WO2017148432A1 - Procédé et kit de test pour prédire une réponse à un traitement par ifnα d'un patient atteint d'hépatite b chronique - Google Patents

Procédé et kit de test pour prédire une réponse à un traitement par ifnα d'un patient atteint d'hépatite b chronique Download PDF

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WO2017148432A1
WO2017148432A1 PCT/CN2017/075542 CN2017075542W WO2017148432A1 WO 2017148432 A1 WO2017148432 A1 WO 2017148432A1 CN 2017075542 W CN2017075542 W CN 2017075542W WO 2017148432 A1 WO2017148432 A1 WO 2017148432A1
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sample
marker
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ifnα
pbmc
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葛胜祥
王邵娟
熊丽霞
刘永亮
夏宁邵
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厦门大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • 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
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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 invention relates to markers for predicting the response of chronic hepatitis B patients to IFN[alpha] treatment.
  • the invention also relates to a method for predicting the response of a chronic hepatitis B patient to IFN[alpha] treatment comprising the step of determining the level of expression of such a marker in PBMC of a chronic hepatitis B patient.
  • the invention also relates to a kit for use in the above method.
  • Hepatitis B virus infection is one of the most important public health problems in the world. There are currently more than 350 million chronic hepatitis B virus infections worldwide. Chronic hepatitis B virus infection can cause chronic liver disease such as Chronic hepatitis B (CHB), Liver cirrhosis (LC) and Hepatocellular carcinoma (HCC). Hepatitis virus infection and related diseases caused by death are more than 1 million people worldwide each year.
  • CHB Chronic hepatitis B
  • LC Liver cirrhosis
  • HCC Hepatocellular carcinoma
  • Chronic HBV infection is a dynamic process, and the natural history of infection can be divided into four different stages, which are not necessarily inextricably linked: 1) immune tolerance stage, characterized by HBeAg positive, active HBV DNA replication, ALT The level is normal or low, generally no fibrosis; 2) active immune stage / immune clearance stage, characterized by HBeAg positive but its level begins to decline, HBV DNA replication level decreases, serum HBsAg levels decrease, ALT continues to rise or fluctuation.
  • HBeAg-negative hepatitis which belongs to the late stage of chronic hepatitis B, is characterized by periodic fluctuations in HBV DNA and ALT levels and active hepatitis.
  • the HBV virus which was previously variant of the C region and/or the basic core promoter (BCP), is predominantly unable to express or express very low levels of HBeAg.
  • HBV virus has no direct pathogenic effect on host cells, and its main pathogenic mechanism is that HBV virus causes host immune response, and immune response causes immune liver tissue damage.
  • the treatment of chronic HBV infection requires different antiviral treatments depending on the individual patient.
  • the purpose of antiviral therapy is to continue to suppress HBV virus, Prevent the development or transformation of hepatitis into fibrosis, cirrhosis and even liver cancer, prolong the survival of patients and improve the quality of life.
  • nucleoside analogues lamivudine (LAM), telbivudine (LdT), emtricitabine (FTC), entecavir (ETV), Defovir (ADV), tenofovir (TDF); two interferons: common interferon (IFN ⁇ ) and pegylated interferon (PEG-IFN ⁇ ).
  • LAM lamivudine
  • LdT telbivudine
  • FTC emtricitabine
  • ETV entecavir
  • ADV Defovir
  • TDF tenofovir
  • IFN ⁇ common interferon
  • PEG-IFN ⁇ pegylated interferon
  • IFN ⁇ was first approved by the FDA for the treatment of chronic hepatitis B.
  • PEG-IFN ⁇ is also the drug of choice for the treatment of chronic hepatitis B, with antiviral and immunomodulatory effects. effect.
  • the main advantage of IFN ⁇ is its non-resistance and immune-mediated control of HBV infection, resulting in a higher chance of HBeAg seroconversion, a more sustained virological response, and HBsAg clearance at the end of treatment.
  • the level of HBV DNA in the medium is maintained at the lower limit of detection.
  • Antiviral therapy must ensure a certain degree of virological suppression and can produce biochemical remission, histological recovery and prevention of complications.
  • the ideal end point for HBV treatment is the disappearance of HBsAg, but existing antiviral drugs are difficult to achieve this goal, so a more realistic treatment endpoint is the ability to induce a sustained virological suppression state.
  • Antiviral responses can be divided into biochemical levels, serum levels, virological levels, and histological levels. All responses can be assessed during and after treatment. Clinical studies have shown that the response rate of CHB patients treated with IFN ⁇ for 6 months is only 25-40%. Determining the ability of CHB patients to respond to HBV-specific immune responses may be able to predict the expected efficacy of CHB patients receiving treatment.
  • markers that are capable of predicting the response of CHB patients to IFN[alpha] treatment in a simple, convenient, rapid, and highly accurate and specific manner.
  • IFN ⁇ or " ⁇ -type interferon” as used herein includes all natural or recombinant alpha-type interferons, particularly preferably human alpha-type interferons, such as recombinant human alpha-type interferons, including but not limited to IFN ⁇ -1b (eg available from Schering Corporation, Kenilworth, NJ) Interferon), IFN ⁇ -2a (eg available from Hoffmann-La Roche, Nutley, NJ) Interferon) or IFN ⁇ -2b (eg available from Schering Corporation, Kenilworth, NJ) Interferon); for example a mixture of natural alpha interferons, including but not limited to IFN ⁇ -n1 (eg available from Sumitomo, Japan) Or Glaxo-Wellcome Ltd., London, Great Britain Interferon alpha-n1) or IFNa-n3 (eg available from Alferon of Interferon Sciences) Interferon).
  • IFN ⁇ -1b eg available from Schering Corporation, Kenilworth, NJ
  • the term “IFN ⁇ ” or “ ⁇ -type interferon” also includes any substance having biological activity of IFN ⁇ , such as a mutant or modified IFN ⁇ , such as a PEG derivative of IFN ⁇ (PEG-IFN ⁇ ).
  • the term “IFN ⁇ ” or “ ⁇ -type interferon” is not limited by any particular source of acquisition, may be obtained from commercially available sources or may be produced by conventional techniques known to those skilled in the art, including However, it is not limited to the biological source extraction method and the genetic engineering extraction method, which are described in detail, for example, in “Pestka S. Arch Biochem Biophys. 1983 Feb 15; 221(1): 1-37” (which is incorporated herein by reference).
  • HBV antigen refers to a protein present in hepatitis B virus (HBV) that is capable of eliciting an immune response in the body, including hepatitis B virus core antigen (HBcAg), hepatitis B virus surface. Antigen (HBsAg) and hepatitis B virus E antigen (HBeAg).
