 9118m/z, 9409m/z, 9700m/z.
  • BE-V 2.0 software (Beijing Yixin Bochuang Biotechnology Co., Ltd.) processed the spectrum.
  • Spectrum processing includes variance stabilization, smoothing and noise reduction, baseline removal, peak intensity normalization, spectral averaging, spectral alignment, spectral peak identification, spectral peak binning, etc.
  • the resulting matrix is used in the following analysis. After log2 transformation, the peak intensity matrix was quantile normalized. In all samples, missing values are filled with the minimum value. 41 samples were randomly selected for model building, and the remaining 10 samples were used for model validation.
  • the peaks of the training set were analyzed using the following three machine learning methods: Partial Least Squares Regression Analysis (PLS-DA), Volcano Plot Analysis (Volcano Plot) and Pairwise t test method.
  • PLS-DA Partial Least Squares Regression Analysis
  • Volcano Plot Volcano Plot
  • Pairwise t test method select the peak with vip greater than 2; in the volcano map analysis method, set Fc greater than 1.6 and P equal to 0.05; in the paired t test method, select the peak with P value less than 0.05 and the proportion greater than 1.2 .
  • PLS-DA partial least squares regression analysis
  • neutralizing antibody-positive serum Compared with neutralizing antibody-negative samples, neutralizing antibody-positive serum showed significant down-regulation of peak intensities at 6609m/z, 6980m/z, 13929m/z, 13939m/z, and 14083m/z; There is a clear peak intensity upregulation at z. See Figure 5 for details on the up-down-regulation relationship of neutralizing antibody-related characteristic peaks.
  • Example 2 Three serum samples with higher characteristic peak intensities were selected, and the characteristic peaks determined in Example 2 were identified by secondary mass spectrometry. After the samples were reduced by DTT, they were separated by tricine-SDS-PAGE. Each band was identified by secondary mass spectrometry after in-gel digestion.
  • the nano-LC-MS/MS platform was used for peptide sequence identification, including nanoflow HPLC (Thermo Fisher Scientific, USA) and Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA).
  • the ion mode is positive ion mode, and the scanning range is 300-1400m/z.
  • the resolution of the primary mass spectrometer is 70,000, and the resolution of the secondary mass spectrometry is 17,500.
  • Liquid phase analysis column model: Exsil Pure 120 C18 (Dr.Maisch GmbH, USA); specification: 360 ⁇ m ⁇ 12cm; inner diameter: 150 ⁇ m; particle: 1.9um.
  • Elution mode mobile phase from 7% B solution (80% acetonitrile, 0.1% formic acid) to 45% B solution, linear elution. Flow rate: 600 nl/min; total time 38 minutes.
  • Example 1 the method was tested to analyze the immune response fingerprints of the saliva samples of the new crown vaccine recipients, to confirm that the immune response fingerprints of the present invention are also applicable to other body fluid samples.
  • the original data of MALDI-TOF was processed by BE1.0 software (Beijing Yixin Bochuang Biotechnology Co., Ltd.). Spectrum processing includes variance stabilization, smoothing and noise reduction, baseline removal, peak intensity normalization, spectral averaging, spectral alignment, spectral peak identification, spectral peak binning, etc. Keep the peaks whose peak frequency is not lower than 25% in the group. Finally, the resulting matrix is used in the following analysis. After log2 transformation, the peak intensity matrix was quantile normalized. In all samples, missing values are filled with the minimum value.
  • peaks in the training set were analyzed with a paired t-test.
  • the peak with P value less than 0.01 was selected as the characteristic peak to establish the model.
  • the mean-variance diagrams of each characteristic peak in the two groups of samples are shown in Figure 7.
  • the samples in the left column have been vaccinated with 2 doses; the samples in the right column are those who have not been vaccinated or only received 1 dose of vaccine.
  • the horizontal axis is the group name, and the vertical axis is the peak intensity.
  • the middle horizontal line represents the mean intensity value of the characteristic peak of this group of data; the upper and lower horizontal lines represent the degree of dispersion of the characteristic peak intensity of this group of samples.
  • FIG. 8 See Figure 8 for the principal component analysis (PCA) diagram of the model.
  • the green dots in the figure represent the samples that have completed 2 injections of the vaccine, and the red dots represent the samples that have not completed the complete injection.
  • the figure shows the projection distribution of two sets of sample points in three-dimensional space. The more dispersed the distribution of sample points between the two groups, the greater the discriminative potential of the representative model.
  • KNN K-nearest algorithm
  • SVM support vector machine
  • the 22 samples from the verification group were collected by the same method for immune response fingerprinting, and the results were imported into the KNN model.
  • the model verification results are shown in the table below.
  • Immunoreactive fingerprinting of nasal swab samples from unvaccinated and vaccinated children Through immune response fingerprinting, the changes of nasal mucosal polypeptides during immunization are evaluated, and the immune response of vaccines is evaluated.
  • Clin-TOF mass spectrometer (Beijing Yixin Bochuang Biotechnology Co., Ltd.) was used. Set the appropriate laser energy to collect a certain point of the crystallization point of the sample. For each sample point, 50 laser bombardment positions were selected, and each position was bombarded 10 times, that is, 500 laser bombardments were performed on each sample crystallization point to collect spectra. Laser frequency: 30Hz. Data collection range: 2 ⁇ 20KDa. Before the crystallization point of each sample is collected, external standard calibration is performed with a standard, and the average molecular weight deviation is less than 500ppm.
  • the raw data of MALDI-TOF were processed with BE-V 2.0 software (Beijing Yixin Bochuang Biotechnology Co., Ltd.). Spectrum processing includes variance stabilization, smoothing and noise reduction, baseline removal, peak intensity normalization, spectral averaging, spectral alignment, spectral peak identification, spectral peak binning, etc. Keep the peaks whose peak frequency is not lower than 25% in the group. Finally, the resulting matrix is used in the following analysis. After log2 transformation, the peak intensity matrix was quantile normalized. In all samples, missing values are filled with the minimum value.
  • the peaks of the training set were analyzed using the following two machine learning methods: Partial Least Squares Regression Analysis (PLS-DA) and Volcano Plot Analysis (Volcano Plot). Select vip greater than 1.5 peaks in the partial least squares regression analysis method (accompanying drawing 10); set Fc greater than 1.5 and P equal to 0.05 in the volcano map analysis method (accompanying drawing 11). Through the analysis of the difference peaks among the groups, the common difference peaks were selected as the characteristic peaks to establish the model.
  • PLS-DA Partial Least Squares Regression Analysis
  • Volcano Plot Volcano Plot
  • the characteristic peak m/z is as follows: 2061m/z, 2034m/z, 2176m/z, 2043m/z, 2131m/z, 2079m/z, 2292m/z, 2233m/z, 2087m/z, 5336m/z, 2120m/z , 2345m/z, 3687m/z, 2320m/z, 2057m/z, 2385m/z, 4061m/z, 4110m/z, 4961m/z, 3313m/z, 2850m/z, 2408m/z, 3665m/z, 2219m/z, 5853m/z, 2167m/z.
  • the characteristic peaks are 2034m/z, 2079m/z, 2233m/z, 2120m/z, 2345m/z, 2320m/z, 2057m/z, 2385m/z, 4061m/z, 4961m/z, 2850m/z, 2408m/z z, 5853m/z, 2167m/z up, while 2061m/z, 2176m/z, 2043m/z, 2131m/z, 2292m/z, 2087m/z, 5336m/z, 3687m/z, 4110m/z, 3313m/z Down-regulation of z, 3665m/z, and 2219m/z indicates that the subject has been vaccinated against influenza.
