WO2022089426A1 - Method for analyzing peripheral blood sample and kit - Google Patents

Method for analyzing peripheral blood sample and kit Download PDF

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WO2022089426A1
WO2022089426A1 PCT/CN2021/126426 CN2021126426W WO2022089426A1 WO 2022089426 A1 WO2022089426 A1 WO 2022089426A1 CN 2021126426 W CN2021126426 W CN 2021126426W WO 2022089426 A1 WO2022089426 A1 WO 2022089426A1
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peripheral blood
blood sample
lymphocyte
gene
vaccination
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PCT/CN2021/126426
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French (fr)
Chinese (zh)
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黄丹
梁广锡
邓亮生
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细胞图谱有限公司
香港中文大学
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present application relates to the field of biological detection and analysis, in particular to a peripheral blood sample analysis method and corresponding kit and application, which can be used to evaluate the effectiveness of vaccines and the effect of immunotherapy.
  • Peripheral blood is a mixture of various components, including plasma and blood cells, and is easily obtained from the human body, mainly from fingertips, earlobes, and elbow meridians.
  • Plasma components include metabolites, wastes, hormones, ions, proteins, etc.
  • blood cells include red blood cells, white blood cells, and platelets.
  • peripheral blood is often used for routine blood, biochemical and serum immunological tests to diagnose or identify certain diseases. Morphological and cytological examinations of peripheral blood are of great significance for the diagnosis of various blood diseases.
  • Gene expression of a single cell type is a good biomarker.
  • traditional methods require prior isolation of subsets of specified cell types.
  • a method called single cell RNA-sequencing (scRNA-seq) has also made it possible to obtain gene expression information from single cells.
  • Single-cell RNA sequencing generates gene expression data for each single cell by using expensive equipment and reagents. This expensive technology is generally only used in research and is not suitable for widespread clinical application.
  • Some methods can directly measure genes characteristic of selected subpopulations of single cells in a sample of cell mixtures without isolating the subpopulations of cells of interest. It also avoids the use of expensive equipment for single-cell RNA-seq.
  • This new assay is called the "Direct Leukocyte Subpopulation Transcript Abundance Assay, or Direct LS-TA assay” assay.
  • Immunology is an important research aspect of biology. Through immunological research, the immune system of human and animal bodies has been further understood, and more substances, means and methods have been provided for disease prevention and treatment. Immunology has made great progress in recent years, and various vaccines have been emerging. Vaccines to prevent disease or vaccine to treat disease is a rapidly developing aspect. However, the effects of vaccines after use are uncertain, and it is a very important issue to obtain the effect of vaccine use or confirm the effect of vaccine use in time.
  • Vaccination is a good way to control infection. Vaccines cause the body to prime the acquired (also known as adaptive or specific) immune system to produce antibodies against the pathogen before infection. In individuals who have been vaccinated, the pathogen will be rapidly controlled and the symptoms of infection will be greatly reduced or even asymptomatic. Common examples include vaccination against hepatitis and influenza viruses. Because of the frequent changes in influenza strains, annual vaccinations against different influenza strains are routinely performed in many locations. COVID-19 is now a new pandemic infection and vaccine development is underway.
  • the present application provides an analytical method and corresponding kits and applications for direct measurement of cell-type informative genes of specific B lymphocyte types in samples of various cell mixtures (eg, peripheral blood).
  • Gene) transcript abundance Transcript Abundance, TA
  • Marker parameters are used to predict the effectiveness of vaccination and the effect of immunotherapy (eg, cancer immunotherapy), thereby facilitating early or rapid judgment.
  • one aspect of the present application provides a peripheral blood sample analysis method, comprising determining the transcript abundance of at least one B lymphocyte target gene and the transcript abundance of at least one B lymphocyte reference gene in the peripheral blood sample degree; the B lymphocyte target gene is selected from TNFRSF17, TNFRSF13B, TXNDC5, ? and? At least one of the B lymphocyte reference genes is selected from TNFRSF13C and FCRLA.
  • the present application also provides the use of a reagent component for determining the abundance of gene transcripts in the preparation of a kit for analyzing a peripheral blood sample, wherein the B lymphocyte target gene is selected from TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, so The B lymphocyte reference gene is selected from TNFRSF13C and FCRLA.
  • the above method may comprise the steps of:
  • the peripheral blood sample is a peripheral blood sample before or after vaccination.
  • the peripheral blood sample can be a sample 5-12 days after vaccination.
  • the method further comprises comparing the biomarker parameter of the peripheral blood sample to a cutoff value.
  • the comparison can be used, for example, to predict the effect of the vaccination-induced antibody production and/or cancer immunotherapy.
  • the peripheral blood sample includes a first peripheral blood sample obtained before vaccination, and a second peripheral blood sample obtained after vaccination (eg, 5-12 days after vaccination); the biomarker parameters A first biomarker parameter obtained from the first peripheral blood sample and a second biomarker parameter obtained from the second peripheral blood sample are included.
  • the method may further comprise calculating a change in the first biomarker parameter and the second biomarker parameter.
  • the change value can be used, for example, to predict the effect of antibody production and/or immunotherapy (eg, cancer immunotherapy) resulting from the vaccination.
  • the calculation and comparison of the biomarker parameters can be performed by, for example, a computer system, for example by a computer.
  • the vaccine is a prophylactic or therapeutic vaccine.
  • it could be a vaccine to prevent infectious diseases or a vaccine to treat cancer.
  • the vaccine is an existing vaccine already on the market, or a new vaccine.
  • the application provides a kit comprising reagent components for quantifying the transcript abundance of a gene selected from at least one of TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, and TNFRSF13C and FCRLA at least one of them.
  • a gene selected from at least one of TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, and TNFRSF13C and FCRLA at least one of them.
  • Other genes may be selected without departing from the spirit and essence of the present invention.
  • the kit contains at least one of the primers having the following sequences:
  • the present application provides a reagent composition or kit for determining the abundance of gene transcripts for predicting vaccination efficacy and/or immunotherapy efficacy, wherein the gene is selected from TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN at least one of TNFRSF13C and FCRLA.
  • the application provides the use of a reagent composition for determining the abundance of a gene transcript in the preparation of a kit or a medicament for predicting the effectiveness of vaccination and/or the effect of immunotherapy, wherein the gene is selected from TNFRSF17 , at least one of TNFRSF13B, TXNDC5, and JCHAIN, and at least one of TNFRSF13C and FCRLA.
  • the reagent component comprises a primer, and the sequence of the primer is shown in any one of SEQ ID NOs: 1-14.
  • the gene is obtained from a peripheral blood sample, preferably B lymphocytes from the peripheral blood sample.
  • peripheral blood samples may be obtained at least once, eg, once, twice, or multiple times.
  • the method disclosed in CN103764848B can be used to determine the abundance of transcripts of reference genes and target genes of B lymphocytes in peripheral blood.
  • the methods of the invention are used to analyze the immune response elicited by vaccination to assess the effectiveness of a vaccine or for the assessment of the efficacy of immunotherapy (eg, cancer immunotherapy).
  • immunotherapy eg, cancer immunotherapy
  • the effectiveness of traditional vaccines or the prognosis of immunotherapy is usually assessed by antibody titration confirmation, which can only be carried out after the antibody is produced, usually after 28 days.
  • the existence of this traditional method is limited by the biological time limit of antibody production, especially for acute infectious diseases and fast-developing disease treatment, the confirmation time is too long.
  • the methods of the present application significantly shorten the time for vaccine efficacy confirmation or prognostic assessment of immunotherapy.
  • specific B lymphocyte target genes and reference genes are screened in the present invention, and by analyzing and determining the transcripts of these specific genes, the time for confirming the effectiveness of vaccines or evaluating the prognosis of immunotherapy can be greatly advanced. As early as within 28 days of vaccine administration, such as three weeks, such as two weeks, such as one week, or even within a few days, or less.
  • Figure 1 shows the detection results of biomarkers obtained by "Direct B Lymphocyte Subgroup Transcript Abundance" ("Direct B Lymphocyte LS-TA” assay, referred to as “Direct B Lymphocyte LS-TA”) and traditional methods Correlation of target gene expression detected in isolated and purified B lymphocytes.
  • the X-axis in Figure 1 shows the gene expression of TNFRSF17 assayed in isolated and purified B lymphocytes using RPL32 as the conventional housekeeping gene, where the X-axis is the gold standard.
  • Fig. 2 is the first embodiment of the method for evaluating the efficacy of vaccine use according to the present invention. This protocol requires only one sample.
  • Fig. 3 is a second embodiment of the method for evaluating the efficacy of vaccine use according to the present invention. This protocol requires two samples before and after vaccination.
  • Figure 4A shows the PBMC data at day 7 used to obtain raw values for the "direct B lymphocyte LS-TA" biomarker parameter of TNFRSF17 for NR and R subjects.
  • the MoM values after conversion are shown in Figure 4B.
  • Figure 5A shows the ROC analysis (receiver operating characteristic curve, receiver operating characteristic curve) of the TNFRSF17 value of the "direct B lymphocyte LS-TA" PBMC sample on day 7 to determine the "direct B lymphocyte LS-TA” "The ability to make early predictions after vaccination.
  • the area under the curve (AUC) was 0.85, indicating good predictive power for this biomarker parameter.
  • Figure 5B shows that the ROC analysis was performed on the dataset GSE59635 of another influenza vaccination experiment using a similar method, and the AUC also reached 0.73.
  • Figure 6A shows the increase in "direct B lymphocyte-LS-TA" of TNFRSF17.
  • Figure 6B shows the same data as Figure 6A converted to MoM representation.
  • the increments of biomarker parameters D7-D0
  • the R group had a higher incremental change, which ranged from -0.13 to 3.39 with a median increment of 1.11, and the incremental change was significantly different between the NR and R groups (paired test, p-value ⁇ 0.01).
  • Figure 7A shows that ROC analysis was performed to determine the ability of the increase in TNFRSF17 to discriminate between R and NR groups using "direct B lymphocyte LS-TA" from day 0 to day 7.
  • Figure 7B shows an AUC of 0.7 for the increment (D7-D0) of "direct B lymphocyte LS-TA" for TNFRSF17 in the dataset GSE59635 PBMC samples.
  • Figures 8A and 8B in dataset GSE136163, used results for whole blood samples from yellow fever vaccinators. Of these, 2 vaccinators were classified as NR because they did not have more than a 4-fold increase in neutralizing antibody (nab) titers.
  • the median change in "direct B lymphocyte LS-TA" for TNFRSF17 in the NR group (D7-D0) was negative (-0.28).
  • the D7-D0 "direct B lymphocyte LS-TA" increase of TNFRSF17 in group R was higher (median 0.38).
  • Figure 8C shows that ROC analysis was performed and the AUC of "direct B lymphocyte-LS-TA" TNFRSF17 on day 7 alone was 0.82.
  • Figure 8D shows an AUC of 0.88 for the increment of "direct B lymphocyte-LS-TA" TNFRSF17 on days 0 and 7.
  • Figure 9A shows the increase in biomarkers from D0 to D7 in the NR and R groups when using the TXNDC5 gene data from dataset GSE59635. Differences between the two groups (NR and R) were significant (paired test, p-value ⁇ 0.01).
  • Figure 9B shows the ROC assayed for the increase in TXNDC5 using "direct B lymphocyte LS-TA" with an AUC of 0.77.
  • Figure 9C shows that the "direct B lymphocyte LS-TA" assay of TNFRSF13B relative to TNFRSF13C using the dataset GSE59635, using the seventh day samples alone, determined that responders (group R) had higher biomarker parameters (p-values). ⁇ 0.05).
  • Figure 10A shows that the R group had a higher increase in "direct B lymphocyte LS-TA" of TNFRSF17:FCRLA than the NR group (paired test, p-value ⁇ 0.001).
  • Figure 10B shows the incremental AUC of "direct B lymphocyte LS-TA" (TNFRSF13C, FCRLA) in ROC analysis was 0.82.
  • FIG 11A shows that patients who were able to survive for a long time had lower "direct B lymphocyte LS-TA" biomarker parameters before vaccination (P value ⁇ 0.05).
  • Figure 12A is the results of survival analysis.
  • Cox regression Cox regression
  • the ranges are divided into (a) low (indicator below the 25th percentile), (b) intermediate (25th-75th percentile), and (c) high (above the 75th percentile) .
  • the survival curves of the three groups were significantly different.
  • the group with the worst survival rate was the group with high "direct B lymphocyte LS-TA" index, and most patients died within almost a year (p value ⁇ 0.01).
  • Figure 12B shows that only comparing the two groups with low (below 25th percentile) and high (above 75th percentile) biomarker parameters with each other, the difference in survival curves is more pronounced (p-value ⁇ 0.001).
  • Figure 13A shows a meta-analysis Forest Plot of the seventh day expression of the biomarker (JCHAIN/TNFRSF13C) in the NR and R groups.
  • Figure 13B shows a meta-analysis forest plot of the increase in biomarker (JCHAIN/TNFRSF13C) from day 0 to day 7 in the NR and R groups.
  • the traditional method to measure the gene expression of specific cells in peripheral blood is to first isolate specific cell subsets and then determine their gene expression levels.
  • Cellular expression levels derived from traditional methods serve as the gold standard.
  • the present application provides methods for the direct determination of gene expression in specific cells (eg, B lymphocytes) in peripheral blood samples (eg, peripheral blood mononuclear cells (PBMCs)) without the need to purify the specific cells from other blood cells.
  • the utility of the new method can be determined by comparing the gold standard obtained by the traditional method and the gene expression levels of specific cells obtained by the new method in this paper and their correlations.
  • transcript abundance (TA) of a single cell type can be determined directly in a sample of a cell mixture of peripheral blood (eg, PBMC) without the need for subpopulation isolation. It applies to a group of genes called B cell information genes. The relative values of two B cell informative genes (target gene and stably expressed reference gene) obtained in PBMC were used as new biomarker parameters to represent target gene expression in purified B lymphocytes.
  • This method of directly determining the TA of leukocyte subsets in peripheral blood samples without the tedious process of cell isolation is called the direct LS-TA method.
  • the method can be applied to gene expression datasets collected in influenza vaccination trials for early prediction of seroconversion.
  • the present application provides novel, easy-to-analyze peripheral blood biomarkers that can be readily incorporated into routine clinical laboratory tests, or used in routine clinical research.
  • the peripheral blood biomarkers can predict early seroconversion status after influenza vaccination (eg, antibody production after vaccination can be predicted on day 7, etc.). Since the pathways of acquired immunity are shared by vaccination against influenza and COVID-19, these biomarkers can also be used to predict seroconversion in new COVID-19 vaccines.
  • Direct B lymphocyte LS-TA will be a useful test for implementing personalized vaccination.
  • peripheral blood sample has its usual meaning in the field of medical testing, mainly referring to blood samples taken from fingertips, earlobes, arm veins, etc.
  • it can be a whole blood sample (Whole Blood, WB) or a mixture of multiple types of monocytes in peripheral blood (Peripheral Blood Mononuclear Cells, PBMC).
  • WB Whole Blood
  • PBMC Peripheral Blood Mononuclear Cells
  • direct measurement refers to measurement without isolation of specific cells (eg, B lymphocytes) therein. That is, direct measurement is performed without isolating the specific cells to be assayed (eg, B lymphocytes) from the blood sample.
  • gene expression level refers to the gene expression level of cells (eg, B lymphocytes), with RNA as the measurement object, specifically mRNA as the measurement object, which is the quantitative level of messenger ribonucleic acid.