  • HBV hepatitis B virus
  • HBcAg when referring to the amino acid sequence of HBcAg, the description is made with reference to the sequence shown in SEQ ID NO:38.
  • mutations or mutations including but not limited to, substitutions, deletions and/or additions, such as different genotypes, subtypes or different serotypes
  • HBcAg shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 38, as well as natural or artificial variants thereof.
  • sequence fragment of HBcAg when describing a sequence fragment of HBcAg, it includes not only the sequence fragment of SEQ ID NO: 38 but also the corresponding sequence fragment in its native or artificial variant.
  • amino acid residues 1-183 of HBcAg includes the amino acid residues 1-183 of SEQ ID NO: 38, and the corresponding fragments thereof (natural or artificial).
  • corresponding segment means a segment located at an equivalent position in the sequence to be compared when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.
  • HBecAg is not limited by any particular method of synthesizing a protein, and can be produced by conventional techniques known to those skilled in the art, such as DNA recombination techniques or chemical synthesis techniques.
  • the expression "antigenic fragment of HBcAg” refers to a fragment of an amino acid sequence (ie, a polypeptide) obtained by truncating an HBcAg protein, the fragment having the same biological activity as the corresponding full-length protein, That is, PBMC from chronic hepatitis B patients can be stimulated and activated.
  • the amino acid sequence fragment shown by SEQ ID NO: 3-37 in the present invention is an antigenic fragment of HBcAg.
  • the antigenic fragment is not limited by any particular method of synthesizing the polypeptide, and can be produced by conventional techniques known to those skilled in the art, such as DNA recombination techniques or chemical synthesis techniques.
  • HBcAg or an antigenic fragment thereof can be obtained by a DNA recombination technique, for example, by encoding using a cell-free expression system.
  • Cell-free expression systems include, for example, reticulocyte-based lysate-based expression systems, wheat germ extract-based expression systems, and E. coli extract-based expression systems); or by using in vivo expression Systems (eg, E. coli prokaryotic expression systems, yeast eukaryotic expression systems) are obtained from polynucleotides encoding these proteins or polypeptides.
  • HBcAg or an antigenic fragment thereof can be produced by chemical synthesis.
  • Methods for chemical total synthesis of proteins or polypeptides are well known in the art (see, for example, Raibaut L, et al., Top Curr Chem. 2015; 363: 103-54; Thapa P, et al. Molecules.
  • SPPS Solid Phase Peptide Synthesis
  • NCL Native Chemical Ligation
  • TAEC Transfer Active Ester Condensation
  • an antigenic fragment having an amino acid sequence as shown in SEQ ID NO: 3-37, respectively means an antigenic fragment having the amino acid sequence of SEQ ID NO: 3, having SEQ An antigenic fragment of the amino acid sequence shown by ID NO: 4, a combination of an antigenic fragment having the amino acid sequence of SEQ ID NO: 36, and an antigenic fragment having the amino acid sequence of SEQ ID NO: 37.
  • immunological assay refers to an assay that utilizes specific interaction/binding affinity between antigen-antibodies, which is generally useful for detecting the presence of a particular antigen or antibody in a sample or Level.
  • immunological assays are well known to those skilled in the art and include, but are not limited to, ELISA assays, Elispot assays Measurement, Western blotting, surface plasmon resonance, and the like.
  • ELISA assays Elispot assays Measurement, Western blotting, surface plasmon resonance, and the like.
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as K and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain CL.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with high denaturation (referred to as complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy/light chain pair form an antibody binding site, respectively.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342: 878-883.
  • the term "antibody” is not limited by any particular method of producing antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD, IgE or IgM antibodies for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • an "antigen-binding fragment" of an antibody refers to one or more portions of a full length antibody that retain the ability to bind to the same antigen (eg, OAS2 or USP18) to which the antibody binds, capable of Intact antibodies compete for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 Paul, W., ed., 2nd Ed., Raven Press, N. Y. (1989), which is incorporated herein by reference in its entirety for all purposes.
  • Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabody Diabody and a polypeptide comprising at least a portion of an antibody sufficient to confer specific ability to bind the antigen to the polypeptide.
  • CDR complementarity determining region
  • the term "Aptamer” means capable of high affinity and high specificity.
  • G-tetramer (G) -tetramer) A thermodynamically stable three-dimensional structure that specifically binds to a target protein of interest or other biological target molecule by, for example, structural complementation, base stacking force, van der Waals force, hydrogen bonding, or electrostatic interaction.
  • the aptamer may be DNA or RNA, and may also contain a nucleic acid analog (eg, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a glycol nucleic acid (GNA), or a threose nucleic acid (TNA).
  • a nucleic acid analog eg, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a glycol nucleic acid (GNA), or a threose nucleic acid (TNA).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • targeting polypeptide refers to a polypeptide molecule that can specifically bind to a target protein of interest.
  • the targeting polypeptide may comprise a natural amino acid, a synthetic amino acid or an amino acid mimetic that functions in a manner similar to a naturally occurring amino acid.
  • Naturally occurring amino acids are those encoded by the genetic code and those amino acids that are later modified, for example, hydroxyproline, ⁇ -hydroxyglutamate, O-phosphoserine, phosphothreonine or phosphotyrosine.
  • polypeptide solution it binds the target protein can be used to target affinity dissociation equilibrium constant (i.e., K D value) Describe.
  • K D value affinity dissociation equilibrium constant
  • K D value is greater than about 10 -3 M
  • K D value is usually considered to represent a non-binding or non-specific binding.
  • a targeting polypeptide that specifically binds to the target protein can be obtained by methods known to those skilled in the art, such as by phage display technology or protein microarray technology.
  • the expression “response to IFN[alpha] treatment” refers to a condition in which a chronic hepatitis B patient develops a virological response or a serological response after treatment with IFN/PEG-IFN.
  • the virological response means HBV DNA ⁇ 2000 IU/ml 6 months after treatment and 6 to 12 months after withdrawal;
  • the serological response means HBeAg is negative and anti-HBe appears (see, EASL Clinical Practice Guidelines for Chronic Hepatitis B (2012 Edition)).
  • the expression “non-responsive to IFN ⁇ treatment” refers to a condition in which a chronic hepatitis B patient does not satisfy the conditions described above after receiving IFN/PEG-IFN treatment.
  • the term "statistical analysis value” refers to a value obtained by statistically analyzing the detection results obtained by various detection methods.
  • a variety of statistical analysis methods are known in the art (see, for example, PCT International Application WO2009064901, which is incorporated herein by reference), and including but not limited to the linear combination of the test results, the linear regression model, the logistic regression model, the linear discriminant analysis ( LDA) model, nearest neighbor model or microarray predictive analysis (PAM).
