WO2017148432A1 - Method and test kit for predicting ifnα treatment response of chronic hepatitis b patient - Google Patents

Abstract

The present invention provides a marker for predicting an IFNα treatment response of a chronic hepatitis B patient. The invention further provides a method for predicting the IFNα treatment response of the chronic hepatitis B patient. The method comprises measuring an expression level of the marker in PBMC of the chronic hepatitis B patient. The invention also provides a kit for using the method.

Description

Method and kit for predicting the response of chronic hepatitis B patients to IFNα therapy Technical field

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.

Background technique

Hepatitis B virus infection, especially chronic 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.

Most patients with chronic HBV infection show no obvious clinical symptoms, and about 10-30% of them develop cirrhosis or liver cancer. 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. The CTL response in this phase of the patient is activated, prone to moderate or severe inflammatory response and liver fibrosis with varying durations; 3) inactive carrier phase, where HBV DNA is in a low replication phase with very low DNA levels Even if it is not detected, HBsAg levels are significantly reduced, ALT levels are normal, liver tissue has no or only mild inflammation, HBeAg-negative, anti-HBe-positive, this period means that the patient's HBV infection has been immuno-controlled, also indicating a Long-term and good prognosis, most patients have a low incidence of cirrhosis and even liver cancer; 4) 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.

There are currently 8 drugs available for CHB antiviral therapy, including 6 nucleoside analogues: lamivudine (LAM), telbivudine (LdT), emtricitabine (FTC), entecavir (ETV), Defovir (ADV), tenofovir (TDF); two interferons: common interferon (IFNα) and pegylated interferon (PEG-IFNα). The primary role of nucleoside analogs is to inhibit the replication of HBV virus by inhibiting the activity of HBV polymerase. In 1976, Greenberg first reported the efficacy of IFNα in the treatment of chronic hepatitis B. IFNα was first approved by the FDA for the treatment of chronic hepatitis B. PEG-IFNα is also the drug of choice for the treatment of chronic hepatitis B, with antiviral and immunomodulatory effects. effect. The main advantage of IFNα 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 IFNα 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. For a long time, due to the lack of a well-defined anti-HBV immune response index, serum ALT levels in CHB patients have been used as an indirect surrogate for measuring host immunity against HBV, but the prediction results are not satisfactory. If treatment response prediction can be performed before treatment, it is of great significance to optimize drug selection, improve treatment compliance and ensure efficacy.

Thus, there is a need in the art for 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.

Summary of the invention

In the present invention, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise stated. Moreover, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are routine steps that are widely used in the corresponding art. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

The term "IFNα" or "α-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 IFNα-1b (eg available from Schering Corporation, Kenilworth, NJ)

Interferon), IFNα-2a (eg available from Hoffmann-La Roche, Nutley, NJ)

Interferon) or IFNα-2b (eg available from Schering Corporation, Kenilworth, NJ)

Interferon); for example a mixture of natural alpha interferons, including but not limited to IFNα-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). In the present invention, the term "IFNα" or "α-type interferon" also includes any substance having biological activity of IFNα, such as a mutant or modified IFNα, such as a PEG derivative of IFNα (PEG-IFNα). In the present invention, the term "IFNα" or "α-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).

As used herein, the term "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).

As used herein, the term "HBcAg" refers to the core antigenic protein of hepatitis B virus (HBV), which is well known to those skilled in the art (see, for example, GENBANK Accession No.: CAM31905.1).

In the present invention, when referring to the amino acid sequence of HBcAg, the description is made with reference to the sequence shown in SEQ ID NO:38. However, it will be understood by those skilled in the art that mutations or mutations (including but not limited to, substitutions, deletions and/or additions, such as different genotypes, subtypes or different serotypes) may be naturally occurring or artificially introduced in the amino acid sequence of HBcAg. , serum subtype of HBcAg), without affecting its biological function. Thus, in the present invention, the term "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. Also, 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. For example, the expression "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). In the present invention, the expression "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.

In the present invention, the term "HBcAg" 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.

As used herein, 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. For example, the amino acid sequence fragment shown by SEQ ID NO: 3-37 in the present invention is an antigenic fragment of HBcAg. In the present invention, 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.