  • RNA as the measurement object
  • mRNA as the measurement object
  • TA transcript abundance
  • vaccine refers to a biological product, which is made against pathogenic microorganisms or their proteins (polypeptides, peptides), polysaccharides or nucleic acids by artificial attenuation, inactivation, lysis, genetic recombination, purification, etc., and can induce The body produces corresponding protective immune substances, which are used to prevent and control the occurrence and prevalence of corresponding diseases, or can regulate the specific immune responses of certain harmful factors (such as smoking) and physiological states (such as contraception) in the body, so as to achieve Biological products for the purpose of treating or eliminating harmful factors and preventing fertility.
  • a vaccine is a substance that stimulates the body to carry out an immune response, which is used to eliminate specific infected organisms and specific cells, and play a preventive or therapeutic role.
  • Vaccine ingredients include active ingredients and auxiliary ingredients.
  • the "vaccine” used in this application includes the active ingredient of the vaccine, as well as the composition or preparation to which other substances are added, that is, a single ingredient or composition or preparation suitable for stimulating the immune response of the human and animal body.
  • Vaccine refers to the substance referred to in any of the above situations. In simple terms, it is an immune response stimulator, or a substance that induces a beneficial immune response.
  • cancer refers to a class of diseases in which oncogene expression or expansion of cancer cells occurs or the development of solid tumors. Any cancer diagnosed clinically by histology, immunology, protein assay is within the meaning of this term.
  • cancer immunotherapy also known as cancer immunotherapy, is a new generation of cancer treatment options (Wikipedia: (“Cancer Immunotherapy” 2020)), which use the body's immune function to attack tumor cells.
  • Wikipedia (“Cancer Immunotherapy” 2020)
  • cancer vaccine (cancer vaccine) is a new treatment.
  • Cancer vaccines here do not refer to preventive vaccines that individuals receive before cancer, such as the hepatitis B virus (HBV) vaccine that can prevent liver cancer and the human papilloma virus (HPV) vaccine that prevents cervical cancer, but refers to the onset of cancer Afterwards, cancer cells possess some characteristic antigens that can be directed against these antigens and cancer cells by stimulating an autologous immune response. Different treatment regimens use different candidate antigens.
  • An example here is the use of personalized peptide vaccination (PPV) to treat prostate cancer (Araki et al. 2015).
  • sample of cell mixture is a mixture of cells from an individual (eg, a human body).
  • the cell mixture sample can be derived from peripheral blood, eg, a peripheral blood sample that has not been processed in any way.
  • cell-type informative genes refers to genes that are specifically expressed by a specific cell (eg, B lymphocyte) type, and the activity of the characteristic gene can be determined by measuring its transcript abundance. Characterized genes can include target genes and reference genes. Since the target specific cells used in this patent application are B cells, B cell characteristic genes can also be called B cell information genes. In this patent application, characteristic gene and informative gene can be used interchangeably.
  • biomarker and “biomarker” refer to the amount of messenger RNA in gene expression of a particular cell (eg, B lymphocyte), and both relative and absolute amounts can be used to record biomarker status.
  • a particular cell eg, B lymphocyte
  • biomarkers and biomarkers are used interchangeably.
  • LS Leukocyte Subpopulation
  • B lymphocyte subsets include many types of cells.
  • Peripheral blood is a typical sample of a mixture of multiple types of cells, including various leukocyte subsets, such as neutrophils, lymphocytes and monocytes.
  • Transcript Abundance refers to the gene expression level (gene expression level) obtained by testing a sample.
  • Transcript refers to the product after gene transcription, usually RNA.
  • RNA messenger RNA
  • mRNA messenger RNA
  • direct B lymphocyte subpopulation transcript abundance assay
  • direct B lymphocyte leukocyte subpopulation transcript abundance assay
  • direct B lymphocyte LS-TA assay
  • the target cell subset discussed in the present invention is B lymphocytes in peripheral blood. So the test is called “direct B lymphocyte subset transcript abundance” test.
  • the calculation of "direct B lymphocyte LS-TA" requires the use of target genes characteristic of cell subsets and reference genes characteristic of cell subsets.
  • subpopulation informative target gene refers to a gene whose majority of the transcripts in a multicellular mixed sample (eg WB, PBMC) are derived from the specified target cell subpopulation , that is, produced by this cell subset.
  • WB multicellular mixed sample
  • PBMC PBMC
  • subpopulation informative reference gene means that in a multicellular mixed sample (eg WB, PBMC), most of its transcripts are from the specified target cell subpopulation, and Genes that are stably expressed in target cells at the same time.
  • the inventors of the present application proposed to directly measure the gene expression of specific cells (eg B lymphocytes) from various peripheral blood samples and unexpectedly found that these gene expressions are of great value in predicting the effects of vaccination and cancer immunotherapy.
  • specific cells eg B lymphocytes
  • the methods described herein also include comparing the biomarker parameter of the peripheral blood sample to a cutoff value. In some embodiments, the comparison is used to predict the effect of vaccination-induced antibody production and/or immunotherapy.
  • critical value can be defined in several ways.
  • the critical value can be defined as a value outside the reference interval of the control group.
  • the reference interval for the control group usually takes the middle 95% distribution of the control group. Values outside this range can be used as thresholds to define abnormally low or abnormally high results.
  • the critical value can also be best defined from the ROC chart, as shown in Figure 5A. The value labeled at each point on the ROC curve represents a potential cutoff, and the Y- and X-axes show the relative sensitivity and specificity using this cutoff, respectively.
  • the cutoff value was defined by the 75th percentile value of the direct LS-TA value of the ratio of TNFRSF17 to FCRLA ( Figure 9B), and cancer patients with direct LS-TA greater than this cutoff value ( Figure 9B) Solid line) had a poorer response to cancer vaccine treatment and a shorter survival compared with other patients.
  • the types of blood samples obtained include whole blood (WB) and peripheral blood mononuclear cells (PBMC). Specific cell types that were further isolated and purified were also included in some datasets, such as isolated and purified B lymphocytes (eg GSE45764) or T lymphocytes.
  • WB whole blood
  • PBMC peripheral blood mononuclear cells
  • the standard definition of a responder (R) after vaccination is based on the titer of antibodies against a specific antigen in serum, such as after influenza vaccination, the level of hemagglutination-inhibiting (HI) antibodies (also known as HI titer, titer in hemagglutination) A significant increase in inhibition assay) indicates that the recipient is responding to the vaccine.
  • This criterion is determined by performing hemagglutination inhibition (HI) on the serum of subjects before vaccination and twenty-eight days after vaccination.
  • CHMP European Committee for Human Medicinal Products
  • Direct B Lymphocyte Transcript Abundance (“Direct B Lymphocyte-LS-TA”, referred to as “Direct B Lymphocyte LS-TA”) biomarker parameters
  • a biomarker parameter is calculated by using the relative TA values of B lymphocyte target genes (such as TNFRSF17) and B lymphocyte characteristic reference genes (such as TNFRSF13C) in the cell mixture sample to evaluate the gene expression of B lymphocytes after isolation and purification.
  • B lymphocyte target genes such as TNFRSF17
  • B lymphocyte characteristic reference genes such as TNFRSF13C
  • the designated target gene was TNFRSF17.
  • the designated B lymphocyte reference gene is TNFRSF13C. "Direct B lymphocyte LS-TA" is indicated on the Y-axis.
  • conventional housekeeping genes were only used to normalize the gene expression results of isolated and purified B lymphocyte samples.
  • Conventional housekeeping genes include RPL31, RPL32, ACTB, GAPDH and UBC. Selection of conventional housekeeping genes is described in Eisenberg and Levanon 2013, also available on websites such as https://www.tau.ac.il/ ⁇ elieis/HKG/.
  • the X-axis of Figure 1 shows the gene expression of TNFRSF17 (gold standard) assayed in isolated and purified B lymphocytes, using RPL32 as the conventional housekeeping gene.
  • Conventional housekeeping genes are used by manufacturers only for calibration gold standard and validation purposes, not in the kits of the present invention or embodiments thereof.
  • results of the "direct B lymphocyte LS-TA" biomarkers of the present invention (relative value of TNFRSF17:TNFRSF13C) measured in PBMC cell mixture samples compared with traditional methods in the separation and purification of B lymphocytes
  • the target gene expression (gold standard) detected in cells was well correlated (correlation coefficient, R>0.9).
  • B lymphocyte target genes including TNFRSF13B, TXNDC5, JCHAIN
  • B lymphocyte reference genes including TNFRSF13C, FCRLA
  • the "direct B lymphocyte-LS-TA" marker will be used to predict the status of antibody production following vaccination.
  • Two schemes will be used here. The first scheme requires only one sample. The implementation process is shown in Figure 2. The second scheme requires two samples before and after vaccination. The implementation process is shown in Figure 3.
  • This biomarker parameter represents the gene expression level of the B lymphocyte gene TNFRSF17 in the cell mixture sample of PBMC. Because this biomarker can be obtained without prior isolation of B lymphocytes, it is labeled "Direct B lymphocyte transcript abundance" (“Direct B lymphocyte-LS-TA”) in the graph.
  • the "direct B lymphocyte-LS-TA" biomarker parameter of the B lymphocyte target gene TNFRSF17 calculated using the ratio of target genes characteristic of cell subsets to reference genes characteristic of cell subsets, can be expressed as:
  • TNFRSF17 (TNFRSF17 in PBMC)/(TNFRSF13C in PBMC)
  • This biomarker parameter can also be logarithmically transformed, which can also be expressed as:
  • the median (Multiple of median of a reference group, MoM) relative to the normal control group is a commonly used standardization method.
  • MoM Multiple of median of a reference group
  • data from pre-vaccination (day 0) were used to define the median of the normal control group.
  • Results for all individuals (including NR and R) were then expressed as multiples of this normal median. So this data representation is called the multiple of the control group median (MoM).
  • MoM multiple of the control group median
  • the benefit of using MoM is that it removes the unit limitation of the data, allowing comparison of results from different detection schemes.
  • ROC analysis (receiver operating characteristic curve, receiver operating characteristic curve) was performed on the TNFRSF17 value of "direct B lymphocyte LS-TA" in PBMC samples on day 7 to determine the effect of "direct B lymphocyte LS-TA” on the vaccine Ability to make early predictions of vaccination effectiveness (Figure 5A).
  • the area under the curve (AUC) was 0.85, indicating that this biomarker parameter has good predictive power.
  • the data set (GSE29617) was used to calculate the increment of "direct B lymphocyte-LS-TA” on day 0 and day 7, using the data of day 7 "direct B lymphocyte-LS-TA” TNFRSF17 value and The "direct B lymphocyte-LS-TA” TNFRSF17 value on day 0 (baseline, pre-vaccination), the delta change value (delta, delta) obtained by subtracting the two values, can be used to predict subsequent antibody production state. That is, the present embodiment executes the flow of FIG. 3 .
  • D7-D0 changes in "direct B lymphocyte-LS-TA" of TNFRSF17 in whole blood samples and the ability to effectively differentiate between R and NR groups also in other vaccinations
  • TXNDC5 and TNFRSF13B genes are two other target genes available for "direct B lymphocyte-LS-TA".
  • the relative expression of TXNDC5 and TNFRSF13B relative to TNFRSF13C was used as a biomarker.
  • FIG. 9A shows the increase in biomarkers from D0 to D7 in the NR and R groups. Differences between the two groups (NR and R) were significant (paired test, p-value ⁇ 0.01). And the ROC of the increment of "direct B lymphocyte-LS-TA" TXNDC5 was analyzed, and its AUC was 0.77 (Fig. 9B).
  • FCRLA can also be used as another B lymphocyte reference gene.
  • Figures 10A and 10B show the results and discrimination performance of "direct B lymphocyte-LS-TA" using TNFRSF17 and FCRLA.
  • Figure 10A shows that the R group had a higher increase in "direct B lymphocyte-LS-TA" of TNFRSF17:FCRLA than the NR group (paired test, p-value ⁇ 0.001).
  • Figure 10B shows that the incremental AUC for "direct B lymphocytes-LS-TA" (TNFRSF13C, FCRLA) in ROC analysis was 0.82 ( Figure 10B).
  • the GSE53922 database contains PMBC gene expression, prognosis and overall survival (OS) data in peripheral blood multitype monocyte samples from 112 prostate patients before cancer vaccination. Patients were divided into two groups, the first group was patients who survived less than one year (365 days) after treatment. The other group consists of patients with better treatment effect and survival for more than one year after treatment.
  • the "direct B lymphocyte-LS-TA" biomarker parameters were then compared between the two groups of patients before PPV vaccination. In this example, the "direct B lymphocyte-LS-TA" results were calculated using the relative values of the two genes TNFRSF17 and FCRLA in PBMC samples.
  • Figures 11A and 11B show that the group of patients with better treatment effect had lower "direct B lymphocyte-LS-TA” biomarker parameters before vaccination.
  • Figure 11A shows that patients with long-term survival had lower "direct B lymphocyte-LS-TA” biomarker parameters before vaccination (P value ⁇ 0.05).
  • Figure 12A is a survival analysis, using Cox regression (Cox regression) to calculate the survival curve (survival curve) of the three groups of patients, according to the patients before vaccine injection
  • "direct B lymphocytes-LS-TA" biomarker Objects were divided into three groups using quantile ranges as (a) low (indicators below the 25th percentile), (b) intermediate (25th-75th percentile), and (c) high ( above the 75th percentile).
  • the survival curves of the three groups were significantly different.
  • the group with the worst survival rate was the group with high "direct B lymphocyte-LS-TA" index before vaccination, and most patients died within one year (p value ⁇ 0.01).
  • the JCHAIN gene is also a target gene that can be used for "direct B lymphocyte-LS-TA".
  • a meta-analysis of influenza vaccine recipients was performed using the relative expression of JCHAIN relative to TNFRSF13C as a biomarker.
  • FIG. 13 shows a meta-analyzed Forest Plot of day 7 expression of the biomarker (JCHAIN/TNFRSF13C) in NR and R groups, while Figure 13B shows this biomarker in NR and R groups from day 0 to Meta-analysis forest plot of day 7 increments.
  • JCHAIN/TNFRSF13C the biomarker
  • This application provides some examples of primers that can be used in quantitative PCR (qPCR) for reference. They can be used in the presence of SYBR Green in qPCR reactions to obtain threshold cycle (CT) data that can be used to determine delta-CT, delta-delta CT or efficiency-corrected delta-CT by relative quantification assays as biological Flag parameters. Quantify RNA transcript abundance in blood samples (Dorak 2007). Other quantitative methods can also be implemented.
  • qPCR quantitative PCR
  • RNA is extracted from various blood samples using Trizol or similar reagents. Commercial kits are also available for column-based RNA extraction. Then, the RNA is reverse transcribed into cDNA by reverse transcriptase. Specific genes are quantified by user-selected methods, including qPCR, RNA sequencing, DNA microarrays (gene chips), branched chain DNA assays (branched chain DNA, bDNA assay, US8426578B2, US7927798B2), nano-reporter probe assays (quantification) Assay using nanoreporters, US8415102B2), digital PCR (US10465238B2) or hybridization.
  • cDNA samples using this method can be used for TA of both B lymphocyte target genes (such as TNFRSF17, TNFRSF13B, TXNDC5 or JCHAIN) and B lymphocyte reference genes (such as TNFRSF13C, FCRLA, CD79A, CD79B or MS4A1)
  • B lymphocyte target genes such as TNFRSF17, TNFRSF13B, TXNDC5 or JCHAIN
  • B lymphocyte reference genes such as TNFRSF13C, FCRLA, CD79A, CD79B or MS4A1
  • Biomarker parameters were calculated from delta-CT, delta-delta CT or efficiency-corrected delta-CT of qPCR results for such B lymphocyte informative gene pairs.
  • the biomarker parameters generated by this "direct B lymphocyte-LS-TA" analysis provide an indication of B lymphocyte gene expression levels in samples of various cell mixtures in blood (e.g., PBMC, WB) without prior isolation of B lymphocytes cell.