  • LDA linear discriminant analysis
  • PAM microarray predictive analysis
  • the statistical analysis value is a value obtained by statistical analysis by a logistic regression model.
  • Logistic regression models are described in detail, for example, in Hu Chunyan. Four tumor markers are in Combined detection in ovarian cancer serum [D]. Guangzhou: Sun Yat-sen University, 2008: 1-39", which is incorporated herein by reference in its entirety.
  • the term "reference value" refers to a value that reflects the condition of a population of CHB patients who are not responsive to IFN[alpha] treatment.
  • the reference value includes, for example, a marker level in a CHB patient population sample according to a CHB patient population sample that is not responsive to IFN ⁇ treatment or a marker in a CHB patient population sample that does not respond to IFN ⁇ treatment before and after IFN ⁇ and/or HBV antigen stimulation.
  • a level of change in the level (relative expression level), a normal value or range of values determined; and a test value obtained from a sample of a population of CHB patients who did not respond to treatment with IFN ⁇ (eg, the level of the marker described above) Change factor)
  • a test value obtained from a sample of a population of CHB patients who did not respond to treatment with IFN ⁇ (eg, the level of the marker described above) Change factor)
  • a value obtained by statistical analysis statistic analysis value.
  • Methods for determining optimal diagnostic cutoff values are well known in the art and include, but are not limited to, Receiver Operating Characteristic (ROC) curve analysis, which is described in detail, for example, in "Habibzadeh F, et al., Biochem.
  • Cycle threshold refers to the number of cycles experienced by a fluorescent signal within each reaction tube when it reaches a set threshold in a real-time PCR assay.
  • Ct value of each template There is a linear relationship between the Ct value of each template and the logarithm of the initial copy number of the template. The more the starting copy number, the smaller the Ct value.
  • a standard curve can be made using a standard of known starting copy number, where the logarithm of the starting copy number is plotted on the abscissa and the Ct value is taken as the ordinate. Therefore, as long as the Ct value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.
  • the term "comparative Ct method” is a method of relative quantitative analysis of mRNA well known in the art, which is described in detail, for example, in Livak KJ, et al. Methods. 2001 Dec; 25(4): 402-8 ( It is incorporated herein by reference).
  • quantitative PCR quantitative PCR
  • each PCR cycle doubles the amplification product
  • the Ct value obtained during the exponential phase of the PCR reaction can reflect the copy number of the starting template, so
  • the difference in Ct values of different samples to be tested is one cycle, which means that their starting template copy number has a difference of 2 times. Based on this, by comparing the Ct values of different samples, the relative expression level of the target gene in the sample to be tested can be judged.
  • normalization refers to the elimination of the possible variations in the various steps of the detection method employed by comparing the expression levels of the same gene in different samples. The difference in expression caused by the yield.
  • Methods of normalization are known in the art, including but not limited to The method of PCT International Application No. WO2013068422A1 or Chinese Patent Application No. 201280035399.6, for example, a comparative Ct method based on an internal reference gene.
  • the term "expression level" refers to a measurable amount of a gene product produced by a gene of interest in a sample from a subject, wherein the gene product can be a transcription product or a translation product.
  • the expression "determining the expression level of a gene of interest” may mean determining the level of the mRNA or fragment of the mRNA of the gene, or the cDNA of the gene or the fragment of the cDNA, or is encoded by the gene. The level of the protein or its polypeptide fragment.
  • PBMC Peripheral blood mononuclear cell
  • T cells lymphocytes
  • B cells lymphocytes
  • NK cells monocytes or dendritic cells.
  • Methods for obtaining PBMC from peripheral blood are well known in the art and include, but are not limited to, Ficoll stratified liquor or Percoll stratified liquor.
  • reagent for isolating PBMC means an agent which is required in the method for obtaining PBMC described above, for example, a polysucrose-transfloxacin for Ficoll stratified liquid method. Amine solution.
  • PBMC separation device means a device capable of separating PBMC from a sample (for example, peripheral blood) from a subject, such devices are known in the art including, but not limited to, detailed Cell separation devices are described in Chinese Patent Application Nos. CN1958776A, CN102286360A, and CN105132278A, all of which are incorporated herein by reference.
  • peripheral blood leukocyte layer refers to a component formed by peripheral anticoagulation after natural sedimentation, centrifugation or density gradient centrifugation, mainly by white blood cells (including peripheral blood mononuclear cells) and Platelet composition. After anticoagulation, the upper layer of plasma, the lower layer of red blood cells, and a thin layer of white film between them, which accounts for about 1% of the total blood volume, are called the white film layer.
  • the term "subject” includes, but is not limited to, various animals, with humans being particularly preferred.
  • the term "diluent" is preferably an electrolyte solution capable of maintaining cell osmotic pressure, and if necessary, the solution also has a function of maintaining physiological pH.
  • solutions are well known in the art and include, but are not limited to, Alsever's solution, Earle's Balanced Salt Solution (EBSS), Gey's Balanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS), Phosphate Buffer (PBS), Dulbose Phosphate Buffer (DPBS), Puck's Balanced Salt Solution, Ringer's Balanced Salt Solution (RBSS), Simm's Balanced Salt Solution (SBSS), TRIS Buffer (TBS), Tyrode's Balanced Salt Solution (TBSS), Physiology Salt water or Ringer's Solution.
  • the diluent is a phosphate buffer or physiological saline.
  • anticoagulant refers to an agent or substance that is capable of preventing blood from clotting, such Substances are well known in the art and include, but are not limited to, heparin, EDTA, oxalates (e.g., sodium oxalate, potassium oxalate, ammonium oxalate), sodium citrate (sodium citrate).
  • the term "culture fluid” or “medium” refers to a nutrient that is capable of maintaining cellular activity.
  • the nutrients contain amino acids, vitamins, carbohydrates, inorganic salts and the like.
  • Such nutrients are well known in the art and include, but are not limited to, RPMI-1640 medium or DMEM medium.
  • the purpose of adding a culture solution or a medium to a sample from the subject is to maintain the activity of cells, particularly PBMC, in the sample.
  • Methods for maintaining the activity of cells in blood components are well known in the art, and those skilled in the art can select according to actual needs.
  • a culture solution when the sample is whole blood, a culture solution may be added, for example, adding a suitable amount of glucose, sodium chloride, potassium chloride, etc. in a phosphate buffer or physiological saline; In some embodiments, the culture solution is added with a suitable amount of glucose and potassium chloride in a phosphate buffer.
  • a medium such as a cell culture medium, may be added, for example, suitable for maintaining blood cells, particularly PBMC. Active cell culture medium, such as RPMI-1640 medium or DMEM medium.