In the present invention, HBcAg or an antigenic fragment thereof (for example, an antigenic fragment having an amino acid sequence as shown in SEQ ID NOs: 3-37, respectively) can be obtained by a DNA recombination technique, for example, by encoding using a cell-free expression system. Polynucleotide acquisition of these proteins or polypeptides (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. Alternatively, HBcAg or an antigenic fragment thereof (for example, an antigenic fragment having an amino acid sequence as shown in SEQ ID NOs: 3-37, respectively) 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. 2014; 19 ( 9): 14461-83; Dawson PE, et al., Science, 1994; 266 (5186): 776-9; and Wang P, et al., Tetrahedron Lett, 1998, 39(47): 88711-14; Incorporated herein by reference), and including but not limited to: Solid Phase Peptide Synthesis (SPPS) or liquid phase fractional synthesis techniques (eg, Native Chemical Ligation (NCL), azide Azide method, Transfer Active Ester Condensation (TAEC)).

As used herein, the expression "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.

As used herein, the term "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. Such 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. For a detailed description of immunological assays, see, for example, Fundamental Immunology, Ch. 7 Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989).

As used herein, the term "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 μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, 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). 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. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal 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.

As used herein, 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. In some cases, 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.

As used herein, the term "Aptamer" means capable of high affinity and high specificity. A single-stranded oligonucleotide that binds to a target protein of interest or other biological target molecule that folds to form, for example, Stem-Loop, Hairpin, Pseudoknot, or 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). Methods for obtaining nucleic acid aptamers that bind to a particular target protein are well known in the art, such as SELEX (Systematic evolution of ligands by exponential enrichment) screening techniques.

As used herein, the term "targeting polypeptide" refers to a polypeptide molecule that can specifically bind to a target protein of interest. In the present invention, 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, γ-hydroxyglutamate, O-phosphoserine, phosphothreonine or phosphotyrosine. In the present invention, it can be determined based on affinity to target "specificity" between the polypeptide and its target protein, polypeptide solution it binds the target protein can be used to target affinity dissociation equilibrium constant (i.e., K _D value) Describe. The lower the K _D value, the stronger the binding strength between the target protein and the targeting polypeptide binds. Generally known in the art, is greater than about 10 ^-3 M K _D value is usually considered to represent a non-binding or non-specific binding. Depending on the particular target protein of interest, 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.

As used herein, 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. Wherein, 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)). Accordingly, the expression "non-responsive to IFNα 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.

As used herein, 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). In general, it is particularly preferred that 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.

As used herein, the term "reference value" (also referred to as the optimal diagnostic cut-off value) refers to a value that reflects the condition of a population of CHB patients who are not responsive to IFN[alpha] treatment. In the present invention, 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 IFNα treatment or a marker in a CHB patient population sample that does not respond to IFNα treatment before and after IFNα 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 IFNα (eg, the level of the marker described above) Change factor) A value obtained by statistical analysis (statistical 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. Med (Zagreb).2016;26(3):297-307" and "Chen Weizhong.Selection of the best working point in the ROC curve[J].China Health Statistics,2006,23:157-158", all of which This is incorporated herein by reference.

As used herein, the term "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. 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.

As used herein, 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). In the real-time PCR (quantitative PCR) reaction, it can be assumed that each PCR cycle doubles the amplification product, and 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.

As used herein, the term "normalization" or "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.

As used herein, 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. Thus, 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.

As used herein, the term "Peripheral blood mononuclear cell (PBMC)" refers to a generic term for cells having a single cell nucleus in peripheral blood, including but not limited to lymphocytes (T cells, B cells, 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. In the present invention, the term "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. In the present invention, the term "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.

As used herein, the term "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.

As used herein, the term "subject" includes, but is not limited to, various animals, with humans being particularly preferred.

In the present invention, 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. Such 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. In certain embodiments, the diluent is a phosphate buffer or physiological saline.

As used herein, the term "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).