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Abstract

Provided in the present application is a method for analyzing a peripheral blood sample, the method comprising determining an abundance of the transcript of at least one B lymphocyte target gene in the peripheral blood sample and an abundance of the transcript of at least one B lymphocyte reference gene. The B lymphocyte target gene is selected from TNFRSF17, TNFRSF13B, TXNDC5, and JCHAIN, and the B lymphocyte reference gene is selected from TNFRSF13C and FCRLA. The method can be used in the early stages of vaccination to predict the effectiveness of vaccinations and the effect of immunotherapy. Further provided in the present invention are a kit for implementing the above-mentioned method, and the related use.

Description

外周血样本分析方法和试剂盒Peripheral blood sample analysis method and kit 技术领域technical field
本申请涉及生物检测和分析领域，具体涉及一种外周血样本分析方法及相应的试剂盒和应用，可用于评估疫苗的有效性和免疫治疗的效果。The present application relates to the field of biological detection and analysis, in particular to a peripheral blood sample analysis method and corresponding kit and application, which can be used to evaluate the effectiveness of vaccines and the effect of immunotherapy.
背景技术Background technique
外周血的检测和分析是医学检查的一个重要方面。外周血是多种成分混合物，包括血浆和血细胞，易于从人体获得，主要从指尖、耳垂、肘部经脉抽取获得。血浆成分有代谢物、废物、激素、离子、蛋白质等，血细胞包括红细胞、白细胞、血小板等。临床上常应用外周血进行血常规、生化、血清免疫学检验，以此来诊断或者鉴别某些疾病。外周血的形态学检查和细胞学检查，对于各种血液病的诊断有重要意义。The detection and analysis of peripheral blood is an important aspect of medical examination. Peripheral blood is a mixture of various components, including plasma and blood cells, and is easily obtained from the human body, mainly from fingertips, earlobes, and elbow meridians. Plasma components include metabolites, wastes, hormones, ions, proteins, etc., and blood cells include red blood cells, white blood cells, and platelets. Clinically, peripheral blood is often used for routine blood, biochemical and serum immunological tests to diagnose or identify certain diseases. Morphological and cytological examinations of peripheral blood are of great significance for the diagnosis of various blood diseases.
单个细胞类型(single cell type)的基因表达是良好的生物标志物。为了获得外周血中单种细胞亚群的基因表达水平，传统方法需要事先分离指定细胞类型的亚群。最近，一种称为单细胞RNA测序(single cell RNA-sequencing，scRNA-seq)的方法也可能获得单细胞的基因表达信息。单细胞RNA测序通过使用价钱昂贵的设备和试剂，生成每个单细胞基因表达数据，这种昂贵的技术一般只在研究中使用，不适宜在临床中广泛应用。Gene expression of a single cell type is a good biomarker. To obtain gene expression levels of individual cell subsets in peripheral blood, traditional methods require prior isolation of subsets of specified cell types. More recently, a method called single cell RNA-sequencing (scRNA-seq) has also made it possible to obtain gene expression information from single cells. Single-cell RNA sequencing generates gene expression data for each single cell by using expensive equipment and reagents. This expensive technology is generally only used in research and is not suitable for widespread clinical application.
一些方法可以直接测量细胞混合物样本中选定的单细胞亚群的特征性基因，而无需分离目标细胞亚群。它还避免了使用单细胞RNA-seq的昂贵设备。具体参见专利文献CN103764848B(申请日2012年7月23日，公开日2014年4月30日)；和US9589099B2(申请日2012年7月20日，公开日2017年3月7日)。这种新的检测方法称为“直接白 血细胞亚群的转录本丰度”检测(Direct Leukocyte Subpopulation Transcript Abundance assay,或简称Direct LS-TA assay)。这些方法提供了不用事先对细胞进行分离而直接进行白血细胞亚群的转录本丰度测量的技术，测量速度和成本上具有很大优势。Some methods can directly measure genes characteristic of selected subpopulations of single cells in a sample of cell mixtures without isolating the subpopulations of cells of interest. It also avoids the use of expensive equipment for single-cell RNA-seq. For details, please refer to patent documents CN103764848B (application date July 23, 2012, publication date April 30, 2014); and US9589099B2 (application date July 20, 2012, publication date March 7, 2017). This new assay is called the "Direct Leukocyte Subpopulation Transcript Abundance Assay, or Direct LS-TA assay" assay. These methods provide a technique for directly measuring transcript abundance of white blood cell subsets without prior isolation of cells, with great advantages in measurement speed and cost.
免疫学是生物学的一个重要研究方面。通过免疫学研究，对人体和动物体的免疫***有了进一步认识，对疾病预防和治疗提供了更多物质、手段和方法。免疫学近年有很大发展，各类疫苗不断涌现。疫苗预防疾病或者疫苗治疗疾病是当前一个发展较快的方面。但是，疫苗使用后的效果存在不确定性，及时获得疫苗使用效果或者确认疫苗使用效果是一个很重要的议题。Immunology is an important research aspect of biology. Through immunological research, the immune system of human and animal bodies has been further understood, and more substances, means and methods have been provided for disease prevention and treatment. Immunology has made great progress in recent years, and various vaccines have been emerging. Vaccines to prevent disease or vaccine to treat disease is a rapidly developing aspect. However, the effects of vaccines after use are uncertain, and it is a very important issue to obtain the effect of vaccine use or confirm the effect of vaccine use in time.
传染病是人类和动物的一个重大敌人。通过疫苗接种是控制感染的好方法。疫苗会使身体在感染之前启动获得性(又称为适应性或特异性)免疫***，以产出针对病原体的抗体。在已经接种疫苗的个体中，病原体将得到快速控制，感染症状会大大减轻甚至无症状。常见的例子包括针对肝炎病毒和流感病毒的疫苗接种。由于流行性感冒病毒株频繁变化，因此在许多地方通常对不同的流行性感冒病毒株进行年度疫苗接种。现时COVID-19是一种新的大流行感染，疫苗的开发也在进行中。Infectious diseases are a major enemy of humans and animals. Vaccination is a good way to control infection. Vaccines cause the body to prime the acquired (also known as adaptive or specific) immune system to produce antibodies against the pathogen before infection. In individuals who have been vaccinated, the pathogen will be rapidly controlled and the symptoms of infection will be greatly reduced or even asymptomatic. Common examples include vaccination against hepatitis and influenza viruses. Because of the frequent changes in influenza strains, annual vaccinations against different influenza strains are routinely performed in many locations. COVID-19 is now a new pandemic infection and vaccine development is underway.
在针对流感病毒进行疫苗接种的情况下，并非所有接种疫苗的接种者都免受随后的感染，也不是每一个接种者都有抗体产生，这种保护不佳(低抗体产生)的人称为无反应者(non-responder，NR)。另一方面，疫苗接种的反应者(Responder，R)会产生针对抗原的抗体。抗体的产生是以针对特定抗原的抗体滴度(Antibody Titer)来衡量的，需要在接种后28天的血液样本中进行检测。一般来说，流感疫苗接种后的反应者比率少于接种者的50％。此外，获得性免疫***需要较长时间产生针对新抗原的抗体，所以一般在疫苗接种后28天才分析抗体滴度的结果。以抗体出现为生物学标志物的测试确认试验，需要在接种后至少28天才能获得结果。In the case of vaccination against the influenza virus, not all vaccinated persons are protected from subsequent infections, nor do every vaccinated person develop antibodies, a person with poor protection (low antibody production) is called no Responder (non-responder, NR). On the other hand, vaccinated responders (Responder, R) develop antibodies against the antigen. Antibody production is measured as an antibody titer against a specific antigen (Antibody Titer), which needs to be detected in blood samples 28 days after vaccination. In general, the responder rate after influenza vaccination is less than 50% of those who receive it. In addition, the acquired immune system takes a longer time to produce antibodies against neoantigens, so the results of antibody titers are generally analyzed only 28 days after vaccination. Tests confirming the presence of antibodies as biomarkers require results at least 28 days after vaccination.
所以，疫苗使用需要配合有效检测才能真正做到及时预防和治疗疾病。快速和早期检测疫苗接种效果是疫苗使用中一个急需解决的问 题。Therefore, the use of vaccines needs to be combined with effective testing in order to truly prevent and treat diseases in a timely manner. Rapid and early detection of vaccination efficacy is an urgent issue in vaccine use.
因此，需要一种新的简单而快速的外周血分析方法，使得可以在预防性或治疗性疫苗接种后的早期进行，以评估疫苗接种的有效性和免疫治疗的效果。Therefore, there is a need for a new simple and rapid peripheral blood analysis method that can be performed early after prophylactic or therapeutic vaccination to assess the effectiveness of vaccination and the effect of immunotherapy.
发明内容SUMMARY OF THE INVENTION
总体而言，本申请提供了一种分析方法及相应的试剂盒和应用，可通过直接测量各种细胞混合物样本(例如外周血)中的特定B淋巴细胞类型的特征性基因(cell-type informative gene)的转录本丰度(Transcript Abundance，TA)来直接评估B淋巴细胞基因的表达，从而避免事先分离B淋巴细胞，也不需要使用昂贵的设备进行单细胞RNA测序，通过该方法获得的生物标志物参数，用于预测接种疫苗的有效性和免疫治疗(例如癌症免疫治疗)的效果，从而有利于早期或者快速作出判断。In general, the present application provides an analytical method and corresponding kits and applications for direct measurement of cell-type informative genes of specific B lymphocyte types in samples of various cell mixtures (eg, peripheral blood). Gene) transcript abundance (Transcript Abundance, TA) to directly assess B lymphocyte gene expression, thereby avoiding the need to separate B lymphocytes in advance, and does not require the use of expensive equipment for single-cell RNA sequencing. Marker parameters are used to predict the effectiveness of vaccination and the effect of immunotherapy (eg, cancer immunotherapy), thereby facilitating early or rapid judgment.
具体地，本申请一方面提供了一种外周血样本分析方法，包括测定外周血样本中的至少一种B淋巴细胞靶基因的转录本丰度和至少一种B淋巴细胞参考基因的转录本丰度；所述B淋巴细胞靶基因选自TNFRSF17、TNFRSF13B、TXNDC5、？和？中的至少一种，所述B淋巴细胞参考基因选自TNFRSF13C和FCRLA。本申请还提供了用于测定基因转录本丰度的试剂成分在制备用于外周血样本分析方法的试剂盒中的用途，所述B淋巴细胞靶基因选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN，所述B淋巴细胞参考基因选自TNFRSF13C和FCRLA。Specifically, one aspect of the present application provides a peripheral blood sample analysis method, comprising determining the transcript abundance of at least one B lymphocyte target gene and the transcript abundance of at least one B lymphocyte reference gene in the peripheral blood sample degree; the B lymphocyte target gene is selected from TNFRSF17, TNFRSF13B, TXNDC5, ? and? At least one of the B lymphocyte reference genes is selected from TNFRSF13C and FCRLA. The present application also provides the use of a reagent component for determining the abundance of gene transcripts in the preparation of a kit for analyzing a peripheral blood sample, wherein the B lymphocyte target gene is selected from TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, so The B lymphocyte reference gene is selected from TNFRSF13C and FCRLA.
在不背离本发明精神和实质的情况下，上述参考基因和靶基因还可以选择其他基因。Without departing from the spirit and essence of the present invention, other genes can also be selected for the above-mentioned reference genes and target genes.
在一种实施方案中，上述方法可以包括如下步骤：In one embodiment, the above method may comprise the steps of:
a).采集所述外周血样本；a). Collecting the peripheral blood sample;
b).测定所述外周血样本的所述至少一种B淋巴细胞靶基因的转录本丰度，以获得第一量；b). Determining the transcript abundance of the at least one B lymphocyte target gene in the peripheral blood sample to obtain a first amount;
c).测定所述外周血样本的所述至少一种B淋巴细胞参考基因的 转录本丰度，以获得第二量；c). Determining the transcript abundance of the at least one B lymphocyte reference gene of the peripheral blood sample to obtain a second amount;
d).计算生物标志物参数，即所述第一量与所述第二量的相对值。d). Calculating biomarker parameters, ie relative values of the first amount and the second amount.
在一种实施方案中，所述外周血样本为接种疫苗之前或接种疫苗之后的外周血样本。例如，所述外周血样本可以是接种疫苗5-12天后的样本。In one embodiment, the peripheral blood sample is a peripheral blood sample before or after vaccination. For example, the peripheral blood sample can be a sample 5-12 days after vaccination.
在一种实施方案中，所述方法还包括将所述外周血样本的所述生物标志物参数与临界值进行比较。所述比较例如可以用于预测所述接种疫苗导致的抗体产生和/或癌症免疫治疗的效果。In one embodiment, the method further comprises comparing the biomarker parameter of the peripheral blood sample to a cutoff value. The comparison can be used, for example, to predict the effect of the vaccination-induced antibody production and/or cancer immunotherapy.
在一种实施方案中，所述外周血样本包括接种疫苗之前获取的第一外周血样本，和接种疫苗后(例如接种5-12天后)获取的第二外周血样本；所述生物标志物参数包括由所述第一外周血样本获得的第一生物标志物参数和由所述第二外周血样本获得的第二生物标志物参数。在进一步的实施方案中，所述方法还可包括计算所述第一生物标志物参数和第二生物标志物参数的变化值。所述变化值例如可以用于预测所述接种疫苗所导致的抗体产生和/或免疫治疗(例如癌症免疫治疗)的效果。In one embodiment, the peripheral blood sample includes a first peripheral blood sample obtained before vaccination, and a second peripheral blood sample obtained after vaccination (eg, 5-12 days after vaccination); the biomarker parameters A first biomarker parameter obtained from the first peripheral blood sample and a second biomarker parameter obtained from the second peripheral blood sample are included. In a further embodiment, the method may further comprise calculating a change in the first biomarker parameter and the second biomarker parameter. The change value can be used, for example, to predict the effect of antibody production and/or immunotherapy (eg, cancer immunotherapy) resulting from the vaccination.
在一种实施方案中，所述生物标志物参数的计算和比较可以通过例如计算器***来进行，例如通过计算机来进行。In one embodiment, the calculation and comparison of the biomarker parameters can be performed by, for example, a computer system, for example by a computer.
在一种实施方案中，所述疫苗是预防性疫苗或者治疗性疫苗。例如，可以是预防传染病的疫苗或治疗癌症的疫苗。In one embodiment, the vaccine is a prophylactic or therapeutic vaccine. For example, it could be a vaccine to prevent infectious diseases or a vaccine to treat cancer.
在一种实施方案中，所述疫苗是已经上市的现有疫苗，也可以是新疫苗。In one embodiment, the vaccine is an existing vaccine already on the market, or a new vaccine.
另一方面，本申请提供了一种试剂盒，其包含用于定量基因转录本丰度的试剂成分，所述基因可以选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN中的至少一种，以及TNFRSF13C和FCRLA中的至少一种。在不背离本发明精神和实质的情况下，可以选择其他基因。In another aspect, the application provides a kit comprising reagent components for quantifying the transcript abundance of a gene selected from at least one of TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, and TNFRSF13C and FCRLA at least one of them. Other genes may be selected without departing from the spirit and essence of the present invention.