  • the inventors of the present application after tracking the response of chronic hepatitis B patients to IFN ⁇ treatment, unexpectedly found that the level of OAS2 in the pre-treatment PBMC samples of patients treated with IFN ⁇ was significantly higher than that of patients who did not respond to IFN ⁇ treatment. Differences, or pre-treatment PBMC samples from patients treated with IFN ⁇ , showed significant differences in OAS2 and/or UPS18 levels compared to patients who did not respond to IFN ⁇ treatment after in vitro induction, so OAS2 and/or UPS18 could be used to predict CHB A marker of IFN ⁇ therapeutic response in patients.
  • the field has long lacked a well-defined anti-HBV immune response index to predict the response of CHB patients to IFN ⁇ treatment. Based on this finding, the inventors have developed a new method for predicting the therapeutic effect of IFN ⁇ on CHB patients or the response of CHB patients to IFN ⁇ treatment.
  • the invention provides a kit comprising a first reagent capable of detecting a level of expression of a marker, optionally further comprising an IFN alpha and/or a stimulator; wherein the marker is selected from OAS2 or In USP 18, the stimulator is selected from the group consisting of an HBV antigen, an antigenic fragment of an HBV antigen, or any combination thereof.
  • the HBV antigen is HBcAg.
  • the HBcAg has the amino acid sequence set forth in SEQ ID NO:38.
  • the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-37.
  • the stimuli comprise as shown in SEQ ID NOs: 3-37, respectively.
  • the first agent is an agent capable of detecting the mRNA level of the marker.
  • agents are well known in the art and include, but are not limited to, nucleic acid probes that specifically bind to a sequence of interest, primers that amplify a target sequence, non-specific fluorescent dyes (eg, SYBR Green I), or combinations thereof.
  • the nucleic acid probe can be a single-labeled nucleic acid probe, such as a radionuclide (eg, 32 P, 3 H, 35 S, etc.) labeled probe, a biotinylated probe, or a horseradish.
  • the nucleic acid probe may also be a double-labeled nucleic acid Probes, such as Taqman probes, molecular beacons, displacement probes, scorpion primer probes, QUAL probes, FRET probes, and the like.
  • the first reagent comprises a Taqman probe.
  • the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1.
  • the cDNA of USP18 has the nucleotide sequence set forth in SEQ ID NO:2.
  • the first agent is an agent capable of detecting the protein level of the marker.
  • agents are well known in the art and include, but are not limited to, antibodies, targeting polypeptides or nucleic acid aptamers that are capable of specifically binding to OAS2 or USP18 proteins.
  • such agents carry a detectable label, such as an enzyme (eg, horseradish peroxidase, alkaline phosphatase, etc.), a radionuclide (eg, 3 H, 125 I, 35 S, 14) C, 32 P, etc.), fluorescent dyes (such as FITC, TRITC, PE, Texas Red, quantum dots, Cy7, Alexa 750, etc.), acridine esters, magnetic beads (for example, ), colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the above-described label modified avidin (eg, streptavidin).
  • an enzyme eg, horseradish peroxidase, alkaline phosphatase, etc.
  • a radionuclide eg, 3 H, 125 I, 35 S, 14
  • fluorescent dyes such as FITC, TRITC, PE, Texas Red, quantum dots, Cy7, Alex
  • the first agent determines the protein level of OAS2 or USP18 in the sample by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • the first agent comprises an antibody against an OAS2 or USP18 protein or an antigen binding fragment thereof.
  • kits of the invention comprise a first agent capable of detecting the level of expression of OAS2, optionally further comprising IFN ⁇ and/or a stimulator.
  • kits of the invention comprise a first agent and IFN[alpha] capable of detecting USP18 expression levels, optionally further comprising an irritant.
  • the kit of the present invention further comprises a second reagent for pretreating a sample containing PBMC, wherein the second reagent comprises one or more reagents selected from the group consisting of: a diluent for the sample (eg, phosphate buffer or saline); an anticoagulant (eg, heparin) used to prevent blood clotting; used to maintain A reagent for PBMC activity (for example, a culture medium or a culture solution); and an agent for isolating PBMC (for example, a lymphocyte separation solution such as a sucrose-diammonia solution).
  • a diluent for the sample eg, phosphate buffer or saline
  • an anticoagulant eg, heparin
  • a reagent for PBMC activity for example, a culture medium or a culture solution
  • an agent for isolating PBMC for example, a lymphocyte separation solution such as a sucrose-diammonia solution.
  • the second agent comprises an agent (eg, a culture medium or culture medium) for maintaining PBMC activity, and/or an agent for isolating PBMC (eg, a lymphocyte separation solution, eg, Polysucrose - diatrizoate solution).
  • an agent eg, a culture medium or culture medium
  • an agent for isolating PBMC eg, a lymphocyte separation solution, eg, Polysucrose - diatrizoate solution.
  • the kit of the invention further comprises a blood collection device (eg, a pyrogen-free vacuum blood collection tube) and/or a PBMC separation device (eg, a PBMC cell separation tube).
  • a blood collection device eg, a pyrogen-free vacuum blood collection tube
  • a PBMC separation device eg, a PBMC cell separation tube
  • the kit of the present invention further comprises an agent capable of detecting the expression level of the internal reference gene, the expression of the internal reference gene in each tissue and cell is relatively constant, and does not under the condition of the treatment factor Expression changes occur, such as a housekeeping gene; typically, the housekeeping gene encodes proteins necessary for maintaining essential cellular life activities including, but not limited to, ⁇ -actin, GAPDH, 18S rRNA, ⁇ 2-MG, UBC, or ⁇ -tubulin.
  • the agent capable of detecting the expression level of an internal reference gene is an agent capable of detecting the mRNA level of the internal reference gene.
  • the agent capable of detecting the expression level of an internal reference gene is an agent capable of detecting the protein level of the internal reference gene.
  • the invention relates to the use of an agent capable of detecting the expression level of a marker for predicting the therapeutic effect of IFN ⁇ on a subject having chronic hepatitis B or the subject A response to IFN[alpha] treatment; wherein the marker is selected from the group consisting of OAS2 or USP18.
  • the kit further comprises an IFN alpha and/or a stimulator selected from the group consisting of an HBV antigen, an antigenic fragment of an HBV antigen, or any combination thereof.
  • the HBV antigen is HBcAg.
  • the HBcAg has the amino acid sequence set forth in SEQ ID NO:38.
  • the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-37.
  • the stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 3-37, respectively.
  • the agent capable of detecting the level of expression of a marker is an agent capable of detecting the mRNA level of the marker.
  • agents are well known in the art and include, but are not limited to, nucleic acid probes that specifically bind to a sequence of interest, primers that amplify a target sequence, non-specific fluorescent dyes (eg, SYBR Green I), or combinations thereof.