  • the 12 samples from the verification group were collected by the same method for immune response fingerprinting, and the results were imported into the PLS-DA model.
  • the model verification results are shown in the table below.

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Abstract

一种评价个体是否完成疫苗接种或接种人体免疫变化的方法，通过MALDI-TOF MS质谱技术检测人体接种疫苗前后的体液，分别获得由5~200个质谱峰组成的免疫反应指纹谱，基于未发生免疫反应和已发生免疫反应的指纹谱的变化，结合机器学习的方法建立模型分类器，分析待测人体血清样本中的分子的免疫变化，可判断该样本是否接种过疫苗和产生免疫保护效果，从而评价疫苗质量和/或疫苗保护效期。可以同时检测体液中多种分子免疫反应的变化，突破了传统单一标志物如中和抗体的监测思路，在保证高特异性的基础上，有效地提高了检测的准确度和灵敏度，简化实验操作，缩短检测时间，降低检测成本。

Description

一种评价个体是否完成疫苗接种或个体免疫变化的方法 技术领域
本发明属于生物技术领域，涉及一种利用飞行时间质谱技术基于免疫反应指纹谱来评价疫苗质量和疫苗保护效期的方法，属于疫苗检测领域。
背景技术
免疫效果监测是指通过检测机体内相关抗体水平是否达到预防疾病所需的最低水平，从而判断个体的免疫保护力。成功有效的预防接种或者自然感染后体内均会产生IgG抗体。但每个机体内在情况、机体对疫苗的反应以及机体的代谢等状况是不同的，所以预防接种后不同机体产生的抗体水平、抗体在体内衰减的速度以及存留时间也有所不同，针对相应疾病的保护力也会有差异。
因此，及时对完成基础免疫的人群进行体内保护性抗体检测，提示感染风险或免疫补种，能帮助预防疾病。
评价免疫效果的传统技术，是针对检测机体相关抗体或关联的细胞因子、病原体水平，即需要检测体内的相关抗体或细胞因子是否达到一定水平，或病原体是否下降到预期水平。
例如，彭怡(《中国学校卫生》，2000年第21卷第1期)报道了结核免疫水平监测方法，该方法利用卡介苗纯蛋白衍生物(PPD)作为指标物，通过PPD试验阳转率的卡痕反应，来检测接种者结核免疫水平，取得良好的效果。
耿丽娜(《中国校医》，2017年第31卷第10期)报道了采用酶联免疫吸附试验检测血清中风疹抗体浓度，通过软件分析人群总体抗体水平、评价人群免疫力和年龄、疫苗接种对抗体水平的影响，也取得良好的效果。
然而，这种评价技术需要针对特定抗体或细胞因子及病原体来预先设计待检标志物，例如酶联免疫吸附检测是利用病毒的敏感性和特异性来获取检测结果，颗粒凝集检测法是通过培养病毒来观察病毒的变化过程，快速诊断试剂检测法是利用病毒与试剂之间发生的化学反应来取得实验结果，抗体确认实验检测法是利用放射免疫沉淀和免疫印迹使用来取得抗体检测结果。因此，传统疫苗效果的评价技术只能评价单一指标的免疫效果，不具备通用性。
随着技术的发展，现有出现了利用多肽指纹图谱技术来检测免疫后的抗体水平。然而，该技术仍然存在如上问题，即需要预先确定机体产生的相关抗体或其片段的特征多肽，并以此为标志物进行检测。
因此，目前需要一种评价疫苗效果的新方法，该方法不针对与抗体或其片段相关的特定标志物，而是监测多种疫苗免疫反应后体液中分子免疫变化的情况，具有很高的通用性。
发明内容
本发明第一原理在于，通常接种DNA或RNA病毒疫苗2-4周内，即可产生有效滴度的抗体。然而，发明人通过质谱试验发现，接种后机体所产生的免疫反应的所出现的特征蛋白片段或特征多肽片段，其中一些特征片段并不专属于机体产生的抗体或抗体片段。通过优化筛选所得到的评价体系，并根据DNA或RNA病毒类型的产生抗体的各个已知时点，筛选疫苗免疫效应相关的非抗体的特征多肽片段，并由此建立特征质谱模型，就可以建立起针对该疫苗的免疫效果的评价方 法。由于该方法根据疫苗接种前后的体内特征多肽而非特征抗体片段的差异，因此能快速而精准地评价机体免疫反应的变化。
本发明第二原理在于，通过上述原理和方法，针对不同疫苗筛选得到的特征蛋白或多肽片段组合物，通过分析其蛋白序列，从而构建用于检测或评价该疫苗免疫效果的质谱模型。
因此本发明的第一个目的是提供制备用于评价多种疫苗免疫效应的质谱模型的构建方法，包括：
(1)在疫苗接种前后多个时间点采集多名志愿者的体液样本，进行低温冷冻备用，其中所述体液样本选自血清、唾液、血浆、全血、唾液、尿液、组织液、关节液、鼻咽拭子提取液或脑积液。
(2)对体液样本进行质谱预处理；
(3)对预处理过的体液样本进行飞行时间质谱检测读取，获得由5～200个特征多肽/蛋白峰的质荷比、分子质量、质谱峰面积、质谱峰强度或相对强度数据所组成的免疫指纹表征谱；
(4)对所有样本的免疫指纹表征谱进行标准化处理，包括选择分子质量区间、谱图归一化、谱图对齐、峰对齐、归一化、过滤低频峰，并导出数据；
(5)对所得数据进行质控处理，筛选出具有特定质荷比的特征多肽，并根据这些质荷比数据和质谱峰强度或相对强度数据，通过计算机软件进行汇总和整理，建立免疫反应的标准指纹谱模型；
在一个实施方案中，所述软件是发明人自行研究开发的BE-V 2.0软件；
其中，步骤(1)中，当所述疫苗需要接种1针时，时间点为接种前1天、接种后第3周；
当所述疫苗需要接种2针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，其中两针之间间隔至少21天；
当所述疫苗需要接种3针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，第三针接种后第1周、第三针接种后第3周，其中两针之间间隔至少21天；以及，
所述步骤(5)所述的质控处理，选取某一类体液样本(血清、唾液等)中固有的一组特征峰作为质控内标峰，其中所述内标峰m/z如下：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z，并且谱图质量控制条件为：在单个样本的谱图中，质控内标峰出现数量不低于质控内标峰总数的70％且内标峰分子量偏移偏差小于0.002时或偏移范围不超过2‰视为质控合格。
在上述的实施方案中，所述需要接种1针的疫苗是腺病毒载体疫苗，接种2针的疫苗是灭活疫苗，接种3针的疫苗是重组蛋白疫苗。
在一个实施方案中，其中步骤(2)预处理的方法包括使用样本处理液(北京毅新博创生物科技有限公司)稀释稳定样品中的蛋白或多肽。
在一个实施方案中，其中所述步骤(2)采用免疫反应指纹谱试剂盒(北京毅新博创生物科技有限公司，货号：1010307)对体液样本中的蛋白或多肽进行稀释和读取，获得多肽指纹图谱。
在一个实施方案中，所述步骤(5)所述的质控处理，对于空白基质，用相同的质谱参数检测空白基质结晶点，若出现明显质谱峰则认为基质溶液质量不合格。
在一个实施方案中，所述飞行时间质谱法所用的检测仪器为MALDI-TOF MS。
在上述任一项实施方案中，所述需要接种1针的疫苗是腺病毒载体疫苗，接种2针的疫苗是灭活疫苗，接种3针的疫苗是重组蛋白疫苗。
在上述任一实施方案中，其中所述疫苗是针对DNA病毒疾病的疫苗或RNA病毒疾病的疫苗。
在一个具体实施方案中，所述DNA病毒包括乙肝病毒，疱疹病毒、风疹病毒、人乳头瘤病毒。
在另一具体实施方案中，所述RNA病毒包括艾滋病病毒/丙型肝炎病毒，乙型脑炎病毒/全部流感病毒、SARS病毒、MERS病毒、新型冠状病毒。