在一种实施方案中，所述试剂盒中含有具有如下序列的引物中的至少一种:In one embodiment, the kit contains at least one of the primers having the following sequences:
Figure PCTCN2021126426-appb-000001
Figure PCTCN2021126426-appb-000001
Figure PCTCN2021126426-appb-000002
Figure PCTCN2021126426-appb-000002
另一方面，本申请提供了测定基因转录本丰度的试剂成分或试剂盒，其用于预测疫苗接种有效性和/或免疫治疗效果，其中所述基因选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN中的至少一种，以及TNFRSF13C和FCRLA中的至少一种。再一方面，本申请提供了用于测定基因转录本丰度的试剂成分在制备用于预测疫苗接种有效性和/或免疫治疗效果的试剂盒或药物中的应用，其中所述基因选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN中的至少一种，以及TNFRSF13C和FCRLA中的至少一种。In another aspect, the present application provides a reagent composition or kit for determining the abundance of gene transcripts for predicting vaccination efficacy and/or immunotherapy efficacy, wherein the gene is selected from TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN at least one of TNFRSF13C and FCRLA. In yet another aspect, the application provides the use of a reagent composition for determining the abundance of a gene transcript in the preparation of a kit or a medicament for predicting the effectiveness of vaccination and/or the effect of immunotherapy, wherein the gene is selected from TNFRSF17 , at least one of TNFRSF13B, TXNDC5, and JCHAIN, and at least one of TNFRSF13C and FCRLA.
在具体的实施方案中，所述试剂成分包括引物，所述引物的序列如SEQ ID NOs:1-14任一项所示。In a specific embodiment, the reagent component comprises a primer, and the sequence of the primer is shown in any one of SEQ ID NOs: 1-14.
在具体的实施方案中，所述基因获自外周血样本，优选获自所述外周血样本的B淋巴细胞。在一种实施方案中，可以获得至少一次外周血样本，例如一次，两次，或者多次。In a specific embodiment, the gene is obtained from a peripheral blood sample, preferably B lymphocytes from the peripheral blood sample. In one embodiment, peripheral blood samples may be obtained at least once, eg, once, twice, or multiple times.
在一种实施方案中，测定外周血中的B淋巴细胞的参考基因和靶基因的转录本的丰度可以采用CN103764848B所公开的方法。In one embodiment, the method disclosed in CN103764848B can be used to determine the abundance of transcripts of reference genes and target genes of B lymphocytes in peripheral blood.
在一种实施方案中，本发明的方法用于分析疫苗接种所引起的免疫反应，从而评价疫苗的有效性或用于免疫治疗(例如癌症免疫治疗)的效果评估。In one embodiment, the methods of the invention are used to analyze the immune response elicited by vaccination to assess the effectiveness of a vaccine or for the assessment of the efficacy of immunotherapy (eg, cancer immunotherapy).
传统疫苗的有效性或免疫疗法的预后评估多采用抗体滴定确认的方法，需要等抗体产生后才能进行，一般需要28天后进行。这种传统方法存在受制于抗体产生的生物学时限限制，尤其对急性传染病和发展快速的疾病治疗，确认时间太长。相比之下，本申请的方法显著缩短了疫苗有效性确认或免疫疗法的预后评估的时间。例如，本发明筛选得到了特定的B淋巴细胞靶基因和参考基因，通过对于这些特定基因的转录本进行分析测定，可以将对于疫苗有效性确认或免疫疗法的预后评估的时间大幅提前，例如可以提前至疫苗施用后的28天内，例如三个星期，例如两个星期，例如一个星期，甚至几天内，或者更短时间。The effectiveness of traditional vaccines or the prognosis of immunotherapy is usually assessed by antibody titration confirmation, which can only be carried out after the antibody is produced, usually after 28 days. The existence of this traditional method is limited by the biological time limit of antibody production, especially for acute infectious diseases and fast-developing disease treatment, the confirmation time is too long. In contrast, the methods of the present application significantly shorten the time for vaccine efficacy confirmation or prognostic assessment of immunotherapy. For example, specific B lymphocyte target genes and reference genes are screened in the present invention, and by analyzing and determining the transcripts of these specific genes, the time for confirming the effectiveness of vaccines or evaluating the prognosis of immunotherapy can be greatly advanced. As early as within 28 days of vaccine administration, such as three weeks, such as two weeks, such as one week, or even within a few days, or less.
附图的简要说明Brief Description of Drawings
下面将结合附图对本发明作进一步说明，附图中：The present invention will be further described below in conjunction with the accompanying drawings, in which:
图1显示利用“直接B淋巴细胞亚群转录本丰度”检测(“Direct B Lymphocyte LS-TA”assay,简称“直接B淋巴细胞LS-TA”)获得的生物标志物的检测结果与传统方法在分离纯化B淋巴细胞中检测到的靶基因表达的相关性。图1中的X轴显示在分离纯化的B淋巴细胞中检定TNFRSF17的基因表达，使用了RPL32作为常规管家基因,这里X轴是黄金标准。如图1的Y轴所示，在PBMC中利用“直接B淋巴细胞LS-TA”获得的生物标志物的检测结果(TNFRSF17:TNFRSF13C的相对值)与传统方法在分离纯化B淋巴细胞中检测到的靶基因表达有很好的相关性(相关系数,R>0.9)。Figure 1 shows the detection results of biomarkers obtained by "Direct B Lymphocyte Subgroup Transcript Abundance" ("Direct B Lymphocyte LS-TA" assay, referred to as "Direct B Lymphocyte LS-TA") and traditional methods Correlation of target gene expression detected in isolated and purified B lymphocytes. The X-axis in Figure 1 shows the gene expression of TNFRSF17 assayed in isolated and purified B lymphocytes using RPL32 as the conventional housekeeping gene, where the X-axis is the gold standard. As shown in the Y-axis of Figure 1, the detection results of biomarkers (the relative value of TNFRSF17:TNFRSF13C) obtained by "direct B lymphocyte LS-TA" in PBMC were compared with those detected in the isolation and purification of B lymphocytes by traditional methods. There was a good correlation between the target gene expression (correlation coefficient, R>0.9).
图2是本发明的疫苗使用效果评价方法的第一种实施方案。该方案只需要一个样本。Fig. 2 is the first embodiment of the method for evaluating the efficacy of vaccine use according to the present invention. This protocol requires only one sample.
图3是本发明的疫苗使用效果评价方法的第二种实施方案。该方案需要疫苗接种前、接种后的两个样本。Fig. 3 is a second embodiment of the method for evaluating the efficacy of vaccine use according to the present invention. This protocol requires two samples before and after vaccination.
图4A显示第7天的PBMC数据用于获得NR和R受试者的TNFRSF17的“直接B淋巴细胞LS-TA”生物标志物参数的原始值。图4B中显示为转换之后的MoM值。Figure 4A shows the PBMC data at day 7 used to obtain raw values for the "direct B lymphocyte LS-TA" biomarker parameter of TNFRSF17 for NR and R subjects. The MoM values after conversion are shown in Figure 4B.
图5A显示通过对第7天PBMC样本，“直接B淋巴细胞LS-TA” 的TNFRSF17值进行ROC分析(receiver operating characteristic curve，接收器工作特性曲线)，用以确定“直接B淋巴细胞LS-TA”对疫苗接种后进行早期预测的能力。曲线下面积(AUC)为0.85，表明该生物标记参数具有不错的预测能力。Figure 5A shows the ROC analysis (receiver operating characteristic curve, receiver operating characteristic curve) of the TNFRSF17 value of the "direct B lymphocyte LS-TA" PBMC sample on day 7 to determine the "direct B lymphocyte LS-TA" "The ability to make early predictions after vaccination. The area under the curve (AUC) was 0.85, indicating good predictive power for this biomarker parameter.
图5B显示用类似的方法，对另一个流感接种实验的数据集GSE59635进行了ROC分析，AUC也达到0.73。Figure 5B shows that the ROC analysis was performed on the dataset GSE59635 of another influenza vaccination experiment using a similar method, and the AUC also reached 0.73.
图6A显示TNFRSF17的“直接B淋巴细胞-LS-TA”的增量。Figure 6A shows the increase in "direct B lymphocyte-LS-TA" of TNFRSF17.
图6B显示为与图6A相同的数据转换为MoM表示。在NR组中，生物标志物参数(D7-D0)的增量范围为从-1.01到0.67，增量中位数为0.066。反之，R组有更高的增量变化，其增量范围为从-0.13到3.39，增量中位数为1.11，NR组和R组之间的增量变化显著不同(配对测试，p值<0.01)。Figure 6B shows the same data as Figure 6A converted to MoM representation. In the NR group, the increments of biomarker parameters (D7-D0) ranged from -1.01 to 0.67, with a median increment of 0.066. Conversely, the R group had a higher incremental change, which ranged from -0.13 to 3.39 with a median increment of 1.11, and the incremental change was significantly different between the NR and R groups (paired test, p-value <0.01).
图7A显示进行了ROC分析以确定第0天至第7天利用“直接B淋巴细胞LS-TA”检测TNFRSF17的增量在辨别R和NR组的能力。增量值的分辨能力也有高的AUC(AUC＝0.85)，和单独使用第7天的“直接B淋巴细胞LS-TA”TNFRSF17的AUC差不多。Figure 7A shows that ROC analysis was performed to determine the ability of the increase in TNFRSF17 to discriminate between R and NR groups using "direct B lymphocyte LS-TA" from day 0 to day 7. The resolving power of the incremental values also had a high AUC (AUC=0.85), similar to the AUC of the "direct B lymphocyte LS-TA" TNFRSF17 on day 7 alone.
图7B显示在数据集GSE59635PBMC样本中，TNFRSF17的“直接B淋巴细胞LS-TA”的增量(D7-D0)的AUC为0.7。Figure 7B shows an AUC of 0.7 for the increment (D7-D0) of "direct B lymphocyte LS-TA" for TNFRSF17 in the dataset GSE59635 PBMC samples.
图8A和图8B在数据集GSE136163中，使用了针对黄热病接种者的全血样本的结果。其中，2个接种者被归为NR，因为它们的中和抗体(nab)滴度没有超过4倍的增加。NR组的TNFRSF17的“直接B淋巴细胞LS-TA”的中位数变化(D7-D0)为负数(-0.28)。相反地，R组的TNFRSF17的D7-D0“直接B淋巴细胞LS-TA”的增幅较高(中位数为0.38)。Figures 8A and 8B, in dataset GSE136163, used results for whole blood samples from yellow fever vaccinators. Of these, 2 vaccinators were classified as NR because they did not have more than a 4-fold increase in neutralizing antibody (nab) titers. The median change in "direct B lymphocyte LS-TA" for TNFRSF17 in the NR group (D7-D0) was negative (-0.28). Conversely, the D7-D0 "direct B lymphocyte LS-TA" increase of TNFRSF17 in group R was higher (median 0.38).
图8C显示进行了ROC分析，单独使用第7天的“直接B淋巴细胞-LS-TA”TNFRSF17的AUC为0.82。Figure 8C shows that ROC analysis was performed and the AUC of "direct B lymphocyte-LS-TA" TNFRSF17 on day 7 alone was 0.82.
图8D显示第0天和第7天的“直接B淋巴细胞-LS-TA”TNFRSF17的增量的AUC为0.88。Figure 8D shows an AUC of 0.88 for the increment of "direct B lymphocyte-LS-TA" TNFRSF17 on days 0 and 7.
图9A显示：在使用数据集GSE59635中的TXNDC5基因数据时，NR和R组中生物标志物从D0到D7的增量。两组(NR和R)之间的差 异是显著的(配对检验，p值<0.01)。Figure 9A shows the increase in biomarkers from D0 to D7 in the NR and R groups when using the TXNDC5 gene data from dataset GSE59635. Differences between the two groups (NR and R) were significant (paired test, p-value < 0.01).
图9B显示分析利用“直接B淋巴细胞LS-TA”检测TXNDC5的增量的ROC，其AUC为0.77。Figure 9B shows the ROC assayed for the increase in TXNDC5 using "direct B lymphocyte LS-TA" with an AUC of 0.77.
图9C显示使用数据集GSE59635，单独使用第七天的样本，进行TNFRSF13B相对于TNFRSF13C的“直接B淋巴细胞LS-TA”检测，确定反应者(R组)有更高的生物指标参数(p值<0.05)。Figure 9C shows that the "direct B lymphocyte LS-TA" assay of TNFRSF13B relative to TNFRSF13C using the dataset GSE59635, using the seventh day samples alone, determined that responders (group R) had higher biomarker parameters (p-values). <0.05).
图9D显示ROC分析，说明这个指标能够有效辨别两组(AUC＝0.81)。Figure 9D shows the ROC analysis, demonstrating that this metric is effective in discriminating the two groups (AUC=0.81).
图10A显示R组比NR组具有更高的TNFRSF17：FCRLA的“直接B淋巴细胞LS-TA”的增量(配对测试，p值＜0.001)。Figure 10A shows that the R group had a higher increase in "direct B lymphocyte LS-TA" of TNFRSF17:FCRLA than the NR group (paired test, p-value < 0.001).
图10B显示ROC分析中，“直接B淋巴细胞LS-TA”(TNFRSF13C，FCRLA)的增量的AUC为0.82。Figure 10B shows the incremental AUC of "direct B lymphocyte LS-TA" (TNFRSF13C, FCRLA) in ROC analysis was 0.82.
图11A显示能够长时间存活的病人在疫苗接种前的“直接B淋巴细胞LS-TA”生物标志物参数较低(P值<0.05)。图11B显示ROC分析表示，这个检测有一定的预测能力(AUC＝0.63)。Figure 11A shows that patients who were able to survive for a long time had lower "direct B lymphocyte LS-TA" biomarker parameters before vaccination (P value < 0.05). Figure 11B shows that the ROC analysis indicated that this assay had some predictive power (AUC=0.63).
图12A是生存分析(survival analysis)结果。使用考克斯回归(Cox regression)计算三组病人的存活曲线(survival curve)，把病人依据疫苗注射前的“直接B淋巴细胞LS-TA”生物标志物参数分为三组，使用分位数范围分为(a)低(指标低于25百分位)，(b)中间区域(25百分位-75百分位)，和(c)高(高于75百分位)的三组。三组生存曲线有明显的差别。存活机率最差的是“直接B淋巴细胞LS-TA”指标高的一组，大部分病人差不多都在一年内死亡(p值<0.01)。Figure 12A is the results of survival analysis. Cox regression (Cox regression) was used to calculate the survival curves of the three groups of patients, and the patients were divided into three groups according to the "direct B lymphocyte LS-TA" biomarker parameters before vaccine injection, using quantiles The ranges are divided into (a) low (indicator below the 25th percentile), (b) intermediate (25th-75th percentile), and (c) high (above the 75th percentile) . The survival curves of the three groups were significantly different. The group with the worst survival rate was the group with high "direct B lymphocyte LS-TA" index, and most patients died within almost a year (p value < 0.01).
图12B显示只把生物标志物参数指标低(低于25百分位)和指标高(高于75百分位)的两组互相比较，存活曲线的差别更加明显(p值<0.001)。存活机率最差的是高“直接B淋巴细胞LS-TA”指标的一组，死亡危险几率(也叫作风险率或者风险比，Hazard ratio)比生物标志物参数指标低的那一组高出差不多三倍(危险几率＝2.95，置信区间＝1.6-5.6)。Figure 12B shows that only comparing the two groups with low (below 25th percentile) and high (above 75th percentile) biomarker parameters with each other, the difference in survival curves is more pronounced (p-value < 0.001). The group with the worst survival rate was the group with high "direct B lymphocyte LS-TA" index, and the risk of death (also called hazard ratio or hazard ratio, Hazard ratio) was higher than that of the group with low biomarker parameter index. Almost tripled (hazard odds = 2.95, confidence interval = 1.6-5.6).
图13A显示了NR和R组中生物标志物(JCHAIN/TNFRSF13C)第七天表达的荟萃分析森林图(Forest Plot)。Figure 13A shows a meta-analysis Forest Plot of the seventh day expression of the biomarker (JCHAIN/TNFRSF13C) in the NR and R groups.
图13B显示了NR和R组中生物标志物(JCHAIN/TNFRSF13C)从第0天到第7天的增量的荟萃分析森林图。Figure 13B shows a meta-analysis forest plot of the increase in biomarker (JCHAIN/TNFRSF13C) from day 0 to day 7 in the NR and R groups.