  • the nucleic acid probe can be a single-labeled nucleic acid probe, such as a radionuclide (eg, 32 P, 3 H, 35 S, etc.) labeled probe, a biotinylated probe, or a horseradish.
  • the nucleic acid probe may also be a double-labeled nucleic acid Probes, such as Taqman probes, molecular beacons, displacement probes, scorpion primer probes, QUAL probes, FRET probes, and the like.
  • the agent capable of detecting the expression level of a marker comprises a Taqman probe.
  • the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1.
  • the cDNA of USP18 has the nucleotide sequence set forth in SEQ ID NO:2.
  • the mRNA level of the marker can be determined using mRNA detection methods known in the art, such as fluorescent quantitative PCR, Northern blotting, in situ hybridization, or including mRNA amplification (eg, reverse transcription PCR) and the mRNA amplification.
  • a method of quantifying product eg, electrophoresis and staining.
  • the agent capable of detecting the expression level of a marker quantifies the mRNA level of the marker by PCR.
  • the agent capable of detecting the expression level of a marker determines the mRNA level of the marker by real-time PCR.
  • the agent capable of detecting the level of expression of a marker is an agent capable of detecting the protein level of the marker.
  • agents are well known in the art and include, but are not limited to, antibodies, targeting polypeptides or nucleic acid aptamers that are capable of specifically binding to OAS2 or USP18 proteins.
  • such agents carry a detectable label, such as an enzyme (eg, horseradish peroxidase, alkaline phosphatase, etc.), a radionuclide (eg, 3 H, 125 I, 35 S, 14) C, 32 P, etc.), fluorescent dyes (such as FITC, TRITC, PE, Texas Red, quantum dots, Cy7, Alexa 750, etc.), acridine esters, magnetic beads (for example, ), colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the above-described label modified avidin (eg, streptavidin).
  • an enzyme eg, horseradish peroxidase, alkaline phosphatase, etc.
  • a radionuclide eg, 3 H, 125 I, 35 S, 14
  • fluorescent dyes such as FITC, TRITC, PE, Texas Red, quantum dots, Cy7, Alex
  • the agent capable of detecting the expression level of a marker determines the protein level of OAS2 or USP18 in the sample by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • the agent capable of detecting the expression level of a marker comprises an antibody against an OAS2 or USP18 protein or an antigen-binding fragment thereof.
  • the kit further comprises an agent capable of detecting the expression level of the internal reference gene, the expression of the internal reference gene in each tissue and cell is relatively constant, and does not occur under the conditions of the treatment factor Expression alterations, such as housekeeping genes; typically, the housekeeping genes encode proteins necessary for maintaining essential life activities of the cell including, but not limited to, ⁇ -actin, GAPDH, 18S rRNA, ⁇ 2-MG, UBC or ⁇ -tubulin.
  • the agent capable of detecting the level of expression of an internal reference gene is capable of detecting the internal reference group Reagents for mRNA levels.
  • the agent capable of detecting the expression level of an internal reference gene is an agent capable of detecting the protein level of the internal reference gene.
  • the kit further comprises one or more reagents or devices selected from 1) to 6):
  • a diluent for diluting the sample such as phosphate buffer or physiological saline
  • an anticoagulant for preventing blood coagulation such as heparin
  • an agent for maintaining the activity of PBMC such as a medium or a culture solution
  • an agent for isolating PBMC such as a lymphocyte separation solution, such as a sucrose-diammonia solution;
  • a blood collection device such as a pyrogen-free vacuum blood collection tube
  • PBMC separation device such as a PBMC cell separation tube.
  • the kit includes an agent (eg, a culture medium or culture medium) for maintaining PBMC activity, and/or an agent for isolating PBMC (eg, a lymphocyte separation solution, such as a poly Sucrose - diatrizoate solution).
  • an agent eg, a culture medium or culture medium
  • an agent for isolating PBMC eg, a lymphocyte separation solution, such as a poly Sucrose - diatrizoate solution.
  • the kit predicts the therapeutic effect of IFNa on a subject having chronic hepatitis B or the subject's response to IFN[alpha] treatment by a method comprising the steps of:
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or a peripheral blood white layer.
  • PBMC peripheral blood mononuclear cells
  • the expression level when the expression level is greater than the reference value, or when the statistical analysis value of the expression level is greater than the reference value, indicating that the subject is responsive to IFN[alpha] treatment Suitably receiving IFN ⁇ treatment; when the expression level is not greater than the reference value, or when the statistical analysis value of the expression level is not greater than the reference value, indicating that the subject does not respond to IFN ⁇ treatment It is not suitable for IFN ⁇ treatment.
  • step (2) the expression levels are statistically analyzed using a logistic regression model.
  • the expression level of the marker to be tested in different samples can usually be used. Normalization is performed, for example, by comparing the expression level value of the marker to be tested in the sample with the expression level value of the reference gene.
  • the expression level of the marker is a normalized expression level.
  • the normalized expression level can be obtained by an exemplary method of determining the expression level of the internal reference gene in a sample from the subject using an agent capable of detecting the expression level of the internal reference gene, and expressing the marker The level is compared to the expression level of the reference gene to obtain a normalized expression level of the marker.
  • the method further comprises one or more of the following steps: (a) obtaining a sample from the subject using a blood collection device; (b) using An anticoagulant treats a blood collection device or a sample from the subject; (c) treats a sample from the subject using an agent for maintaining PBMC activity; (d) dilutes from the subject using a diluent And (e) separating the PBMC from the sample from the subject using a reagent for separating PBMC or a PBMC separation device.
  • the kit predicts the therapeutic effect of IFNa on a subject having chronic hepatitis B or the subject's response to IFN[alpha] treatment by a method comprising the steps of:
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or a peripheral blood white layer.
  • PBMC peripheral blood mononuclear cells
  • the relative expression level when the relative expression level is less than the reference value, or when the statistical analysis value of the relative expression level is less than the reference value, indicating that the subject is to be treated with IFN ⁇ Generating a response suitable for IFN ⁇ treatment; when the relative expression level is not less than the reference value, or when the statistical analysis value of the relative expression level is not less than the reference value, indicating that the subject does not IFN ⁇ treatment produces a response that is not suitable for IFN ⁇ treatment.
  • step (2) a change in the mRNA level of the marker in the test sample compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method. That is, the relative expression level of the mRNA of the marker in the sample to be tested.
  • step (3) the relative expression levels are statistically analyzed using a logistic regression model.
  • the expression level of the marker to be tested in different samples can usually be used. Normalization is performed, for example, by comparing the expression level value of the marker to be tested in the sample with the expression level value of the reference gene.
  • the expression level of the marker is a normalized expression level.
  • the normalized expression level can be obtained by an exemplary method of determining the expression level of the internal reference gene in a sample from the subject using an agent capable of detecting the expression level of the internal reference gene, and expressing the marker The level is compared to the expression level of the reference gene to obtain a normalized expression level of the marker.