在上述任一实施方案中，所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13761m/z、13882m/z、13939m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：3198m/z、3213m/z、6609m/z。
在一个特别优选的实施方案中，其中所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z，并且所述多肽序列选自：
Figure PCTCN2022143141-appb-000001
在优选的实施方案中，其中所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z。
本发明的第二个发明目的在于通过上述方法，所构建的基于免疫反应指纹谱表征人体免疫变化的质谱模型。
在一个实施方案中，当所述疫苗需要接种1针时，时间点为接种前1天、接种后第3周；
当所述疫苗需要接种2针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，其中两针之间间隔至少21天；
当所述疫苗需要接种3针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，第三针接种后第1周、第三针接种后第3周，其中两针之间间隔至少21天；以及，
所述步骤(5)所述的质控处理，选取某一类体液样本(血清、唾液等)中固有的一组特征峰作为质控内标峰，其中所述内标峰m/z如下：6426m/z、6623m/z、8753m/z、8785m/z、 8904m/z、9118m/z、9409m/z、9700m/z，并且谱图质量控制条件为：在单个样本的谱图中，质控内标峰出现数量不低于质控内标峰总数的70％且内标峰分子量偏移偏差小于0.002时或偏移范围不超过2‰视为质控合格。
在一个实施方案中，所述模型的检测方法为飞行时间质谱法，检测仪器为MALDI-TOF MS。
在具体的实施方案中，所述模型由免疫反应指纹图谱制备而成。该模型包括5～200个特征多肽/蛋白峰的质荷比、分子质量、质谱峰面积、质谱峰强度或相对强度数据。
在上述任一实施方案中，其中所述疫苗是针对DNA病毒疾病的疫苗或RNA病毒疾病的疫苗。
在一个具体实施方案中，所述DNA病毒包括乙肝病毒，疱疹病毒、风疹病毒、人乳头瘤病毒。
在另一具体实施方案中，所述RNA病毒包括艾滋病病毒/丙型肝炎病毒，乙型脑炎病毒/全部流感病毒、SARS病毒、MERS病毒、新型冠状病毒。
在优选的实施方案中，其中所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13761m/z、13882m/z、13939m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：3198m/z、3213m/z、6609m/z。
在一个特别优选的实施方案中，其中所述特征多肽片段包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z，并且所述多肽序列选自：
Figure PCTCN2022143141-appb-000002
在优选的实施方案中，其中所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z，。
本发明第三个目的通过上述构建质谱模型的方法，所得到的用于评价疫苗免疫效应的特征多肽组合物。
在一个实施方案中，所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z。
在一个特别优选的实施方案中，其中所述特征多肽片段序列为：
m/z 基因名称 蛋白名称
3025 CD99 CD99 antigen(75-105)
6609 CD93 Complement component C1q receptor(242-303)
13882 PPBP Platelet basic protein
14044 HBD Hemoglobin subunit delta(19-147)
14092 MBL2 Mannose-binding protein C(100-227)
14150 CD59 CD59 glycoprotein
15124 HBA1 Hemoglobin subunit alpha(2-142)
15868 HBB Hemoglobin subunit beta(2-147)
28195 LRG1 Leucine-rich alpha-2-glycoprotein(94-347)
在优选的实施方案中，其中所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3484m/z；上调表达的特征峰为：3799m/z、3878m/z、4176m/z、4186m/z、4609m/z。
本发明第四个发明目的是提供一种用于评价免疫反应指纹谱的试剂盒，包括：
样本处理液；
内标峰特征片段：：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z；以及，
通过上述构建质谱模型的方法，所得到的用于评价疫苗免疫效应的特征多肽组合物。
在一个实施方案中，所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z。
在一个特别优选的实施方案中，其中所述特征多肽片段序列为：
Figure PCTCN2022143141-appb-000003
在优选的实施方案中，其中所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3484m/z；上调表达的特征峰为：3799m/z、3878m/z、4176m/z、4186m/z、4609m/z。
在另一个实施方案中，该试剂盒还包括保证质谱仪所测分子量准确的标准质谱样品管。该样品管既可以是含有单一特征多肽的多种样品管，也可以是含有多种特征多肽的一种样品管。所述 标准样品管中的样品用于与待测样品进行质谱时进行平行质谱测试，以判断待测样品分子量信息是否准确可靠。
在另一个实施方案中，该试剂盒可含有上述免疫反应指纹图谱的标准数据库的软件或芯片。可用于待测样品进行质谱时提供标准数据或曲线的比对，以判断待测样品中特征多肽的表达状况。
本发明第五个目的是通过上述质谱模型、特征多肽组合物或试剂盒，用于评价个人是否完成疫苗接种或个体化疫苗接种效果的用途。
本发明第六个目的是提供一种用于评价疫苗质量，和/或，分析疫苗保护效果时间序列或疫苗保护的效期，和/或，疫苗保护时间中的方法。
在一个实施方案中，所述方法包括：
(1)在疫苗接种前后多个时间点采集多名已知志愿者的体液样本，对体液样本进行质谱预处理，其中所述体液样本选自血清、唾液、血浆、全血、唾液、尿液、组织液、关节液、鼻咽拭子提取液或脑积液；
(2)对预处理过的体液样本进行飞行时间质谱检测读取，获得由5～200个特征多肽/蛋白峰的质荷比、分子质量、质谱峰面积、质谱峰强度或相对强度数据所组成的免疫指纹表征谱；
(3)对所有样本的免疫指纹表征谱进行标准化处理，包括选择分子质量区间、谱图归一化、谱图对齐、峰对齐、归一化、过滤低频峰，并导出数据；
(4)对所得数据进行质控处理，筛选出具有特定质荷比的特征多肽，并根据这些质荷比数据和质谱峰强度或相对强度数据，通过计算机软件进行汇总和整理，建立免疫反应的标准指纹谱模型；