具体实施方案specific implementation
传统方法测定外周血中特定细胞的基因表达是首先分离特定细胞亚群,然后确定其基因表达水平。传统方法得出的细胞表达水平作为黄金标准。本申请提供了直接测定外周血样本(例如外周血单核细胞(PBMC))中特定细胞(例如B淋巴细胞)基因表达的方法，而不需要从其它血细胞中纯化特定细胞。比较传统方法得出的黄金标准和本文新方法得出的特定细胞基因表达水平以及它们的相关性,这样能确定新方法的效用。The traditional method to measure the gene expression of specific cells in peripheral blood is to first isolate specific cell subsets and then determine their gene expression levels. Cellular expression levels derived from traditional methods serve as the gold standard. The present application provides methods for the direct determination of gene expression in specific cells (eg, B lymphocytes) in peripheral blood samples (eg, peripheral blood mononuclear cells (PBMCs)) without the need to purify the specific cells from other blood cells. The utility of the new method can be determined by comparing the gold standard obtained by the traditional method and the gene expression levels of specific cells obtained by the new method in this paper and their correlations.
利用本申请的方法，能够直接在外周血(例如，PBMC)的细胞混合物样本中测定单细胞型(例如B淋巴细胞)的转录本丰度(TA)，而不需要亚群分离。它适用于称为B细胞信息基因的一组基因。将在PBMC中获得的两个B细胞信息基因(靶基因和稳定表达的参考基因)的相对值用作新的生物标志物参数以代表纯化的B淋巴细胞中的靶基因表达。这种排除细胞分离的繁琐过程而直接测定外周血样品中白细胞亚群的TA的方法被称为直接LS-TA方法。例如，该方法可以被应用于在流感疫苗接种试验中收集的基因表达数据集来对血清转化进行早期预测。Using the methods of the present application, transcript abundance (TA) of a single cell type (eg, B lymphocytes) can be determined directly in a sample of a cell mixture of peripheral blood (eg, PBMC) without the need for subpopulation isolation. It applies to a group of genes called B cell information genes. The relative values of two B cell informative genes (target gene and stably expressed reference gene) obtained in PBMC were used as new biomarker parameters to represent target gene expression in purified B lymphocytes. This method of directly determining the TA of leukocyte subsets in peripheral blood samples without the tedious process of cell isolation is called the direct LS-TA method. For example, the method can be applied to gene expression datasets collected in influenza vaccination trials for early prediction of seroconversion.
本申请提供了新的、易于分析的外周血生物标志物，其可以很容易地纳入常规临床实验室检查，或用于常规临床研究。所述外周血生物标志物可以预测流感疫苗接种后的早期血清转化状态(例如在第7天就可以预测疫苗接种后的抗体产生等)。由于获得性免疫的途径是针对流感和COVID-19的疫苗接种所共有的，因此这些生物标志物也可用于预测新的COVID-19疫苗的血清转化。直接B淋巴细胞LS-TA将成为实施个性化疫苗接种的有用检测。The present application provides novel, easy-to-analyze peripheral blood biomarkers that can be readily incorporated into routine clinical laboratory tests, or used in routine clinical research. The peripheral blood biomarkers can predict early seroconversion status after influenza vaccination (eg, antibody production after vaccination can be predicted on day 7, etc.). Since the pathways of acquired immunity are shared by vaccination against influenza and COVID-19, these biomarkers can also be used to predict seroconversion in new COVID-19 vaccines. Direct B lymphocyte LS-TA will be a useful test for implementing personalized vaccination.
术语及定义Terms and Definitions
术语“外周血样本”具有医学检验领域的通常含义，主要指取自 指点、耳垂、手臂静脉等处的血液样本。例如，可以全血样本(Whole Blood，WB)，也可以是外周血中多类型单核细胞的混合物样本(Peripheral Blood Mononuclear Cells，PBMC)。The term "peripheral blood sample" has its usual meaning in the field of medical testing, mainly referring to blood samples taken from fingertips, earlobes, arm veins, etc. For example, it can be a whole blood sample (Whole Blood, WB) or a mixture of multiple types of monocytes in peripheral blood (Peripheral Blood Mononuclear Cells, PBMC).
术语“直接测量”是指未经分离出其中的特定细胞(例如B淋巴细胞)而进行测量。也就是从血液样本中未经分离出要测定的特定细胞(例如B淋巴细胞)而进行直接测量。The term "direct measurement" refers to measurement without isolation of specific cells (eg, B lymphocytes) therein. That is, direct measurement is performed without isolating the specific cells to be assayed (eg, B lymphocytes) from the blood sample.
术语“基因表达水平”是指细胞(例如B淋巴细胞)的基因表达水平，以RNA为测量对象，具体是mRNA为测量对象，就是信使核糖核酸的数量水平。换句话说，是测量样本中细胞(例如B淋巴细胞)的基因的mRNA水平。在技术上，它也被称为转录本丰度(TA)。The term "gene expression level" refers to the gene expression level of cells (eg, B lymphocytes), with RNA as the measurement object, specifically mRNA as the measurement object, which is the quantitative level of messenger ribonucleic acid. In other words, the mRNA levels of genes in cells (eg B lymphocytes) in the sample are measured. Technically, it is also known as transcript abundance (TA).
术语“疫苗”指一种生物制品，是针对疾病的病原微生物或其蛋白质(多肽、肽)、多糖或者核酸，通过人工减毒、灭活、裂解、基因重组、提纯等方法制成，可以诱导机体产生相应的保护性的免疫物质，用于预防、控制相应疾病的发生和流行，或者可以调控机体内某一致害因素(如吸烟)和生理状态(如避孕)的特异性免疫反应，从而达到治疗或消除致害因素、避免生育为目的的生物制品。The term "vaccine" refers to a biological product, which is made against pathogenic microorganisms or their proteins (polypeptides, peptides), polysaccharides or nucleic acids by artificial attenuation, inactivation, lysis, genetic recombination, purification, etc., and can induce The body produces corresponding protective immune substances, which are used to prevent and control the occurrence and prevalence of corresponding diseases, or can regulate the specific immune responses of certain harmful factors (such as smoking) and physiological states (such as contraception) in the body, so as to achieve Biological products for the purpose of treating or eliminating harmful factors and preventing fertility.
从功能上理解，疫苗就是刺激身体进行免疫反应，用于消灭特定感染生物、特定细胞，起到预防或者治疗作用的物质。疫苗成分包括活性成分和辅助成分。From a functional understanding, a vaccine is a substance that stimulates the body to carry out an immune response, which is used to eliminate specific infected organisms and specific cells, and play a preventive or therapeutic role. Vaccine ingredients include active ingredients and auxiliary ingredients.
本申请中的所用的“疫苗”包括疫苗活性成分，以及加入了其他物质的组合物或者制剂，也就是适合用于人体和动物体刺激免疫反应的单一成分或者组合物或者制剂。疫苗所指代的是上述任何一种情形所指的物质。简单来说，是免疫反应刺激物，或者诱导有益免疫反应的物质。The "vaccine" used in this application includes the active ingredient of the vaccine, as well as the composition or preparation to which other substances are added, that is, a single ingredient or composition or preparation suitable for stimulating the immune response of the human and animal body. Vaccine refers to the substance referred to in any of the above situations. In simple terms, it is an immune response stimulator, or a substance that induces a beneficial immune response.
术语“癌症”是指一类疾病，即出现了癌基因表达或者有癌细胞扩增生长或者出现了实体瘤。临床上通过组织学、免疫学、蛋白质测定所做诊断的任何癌症，均在本词语含义内。The term "cancer" refers to a class of diseases in which oncogene expression or expansion of cancer cells occurs or the development of solid tumors. Any cancer diagnosed clinically by histology, immunology, protein assay is within the meaning of this term.
术语“癌症免疫治疗”又称为癌症免疫疗法，是新一代的癌症治疗方案(Wikipedia：(“癌症免疫疗法”2020))，此类疗法利用身体的 免疫功能，去攻击肿瘤细胞。有不同的治疗方案去刺激机体的免疫效能。最常见的是使用PD-1或PD-L1抑制剂在大肠癌治疗就有很好的成效。癌症疫苗(cancer vaccine)是一个新的治疗手段。这里的癌症疫苗并不是指那些个体未出现癌症之前接种的预防性疫苗，例如可以预防肝癌的乙肝病毒(HBV)疫苗和预防子***的人类乳突病毒(HPV)疫苗，而是指癌症出现之后，癌细胞拥有一些特征的抗原，可以通过刺激自体产生免疫反应来针对这些抗原和癌细胞。不同的治疗方案使用不同的候选抗原。本文的例子是使用接种个性化肽疫苗(Personalized peptide vaccination，PPV)治疗***癌(Araki et al.2015)。The term "cancer immunotherapy", also known as cancer immunotherapy, is a new generation of cancer treatment options (Wikipedia: ("Cancer Immunotherapy" 2020)), which use the body's immune function to attack tumor cells. There are different treatment options to stimulate the body's immune function. The most common is the use of PD-1 or PD-L1 inhibitors in the treatment of colorectal cancer with good results. Cancer vaccine (cancer vaccine) is a new treatment. Cancer vaccines here do not refer to preventive vaccines that individuals receive before cancer, such as the hepatitis B virus (HBV) vaccine that can prevent liver cancer and the human papilloma virus (HPV) vaccine that prevents cervical cancer, but refers to the onset of cancer Afterwards, cancer cells possess some characteristic antigens that can be directed against these antigens and cancer cells by stimulating an autologous immune response. Different treatment regimens use different candidate antigens. An example here is the use of personalized peptide vaccination (PPV) to treat prostate cancer (Araki et al. 2015).
术语“细胞混合物样本”是来自个体(例如人体)的细胞混合物。典型地，该细胞混合物样本可以来自外周血，例如可以是未经任何处理的外周血样本。The term "sample of cell mixture" is a mixture of cells from an individual (eg, a human body). Typically, the cell mixture sample can be derived from peripheral blood, eg, a peripheral blood sample that has not been processed in any way.
术语“特征性基因(cell-type informative genes)”，是指特定细胞(例如B淋巴细胞)类型所特定表达的基因，测定其转录本丰度可以确定该特征性基因的活性。特征性基因可以包括靶基因和参考基因。由于在本专利申请使用的目标特定细胞是B细胞,B细胞特征性基因又可称为B细胞信息基因。在本专利申请中，特征性基因与信息基因可以互换使用。The term "cell-type informative genes" refers to genes that are specifically expressed by a specific cell (eg, B lymphocyte) type, and the activity of the characteristic gene can be determined by measuring its transcript abundance. Characterized genes can include target genes and reference genes. Since the target specific cells used in this patent application are B cells, B cell characteristic genes can also be called B cell information genes. In this patent application, characteristic gene and informative gene can be used interchangeably.
术语“生物标志物”和“生物指标”(biomarker)是指特定细胞(例如B淋巴细胞)的基因表达中的信使核糖核酸的量，包括相对量和绝对量可以用于记录生物标志物状态。在本专利申请中，生物标志物与生物指标可以互换使用。The terms "biomarker" and "biomarker" refer to the amount of messenger RNA in gene expression of a particular cell (eg, B lymphocyte), and both relative and absolute amounts can be used to record biomarker status. In this patent application, biomarkers and biomarkers are used interchangeably.
术语“白血球细胞亚群(Leukocyte Subpopulation，LS)”包括B淋巴细胞亚群。白血球细胞包括多类型细胞。外周血是典型的多类型细胞混合物样本，包含了各种白血球细胞亚群，例如嗜中性粒细胞(neutrophil)，淋巴细胞(lymphocyte)和单核细胞(monocyte)等等。The term "Leukocyte Subpopulation (LS)" includes B lymphocyte subsets. White blood cells include many types of cells. Peripheral blood is a typical sample of a mixture of multiple types of cells, including various leukocyte subsets, such as neutrophils, lymphocytes and monocytes.
术语“转录本丰度(Transcript Abundance，TA)”是指通过检测样本得出的基因表达量(gene expression level)。转录本是指基因转录 (gene transcription)之后的产物,一般为RNA。例如蛋白质编码的基因会产出信使RNA(messanger RNA,mRNA)。The term "Transcript Abundance (TA)" refers to the gene expression level (gene expression level) obtained by testing a sample. Transcript refers to the product after gene transcription, usually RNA. For example, protein-coding genes produce messenger RNA (mRNA).
术语“直接B淋巴细胞亚群转录本丰度”检测(“Direct B lymphocyte leukocyte subpopulation transcript abundance”assay,简称“直接B淋巴细胞LS-TA”检测)是一种新型的生物指标参数检测方案，针对多类型细胞混合物样本，不需要分离纯化当中的目标细胞类型亚群，就可以直接从混合物样本中评估目标细胞亚群的平均基因表达。本发明论述的目标细胞亚群就是在外周血的B淋巴细胞。所以该检测名为“直接B淋巴细胞亚群转录本丰度”检测。“直接B淋巴细胞LS-TA”的计算需要使用细胞亚群特征性靶基因和细胞亚群特征性参考基因。The term "direct B lymphocyte subpopulation transcript abundance" assay ("Direct B lymphocyte leukocyte subpopulation transcript abundance" assay, referred to as "direct B lymphocyte LS-TA" assay) is a new type of biomarker parameter detection scheme. For multi-type cell mixture samples, the average gene expression of target cell subsets can be assessed directly from the mixture samples without the need to isolate and purify the target cell type subsets. The target cell subset discussed in the present invention is B lymphocytes in peripheral blood. So the test is called "direct B lymphocyte subset transcript abundance" test. The calculation of "direct B lymphocyte LS-TA" requires the use of target genes characteristic of cell subsets and reference genes characteristic of cell subsets.
在一些实施方案中，直接B淋巴细胞LS-TA值可以通过使用细胞亚群特征性靶基因和细胞亚群特征性参考基因的比率计算得到，例如某一靶基因的“直接B淋巴细胞-LS-TA”＝(PBMC中的该靶基因)/(PBMC中的相应参考基因)。在另一些实施方案中，使用log(比率)来计算直接B淋巴细胞LS-TA值。例如，log(“直接B淋巴细胞-LS-TA”TNFRSF17)＝log(PBMC中的TNFRSF17)-log(PBMC中的TNFRSF13C)。In some embodiments, the direct B lymphocyte LS-TA value can be calculated by using the ratio of a target gene characteristic of a cell subset to a reference gene characteristic of a cell subset, such as the "direct B lymphocyte-LS" of a target gene -TA"=(this target gene in PBMC)/(corresponding reference gene in PBMC). In other embodiments, the log(ratio) is used to calculate direct B lymphocyte LS-TA values. For example, log("direct B lymphocyte-LS-TA" TNFRSF17) = log(TNFRSF17 in PBMC) - log(TNFRSF13C in PBMC).
术语“细胞亚群特征性靶基因(subpopulation informative target gene)”是指在多细胞混合样本(例如WB，PBMC)中，其大部份的转录本都是来自于指定的目标细胞亚群的基因，亦即由该细胞亚群产生。例如，来自下列实施例中的B淋巴细胞的基因。The term "subpopulation informative target gene" refers to a gene whose majority of the transcripts in a multicellular mixed sample (eg WB, PBMC) are derived from the specified target cell subpopulation , that is, produced by this cell subset. For example, genes from B lymphocytes in the following examples.
术语“细胞亚群特征性参考基因(subpopulation informative reference gene)”是指在多细胞混合样本(例如WB，PBMC)中，其大部份的转录本都是来自于指定的目标细胞亚群，并且同时在目标细胞中稳定表达的基因。The term "subpopulation informative reference gene" means that in a multicellular mixed sample (eg WB, PBMC), most of its transcripts are from the specified target cell subpopulation, and Genes that are stably expressed in target cells at the same time.