As used herein, the term "culture fluid" or "medium" refers to a nutrient that is capable of maintaining cellular activity. Usually 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. In the present invention, 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. In some embodiments, 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. In certain embodiments, when the sample is peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes, or other blood components containing PBMC, 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 IFNα treatment, unexpectedly found that the level of OAS2 in the pre-treatment PBMC samples of patients treated with IFNα was significantly higher than that of patients who did not respond to IFNα treatment. Differences, or pre-treatment PBMC samples from patients treated with IFNα, showed significant differences in OAS2 and/or UPS18 levels compared to patients who did not respond to IFNα treatment after in vitro induction, so OAS2 and/or UPS18 could be used to predict CHB A marker of IFNα therapeutic response in patients. Prior to this application, the field has long lacked a well-defined anti-HBV immune response index to predict the response of CHB patients to IFNα treatment. Based on this finding, the inventors have developed a new method for predicting the therapeutic effect of IFNα on CHB patients or the response of CHB patients to IFNα treatment.

Accordingly, in one aspect, 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.

In certain preferred embodiments, the HBV antigen is HBcAg. In certain preferred embodiments, the HBcAg has the amino acid sequence set forth in SEQ ID NO:38. In certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-37.

In a particularly preferred embodiment, the stimuli comprise as shown in SEQ ID NOs: 3-37, respectively. An antigenic fragment of the amino acid sequence.

In certain preferred embodiments, the first agent is an agent capable of detecting the mRNA level of the marker. Such 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. In certain embodiments, 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. Labeling the probe with an oxidase-labeled probe, a digoxigenin-labeled probe or a fluorophore (eg, FITC, FAM, TET, HEX, TAMRA, Cy3, Cy5, etc.); 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. In certain embodiments, the first reagent comprises a Taqman probe. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, the cDNA of USP18 has the nucleotide sequence set forth in SEQ ID NO:2.

In certain preferred embodiments, the first agent is an agent capable of detecting the protein level of the marker. Such 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. In certain embodiments, 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).

In certain preferred embodiments, the first agent determines the protein level of OAS2 or USP18 in the sample by immunological detection. 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, the first agent comprises an antibody against an OAS2 or USP18 protein or an antigen binding fragment thereof.

In certain preferred embodiments, the kits of the invention comprise a first agent capable of detecting the level of expression of OAS2, optionally further comprising IFNα and/or a stimulator.

In certain preferred embodiments, the kits of the invention comprise a first agent and IFN[alpha] capable of detecting USP18 expression levels, optionally further comprising an irritant.

In certain preferred embodiments, 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). In certain preferred embodiments, 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).

In certain preferred embodiments, 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).

In certain preferred embodiments, 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, β-actin, GAPDH, 18S rRNA, β2-MG, UBC, or α-tubulin. In certain preferred embodiments, 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. In certain preferred embodiments, 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.

In another aspect, the invention relates to the use of an agent capable of detecting the expression level of a marker for predicting the therapeutic effect of IFNα 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.

In certain preferred embodiments, 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.

In certain preferred embodiments, the HBV antigen is HBcAg. In certain preferred embodiments, the HBcAg has the amino acid sequence set forth in SEQ ID NO:38. In certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-37.

In a particularly preferred embodiment, the stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 3-37, respectively.

In certain preferred embodiments, the agent capable of detecting the level of expression of a marker is an agent capable of detecting the mRNA level of the marker. Such 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. In certain embodiments, 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. Labeling the probe with an oxidase-labeled probe, a digoxigenin-labeled probe or a fluorophore (eg, FITC, FAM, TET, HEX, TAMRA, Cy3, Cy5, etc.); 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. In certain embodiments, the agent capable of detecting the expression level of a marker comprises a Taqman probe. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, 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). In certain preferred embodiments, the agent capable of detecting the expression level of a marker quantifies the mRNA level of the marker by PCR. In a specific embodiment, the agent capable of detecting the expression level of a marker determines the mRNA level of the marker by real-time PCR.

In certain preferred embodiments, the agent capable of detecting the level of expression of a marker is an agent capable of detecting the protein level of the marker. Such 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. In certain embodiments, 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).

In certain preferred embodiments, 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. 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, 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.

In certain preferred embodiments, 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, β-actin, GAPDH, 18S rRNA, β2-MG, UBC or α-tubulin. In certain preferred embodiments, 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. In certain preferred embodiments, 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.