(5)对待接种的患者，采用步骤(1)-(3)进行处理，而后对所得数据进行质控处理，筛选出具有特定质荷比的特征多肽；
(6)将步骤(5)所得到的具有特定质荷比的特征多肽，与步骤(4)所述的标准指纹谱模型，通过计算机软件来评价疫苗质量，和/或，分析疫苗保护效果时间序列或疫苗保护的效期，和/或，疫苗保护时间；
其中，步骤(1)中，当所述疫苗需要接种1针时，时间点为接种前1天、接种后第3周；
当所述疫苗需要接种2针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，其中两针之间间隔至少21天；
当所述疫苗需要接种3针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，第三针接种后第1周、第三针接种后第3周，其中两针之间间隔至少21天；以及，
所述步骤(4)、步骤(5)所述的质控处理，选取某一类体液样本(血清、唾液等)中固有的一组特征峰作为质控内标峰，其中所述内标峰m/z如下：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z，并且谱图质量控制条件为：在单个样本的谱图中，质控内标峰出现数量不低于质控内标峰总数的70％且内标峰分子量偏移偏差小于0.002时或偏移范围不超过2‰视为质控合格。
在一个实施方案中，所述软件是发明人自行研究开发的BE-V 2.0软件。
在上述的实施方案中，所述需要接种1针的疫苗是腺病毒载体疫苗，接种2针的疫苗是灭活疫苗，接种3针的疫苗是重组蛋白疫苗。
在一个实施方案中，其中步骤(2)预处理的方法包括使用样本处理液(北京毅新博创生物科技有限公司北京毅新博创生物科技有限公司)稀释稳定样品中的蛋白或多肽。
在一个实施方案中，其中所述步骤(2)采用免疫反应指纹谱试剂盒(北京毅新博创生物科技有限公司，货号：1010307)对体液样本中的蛋白或多肽进行稀释和读取，获得多肽指纹图谱。
在一个实施方案中，所述步骤(4)、步骤(5)所述的质控处理，对于空白基质，用相同的质谱参数检测空白基质结晶点，若出现明显质谱峰则认为基质溶液质量不合格。
在一个实施方案中，所述飞行时间质谱法所用的检测仪器为MALDI-TOF MS。
在上述任一项实施方案中，所述需要接种1针的疫苗是腺病毒载体疫苗，接种2针的疫苗是灭活疫苗，接种3针的疫苗是重组蛋白疫苗。
在上述任一实施方案中，其中所述疫苗是针对DNA病毒疾病的疫苗或RNA病毒疾病的疫苗。
在一个具体实施方案中，所述DNA病毒包括乙肝病毒，疱疹病毒、风疹病毒、人乳头瘤病毒。
在另一具体实施方案中，所述RNA病毒包括艾滋病病毒/丙型肝炎病毒，乙型脑炎病毒/全部流感病毒、SARS病毒、MERS病毒、新型冠状病毒。
在上述任一实施方案中，所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13761m/z、13882m/z、13939m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：3198m/z、3213m/z、6609m/z。
在一个特别优选的实施方案中，其中所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z，并且所述多肽序列选自：
Figure PCTCN2022143141-appb-000004
在优选的实施方案中，其中所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z。
技术效果
本发明突破了现有技术仅局限于现有抗体或细胞因子等单一指标评价疫苗免疫反应的技术局限。采用免疫反应指纹图谱法，实现多个特征峰共同监测，提高了免疫效果检测的准确率。
相比传统方法，免疫反应指纹谱法具有操作简单、成本低、速度快、通量高的技术优势。
本发明适用于不同体液的免疫反应指纹谱的表征，为疫苗免疫效果评价提供多条参考依据。
本发明表征疫苗接种人体的免疫变化情况，实时监测疫苗在不同时间序列的保护效果，为精准防疫提供有力证据。
本发明同样适用于动物体液的免疫反应指纹谱表征，可用于临床前的疫苗效果的评价，加快疫苗研发的步伐。
本发明更适用于对于个体疫苗免疫效果的评价，而不局限于对群体免疫反应的分析，可以实现精准防疫。
本发明可以对疫苗接种后不同时间点的免疫反应变化进行连续监测，持续分析疫苗接种后机体的免疫反应变化情况，以及评价个体是否完成疫苗接种或个体化疫苗接种效果。其中，评价标准可以在本发明所得到的指纹图谱对比结果的基础上，通过疫苗现有已知的方式来确定评价标准。
本发明的方法，可以用于分析疫苗保护效果时间序列或疫苗保护的效期，和/或，疫苗保护时间中的用途。其中，评价标准可以在本发明所得到的指纹图谱对比结果的基础上，通过疫苗现有已知的方式来确定评价标准。
附图说明
图1.不同组样本质谱谱图对比。其中，图a为疫苗接种前后免疫反应指纹谱全图；图b为主要特征峰局部放大图。图中横坐标代表各个特征峰的m/z信息，纵坐标代表归一化后的谱峰强度。其中13761m/z、13882m/z、13939m/z、14044m/z、14091m/z、14150m/z、28195m/z特征峰在疫苗接种前后出现显著变化。
图2.新冠疫苗接种相关各特征峰上下调情况。其中图中展示了13个特征峰在接种第0天(n＝95)、第21天(n＝95)、第28天(n＝95)和第42天(n＝95)样本间的相对强度比较。用Wilcoxon检验计算p值。星号表示组间具有统计学意义的差异情况(*P<0.05,**P<0.01,***P<0.001,****P<0.0001)。
图3.主成分分析(PCA图)。其中，
图a为接种新冠灭活疫苗前与接种第一针疫苗后3周免疫反应指纹谱主成分分析图；
图b为接种新冠灭活疫苗前与接种第二针疫苗后1周免疫反应指纹谱主成分分析图；
图c为接种新冠灭活疫苗前与接种第二针疫苗后3周免疫反应指纹谱主成分分析图；
图d为接种第一针疫苗后3周与接种第二针疫苗后1周免疫反应指纹谱主成分分析图；
图e为接种第一针疫苗后3周与接种第二针疫苗后3周免疫反应指纹谱主成分分析图；
图f为接种第二针疫苗后1周与接种第二针疫苗后3周免疫反应指纹谱主成分分析图。
图4.中和抗体阳性样本和中和抗体阴性样本在免疫反应指纹谱模型中的偏最小二乘回归分析法(PLS-DA)图。图中“P”代表中和抗体阳性；“N”代表中和抗体阴性。
图5.中和抗体相关免疫反应指纹谱各特征峰上下调情况。图中展示了中和抗体阴性样本和阳性样本中7个特征峰的相对强度比较。p值由Mann-Whitney检验计算。星号表示组间具有统计学意义的差异(*P<0.05,**P<0.01,***P<0.001,****P<0.0001)。图中“N”代表中和抗体阴性；“P”代表中和抗体阳性。
图6.唾液多肽指纹图谱。其中，图中a曲线为未接种新冠灭活疫苗者唾液免疫多肽指纹表征谱；b曲线为已接种2针新冠灭活疫苗者唾液免疫多肽指纹表征谱。
图7.唾液免疫反应指纹谱各特征峰上下调情况。
图8.唾液免疫反应指纹谱模型主成分分析(PCA)图，图中绿色点代表已完成2针疫苗注射的样本，红色点代表未完成完整注射的样本。图9.流感鼻拭子多肽指纹表征谱。其中，图中a曲线为未接种流感疫苗者鼻拭子多肽指纹表征谱；图中b曲线为已接种流感疫苗者鼻拭子多肽指纹表征谱。图10.流感鼻拭子免疫反应指纹谱模型偏最小二乘判别(PLS-DA)分析图。其中，左侧区域(n)代表未接种流感疫苗者样本分布；右侧区域(p)代表已接种流感疫苗者样本分布。
图11.流感鼻拭子免疫反应指纹谱模型火山图。