本申请的发明人提出了从各种外周血样本中直接测定特定细胞(例如B淋巴细胞)的基因表达，并意外地发现这些基因表达在预测疫苗接种和癌症免疫治疗效果中具有重要价值。The inventors of the present application proposed to directly measure the gene expression of specific cells (eg B lymphocytes) from various peripheral blood samples and unexpectedly found that these gene expressions are of great value in predicting the effects of vaccination and cancer immunotherapy.
本申请所述的方法还包括将外周血样本的所述生物标志物参数与临界值进行比较。在一些实施方案中，所述比较用于预测疫苗接种导致的抗体产生和/或免疫治疗的效果。The methods described herein also include comparing the biomarker parameter of the peripheral blood sample to a cutoff value. In some embodiments, the comparison is used to predict the effect of vaccination-induced antibody production and/or immunotherapy.
术语“临界值”可以使用以下几种方法来定义。(1)临界值可以定义为对照组参考区间之外的值。对照组的参考区间通常取对照组的中间95％分布。超出此范围的值可以作为临界值来定义异常低或异常高的结果。(2)临界值还可以最好从ROC图表中定义，如图5A所示。ROC曲线上每个点上标记的值代表潜在临界值，Y轴和X轴分别显示使用此临界值的相关灵敏度和特异性。因此，当0.6用作TNFRSF17与TNFRSF13C比率的第7天直接LS-TA值的临界值时，它预测疫苗接种反应的灵敏度为～0.8和特异性为～0.85(图5A)。(3)如果没有对照组可用，如实施例7和图12所示，患者组中直接LS-TA数据分布的百分位值可用作临界值。例如，由TNFRSF17与FCRLA比率的直接LS-TA值的第75个百分位值(75th percentile value)定义临界值(图9B)，直接LS-TA大于该临界值的癌症患者(图9B中的实线)与其他患者相比，对癌症疫苗治疗的反应较差，生存期较短。The term "critical value" can be defined in several ways. (1) The critical value can be defined as a value outside the reference interval of the control group. The reference interval for the control group usually takes the middle 95% distribution of the control group. Values outside this range can be used as thresholds to define abnormally low or abnormally high results. (2) The critical value can also be best defined from the ROC chart, as shown in Figure 5A. The value labeled at each point on the ROC curve represents a potential cutoff, and the Y- and X-axes show the relative sensitivity and specificity using this cutoff, respectively. Thus, when 0.6 was used as a cutoff for the day 7 direct LS-TA value for the ratio of TNFRSF17 to TNFRSF13C, it predicted vaccination response with a sensitivity of -0.8 and a specificity of -0.85 (Figure 5A). (3) If no control group is available, as shown in Example 7 and Figure 12, the percentile value of the distribution of direct LS-TA data in the patient group can be used as a cutoff value. For example, the cutoff value was defined by the 75th percentile value of the direct LS-TA value of the ratio of TNFRSF17 to FCRLA (Figure 9B), and cancer patients with direct LS-TA greater than this cutoff value (Figure 9B) Solid line) had a poorer response to cancer vaccine treatment and a shorter survival compared with other patients.
本发明使用的数据集列表:List of datasets used in the present invention:
外周血和特定单细胞类型的基因表达数据集Gene expression datasets for peripheral blood and specific single cell types
为了鉴定可预测疫苗接种反应的单一细胞类型特征性基因，使用了以下从外周血样本获得的基因表达数据集。To identify genes characteristic of a single cell type that predict vaccination response, the following gene expression datasets obtained from peripheral blood samples were used.
这些数据集可从美国国立卫生研究院维护的“基因表达综合”(GEO)中获得。详细信息可在其登录号下获得。获得的血液样本类型包括全血(WB)和外周血单核细胞(PBMC)。在某些数据集中还包括进一步分离和纯化的特定细胞类型，例如分离纯化的B淋巴细胞(例如GSE45764)或T淋巴细胞。These datasets are available from the Gene Expression Omnibus (GEO) maintained by the National Institutes of Health. Details are available under their accession numbers. The types of blood samples obtained include whole blood (WB) and peripheral blood mononuclear cells (PBMC). Specific cell types that were further isolated and purified were also included in some datasets, such as isolated and purified B lymphocytes (eg GSE45764) or T lymphocytes.
Figure PCTCN2021126426-appb-000003
Figure PCTCN2021126426-appb-000003
Figure PCTCN2021126426-appb-000004
Figure PCTCN2021126426-appb-000004
疫苗接种后的反应者(R)的标准定义是根据血清中针对特定抗原的抗体滴度，例如在流感疫苗接种之后，血凝抑制(HI)抗体水平(也称为HI滴度，titer in hemagglutination inhibition assay)的显著增加，代表了接种者对疫苗有反应。该标准是通过对接种前和接种后二十八天的受试者血清进行血凝抑制(HI)测定。欧洲人类医药产品委员会(CHMP)将血清转化/显著增加(seroconversion/significant increase)定义为：(1)HI滴度在接种后至少是1:40和(b)有至少4倍上升(Committee for  Medicinal Products for Human Use.1997；Mo et al.2017)。疫苗接种后的受试者不符合这些标准的情况定义为“无反应者(NR)”。The standard definition of a responder (R) after vaccination is based on the titer of antibodies against a specific antigen in serum, such as after influenza vaccination, the level of hemagglutination-inhibiting (HI) antibodies (also known as HI titer, titer in hemagglutination) A significant increase in inhibition assay) indicates that the recipient is responding to the vaccine. This criterion is determined by performing hemagglutination inhibition (HI) on the serum of subjects before vaccination and twenty-eight days after vaccination. The European Committee for Human Medicinal Products (CHMP) defines seroconversion/significant increase as: (1) an HI titer of at least 1:40 post-vaccination and (b) at least a 4-fold increase (Committee for Medicinal Products for Human Use. 1997; Mo et al. 2017). Post-vaccinated subjects who did not meet these criteria were defined as "non-responders (NR)."
除了流感疫苗之外,在数据集GSE136163中，接种者进行了黄热病疫苗接种。正如其他研究中所使用的反应者定义那样，这里使用了疫苗接种后中和抗体(nab)滴度增加4倍来定义该数据集中的反应者(Casey et al.2019)。在该数据有两个受试者定义为NR。In addition to the influenza vaccine, in dataset GSE136163, the vaccinators were vaccinated against yellow fever. As the responder definition used in other studies, here a 4-fold increase in neutralizing antibody (nab) titers following vaccination was used to define responders in this dataset (Casey et al. 2019). In this data there are two subjects defined as NR.
在本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其他部分、添加物、组分、或步骤。In this specification and claims, the words "including", "comprising" and "containing" mean "including but not limited to" and are not intended to exclude other parts, additives, components, or steps.
应该理解，在本申请的特定方面、实施方案或实施例中描述的特征、特性、组分或步骤，可适用于本文所描述的任何其他的方面、实施方案或实施例，除非与之矛盾。It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application are applicable to any other aspect, embodiment or example described herein unless contradicted by it.
下面将结合本发明实施例中的附图对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。下述实施例仅是说明性的，并不意图限制本申请实施方案的范围或者所附的权利要求的范围。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The following examples are illustrative only and are not intended to limit the scope of the embodiments of the present application or the scope of the appended claims. Based on the embodiments in the present application, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
实施例Example
实施例1Example 1
“直接B淋巴细胞的转录本丰度”(“Direct B Lymphocyte-LS-TA”,简称“直接B淋巴细胞LS-TA”)的生物指标参数"Direct B Lymphocyte Transcript Abundance" ("Direct B Lymphocyte-LS-TA", referred to as "Direct B Lymphocyte LS-TA") biomarker parameters
使用细胞混合物样本中B淋巴细胞靶基因(例如TNFRSF17)和B淋巴细胞特征参考基因(例如TNFRSF13C)的TA相对值来计算一个生物指标参数以评估经过分离纯化后的B淋巴细胞的基因表达量。A biomarker parameter is calculated by using the relative TA values of B lymphocyte target genes (such as TNFRSF17) and B lymphocyte characteristic reference genes (such as TNFRSF13C) in the cell mixture sample to evaluate the gene expression of B lymphocytes after isolation and purification.
在数据集GSE45764中，分别收集了5个接种者在不同天数的多个分离纯化的B淋巴细胞样本以及PBMC样本。PBMC细胞混合物样本中的两个指定的B淋巴细胞特征基因的相对值与纯化的B淋巴细胞 样本中的靶基因的表达(黄金标准)的相关程度提供了对“直接B淋巴细胞LS-TA”方案的性能评估。In the dataset GSE45764, multiple isolated and purified B lymphocyte samples and PBMC samples were collected from 5 vaccinators on different days. The degree to which the relative values of the two specified B lymphocyte signature genes in the PBMC cell mixture sample correlated with the expression of the target gene in the purified B lymphocyte sample (gold standard) provides an insight into the "direct B lymphocyte LS-TA" Program performance evaluation.
在此实施例中，参考图1。指定的靶基因是TNFRSF17。指定的B淋巴细胞参考基因为TNFRSF13C。“直接B淋巴细胞LS-TA”标示在Y轴。In this embodiment, reference is made to FIG. 1 . The designated target gene was TNFRSF17. The designated B lymphocyte reference gene is TNFRSF13C. "Direct B lymphocyte LS-TA" is indicated on the Y-axis.
对于该实施例，常规管家基因只用于标准化已分离纯化的B淋巴细胞样本的基因表达结果。常规管家基因包括RPL31，RPL32，ACTB，GAPDH和UBC。常规管家基因的选择参见Eisenberg and Levanon 2013，也可在网站上获得，例如https://www.tau.ac.il/～elieis/HKG/。图1的X轴显示了分离纯化的B淋巴细胞中检定TNFRSF17的基因表达(黄金标准)，使用RPL32作为常规管家基因。常规管家基因仅由制造商用于校准黄金标准和验证目的，而不是用于本发明的试剂盒或其实施方案中。For this example, conventional housekeeping genes were only used to normalize the gene expression results of isolated and purified B lymphocyte samples. Conventional housekeeping genes include RPL31, RPL32, ACTB, GAPDH and UBC. Selection of conventional housekeeping genes is described in Eisenberg and Levanon 2013, also available on websites such as https://www.tau.ac.il/~elieis/HKG/. The X-axis of Figure 1 shows the gene expression of TNFRSF17 (gold standard) assayed in isolated and purified B lymphocytes, using RPL32 as the conventional housekeeping gene. Conventional housekeeping genes are used by manufacturers only for calibration gold standard and validation purposes, not in the kits of the present invention or embodiments thereof.
如图1的Y轴所示，在PBMC细胞混合物样本中测量的本发明的“直接B淋巴细胞LS-TA”生物标志物的结果(TNFRSF17:TNFRSF13C的相对值)与传统方法在分离纯化B淋巴细胞中检测到的靶基因表达(黄金标准)有很好的相关性(相关系数,R>0.9)。结果证实，该“直接B淋巴细胞LS-TA”生物标志物参数能够评估纯化的B淋巴细胞中的靶基因(TNFRSF17)表达；以及确定了单细胞类型基因表达测定，可以直接从多细胞混合物样本(包括PBMC或WB)获得，而不需要预先分离B淋巴细胞。As shown on the Y-axis of Figure 1, the results of the "direct B lymphocyte LS-TA" biomarkers of the present invention (relative value of TNFRSF17:TNFRSF13C) measured in PBMC cell mixture samples compared with traditional methods in the separation and purification of B lymphocytes The target gene expression (gold standard) detected in cells was well correlated (correlation coefficient, R>0.9). Results confirmed that this "direct B lymphocyte LS-TA" biomarker parameter was able to assess target gene (TNFRSF17) expression in purified B lymphocytes; (including PBMC or WB) without prior isolation of B lymphocytes.
其余的由B淋巴细胞靶基因(包括TNFRSF13B,TXNDC5,JCHAIN)和B淋巴细胞参考基因(包括TNFRSF13C,FCRLA)组成的“直接B淋巴细胞LS-TA”生物标志物也与它们各自的黄金标准有很好的相关性(相关系数,R>0.8)。The remaining "direct B lymphocyte LS-TA" biomarkers, consisting of B lymphocyte target genes (including TNFRSF13B, TXNDC5, JCHAIN) and B lymphocyte reference genes (including TNFRSF13C, FCRLA), are also available with their respective gold standards. Very good correlation (correlation coefficient, R>0.8).
在随后的实施例中，“直接B淋巴细胞-LS-TA”标志物会用于预测疫苗接种后的抗体产生状态。这里会使用两种方案，第一种方案只需要一个样本，实施流程见图2。第二个方案需要疫苗接种前、后两个样本，实施流程见图3。In the examples that follow, the "direct B lymphocyte-LS-TA" marker will be used to predict the status of antibody production following vaccination. Two schemes will be used here. The first scheme requires only one sample. The implementation process is shown in Figure 2. The second scheme requires two samples before and after vaccination. The implementation process is shown in Figure 3.
实施例2Example 2
疫苗接种后第七天的“直接B淋巴细胞LS-TA”能够预测抗体产生反应'Direct B lymphocyte LS-TA' at day 7 post-vaccination predicts antibody-producing responses
在数据集GSE29617中，两个指定的B淋巴细胞特征基因TNFRSF17和TNFRSF13C的转录物丰度(TA)已经对数转化。因此，log(TNFRSF17)减去log(TNFRSF13C)便可得出本申请所需的生物标志物参数(对数值)。该生物标志物参数代表PBMC的细胞混合物样本中的B淋巴细胞基因TNFRSF17的基因表达量。因为这个生物指标，无需事先分离B淋巴细胞即可获得，所以在图标中，它被标记为“直接B淋巴细胞转录本丰度”(“直接B淋巴细胞-LS-TA”)。In dataset GSE29617, the transcript abundances (TA) of two assigned B lymphocyte signature genes, TNFRSF17 and TNFRSF13C, have been log-transformed. Therefore, log(TNFRSF17) minus log(TNFRSF13C) yields the biomarker parameter (log value) required for this application. This biomarker parameter represents the gene expression level of the B lymphocyte gene TNFRSF17 in the cell mixture sample of PBMC. Because this biomarker can be obtained without prior isolation of B lymphocytes, it is labeled "Direct B lymphocyte transcript abundance" ("Direct B lymphocyte-LS-TA") in the graph.
因此，B淋巴细胞靶基因TNFRSF17的“直接B淋巴细胞-LS-TA”生物标志物参数，使用细胞亚群特征性靶基因和细胞亚群特征性参考基因的比率计算可得，可表示为：Therefore, the "direct B lymphocyte-LS-TA" biomarker parameter of the B lymphocyte target gene TNFRSF17, calculated using the ratio of target genes characteristic of cell subsets to reference genes characteristic of cell subsets, can be expressed as:
(“直接B淋巴细胞-LS-TA”TNFRSF17)＝(PBMC中的TNFRSF17)/(PBMC中的TNFRSF13C)("Direct B lymphocyte-LS-TA" TNFRSF17) = (TNFRSF17 in PBMC)/(TNFRSF13C in PBMC)
还可以为此生物标志物参数进行对数变换,即亦可表示为：This biomarker parameter can also be logarithmically transformed, which can also be expressed as:
log(“直接B淋巴细胞-LS-TA”TNFRSF17)＝log(PBMC中的TNFRSF17)-log(PBMC中的TNFRSF13C)log("direct B lymphocyte-LS-TA" TNFRSF17) = log(TNFRSF17 in PBMC) - log(TNFRSF13C in PBMC)
由于用不同的化验检测方法测定会产生不同单位的结果，因此需要一种方法来标准化从多种不同检测方法获得的结果。相对于正常对照组别的中位数(Multiple of median of a reference group，MoM)是一种常用的标准化手法。在该实施例中，接种前(第0天)的数据用于定义正常对照组别的中位数。然后所有个体(包括NR和R)的结果均表示为该正常中值的倍数。所以这种数据表示方法称为对照组中位数的倍数(MoM)。它通常用于未经过大规模测定标准化的检测方法，例如用于确定SARS-CoV感染后不良结局风险的细胞因子测定(Tang et al.2005)。使用MoM的好处是它能够去除数据的单位限制，从而可以比较不同检测方案产生的结果。Since assays with different assay assays produce results in different units, a method is needed to standardize results obtained from multiple different assays. The median (Multiple of median of a reference group, MoM) relative to the normal control group is a commonly used standardization method. In this example, data from pre-vaccination (day 0) were used to define the median of the normal control group. Results for all individuals (including NR and R) were then expressed as multiples of this normal median. So this data representation is called the multiple of the control group median (MoM). It is commonly used in assays that have not been standardized by large-scale assays, such as cytokine assays used to determine the risk of adverse outcomes following SARS-CoV infection (Tang et al. 2005). The benefit of using MoM is that it removes the unit limitation of the data, allowing comparison of results from different detection schemes.