In certain preferred embodiments, the kit further comprises one or more reagents or devices selected from 1) to 6):

1) a diluent for diluting the sample, such as phosphate buffer or physiological saline;

2) an anticoagulant for preventing blood coagulation, such as heparin;

3) an agent for maintaining the activity of PBMC, such as a medium or a culture solution;

4) an agent for isolating PBMC, such as a lymphocyte separation solution, such as a sucrose-diammonia solution;

5) a blood collection device, such as a pyrogen-free vacuum blood collection tube;

6) PBMC separation device, such as a PBMC cell separation tube.

In certain preferred embodiments, 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).

In certain preferred embodiments, 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:

(1) determining the expression level of said marker in a sample from said subject using an agent capable of detecting the expression level of the marker, wherein said marker is OAS2;

(2) comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject will IFNα treatment produces a response;

Wherein 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.

In certain preferred embodiments, 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 IFNα 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 IFNα treatment It is not suitable for IFNα treatment.

In certain preferred embodiments, in step (2), the expression levels are statistically analyzed using a logistic regression model.

In certain preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between the sample and the sample to make the samples comparable, 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. Thus, in step (1), 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. In a specific embodiment, in step (1), 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 substance is compared with the mRNA level of the reference gene to obtain a 2- ^ΔCt value ( ^ΔCt =Ct- ^testable _marker- Ct _{internal reference gene} ), which is the normalized mRNA level of the marker, That is, the expression level of the marker is a multiple of the change in the expression level of the internal reference gene.

In certain preferred embodiments, prior to step (1), 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.

In certain preferred embodiments, 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:

(1) stimulating at least one sample from the subject as a test sample using IFNα, and using a sample that is not stimulated by IFNα as a control sample; or co-stimulation with the IFNα and the stimulator from the subject At least one sample is used as a sample to be tested, and a sample stimulated by the stimulator but not stimulated by IFNα is used as a control sample; wherein the stimulator is selected from the group consisting of an HBV antigen, an antigenic fragment of an HBV antigen, or any combination thereof;

(2) determining the expression level of the marker in each sample in the step (1) using an agent capable of detecting the expression level of the marker, and obtaining the expression level of the marker in the sample to be tested compared to the control sample. a fold change in expression level of the marker, the fold change being used as a relative expression level of the marker in the sample to be tested, wherein the marker is selected from OAS2 or USP18;

(3) comparing the relative expression level with a reference value, or performing statistical analysis on the relative expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject is Will respond to IFNα treatment;

Wherein 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.

In certain preferred embodiments, 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 IFNα Generating a response suitable for IFNα 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 IFNα treatment produces a response that is not suitable for IFNα treatment.

In certain preferred embodiments, in 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.

In certain preferred embodiments, in step (3), the relative expression levels are statistically analyzed using a logistic regression model.

In certain preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between the sample and the sample to make the samples comparable, 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. Thus, in step (2), 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. In a specific embodiment, in step (2), 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.

In certain preferred embodiments, prior to step (1), 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.

In another aspect, the invention provides a method for predicting the therapeutic effect of IFNα on a subject having chronic hepatitis B Or a method of the subject's response to IFNα treatment, or a method of obtaining a result of a diagnostic assay, wherein the diagnostic assay is for predicting a therapeutic effect of IFNα on a subject having chronic hepatitis B or the subject The response to IFNα treatment includes the following steps:

(1) providing a sample from the subject;

(2) determining the expression level of the marker in the sample, wherein the marker is OAS2;

(3) comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject will IFNα treatment produces a response;

Wherein 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.

In certain preferred embodiments, after step (3), 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.

In certain preferred embodiments, 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 IFNα 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 IFNα treatment It is not suitable for IFNα treatment.

In certain preferred embodiments, in step (2), the mRNA level or protein level of the marker is determined.

In certain preferred embodiments, in step (2), 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). In certain preferred embodiments, the mRNA level of the marker is quantified by PCR. In a specific embodiment, the mRNA level of the marker is determined by real-time PCR. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, the cDNA of USP18 has the nucleotide sequence set forth in SEQ ID NO:2.

In certain preferred embodiments, in step (2), the protein level of the marker is determined. In certain preferred embodiments, in step (2), 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 (2), an antibody against OAS2 is used or Its antigen-binding fragment is used to detect the protein level of OAS2.