图12.流感鼻拭子免疫反应指纹谱模型ROC曲线图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1.血清免疫反应指纹谱表征新冠灭活疫苗接种带来的免疫反应变化
试验目的：对未接种新冠灭活疫苗和已接种疫苗不同时间点的志愿者血清进行了免疫反应指纹谱分析。通过免疫反应指纹谱，希望能评价免疫过程中的血清变化，并评定疫苗的免疫效应。
(一)样本收集
95名新冠疫苗接种志愿者参与本次研究。
每名志愿者均在如下4个时间节点采集血清样本：
(1)第一针疫苗前(第1天)；
(2)第一针疫苗后3周(第22+n天)；
(3)第二针疫苗后1周(第29+n天)；
(4)第二针疫苗后3周(第50+n天)；
所有志愿者均为18～59岁健康受试者，且自愿接种新型冠状病毒灭活疫苗。志愿者为男性或非妊娠期、非哺乳期的女性。无重大疾病史、药物过敏史及疫苗接种过敏史。无新冠肺炎病史或感染史，并在采样时持有健康码“绿码”。由此，可以保证所有志愿者体内不会出现近期因疾病而引发的免疫反应。其中，将样本随机分为2份，分别用于模型的建立和验证。其中85例样本用于模型建立，10例样本用于模型验证，以测试质谱模型的灵敏度、特异度、准确率、精密度。
所有样本均由不含添加剂的真空血清采集管采集。采集并分离血清后将血清样本分装冷冻在-80℃低温冰箱中。
血清样品的质谱预处理：在进行质谱检测实验前，从低温冰箱取出分装好的血清样品，放于湿冰上。化冻60-90分钟。
(二)样品准备
将每份样本的5μl血清稀释在45μl样本处理液(北京毅新博创生物科技有限公司)中。然后取出10μl已稀释的血清，与10μl基质溶液(北京毅新博创生物科技有限公司)进行混合。
取出1μl混合液滴加至不锈钢靶板。室温干燥后，将样品进样MALDI-TOF MS质谱仪(Clin-TOF-II；北京毅新博创生物科技有限公司)。每个样品平行测试3次。
(三)质谱数据采集
应用Clin-TOF质谱仪(北京毅新博创生物科技有限公司)。设置合适的激光能量采集样品结晶点的某一个点。每个样品点选取50个激光轰击位置，每个位置轰击10次，即对每个样品结 晶点进行500次激光轰击，收集谱图。激光频率：30Hz。数据收集范围：3～30KDa。在每次采集样品结晶点前用标准品进行外标校准，平均分子量偏差小于500ppm。
实验质控：
(1)用相同的质谱参数检测空白基质结晶点，若出现明显质谱峰则认为基质溶液质量不合格，需更换一支新的基质。
(2)用标准品进行外标校准时需保证不同校准品点的质量偏移不超过500ppm，5个校准品峰必须同时满足要求。
(3)用BE-V 2.0软件(北京毅新博创生物科技有限公司)生成峰列表，并统计出峰数量。若出峰数量低于20个，则需重新采集谱图。
(4)选取8个血清中故有的多肽峰作为内标质控内标峰。若有6～8个内标峰可以被检测到，且内标峰分子量偏移范围不超过2‰则认为谱图合格。否则需重新采集谱图。内标峰m/z如下：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z。
(四)原始数据预处理
MALDI-TOF原始数据用BE-V 2.0软件(北京毅新博创生物科技有限公司)进行内标二次校准。所用内标峰m/z为：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z。然后BE-V 2.0软件(北京毅新博创生物科技有限公司)对谱图进行处理。谱图处理内容包括方差稳定化、平滑降噪、去基线、峰强度归一化、谱图平均、谱图对齐、谱峰识别、谱峰分箱等。保留组内出峰频率不低于25％的峰。最后，将得到的矩阵用于下面的分析。经log2变换后，将峰强度矩阵进行分位数归一化。在所有样本中，缺失值用最小值来填充。
(五)特征蛋白的选择
经过强度归一化和缺失值归一化后，训练组的峰值用如下三种机器学习方法进行分析：偏最小二乘回归分析法(PLS-DA)、火山图分析法(Volcano Plot)和配对t检验法。在偏最小二乘回归分析法中选取vip大于1.8的峰；在火山图分析法中设定Fc大于1.6且P等于0.05；在配对t检验法中，选取P值小于0.05且比例大于1.2的峰。通过对各组之间差异峰进行分析，选取共有差异峰作为特征峰建立模型。
共13个特征峰用于后续的模型分析。其中下调表达的特征峰为：3025m/z、13761m/z、13882m/z、13939m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：3198m/z、3213m/z、6609m/z。
不同组样本的免疫反应指纹谱对比图参见附图1，其中由下至上分别为未接种新冠疫苗的健康人血清图谱、接种过1针灭活疫苗后3周的志愿者血清图谱、接种过2针灭活疫苗后1周的志愿者血清图谱、接种过2针灭活疫苗后3周的志愿者血清图谱)。相比于未接种疫苗的血清，接种1～2针疫苗的血清在3025m/z、13761m/z、13882m/z、13939m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z处出现明显的峰强度下调；而在3198m/z、3213m/z、6609m/z处出现明显的峰强度上调。13个特征峰的上下调情况详见附图2。
(六)模型算法
尝试用4种机器学习方法用训练组数据的多肽指纹图谱特征峰建立模型，通过交叉验证准确率评估模型结果。分析的4中机器学习方法如下：逻辑回归(LR),支持向量机(SVM),随机森林(RF)和偏最小二乘判别分析(PLS-DA)。在比较了四种算法建立模型的预测准确度、灵敏度、 特异度和精密度后发现，随机森林(RF)模型表现最好，其准确度、精密度和灵敏度均超过80％，是最适合的模型算法。因此选取RF模型(灵敏度＝85.7％，特异度＝66.7％，准确率＝80.0％，精密度＝85.7％)应用于对疫苗免疫反应的评价。
(七)新冠疫苗免疫反应的特征多肽的鉴定
选取特征峰强度较高的3个血清样本，对实施例1中所确定的特征峰进行二级质谱鉴定。样本经DTT还原后，用tricine-SDS-PAGE分离。各条带经胶内酶切后进行二级质谱鉴定。
采用nano-LC-MS/MS平台进行多肽序列鉴定，包括nanoflow HPLC(Thermo Fisher Scientific,USA)和Q-Exactive mass spectrometer(Thermo Fisher Scientific,USA)。离子模式为正离子模式，扫描范围为300-1400m/z。一级质谱分辨率为70000，二级质谱分辨率为17500。
液相分析柱：型号：Exsil Pure 120 C18(Dr.Maisch GmbH,USA)；规格：360μm×12cm；内径：150μm；粒：1.9um。洗脱方式：流动相从7％B液(80％乙腈，0.1％甲酸)到45％B液，线性洗脱。流速：600nl/min；总时间38分钟。
鉴定结果见下表1。
表1 特征峰多肽鉴定结果
m/z 基因名称 蛋白名称
3025 CD99 CD99 antigen(75-105)
6609 CD93 Complement component C1q receptor(242-303)
13882 PPBP Platelet basic protein
14044 HBD Hemoglobin subunit delta(19-147)
14092 MBL2 Mannose-binding protein C(100-227)
14150 CD59 CD59 glycoprotein
15124 HBA1 Hemoglobin subunit alpha(2-142)