  • the expression level of the marker is a normalized mRNA level, which can be obtained by the following exemplary method: using the comparative Ct method to measure the marker in the sample to be tested The mRNA level of the analyte is compared to the mRNA level of the internal reference gene to obtain a normalized mRNA level of the marker. Further, a fold change of the normalized mRNA level of the marker in the sample to be tested compared to the normalized mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the sample in the sample to be tested The relative expression level of the mRNA of the marker.
  • the method further comprises one or more of the following steps: (a) obtaining a sample from the subject using a blood collection device; (b) using An anticoagulant treats a blood collection device or a sample from the subject; (c) treats a sample from the subject using an agent for maintaining PBMC activity; (d) dilutes from the subject using a diluent And (e) separating the PBMC from the sample from the subject using a reagent for separating PBMC or a PBMC separation device.
  • the invention provides a method for predicting the therapeutic effect of IFN ⁇ on a subject having chronic hepatitis B Or a method of the subject's response to IFN ⁇ treatment, or a method of obtaining a result of a diagnostic assay, wherein the diagnostic assay is for predicting a therapeutic effect of IFN ⁇ on a subject having chronic hepatitis B or the subject
  • the response to IFN ⁇ treatment includes the following steps:
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or a peripheral blood white layer.
  • PBMC peripheral blood mononuclear cells
  • the method further comprises the step of administering to the subject IFN[alpha] to treat chronic hepatitis B, wherein the subject is judged to be IFN[alpha] therapeutically produced Answer.
  • the expression level when the expression level is greater than the reference value, or when the statistical analysis value of the expression level is greater than the reference value, indicating that the subject is responsive to IFN[alpha] treatment Suitably receiving IFN ⁇ treatment; when the expression level is not greater than the reference value, or when the statistical analysis value of the expression level is not greater than the reference value, indicating that the subject does not respond to IFN ⁇ treatment It is not suitable for IFN ⁇ treatment.
  • step (2) the mRNA level or protein level of the marker is determined.
  • the mRNA level of the marker is determined.
  • the mRNA level of the marker can be determined using mRNA detection methods known in the art, such as fluorescent quantitative PCR, Northern blotting, in situ hybridization, or including mRNA amplification (eg, reverse transcription PCR) and the mRNA amplification. A method of quantifying product (eg, electrophoresis and staining).
  • the mRNA level of the marker is quantified by PCR.
  • the mRNA level of the marker is determined by real-time PCR.
  • the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1.
  • the cDNA of USP18 has the nucleotide sequence set forth in SEQ ID NO:2.
  • the protein level of the marker is determined.
  • the protein level of the marker is determined by immunological detection.
  • the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method.
  • an antibody against OAS2 is used or Its antigen-binding fragment is used to detect the protein level of OAS2.
  • step (3) the expression levels are statistically analyzed using a logistic regression model.
  • the expression level of the marker to be tested in different samples can usually be used. Normalization is performed, for example, by comparing the expression level value of the marker to be tested in the sample with the expression level value of the reference gene.
  • the expression level of the marker is a normalized expression level.
  • the normalized expression level can be obtained by an exemplary method of determining the expression level of the internal reference gene in a sample from the subject using an agent capable of detecting the expression level of the internal reference gene, and expressing the marker The level is compared to the expression level of the reference gene to obtain a normalized expression level of the marker.
  • step (1) prior to step (1), one or more of the following steps are further included: (a) obtaining a sample from the subject; (b) from the subject An anticoagulant such as heparin is added to the sample; (c) PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer) is obtained from a sample from the subject; (d) from the subject A culture medium or a medium is added to the sample of the tester; and, (e) the sample from the subject is diluted.
  • an anticoagulant such as heparin is added to the sample
  • PBMC or a blood component containing PBMC for example, a peripheral blood leukocyte layer
  • the present invention provides a method for predicting a therapeutic effect of IFN ⁇ on a subject having chronic hepatitis B or a response of said subject to IFN ⁇ treatment, or a method of obtaining a result of a diagnostic assay, wherein The diagnostic assay is used to predict the therapeutic effect of IFNa on a subject with chronic hepatitis B or the subject's response to IFN[alpha] treatment, which includes the following steps:
  • step (3) determining the expression level of the marker in each sample in the step (2), and obtaining a change ratio of the expression level of the marker in the sample to be tested compared to the expression level of the marker in the control sample, Using the change factor as a relative expression level of the marker in the sample to be tested, wherein the marker is selected from OAS2 or USP18;
  • the sample comprises peripheral blood mononuclear cells (PBMC), such as whole blood (eg, anticoagulated whole blood), peripheral blood mononuclear cells (PBMC), or a peripheral blood white layer.
  • PBMC peripheral blood mononuclear cells
  • the method further comprises the step of administering to the subject IFN ⁇ to treat chronic hepatitis B, wherein the subject is judged to be IFN ⁇ therapeutically produced Answer.
  • the relative expression level when the relative expression level is less than the reference value, or when the statistical analysis value of the relative expression level is less than the reference value, indicating that the subject is to be treated with IFN ⁇ Generating a response suitable for IFN ⁇ treatment; when the relative expression level is not less than the reference value, or when the statistical analysis value of the relative expression level is not less than the reference value, indicating that the subject does not IFN ⁇ treatment produces a response that is not suitable for IFN ⁇ treatment.
  • the HBV antigen is HBVcAg.
  • the HBVcAg has the amino acid sequence set forth in SEQ ID NO:38.
  • the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-37.
  • the stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 3-37, respectively.
  • the mRNA level of the marker is determined.
  • the mRNA level of the marker can be determined using mRNA detection methods known in the art, such as fluorescent quantitative PCR, Northern blotting, in situ hybridization, or including mRNA amplification (eg, reverse transcription PCR) and the mRNA amplification. A method of quantifying product (eg, electrophoresis and staining).
  • the mRNA level of the marker is quantified by PCR.
  • the mRNA level of the marker is determined by real-time PCR.
  • the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1.
  • the cDNA of USP18 has the nucleotide sequence set forth in SEQ ID NO:2.
  • the protein level of the marker is determined. In some excellent In selected embodiments, in step (3), the protein level of the marker is determined by immunological detection. Further, in certain preferred embodiments, the immunological assay is selected from the group consisting of an ELISA assay, an Elispot assay, a Western blot, or a surface plasmon resonance method. In certain embodiments, in step (3), an antibody against the OAS2 or USP18 protein or antigen-binding fragment thereof is used to detect the protein level of the marker.
  • step (3) a change in the mRNA level of the marker in the test sample compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method. That is, the relative expression level of the mRNA of the marker in the sample to be tested.