接种疫苗后第7天，R和NR组的外周血单核细胞混合样本(PBMC) 中TNFRSF17基因的“直接B淋巴细胞LS-TA”的比较Comparison of "direct B lymphocyte LS-TA" of TNFRSF17 gene in peripheral blood mononuclear cell pooled samples (PBMC) of R and NR groups on day 7 after vaccination
将第7天的PBMC数据用于获得NR和R受试者的TNFRSF17的“直接B淋巴细胞LS-TA”生物标志物参数。在图4A和4B中显示为原始值和转换之后的MoM值。结果证实R和NR组之间存在显著差异(Wilcoxon检验，p值<0.01)。 Day 7 PBMC data were used to obtain "direct B lymphocyte LS-TA" biomarker parameters for TNFRSF17 in NR and R subjects. The original and converted MoM values are shown in Figures 4A and 4B. The results confirmed a significant difference between the R and NR groups (Wilcoxon test, p-value < 0.01).
通过对第7天PBMC样本，“直接B淋巴细胞LS-TA”的TNFRSF17值进行ROC分析(receiver operating characteristic curve，接收器工作特性曲线)，用以确定“直接B淋巴细胞LS-TA”对疫苗接种有效性进行早期预测的能力(图5A)。曲线下面积(AUC)为0.85，表明该生物标志物参数具有良好的预测能力。ROC analysis (receiver operating characteristic curve, receiver operating characteristic curve) was performed on the TNFRSF17 value of "direct B lymphocyte LS-TA" in PBMC samples on day 7 to determine the effect of "direct B lymphocyte LS-TA" on the vaccine Ability to make early predictions of vaccination effectiveness (Figure 5A). The area under the curve (AUC) was 0.85, indicating that this biomarker parameter has good predictive power.
同样地，用类似的方法，对另一个流感接种实验的数据集GSE59635进行了ROC分析，AUC也达到0.73(图5B)。Similarly, ROC analysis was performed on the dataset GSE59635 of another influenza vaccination experiment using a similar method, and the AUC also reached 0.73 (Fig. 5B).
实施例3Example 3
第0天和第7天的“直接B淋巴细胞-LS-TA”TNFRSF17的增量变化值在R和NR组的比较Comparison of incremental changes in TNFRSF17 values of "direct B lymphocytes-LS-TA" in R and NR groups on days 0 and 7
使用数据集(GSE29617)，来计算第0天和第7天“直接B淋巴细胞-LS-TA”的增量，使用了第7天的数据“直接B淋巴细胞-LS-TA”TNFRSF17值和第0天(基线，接种前)的“直接B淋巴细胞-LS-TA”TNFRSF17值，由两个值相减得出的增量变化数值(delta，δ)，可以用来预测随后的抗体产生状态。即本实施例执行图3的流程。The data set (GSE29617) was used to calculate the increment of "direct B lymphocyte-LS-TA" on day 0 and day 7, using the data of day 7 "direct B lymphocyte-LS-TA" TNFRSF17 value and The "direct B lymphocyte-LS-TA" TNFRSF17 value on day 0 (baseline, pre-vaccination), the delta change value (delta, delta) obtained by subtracting the two values, can be used to predict subsequent antibody production state. That is, the present embodiment executes the flow of FIG. 3 .
由于两个值均已进行对数转换，因此减值表示疫苗接种后倍数变化的对数值。它显示为以下公式：Since both values were log-transformed, the impairment represents the log fold change after vaccination. It appears as the following formula:
接种疫苗后TNFRSF17基因的“直接B淋巴细-LS-TA”的增量＝第7天的“直接B淋巴细胞-LS-TA”TNFRSF17-第0天的“直接B淋巴细胞-LS-TA”TNFRSF17Increase in "direct B lymphocyte-LS-TA" of TNFRSF17 gene after vaccination = "direct B lymphocyte-LS-TA" on day 7 TNFRSF17 - "direct B lymphocyte-LS-TA" on day 0 TNFRSF17
TNFRSF17的“直接B淋巴细胞-LS-TA”的增量如图6A所示。相同的数据转换为MoM表示，如图6B所示。在NR组中，生物标志物参数(D7-D0)的增量范围为从-1.01到0.67，增量中位数为0.066。 反之，R组有更高的增量变化，其增量范围为从-0.13到3.39，增量中位数为1.11，NR组和R组之间的增量变化显著不同(配对测试，p值<0.01)。The increase in "direct B lymphocyte-LS-TA" of TNFRSF17 is shown in Figure 6A. The same data were converted to MoM representation as shown in Fig. 6B. In the NR group, the increments of biomarker parameters (D7-D0) ranged from -1.01 to 0.67, with a median increment of 0.066. Conversely, the R group had a higher incremental change, which ranged from -0.13 to 3.39 with a median increment of 1.11, and the incremental change was significantly different between the NR and R groups (paired test, p-value <0.01).
进行了ROC分析以确定第0天至第7天的“直接B淋巴细胞-LS-TA”TNFRSF17的增量区分R和NR组的能力(图7A)。增量值的分辨能力也有高的AUC(AUC＝0.85)，和单独使用第7天的“直接B淋巴细胞-LS-TA”TNFRSF17的AUC差不多(AUC＝0.85，图5A作比较)。ROC analysis was performed to determine the ability of the increase in "direct B lymphocyte-LS-TA" TNFRSF17 from days 0 to 7 to discriminate between R and NR groups (Figure 7A). The resolving power of the incremental values also had a high AUC (AUC = 0.85), comparable to the AUC of the "direct B lymphocyte-LS-TA" TNFRSF17 on day 7 alone (AUC = 0.85, compare Figure 5A).
同样，在数据集GSE59635PBMC样本中，TNFRSF17的“直接B淋巴细胞-LS-TA”的增量(D7-D0)也提供了有用信息，AUC为0.7(图7B)。Likewise, in the dataset GSE59635 PBMC samples, the increment of "direct B lymphocyte-LS-TA" (D7-D0) of TNFRSF17 was also informative, with an AUC of 0.7 (Fig. 7B).
实施例4Example 4
全血样本中TNFRSF17的“直接B淋巴细胞-LS-TA”的D7-D0变化，以及在其他疫苗接种中也能有效区分R和NR两组的能力D7-D0 changes in "direct B lymphocyte-LS-TA" of TNFRSF17 in whole blood samples and the ability to effectively differentiate between R and NR groups also in other vaccinations
在数据集GSE136163中，使用了针对黄热病接种者的全血样本。2个接种者被归为NR，因为它们的中和抗体(nab)滴度没有超过4倍的增加。NR组的TNFRSF17的“直接B淋巴细胞-LS-TA”的中位数变化(D7-D0)为负数(-0.28)。相反地，R组的TNFRSF17的D7-D0“直接B淋巴细胞-LS-TA”的增幅较高(中位数为0.38)。数据显示在图8A和图8B。In dataset GSE136163, whole blood samples from yellow fever vaccinated persons were used. Two vaccinators were classified as NR because they did not have more than a 4-fold increase in neutralizing antibody (nab) titers. The median change in "direct B lymphocyte-LS-TA" (D7-D0) for TNFRSF17 in the NR group was negative (-0.28). Conversely, the D7-D0 "direct B lymphocyte-LS-TA" increase in TNFRSF17 was higher in group R (median 0.38). The data are shown in Figure 8A and Figure 8B.
进行了ROC分析，单独使用第7天的“直接B淋巴细胞-LS-TA”TNFRSF17的AUC为0.82(图8C)，而第0天和第7天的“直接B淋巴细胞-LS-TA”TNFRSF17的增量的AUC比较高，为0.88(图8D)。ROC analysis was performed and the AUC of TNFRSF17 on day 7 alone was 0.82 for "direct B lymphocytes-LS-TA" (Figure 8C), while the "direct B lymphocytes-LS-TA" for day 0 and day 7 The incremental AUC for TNFRSF17 was higher at 0.88 (FIG. 8D).
实施例5Example 5
使用另外两个可用B淋巴细胞特征性靶基因的“直接B淋巴细胞LS-TA”的结果Results of "Direct B Lymphocyte LS-TA" Using Two Additional Available B Lymphocyte Characteristic Target Genes
除TNFRSF17外，TXNDC5和TNFRSF13B基因是可用于“直接B淋巴细胞-LS-TA”的另外两个靶基因。在本实施例中，TXNDC5和 TNFRSF13B相对于TNFRSF13C的相对表达用作生物标志物。In addition to TNFRSF17, the TXNDC5 and TNFRSF13B genes are two other target genes available for "direct B lymphocyte-LS-TA". In this example, the relative expression of TXNDC5 and TNFRSF13B relative to TNFRSF13C was used as a biomarker.
在数据集GSE59635中，包含TXNDC5基因的数据。因此用这一个数据集来分析。图9A显示了NR和R组中生物标志物从D0到D7的增量。两组(NR和R)之间的差异是显著的(配对检验，p值<0.01)。并且分析了“直接B淋巴细胞-LS-TA”TXNDC5的增量的ROC，其AUC为0.77(图9B)。In dataset GSE59635, data for the TXNDC5 gene is included. So use this dataset for analysis. Figure 9A shows the increase in biomarkers from D0 to D7 in the NR and R groups. Differences between the two groups (NR and R) were significant (paired test, p-value < 0.01). And the ROC of the increment of "direct B lymphocyte-LS-TA" TXNDC5 was analyzed, and its AUC was 0.77 (Fig. 9B).
同样地使用数据集GSE59635，单独使用第七天的样本，进行TNFRSF13B相对于TNFRSF13C的“直接B淋巴细胞-LS-TA”检测，确定了反应者(R组)有更高的生物指标参数(图9C，p值<0.05)。ROC分析显示这个指标能够有效地分辨R和NR两组(图9D，AUC＝0.81)。Similarly using dataset GSE59635, using the seventh day samples alone, a "direct B-lymphocyte-LS-TA" assay of TNFRSF13B relative to TNFRSF13C identified higher biomarker parameters in responders (group R) (Fig. 9C, p-value < 0.05). The ROC analysis showed that this index could effectively distinguish the R and NR groups (Fig. 9D, AUC=0.81).
实施例6Example 6
使用另一个B淋巴细胞特征性参考基因FCRLA的“直接B淋巴细胞-LS-TA”的结果Results of "direct B lymphocyte-LS-TA" using another B lymphocyte characteristic reference gene, FCRLA
除了TNFRSF13C，FCRLA还可以用作另一个B淋巴细胞参考基因。图10A和10B显示了使用TNFRSF17和FCRLA得出的“直接B淋巴细胞-LS-TA”的结果和分辨性能。In addition to TNFRSF13C, FCRLA can also be used as another B lymphocyte reference gene. Figures 10A and 10B show the results and discrimination performance of "direct B lymphocyte-LS-TA" using TNFRSF17 and FCRLA.
图10A显示R组比NR组具有更高的TNFRSF17：FCRLA的“直接B淋巴细胞-LS-TA”的增量(配对测试，p值＜0.001)。图10B显示ROC分析中，“直接B淋巴细胞-LS-TA”(TNFRSF13C，FCRLA)的增量的AUC为0.82(图10B)。Figure 10A shows that the R group had a higher increase in "direct B lymphocyte-LS-TA" of TNFRSF17:FCRLA than the NR group (paired test, p-value < 0.001). Figure 10B shows that the incremental AUC for "direct B lymphocytes-LS-TA" (TNFRSF13C, FCRLA) in ROC analysis was 0.82 (Figure 10B).
实施例7Example 7
癌症疫苗(PPV)接种前的“直接B淋巴细胞-LS-TA”能够预测***病人的预后及存活机率"Direct B-lymphocyte-LS-TA" before cancer vaccine (PPV) vaccination can predict prognosis and survival in prostate patients
GSE53922数据库包含112位***病人癌症疫苗接种前，外周血多类型单核细胞样本PMBC基因表达，预后和生存时间(overall survival，OS)的数据。病人被分为两组，第一组为治疗后存活时间少于一年(365天)的病人。另一组为治疗效果较好，治疗后存活多于一年的病人。然后比较两组的病人PPV接种前的“直接B淋巴细胞-LS-TA” 生物标志物参数。在本实施例中，使用PBMC样本中两个基因TNFRSF17和FCRLA的相对值计算得出的“直接B淋巴细胞-LS-TA”的结果。The GSE53922 database contains PMBC gene expression, prognosis and overall survival (OS) data in peripheral blood multitype monocyte samples from 112 prostate patients before cancer vaccination. Patients were divided into two groups, the first group was patients who survived less than one year (365 days) after treatment. The other group consists of patients with better treatment effect and survival for more than one year after treatment. The "direct B lymphocyte-LS-TA" biomarker parameters were then compared between the two groups of patients before PPV vaccination. In this example, the "direct B lymphocyte-LS-TA" results were calculated using the relative values of the two genes TNFRSF17 and FCRLA in PBMC samples.
图11A和图11B显示，治疗效果较好的病人组,在接种疫苗之前有比较低的“直接B淋巴细胞-LS-TA”生物标志物参数。图11A显示能够长时间存活的病人在疫苗接种前的“直接B淋巴细胞-LS-TA”生物标志物参数较低(P值<0.05)。ROC分析表明，该检测具有一定的预测能力(AUC＝0.63)。Figures 11A and 11B show that the group of patients with better treatment effect had lower "direct B lymphocyte-LS-TA" biomarker parameters before vaccination. Figure 11A shows that patients with long-term survival had lower "direct B lymphocyte-LS-TA" biomarker parameters before vaccination (P value < 0.05). ROC analysis showed that the assay had some predictive power (AUC=0.63).
图12A是生存分析(survival analysis)，使用考克斯回归(Cox regression)计算三组病人的存活曲线(survival curve)，把病人依据疫苗注射前的“直接B淋巴细胞-LS-TA”生物标志物分为三组，使用分位数范围分为(a)低(指标低于25百分位)，(b)中间区域(25百分位-75百分位)，和(c)高(高于75百分位)的三组。三组生存曲线有明显的差别。存活机率最差的是接种前“直接B淋巴细胞-LS-TA”指标高的一组，大部分病人差不多都在一年内死亡(p值<0.01)。Figure 12A is a survival analysis, using Cox regression (Cox regression) to calculate the survival curve (survival curve) of the three groups of patients, according to the patients before vaccine injection "direct B lymphocytes-LS-TA" biomarker Objects were divided into three groups using quantile ranges as (a) low (indicators below the 25th percentile), (b) intermediate (25th-75th percentile), and (c) high ( above the 75th percentile). The survival curves of the three groups were significantly different. The group with the worst survival rate was the group with high "direct B lymphocyte-LS-TA" index before vaccination, and most patients died within one year (p value < 0.01).