15868 HBB Hemoglobin subunit beta(2-147)
28195 LRG1 Leucine-rich alpha-2-glycoprotein(94-347)
由表1可知，所鉴定的特征多肽，均来自于已知的细胞因子片段或血红蛋白亚基片段等，均不是抗体或抗体片段。
(八)模型验证
将步骤(七)中验证的多肽指纹图谱，通过BE-V 2.0软件进行处理，构建新冠疫苗的免疫反应的标准质谱模型。
将验证组的10例样本用同样的方法进行免疫反应指纹谱采集，并通过BE-V 2.0软件与上述标准质谱模型进行比较，验证模型灵敏度、特异度、准确率、精密度结果详见下表。
表2.免疫反应指纹谱法模型验证结果
模型名称 灵敏度 特异度 准确率 精密度
RF 0.857 0.667 0.800 0.857
PLS-DA 0.714 0.667 0.700 0.833
Linear SVM 0.714 0.667 0.700 0.833
LR 0.571 0.667 0.600 0.800
(九)新冠免疫多肽指纹谱表征法时间序列分析
利用主成分分析(PCA)方法对接种前和接种后的三个采时间点采集得到的多肽指纹谱进行MALDI-TOF质谱的时间序列分析，结果如附图3所示。第0天的免疫多肽指纹谱可以与第21天、第28天和第42天很好地区分开。说明在第一次注射疫苗后，人血清中的蛋白和多肽即会发生显著的变化。
综上，根据上表所发现的特征多肽已经构建了标准的多肽指纹图谱，预示着可以表征新冠疫苗的免疫效应，以及用于评价疫苗质量，和/或，分析疫苗保护效果时间序列或疫苗保护的效期，和/或，疫苗保护时间。
实施例2.血清免疫反应指纹谱与新冠中和抗体关联性分析
试验目的：虽然接种疫苗后，通过人体产生的抗体(即结合抗体，或免疫球蛋白)的水平可以评价疫苗的免疫效应，但对于某些病毒性抗原进入人体之前或之中，如果体内就存在与病毒内部的颗粒结合的更小蛋白(即中和抗体)，那么可以阻止病毒感染细胞，从而破坏病毒颗粒。因此，临床试验中不仅要测总的抗体水平，更重要的是要单独测中和抗体水平。因此，检测抗体特别是中和抗体(NAbs)的产生对于大多数COVID-19疫苗引发人体对SARS-COV-2的保护机制具有重要意义。在本实施例中，使用实施例1相同的方法，将接种2针新冠灭活疫苗3周后的志愿者血清按是否产生中和抗体分为两组进行免疫反应指纹谱分析，试图证实免疫反应指纹谱(或指纹图谱)可以用于表征疫苗接种者是否会产生中和抗体。
(一)样本收集
51名新冠疫苗接种志愿者参与本次研究。每名志愿者均在第二针疫苗后3周(第50+n天)采集血清样本。
所有志愿者均为18～59岁健康受试者，且自愿接种新型冠状病毒灭活疫苗。志愿者为非妊娠期、非哺乳期的女性。无重大疾病史、药物过敏史及疫苗接种过敏史。无新冠肺炎病史或感染史，并在采样时持有健康码“绿码”。由此，可以保证所有志愿者体内不会出现近期因疾病而引发的免疫反应。
所有样本均由不含添加剂的真空血清采集管采集。采集并分离血清后将血清样本分装冷冻在-80℃低温冰箱中。
血清样品的质谱预处理：在进行质谱检测实验前，从低温冰箱取出分装好的血清样品，放于湿冰上。化冻60-90分钟。
(二)样品准备
将每份样本的5μl血清稀释在45μl样本处理液(北京毅新博创生物科技有限公司)中。然后取出10μl已稀释的血清，与10μl基质溶液(北京毅新博创生物科技有限公司)进行混合。
取出1μl混合液滴加至不锈钢靶板。室温干燥后，将样品进样MALDI-TOF MS质谱仪(Clin-TOF-II；北京毅新博创生物科技有限公司)。每个样品平行测试3次。
(三)质谱数据采集
应用Clin-TOF质谱仪(北京毅新博创生物科技有限公司)。设置合适的激光能量采集样品结晶点的某一个点。每个样品点选取50个激光轰击位置，每个位置轰击10次，即对每个样品结晶点进行500次激光轰击，收集谱图。激光频率：30Hz。数据收集范围：3～30KDa。在每次采集样品结晶点前用标准品进行外标校准，平均分子量偏差小于500ppm。
实验质控：
(1)用相同的质谱参数检测空白基质结晶点，若出现明显质谱峰则认为基质溶液质量不合格，需更换一支新的基质。
(2)用标准品进行外标校准时需保证不同校准品点的质量偏移不超过500ppm，5个校准品峰必须同时满足要求。
(3)用BE-V 2.0软件(北京毅新博创生物科技有限公司)生成峰列表，并统计出峰数量。若出峰数量低于20个，则需重新采集谱图。
(4)选取8个血清中故有的多肽峰作为内标质控内标峰。若有6～8个内标峰可以被检测到，且内标峰分子量偏移范围不超过2‰则认为谱图合格。否则需重新采集谱图。内标峰m/z如下：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z。
(四)原始数据预处理
MALDI-TOF原始数据用BE-V 2.0软件(北京毅新博创生物科技有限公司)进行内标二次校准。所用内标峰m/z为：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z。然后BE-V 2.0软件(北京毅新博创生物科技有限公司)对谱图进行处理。谱图处理内容包括方差稳定化、平滑降噪、去基线、峰强度归一化、谱图平均、谱图对齐、谱峰识别、谱峰分箱等。保留组内出峰频率不低于25％的峰。最后，将得到的矩阵用于下面的分析。经log2变换后，将峰强度矩阵进行分位数归一化。在所有样本中，缺失值用最小值来填充。随机选择41例样本用于建立模型，剩余10例样本用于模型验证。
(五)特征蛋白的选择
经过强度归一化和缺失值归一化后，训练组的峰值用如下三种机器学习方法进行分析：偏最小二乘回归分析法(PLS-DA)、火山图分析法(Volcano Plot)和配对t检验法。在偏最小二乘回归分析法中选取vip大于2的峰；在火山图分析法中设定Fc大于1.6且P等于0.05；在配对t检验法中，选取P值小于0.05且比例大于1.2的峰。通过对各组之间差异峰进行分析，选取共有差异峰作为特征峰建立模型。模型偏最小二乘回归分析法(PLS-DA)图见附图4。相比于中和抗体阴性样本，中和抗体阳性血清在6609m/z、6980m/z、13929m/z、13939m/z、14083m/z处出现明显的峰强度下调；而在3496m/z、9928m/z处出现明显的峰强度上调。中和抗体相关特征峰上下调关系详见附图5。
(六)模型算法
尝试用4种机器学习方法用训练组数据的多肽指纹图谱特征峰建立模型，通过交叉验证准确率评估模型结果。分析的4中机器学习方法如下：逻辑回归(LR),支持向量机(SVM),随机森林(RF)和偏最小二乘判别分析(PLS-DA)。在比较了四种算法建立模型的预测准确度、灵敏度、特异度和精密度后发现，随机森林(RF)模型表现最好，其准确度、精密度和灵敏度均超过80％，是最适合的模型算法。因此选取RF模型(灵敏度＝85.7％，特异度＝66.7％，准确率＝80.0％，精密度＝85.7％)应用于对疫苗免疫反应的评价。
(七)新冠疫苗免疫反应的特征多肽的鉴定
选取特征峰强度较高的3个血清样本，对实施例2中所确定的特征峰进行二级质谱鉴定。样本经DTT还原后，用tricine-SDS-PAGE分离。各条带经胶内酶切后进行二级质谱鉴定。
采用nano-LC-MS/MS平台进行多肽序列鉴定，包括nanoflow HPLC(Thermo Fisher Scientific,USA)和Q-Exactive mass spectrometer(Thermo Fisher Scientific,USA)。离子模式为正离 子模式，扫描范围为300-1400m/z。一级质谱分辨率为70000，二级质谱分辨率为17500。