  • step (4) the relative expression levels are statistically analyzed using a logistic regression model.
  • the expression level of the marker to be tested in different samples can usually be used. Normalization is performed, for example, by comparing the expression level value of the marker to be tested in the sample with the expression level value of the reference gene.
  • the expression level of the marker is a normalized expression level.
  • the normalized expression level can be obtained by an exemplary method of determining the expression level of the internal reference gene in a sample from the subject using an agent capable of detecting the expression level of the internal reference gene, and expressing the marker The level is compared to the expression level of the reference gene to obtain a normalized expression level of the marker.
  • the expression level of the marker is a normalized mRNA level, which can be obtained by the following exemplary method: using the comparative Ct method to measure the marker in the sample to be tested The mRNA level of the analyte is compared to the mRNA level of the internal reference gene to obtain a normalized mRNA level of the marker. Further, a fold change of the normalized mRNA level of the marker in the sample to be tested compared to the normalized mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the sample in the sample to be tested The relative expression level of the mRNA of the marker.
  • step (1) prior to step (1), one or more of the following steps are further included: (a) obtaining a sample from the subject; (b) from the subject An anticoagulant such as heparin is added to the sample; (c) PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer) is obtained from a sample from the subject; (d) from the subject A culture medium or a medium is added to the sample of the tester; and, (e) the sample from the subject is diluted.
  • an anticoagulant such as heparin is added to the sample
  • PBMC or a blood component containing PBMC for example, a peripheral blood leukocyte layer
  • composition comprising a full length or a partial fragment of a HBV core antigen and/or IFN ⁇ .
  • a marker for predicting the therapeutic effect of alpha interferon in a chronic hepatitis B patient wherein the marker is an Interferon-stimulated genes (ISGs).
  • ISGs Interferon-stimulated genes
  • a method of screening for a marker for predicting the therapeutic effect of alpha interferon in a chronic hepatitis B patient comprising the steps of:
  • the difference in the level of ISGs mRNA in the IFN ⁇ treatment response group and the non-response group can be used as the basis for the therapeutic effect.
  • a kit for predicting the therapeutic effect of alpha interferon in chronic hepatitis B patients comprising an HBV core antigen polypeptide segment and an IFN ⁇ inducer, and an agent for detecting markers OAS2 and USP18 levels.
  • kits of item 6 wherein the HBV core antigen polypeptide segment is a polypeptide segment of amino acid 1-183 of the HBV core antigen.
  • the present invention has found through a large number of experiments and repeated exploration that the expression levels of OAS2 and/or USP18 are significantly different between the two groups of CHB patients who respond to IFN ⁇ treatment and those who do not respond to IFN ⁇ treatment, thereby establishing a simple, A method for predicting the therapeutic effect of IFN ⁇ on a subject having chronic hepatitis B or the subject's response to IFN ⁇ treatment is convenient, rapid, and highly accurate and specific.
  • Figure 1 shows the significant difference in OAS2 mRNA levels of PBMCs in CHB patients between the IFN ⁇ treatment response group (Rs) and the non-response group (NRs) under different treatment conditions.
  • Figure 1a shows that PBMC is not induced in vitro, and the mRNA level of OAS2 in the response group is significantly higher than that in the non-responder group.
  • Figure 1b shows that the relative expression level of mRNA of OAS2 in the response group was significantly lower than that in the non-responder group after PBMC was induced by IFN ⁇ in vitro. (Note: * indicates p ⁇ 0.05, **** indicates p ⁇ 0.0005)
  • Figure 2 shows that there is a significant difference in USP18 mRNA levels of PBMCs in CHB patients between the IFN ⁇ treatment response group (Rs) and the non-response group (NRs) under different treatment conditions.
  • Figure 2a shows that the relative expression level of USP18 mRNA in the response group was significantly lower than that in the non-response group after PBMC was induced by IFN ⁇ in vitro
  • Figure 2b shows the relative expression level of USP18 mRNA in the response group after PBMC was induced by IFN ⁇ and HBcAg peptide libraries. Significantly lower than the no response group. (Note: material * indicates p ⁇ 0.001, material ** indicates p ⁇ 0.0005)
  • Figure 3 shows the ROC curves for the OAS2N assay and the OAS2 (IFNa + N-N) assay. The results showed that the OAS2N assay and the OAS2 (IFNa+N-N) assay have good sensitivity and specificity for predicting the response of CHB patients to IFN ⁇ therapy.
  • Figure 4 shows the ROC curve of the OAS2 (IFNa + N-N) assay, the USP18 (IFNa + N-N) assay, and a combination of both.
  • the results showed that the OAS2 (IFNa+N-N) assay, the USP18 (IFNa+N-N) assay, and the combination of the two were very sensitive and specific for predicting the response of CHB patients to IFN ⁇ therapy.
  • lymphocyte separation solution (Ficoll-page Tm PLUS, GE Helathcare Life Sciences) equivalent to whole blood was added.
  • the diluted whole blood is slowly added to the lymphocyte separation liquid along the tube wall to maintain a layered state.
  • the above PBMC cells were seeded in a 24-well plate, 5 x 10 5 cells were added to each well, and 1 ml of RPM1640 medium containing 10% fetal calf serum was added. A total of four wells are set for each specimen, wherein the first well is not added with an inducing substance (the inducing substance includes IFN ⁇ and HBcAg polypeptide library) labeled as N, and the second hole is added with IFN ⁇ (Andafen, Anhui Anke Bioengineering (Group) Co., Ltd.) (1 ⁇ 10 4 IU/mL), a peptide library containing 35 HBcAg antigenic fragments (SEQ ID NO: 3-37) (synthesized by Shanghai Bioengineering Co., Ltd.) (1 ⁇ g/) was added to the third well. Strips/mL, a total of 35), the fourth well was added to the above IFN ⁇ and HBcAg polypeptide libraries. After the culture plate was placed in a carbon dioxide incubator at
  • the present invention has established fluorescence for 17 genes including the USP18, OAS2, LGP1, LAP3, GIP3, CEB1, ATF5, GIP2, OAS3, RPLP2, STXBP5, Viperin, RPS28, PI3KAP1, MX1, DUSP1, ETEF1 and the internal reference ⁇ -actin. Quantitative PCR detection system.
  • Example 1 The PBMC cells obtained in Example 1 were collected by centrifugation, and the supernatant was discarded; 250 ⁇ l of RPMI1640 medium and 750 ⁇ l of Tripure were added to each well and mixed well; 200 ⁇ l of chloroform was added per 1 ml of cells, mixed upside down, and allowed to stand at 4 ° C. Set for 10min.