另外，只把指标低(低于25百分位)和指标高(高于75百分位)的两组互相比较，存活曲线的差别更加明显(图12B，p值<0.001)。存活机率最差的是高“直接B淋巴细胞-LS-TA”指标的一组，死亡危险几率(Hazard ratio)比指标低的那一组高出差不多三倍(Hazard ratio＝2.95，置信区间＝1.6至5.6)。In addition, comparing only the two groups with low index (below 25th percentile) and high index (above 75th percentile) with each other, the difference in survival curves was more pronounced (Fig. 12B, p-value < 0.001). The group with the worst survival rate was the group with high "direct B-lymphocyte-LS-TA" index, and the risk of death (Hazard ratio) was almost three times higher than that of the group with the low index (Hazard ratio=2.95, confidence interval= 1.6 to 5.6).
以上结果显示，如果在PPV癌症疫苗接种前，***病人已经有一定的B淋巴细胞活性(即“LS-TA”指标高的一组)，为他们接种PPV疫苗对癌症的治疗帮助不大，即癌症疫苗对他们没有什么附加价值。反之，如果在PPV癌症疫苗接种前，病人没有(或只有低)的B淋巴细胞活性(即“LS-TA”指标低的一组)，PPV疫苗对他们有帮助的机会更高，导致这一组别的预后存活机率大为上升。The above results show that if prostate patients already have a certain B lymphocyte activity before PPV cancer vaccination (ie a group with a high "LS-TA" index), vaccinating them with PPV vaccine will not help much in cancer treatment, that is, Cancer vaccines have little added value to them. Conversely, if patients had no (or only low) B-lymphocyte activity prior to PPV cancer vaccination (ie, the group with a low "LS-TA" index), the chance that PPV vaccine would help them was higher, leading to this The prognostic probability of survival in the group increased significantly.
实施例8Example 8
使用JCHAIN基因作为B淋巴细胞特征性靶基因的“直接B淋巴细胞LS-TA”的结果Results of "direct B lymphocyte LS-TA" using JCHAIN gene as a characteristic target gene of B lymphocytes
除前面已经提及的B淋巴细胞特征性靶基因外，JCHAIN基因也是可用于“直接B淋巴细胞-LS-TA”的靶基因。在本实施例中，将JCHAIN相对于TNFRSF13C的相对表达用作生物标志物进行了流感疫苗接种者的荟萃分析(meta-analysis)。In addition to the B lymphocyte characteristic target genes already mentioned, the JCHAIN gene is also a target gene that can be used for "direct B lymphocyte-LS-TA". In this example, a meta-analysis of influenza vaccine recipients was performed using the relative expression of JCHAIN relative to TNFRSF13C as a biomarker.
在荟萃分析中(参见图13),使用了7个数据集GSE59654,GSE59635,GSE59743,GSE101709,GSE101710,GSE29617,GSE29614。荟萃分析共包括了65例NR和79例R流感疫苗接种者。图13A显示了NR和R组中生物标志物(JCHAIN/TNFRSF13C)第7天表达的荟萃分析森林图(Forest Plot),而图13B显示了NR和R组中该生物标志物从第0天到第7天的增量的荟萃分析森林图。这两种表达指标的荟萃分析在两组(NR和R)之间的差异是显著的,不论使用固定效应模型和随机效应模型均都得出大于零的标准化均数差(Standardised Mean Difference,SMD)，而且可信区也确认大于零。例如图13A显示疫苗注射后第七天R组的JCHAIN基因的“直接B淋巴细胞LS-TA”比NR组较高,SMD＝0.75(95％可信区＝0.39-1.11)。In the meta-analysis (see Figure 13), 7 datasets GSE59654, GSE59635, GSE59743, GSE101709, GSE101710, GSE29617, GSE29614 were used. A total of 65 NR and 79 R influenza vaccine recipients were included in the meta-analysis. Figure 13A shows a meta-analyzed Forest Plot of day 7 expression of the biomarker (JCHAIN/TNFRSF13C) in NR and R groups, while Figure 13B shows this biomarker in NR and R groups from day 0 to Meta-analysis forest plot of day 7 increments. The difference between the two groups (NR and R) in the meta-analysis of these two expression indicators was significant, both the fixed-effects model and the random-effects model yielded standardized mean differences greater than zero (Standardised Mean Difference, SMD) ), and the trusted zone is also confirmed to be greater than zero. For example, Figure 13A shows that the "direct B lymphocyte LS-TA" of the JCHAIN gene was higher in the R group than in the NR group on the seventh day after vaccine injection, SMD=0.75 (95% confidence interval=0.39-1.11).
实施例9Example 9
定量B淋巴细胞基因转录本丰度的通用实验室程序和试剂盒组成General laboratory procedure and kit composition for quantifying gene transcript abundance in B lymphocytes
在其他实施方案中，本领域技术人员将知道如何设计引物以确定这些B淋巴细胞信息基因的转录本丰度。In other embodiments, those skilled in the art will know how to design primers to determine the transcript abundance of these B lymphocyte informative genes.
本申请提供了一些可用于定量PCR(qPCR)的引物示例，以供参考。它们可以在qPCR反应中存在SYBR Green的情况下使用，以获取阈值循环(CT)数据，该数据可用于通过相对定量测定法确定delta-CT，delta-delta CT或效率校正的delta-CT作为生物标记参数。定量血液样本中RNA的转录本丰度(Dorak 2007)。其他的定量方法也可以实施。This application provides some examples of primers that can be used in quantitative PCR (qPCR) for reference. They can be used in the presence of SYBR Green in qPCR reactions to obtain threshold cycle (CT) data that can be used to determine delta-CT, delta-delta CT or efficiency-corrected delta-CT by relative quantification assays as biological Flag parameters. Quantify RNA transcript abundance in blood samples (Dorak 2007). Other quantitative methods can also be implemented.
通用实验室程序：首先，使用Trizol或类似试剂从各种血液样本中提取RNA。商业试剂盒也可用于基于柱的RNA提取。然后，通过逆转录酶将RNA逆转录成cDNA。特定基因通过用户选择的方法进行定量，包括qPCR、RNA测序、DNA微阵列(基因芯片)、分支链DNA检测法(branched chain DNA,bDNA assay,US8426578B2, US7927798B2),纳米报告探针检测法(quantification using nanoreporters,US8415102B2)、数字PCR(US10465238B2)或杂交来测定。General laboratory procedure: First, RNA is extracted from various blood samples using Trizol or similar reagents. Commercial kits are also available for column-based RNA extraction. Then, the RNA is reverse transcribed into cDNA by reverse transcriptase. Specific genes are quantified by user-selected methods, including qPCR, RNA sequencing, DNA microarrays (gene chips), branched chain DNA assays (branched chain DNA, bDNA assay, US8426578B2, US7927798B2), nano-reporter probe assays (quantification) Assay using nanoreporters, US8415102B2), digital PCR (US10465238B2) or hybridization.
本申请教导了使用该方法的cDNA样本可以同时用于B淋巴细胞的靶基因(如TNFRSF17,TNFRSF13B,TXNDC5或JCHAIN)和B淋巴细胞参考基因(如TNFRSF13C，FCRLA，CD79A，CD79B或MS4A1)的TA的测定，例如可以使用下面列出的引物。生物标志物参数由此类B淋巴细胞信息基因对的qPCR结果的delta-CT，delta-delta CT或效率校正的delta-CT计算可得。这种“直接B淋巴细胞-LS-TA”分析产生的生物标志物参数提供了血液中各种细胞混合物样本(例如PBMC，WB)中B淋巴细胞基因表达水平的指标，而无需事先分离B淋巴细胞。This application teaches that cDNA samples using this method can be used for TA of both B lymphocyte target genes (such as TNFRSF17, TNFRSF13B, TXNDC5 or JCHAIN) and B lymphocyte reference genes (such as TNFRSF13C, FCRLA, CD79A, CD79B or MS4A1) For the assay, the primers listed below can be used, for example. Biomarker parameters were calculated from delta-CT, delta-delta CT or efficiency-corrected delta-CT of qPCR results for such B lymphocyte informative gene pairs. The biomarker parameters generated by this "direct B lymphocyte-LS-TA" analysis provide an indication of B lymphocyte gene expression levels in samples of various cell mixtures in blood (e.g., PBMC, WB) without prior isolation of B lymphocytes cell.
在“直接B淋巴细胞-LS-TA”分析中用于qPCR分析的引物实例列表。List of examples of primers used for qPCR analysis in the "Direct B lymphocyte-LS-TA" assay.
Figure PCTCN2021126426-appb-000005
Figure PCTCN2021126426-appb-000005
Figure PCTCN2021126426-appb-000006
Figure PCTCN2021126426-appb-000006
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Claims (21)

  1. 外周血样本分析方法，其包括测定外周血样本中的至少一种B淋巴细胞靶基因的转录本丰度和至少一种B淋巴细胞参考基因的转录本丰度，其中所述B淋巴细胞靶基因选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN，所述B淋巴细胞参考基因选自TNFRSF13C和FCRLA。A peripheral blood sample analysis method, comprising determining the transcript abundance of at least one B lymphocyte target gene and the transcript abundance of at least one B lymphocyte reference gene in the peripheral blood sample, wherein the B lymphocyte target gene The B lymphocyte reference gene is selected from TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, and the B lymphocyte reference gene is selected from TNFRSF13C and FCRLA.
  2. 根据权利要求1所述的方法，其包括如下步骤：The method of claim 1, comprising the steps of:
    a).采集外周血样本；a). Collect peripheral blood samples;
    b).测定所述外周血样本的至少一种B淋巴细胞靶基因的转录本丰度，以获得第一量；b). Determining the transcript abundance of at least one B lymphocyte target gene in the peripheral blood sample to obtain a first amount;
    c).测定所述外周血样本的至少一种B淋巴细胞参考基因的转录本丰度，以获得第二量；c). Determining the transcript abundance of at least one B lymphocyte reference gene of the peripheral blood sample to obtain a second amount;
    d).计算生物标志物参数，所述参数为所述第一量与所述第二量的相对值。d). Calculating a biomarker parameter, the parameter being the relative value of the first amount and the second amount.
  3. 根据权利要求2所述的方法，其中所述外周血样本为接种疫苗之前或接种疫苗之后获取的外周血样本。The method of claim 2, wherein the peripheral blood sample is a peripheral blood sample obtained before or after vaccination.
  4. 根据权利要求3所述的方法，其中所述外周血样本为接种疫苗5-12天后获取的外周血样本。The method of claim 3, wherein the peripheral blood sample is a peripheral blood sample obtained 5-12 days after vaccination.
  5. 根据权利要求3所述的方法，其中所述方法还包括将所述外周血样本的所述生物标志物参数与临界值进行比较。4. The method of claim 3, wherein the method further comprises comparing the biomarker parameter of the peripheral blood sample to a cutoff value.
  6. 根据权利要求5所述的方法，其中所述比较用于预测疫苗接种导致的抗体产生和/或免疫治疗的效果。6. The method of claim 5, wherein the comparison is used to predict the effect of vaccination-induced antibody production and/or immunotherapy.
  7. 根据权利要求1所述的方法，其中所述外周血样本包括接种疫苗之前获取的第一外周血样本和接种疫苗后获取的第二外周血样本；所述生物标志物参数包括由所述第一外周血样本获得的第一生物标志物参数和由所述第二外周血样本获得的第二生物标志物参数。The method of claim 1, wherein the peripheral blood sample comprises a first peripheral blood sample obtained before vaccination and a second peripheral blood sample obtained after vaccination; the biomarker parameter comprises a A first biomarker parameter obtained from a peripheral blood sample and a second biomarker parameter obtained from the second peripheral blood sample.
  8. 根据权利要求7所述的方法，其中所述第二外周血样本是接种疫苗5-12天后获取的。The method of claim 7, wherein the second peripheral blood sample is obtained 5-12 days after vaccination.
  9. 根据权利要求7所述的方法，其中所述方法还包括计算所述第一生物标志物参数和第二生物标志物参数的变化值。8. The method of claim 7, wherein the method further comprises calculating a change in the first biomarker parameter and the second biomarker parameter.
  10. 根据权利要求9所述的方法，其中所述变化值用于预测疫苗接种导致的抗体产生和/或免疫治疗的效果。The method of claim 9, wherein the change value is used to predict the effect of vaccination-induced antibody production and/or immunotherapy.
  11. 根据权利要求3-10中任一项所述的方法，其中所述疫苗是预防性疫苗或者治疗性疫苗。The method of any one of claims 3-10, wherein the vaccine is a prophylactic vaccine or a therapeutic vaccine.
  12. 根据权利要求11所述的方法，其中所述疫苗是预防传染病的疫苗或治疗癌症的疫苗。The method of claim 11, wherein the vaccine is a vaccine for the prevention of infectious diseases or a vaccine for the treatment of cancer.
  13. 试剂盒，其包含用于定量基因转录本丰度的试剂成分，所述基因选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN中的至少一种，以及TNFRSF13C和FCRLA中的至少一种。A kit comprising reagent components for quantifying transcript abundance of genes selected from at least one of TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, and at least one of TNFRSF13C and FCRLA.
  14. 根据权利要求13所述的试剂盒，其中所述试剂成分包括引物，所述引物的序列如SEQ ID NOs:1-14任一项所示。The kit according to claim 13, wherein the reagent component comprises a primer, and the sequence of the primer is shown in any one of SEQ ID NOs: 1-14.
  15. 根据权利要求13或14所述的试剂盒，其中所述基因获自外周血样本，优选获自所述外周血样本的B淋巴细胞。The kit according to claim 13 or 14, wherein the gene is obtained from a peripheral blood sample, preferably from B lymphocytes of the peripheral blood sample.
  16. 测定基因转录本丰度的试剂成分或试剂盒，其用于预测疫苗接种有效性和/或免疫治疗效果，其中所述基因选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN中的至少一种，以及TNFRSF13C和FCRLA中的至少一种。A reagent composition or kit for determining the abundance of a gene transcript for predicting vaccination effectiveness and/or immunotherapy effect, wherein the gene is selected from at least one of TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN, and TNFRSF13C and At least one of FCRLA.
  17. 根据权利要求16所述的试剂成分或试剂盒，其包括引物，所述引物的序列如SEQ ID NOs:1-10任一项所示。The reagent composition or kit according to claim 16, comprising primers whose sequences are shown in any one of SEQ ID NOs: 1-10.
  18. 根据权利要求16或17所述的试剂成分或试剂盒，其中所述基因获自外周血样本，优选获自所述外周血样本的B淋巴细胞。The reagent composition or kit according to claim 16 or 17, wherein the gene is obtained from a peripheral blood sample, preferably from B lymphocytes of the peripheral blood sample.
  19. 用于测定基因转录本丰度的试剂成分在制备用于预测疫苗接种有效性和/或免疫治疗效果的试剂盒或药物中的应用，其中所述基因选自TNFRSF17、TNFRSF13B、TXNDC5和JCHAIN中的至少一种，以及TNFRSF13C和FCRLA中的至少一种。Use of a reagent composition for determining the abundance of a gene transcript in the preparation of a kit or a medicament for predicting the effectiveness of vaccination and/or immunotherapy, wherein the gene is selected from the group consisting of TNFRSF17, TNFRSF13B, TXNDC5 and JCHAIN at least one, and at least one of TNFRSF13C and FCRLA.
  20. 如权利要求19所述的应用，其中所述试剂成分包括引物，所述引物的序列如SEQ ID NOs:1-14任一项所示。The application of claim 19, wherein the reagent component comprises a primer, and the sequence of the primer is shown in any one of SEQ ID NOs: 1-14.
  21. 如权利要求19或权利要求20所述的应用，其中所述基因获自外周血样本，优选获自所述外周血样本的B淋巴细胞。The use of claim 19 or claim 20, wherein the gene is obtained from a peripheral blood sample, preferably B lymphocytes from the peripheral blood sample.
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