液相分析柱：型号：Exsil Pure 120 C18(Dr.Maisch GmbH,USA)；规格：360μm×12cm；内径：150μm；粒：1.9um。洗脱方式：流动相从7％B液(80％乙腈，0.1％甲酸)到45％B液，线性洗脱。流速：600nl/min；总时间38分钟。
鉴定结果见下表3。
表3.特征峰多肽鉴定结果
m/z 基因名称 蛋白名称
6980 ITM2B Integral membrane protein 2B
6609 CD93 Complement component C1q receptor(242-303)
9928 PF4 Platelet factor 4
14083 MBL2 Mannose-binding protein C
实施例3.唾液免疫反应指纹谱表征新冠灭活疫苗接种带来的免疫反应变化
试验目的：
根据实施例1的方法，测试该方法对新冠疫苗接种者的唾液样本进行免疫反应指纹谱分析，以证实了本发明的免疫反应指纹图谱在其它体液样本中同样适用。
(一)唾液免疫反应指纹谱样本采集与处理
55名新冠疫苗接种志愿者参与本次研究。其中25人已完成2针新冠灭活疫苗接种，其余30人未完成完整接种(未接种新冠疫苗或仅接种1针新冠疫苗)。
所有志愿者均为18～59岁健康受试者，且自愿接种新型冠状病毒灭活疫苗。志愿者为男性或非妊娠期、非哺乳期的女性。无重大疾病史、药物过敏史及疫苗接种过敏史。无新冠肺炎病史或感染史，并在采样时持有健康码“绿码”。由此，可以保证所有志愿者体内不会出现近期因疾病而引发的免疫反应。
所有样本均为自然流出的唾液样本。采集后8小时内进行质谱检测。将样本随机分为2份，分别用于模型的建立和验证。其中33例样本用于模型建立，22例样本用于模型验证。
将每份样本的5μl唾液稀释在45μl样本处理液(北京毅新博创生物科技有限公司)。然后取出10μl已稀释的唾液，与10μl基质溶液(北京毅新博创生物科技有限公司)进行混合。
取出1μl混合液滴加至不锈钢靶板。室温干燥后，将样品进样MALDI-TOF MS质谱仪(Clin-TOF-II；北京毅新博创生物科技有限公司)。每个样品平行测试3次。
(三)质谱数据采集
应用Clin-TOF质谱仪。设置合适的激光能量采集样品结晶点的某一个点。每个样品点选取50个激光轰击位置，每个位置轰击10次，即对每个样品结晶点进行500次激光轰击，收集谱图。激光频率：30Hz。数据收集范围：3～30KDa。在每次采集样品结晶点前用标准品进行外标校准，平均分子量偏差小于500ppm。
实验质控：
(1)用相同的质谱参数检测空白基质结晶点，若出现明显质谱峰则认为基质溶液质量不合格，需更换一支新的基质。
(2)用标准品进行外标校准时需保证不同校准品点的质量偏移不超过500ppm，5个校准品峰必须同时满足要求。
(3)用BE-V 2.0软件(北京毅新博创生物科技有限公司)生成峰列表，并统计出峰数量。若出峰数量不低于20个，则认为谱图合格。否则需重新采集谱图。
得到的唾液多肽指纹图谱如附图6所示，其中，图中a曲线为未接种新冠灭活疫苗者唾液免疫多肽指纹表征谱；b曲线为已接种2针新冠灭活疫苗者唾液免疫多肽指纹表征谱。由图6所示，已接种2针新冠灭活疫苗者唾液免疫多肽指纹表征谱。应当指出的是，虽然图6b，在9000m/z附近出现明显的特征峰，同时在3484m/z以及11000m/z出现明显下降，然而，这仅是出现在个别样本中，不具有统计学意义，因此未被选中用于建立模型)。
(四)原始数据预处理
MALDI-TOF原始数据用BE1.0软件(北京毅新博创生物科技有限公司)对谱图进行处理。谱图处理内容包括方差稳定化、平滑降噪、去基线、峰强度归一化、谱图平均、谱图对齐、谱峰识别、谱峰分箱等。保留组内出峰频率不低于25％的峰。最后，将得到的矩阵用于下面的分析。经log2变换后，将峰强度矩阵进行分位数归一化。在所有样本中，缺失值用最小值来填充。
(五)特征蛋白的选择
经过强度归一化和缺失值归一化后，训练组的峰值用配对t检验法进行分析。选取P值小于0.01的峰作为特征峰建立模型。各个特征峰在2组样本中的均值-方差图详见图7。在均值-方差图中，不同组别的样品的强度均值和方差分别列出。左栏样本已完成2针疫苗接种；右栏样本为未接种疫苗或仅接种1针疫苗样本。横轴为组别名称，纵轴为峰强度。3条横线中，中间横线代表本组数据该特征峰的强度均值；上下两条横线代表本组样本该特征峰强度的离散程度。
结果：相比于未完成完整接种(2针疫苗接种)的样本，完整接种样本的唾液免疫反应指纹谱在3484m/z处出现明显的峰强度下调；而在3799m/z、3878m/z、4176m/z、4186m/z、4609m/z处出现明显的峰强度上调。各特征峰在两种中的表达情况详见附图7。
模型主成分分析(PCA)图见附图8。图中绿色点代表已完成2针疫苗注射的样本，红色点代表未完成完整注射的样本。图中展示了两组样本点在三维空间中的投影分布情况。两组样本点分布越分散，代表模型的区分潜力越大。
(六)模型算法
尝试用3种机器学习方法用训练组数据的多肽指纹图谱特征峰建立模型，通过准确率评估模型结果。分析的3中机器学习方法如下：Fishert线性分类判别,K临近算法(KNN),支持向量机(SVM)。在比较了三种算法建立模型的预测准确度后发现，K临近算法(KNN)模型表现最好，其灵敏度和特异度度均为100％，是最适合的模型算法。因此选取KNN模型应用于对疫苗免疫反应的评价。
(七)模型验证
将验证组的22例样本用同样的方法进行免疫反应指纹谱采集，并将结果导入KNN模型，模型验证结果详见下表。
表4.唾液免疫反应指纹谱模型验证结果
Figure PCTCN2022143141-appb-000005
实施例4.鼻拭子免疫反应指纹谱表征流感疫苗接种带来的免疫反应变化
试验目的：对未接种流感疫苗和已接种流感疫苗的儿童鼻拭子样本进行免疫反应指纹谱分析。通过免疫反应指纹谱，评价免疫过程中鼻粘膜多肽的变化，并评定疫苗的免疫反应。
(一)样本收集
35名志愿者参与本次研究。其中16名志愿者在取样前1～3个月接种过流感疫苗；另外19名志愿者1年内未接种过流感疫苗。所有志愿者均为1～15岁儿童，性别不限。
所有志愿者均采集鼻拭子样本。采集后立即将鼻拭子放入1ml样本稀释液中混合均匀，使样本溶解于稀释液中。样本采集后于48h内进行质谱检测。
(二)样本检测
取出已溶解样本的稀释液10μl，与10μl基质溶液进行混合。
取出1μl混合液滴加至不锈钢靶板。室温干燥后，将样品进样MALDI-TOF MS质谱仪(Clin-TOF-II；北京毅新博创生物科技有限公司)。每个样品平行测试3次。
(三)质谱数据采集
应用Clin-TOF质谱仪(北京毅新博创生物科技有限公司)。设置合适的激光能量采集样品结晶点的某一个点。每个样品点选取50个激光轰击位置，每个位置轰击10次，即对每个样品结晶点进行500次激光轰击，收集谱图。激光频率：30Hz。数据收集范围：2～20KDa。在每次采集样品结晶点前用标准品进行外标校准，平均分子量偏差小于500ppm。
实验质控：
(1)用相同的质谱参数检测空白基质结晶点，若出现明显质谱峰则认为基质溶液质量不合格，需更换一支新的基质。
(2)用标准品进行外标校准时需保证不同校准品点的质量偏移不超过500ppm，5个校准品峰必须同时满足要求。
(3)用BE-V 2.0软件(北京毅新博创生物科技有限公司)生成峰列表，并统计出峰数量。若出峰数量低于20个，则需重新采集谱图。
(四)原始数据预处理