  • RNA samples were determined using a spectrophotometer. 2 ⁇ L RNA sample was used to measure the absorbance of A260 and A280 in a 2 ⁇ L RNase-free water calibration instrument. The ratio of A260 to A280 was measured to evaluate the purity of the RNA sample. After detection, the RNA purity was A260/280 ⁇ 1.80.
  • cDNA is a template in a fluorescent quantitative PCR detection system and stored at -20 °C.
  • Pre-experiment was performed on 18 sets of primers designed by primer software (Table 2 above), and two templates from CHB patients (labeled CHB 1 and CHB 2, respectively) were randomly selected to detect the gene to be tested and the internal reference primer by real-time PCR. Ct value.
  • the results of the identification are shown in Table 3.
  • the Ct values of the primers of 17 sets of test genes and the ⁇ -actin primers of the internal reference were between 15-35, indicating that they could all be effectively amplified; while the Neg group (without template) was not detected.
  • the 18 sets of primers listed in Table 2 above can be used for real-time PCR detection of the corresponding gene to be tested.
  • the invention has systematically tracked 150 chronic hepatitis B specimens, among which 66 patients received IFN ⁇ treatment and 24 weeks of treatment results; among them, 47 patients had no response to IFN ⁇ treatment and 19 patients had IFN ⁇ treatment response.
  • Table 4 shows The characteristic data of the selected study samples in this example showed that there was no significant difference in age and gender between the response group (Rs) and the non-response group (NRs).
  • the results of ALT, AST and HBsAg, HBV DNA, and HBeAg before treatment were also There are no significant differences.
  • Example 1.2 The specific steps of in vitro induction culture were carried out according to Example 1.2, wherein OAS2 (IFNa+NN) The group indicates the relative expression level of OAS2 mRNA in OAS2 relative to the control sample in the sample to be tested; USP18 (IFNa+NN) group and USP18 (IFNa+HBcAg-HBcAg) group respectively represent USP18 in the sample to be tested. Relative mRNA expression levels of USP18 relative to the respective control samples.
  • Figure 1a shows the results of the OAS2N group. The results showed that the OAS2 normalized mRNA levels were significantly higher in the response group than in the non-responder group in the PBMC samples before the IFN ⁇ treatment without any in vitro induced CHB.
  • Figure 1b shows the results of the OAS2 (IFNa+NN) group. The results showed that the relative expression level of OAS2 mRNA in the response group was significantly lower than that in the PBMC samples before the IFN ⁇ treatment. Group of patients.
  • Figure 2a shows the results of the USP18 (IFNa+NN) group. The results showed that the relative expression level of USP18 mRNA in the response group was significantly lower than that in the PBMC samples before the IFN ⁇ treatment. Group of patients.
  • Figure 2b shows the results of the USP18 (IFNa+HBcAg-HBcAg) group. The results showed that CHB patients receiving cultured in vitro via IFN ⁇ and HBcAg peptide libraries (containing HBcAg antigenic fragments shown in SEQ ID NO: 3-37) were accepted. In the PBMC samples before IFN ⁇ treatment, the relative expression level of USP18 mRNA in the response group was significantly lower than that in the non-responder group.
  • OAS2 levels in pre-treatment PBMC samples in patients treated with IFN ⁇ are significantly higher than those in patients not responding to IFN ⁇ treatment; meanwhile, pre-treatment PBMC samples in IFN ⁇ -treated patients are induced in vitro by IFN ⁇ and/or HBcAg. Afterwards, the levels of OAS2 and/or UPS18 were significantly reduced compared to patients who did not respond to IFN[alpha] treatment. Thus OAS2 and/or UPS18 can be used to predict CHB patient response to IFN[alpha] treatment.
  • Example 3 According to the experimental conditions of the OAS2N group and the OAS2 (IFNa+N-N) group in Example 3, the corresponding OAS2N detection method and OAS2 (IFNa+N-N) detection method were respectively established, and Table 6 shows the main steps of the above two methods.
  • the CHB patient samples described in Example 3 were separately tested by the two methods described above, and two diagnostic indicators of the sample were obtained: OAS2 normalized mRNA level and OAS2 mRNA relative expression level.
  • OAS2N assay or OAS2 (IFNa+NN) assay can be used to predict the therapeutic effect of IFN ⁇ on CHB patients or CHB patients on IFN ⁇ therapy. Response, with high sensitivity and specificity.
  • Example 5 Analysis of OAS2 (IFNa+N-N) detection method, USP18 (IFNa+N-N) detection method and ROC curve analysis of combined detection
  • the CHB patient samples described in Example 3 were respectively tested by the two methods described above, and two diagnostic indicators were obtained: the relative expression level of OAS2 mRNA and the relative expression level of USP18 mRNA;
  • the relative expression level of mRNA of OAS2 or the relative expression level of mRNA of USP18 was used as a diagnostic index using SPSS 17.0 software, and the ROC curve was drawn to test the prediction of IFN ⁇ by OAS2 (IFNa+NN) assay or USP18 (IFNa+NN) assay alone.

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  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)

Abstract

La présente invention concerne un marqueur permettant de prédire une réponse à un traitement par IFNα d'un patient atteint d'hépatite B chronique. L'invention concerne en outre un procédé de prédiction de la réponse au traitement par IFNα du patient atteint d'hépatite B chronique. Le procédé comprend la mesure d'un niveau d'expression du marqueur dans les PBMC du patient atteint d'hépatite B chronique. L'invention concerne également un kit permettant de mettre en œuvre le procédé.
PCT/CN2017/075542 2016-03-04 2017-03-03 Procédé et kit de test pour prédire une réponse à un traitement par ifnα d'un patient atteint d'hépatite b chronique WO2017148432A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115679003A (zh) * 2022-10-28 2023-02-03 北京大学 用于预测药物疗效的组合物、系统及用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101821629A (zh) * 2007-09-10 2010-09-01 诺瓦提斯研究基金会弗里德里克·米谢尔生物医学研究所 用于预测患有肝脏病毒性感染的受试者针对抗病毒疗法的应答的方法
CN104603289A (zh) * 2012-06-15 2015-05-06 哈里·斯泰利 检测疾病或病状的方法

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CN102485907B (zh) * 2010-12-06 2014-03-05 北京大学人民医院 干扰素治疗慢性丙型肝炎转归预测基因芯片

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101821629A (zh) * 2007-09-10 2010-09-01 诺瓦提斯研究基金会弗里德里克·米谢尔生物医学研究所 用于预测患有肝脏病毒性感染的受试者针对抗病毒疗法的应答的方法
CN104603289A (zh) * 2012-06-15 2015-05-06 哈里·斯泰利 检测疾病或病状的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115679003A (zh) * 2022-10-28 2023-02-03 北京大学 用于预测药物疗效的组合物、系统及用途

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