MALDI-TOF原始数据用BE-V 2.0软件(北京毅新博创生物科技有限公司)对谱图进行处理。谱图处理内容包括方差稳定化、平滑降噪、去基线、峰强度归一化、谱图平均、谱图对齐、谱峰识别、谱峰分箱等。保留组内出峰频率不低于25％的峰。最后，将得到的矩阵用于下面的分析。经log2变换后，将峰强度矩阵进行分位数归一化。在所有样本中，缺失值用最小值来填充。
(五)特征蛋白的选择
经过强度归一化和缺失值归一化后，训练组的峰值用如下两种机器学习方法进行分析：偏最小二乘回归分析法(PLS-DA)和火山图分析法(Volcano Plot)。在偏最小二乘回归分析法中选取vip大于1.5的峰(附图10)；在火山图分析法中设定Fc大于1.5且P等于0.05(附图11)。通过对各组之间差异峰进行分析，选取共有差异峰作为特征峰建立模型。
共26个特征峰用于后续的模型分析。特征峰m/z如下：2061m/z、2034m/z、2176m/z、2043m/z、2131m/z、2079m/z、2292m/z、2233m/z、2087m/z、5336m/z、2120m/z、2345m/z、3687m/z、2320m/z、2057m/z、2385m/z、4061m/z、4110m/z、4961m/z、3313m/z、2850m/z、2408 m/z、3665m/z、2219m/z、5853m/z、2167m/z。
其中，特征峰2034m/z、2079m/z、2233m/z、2120m/z、2345m/z、2320m/z、2057m/z、2385m/z、4061m/z、4961m/z、2850m/z、2408m/z、5853m/z、2167m/z上调，同时2061m/z、2176m/z、2043m/z、2131m/z、2292m/z、2087m/z、5336m/z、3687m/z、4110m/z、3313m/z、3665m/z、2219m/z下调表示该受试者接种过流感疫苗。
(六)模型算法
尝试用4种机器学习方法用训练组数据的多肽指纹图谱特征峰建立模型，通过准确率评估模型结果。分析的4中机器学习方法如下：Fishert线性分类判别,K临近算法(KNN),支持向量机(SVM)，偏最小二乘回归分析法(PLS-DA)。在比较了4种算法建立模型的预测准确度后发现，偏最小二乘回归分析法(PLS-DA)模型表现最好，其模型准确度为83.3％，是最适合的模型算法。其ROC曲线下面积AUC为0.925(附图12)。因此选取PLS-DA模型应用于对流感疫苗免疫反应的评价。
(七)模型验证
将验证组的12例样本用同样的方法进行免疫反应指纹谱采集，并将结果导入PLS-DA模型，模型验证结果详见下表。
表5.鼻拭子免疫反应指纹谱流感免疫模型验证结果
实际分组 例数 预测为已接种 预测为未接种 预测准确率
已接种流感疫苗 6 4 2 67％
未接种流感疫苗 6 6 0 100％

Claims (10)

  1. 一种评价个体是否完成疫苗接种或个体免疫变化的方法，步骤包括：
    (1)在疫苗接种前后多个时间点采集多名已知志愿者的体液样本，对体液样本进行质谱预处理，其中所述体液样本选自血清、唾液、血浆、全血、唾液、尿液、组织液、关节液或脑积液；
    (2)对预处理过的体液样本进行飞行时间质谱法检测读取，获得由5～200个特征多肽/蛋白峰的质荷比、分子质量、质谱峰面积、质谱峰强度或相对强度数据所组成的免疫指纹表征谱；
    (3)对所有样本的免疫指纹表征谱进行标准化处理，包括选择分子质量区间、谱图均一化、谱图对齐、峰对齐、归一化、过滤低频峰，并导出数据；
    (4)对所得数据进行质控处理，筛选出具有特定质荷比的特征多肽，并根据这些质荷比数据和质谱峰强度或相对强度数据，通过计算机软件进行汇总和整理，建立免疫反应的标准指纹谱模型；
    (5)对待接种的患者，采用步骤(1)-(4)进行处理，而后对所得数据进行质控处理，筛选出具有特定质荷比的特征多肽；
    (6)将步骤(5)所得到的具有特定质荷比的特征多肽，与步骤(4)所述的标准指纹谱模型，通过计算机软件来评价个体是否完成疫苗接种或个体化疫苗接种效果；
    其中，步骤(1)中，当所述疫苗需要接种1针时，时间点为接种前1天、接种后第3周；
    当所述疫苗需要接种2针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，其中两针之间间隔至少21天；
    当所述疫苗需要接种3针时，时间点为第一针接种前1天、第一针接种后第3周、第二针接种后第1周、第二针接种后第3周，第三针接种后第1周、第三针接种后第3周，其中两针之间间隔至少21天；以及，
    所述步骤(4)、步骤(5)所述的质控处理，选取体液样本中固有的一组特征峰作为质控内标峰，其中所述内标峰m/z如下：6426m/z、6623m/z、8753m/z、8785m/z、8904m/z、9118m/z、9409m/z、9700m/z，并且谱图质量控制条件为：在单个样本的谱图中，质控内标峰出现数量不低于质控内标峰总数的70％且内标峰分子量偏移偏差小于0.002时或偏移范围不超过2‰视为质控合格。
  2. 根据权利要求1所述的方法，其中步骤(2)预处理的方法包括使用样本处理液(Bioyong Technologies Inc.)稀释稳定样品中的蛋白或多肽，并采用免疫反应指纹谱试剂盒(北京毅新博创生物科技有限公司，货号：1010307)对体液样本中的蛋白或多肽进行稀释和读取，获得多肽指纹图谱；所述软件是BE-V 2.0软件。
  3. 根据权利要求1或2所述的方法，其中所述步骤(4)、步骤(5)所述的质控处理，对于空白基质，用相同的质谱参数检测空白基质结晶点，若出现明显质谱峰则认为基质溶液质量 不合格。
  4. 根据权利要求3所述的方法，其中所述需要接种1针的疫苗是腺病毒载体疫苗，接种2针的疫苗是灭活疫苗，接种3针的疫苗是重组蛋白疫苗。
  5. 根据权利要求4所述的方法，其中所述疫苗是针对DNA病毒疾病的疫苗或RNA病毒疾病的疫苗。
  6. 根据权利要求5所述的方法，其中所述DNA病毒包括乙肝病毒，疱疹病毒、风疹病毒、人乳头瘤病毒；所述RNA病毒包括艾滋病病毒/丙型肝炎病毒，乙型脑炎病毒/全部流感病毒、SARS病毒、MERS病毒、新型冠状病毒。
  7. 根据权利要求1-6任一项所述的方法，其中，所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人血清，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13761m/z、13882m/z、13939m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：3198m/z、3213m/z、6609m/z。
  8. 根据权利要求7所述的方法，其中所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z，并且所述多肽序列选自：
    Figure PCTCN2022143141-appb-100001
  9. 根据权利要求1-6任一项所述的方法，其中所述疫苗是针对新型冠状病毒疾病的疫苗，所述体液样本为人唾液，所述特定质荷比的特征多肽包括，下调表达的特征峰3025m/z、13882m/z、14044m/z、14092m/z、14150m/z、15124m/z、15868m/z、28195m/z；上调表达的特征峰为：6609m/z。
  10. 根据权利要求1-9任一项所述的方法，其中所述飞行时间质谱法所用的检测仪器为MALDI-TOF MS或Clin-TOF-II。
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