In certain preferred embodiments, in step (3), the expression levels are statistically analyzed using a logistic regression model.

In certain preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between the sample and the sample to make the samples comparable, 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. Thus, in step (2), 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. In a specific embodiment, in step (2), 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 substance is compared with the mRNA level of the reference gene to obtain a 2- ^ΔCt value ( ^ΔCt =Ct- ^testable _marker- Ct _{internal reference gene} ), which is the normalized mRNA level of the marker, That is, the expression level of the marker is a multiple of the change in the expression level of the internal reference gene.

In certain preferred embodiments, 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.

In another aspect, the present invention provides a method for predicting a therapeutic effect of IFNα on a subject having chronic hepatitis B or a response of said subject to IFNα 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:

(1) providing at least two samples from the subject;

(2) stimulating at least one sample as a test sample using IFNα, and using a sample not stimulated by IFNα as a control sample; or co-stiming at least one sample from the subject using IFNα and a stimulator as a test sample a sample, and a sample stimulated by the stimulator but not stimulated by IFNα is used as a control sample; wherein the stimulator is selected from the group consisting of an HBV antigen, an antigenic fragment of an HBV antigen, or any combination thereof;

(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;

(4) comparing the relative expression level with a reference value, or performing statistical analysis on the relative expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject is Will respond to IFNα treatment;

Wherein 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.

In certain preferred embodiments, after step (4), the method further comprises the step of administering to the subject IFNα to treat chronic hepatitis B, wherein the subject is judged to be IFNα therapeutically produced Answer.

In certain preferred embodiments, 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 IFNα Generating a response suitable for IFNα 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 IFNα treatment produces a response that is not suitable for IFNα treatment.

In certain preferred embodiments, in step (2), the HBV antigen is HBVcAg. In certain preferred embodiments, the HBVcAg has the amino acid sequence set forth in SEQ ID NO:38. In certain preferred embodiments, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-37. In a particularly preferred embodiment, the stimulator comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 3-37, respectively.

In certain preferred embodiments, in step (3), 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). In certain preferred embodiments, the mRNA level of the marker is quantified by PCR. In a specific embodiment, the mRNA level of the marker is determined by real-time PCR. In certain embodiments, the cDNA of OAS2 has the nucleotide sequence set forth in SEQ ID NO: 1. In certain embodiments, the cDNA of USP18 has the nucleotide sequence set forth in SEQ ID NO:2.

In certain preferred embodiments, in step (3), 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.

In certain preferred embodiments, in 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.

In certain preferred embodiments, in step (4), the relative expression levels are statistically analyzed using a logistic regression model.

In certain preferred embodiments, in order to eliminate the difference in the initial value of the marker to be tested between the sample and the sample to make the samples comparable, 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. Thus, in step (3), 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. In a specific embodiment, in step (3), 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.

In certain preferred embodiments, 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.

The invention also encompasses the following exemplary embodiments:

1. For the induction of stimulation of interferon-stimulated genes (ISGs) in vitro A composition comprising a full length or a partial fragment of a HBV core antigen and/or IFNα.

2. 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).

3. The marker of item 2, wherein the interferon-inducing gene is selected from the group consisting of OAS2 and USP18.

4. A method of screening for a marker for predicting the therapeutic effect of alpha interferon in a chronic hepatitis B patient, the method comprising the steps of:

1) Collecting whole blood samples before administration of chronic hepatitis B patients and separating PBMC;

2) inducing a PBMC sample with a combination of an HBV core antigen polypeptide segment or an IFNα or HBV core antigen polypeptide segment and IFNα;

3) Collect the induced PBMC samples, extract RNA, and detect the mRNA level of ISGs after reverse transcription;

4) collecting the clinical IFNα treatment response results of the specimens and screening the ISGs;

Among them, comparing the different ISGs mRNA levels under the same induction conditions in the IFNα treatment response group and the non-response group, the difference in the level of ISGs mRNA in the IFNα treatment response group and the non-response group can be used as the basis for the therapeutic effect.

5. The method of clause 4, wherein the HBV core antigen polypeptide segment is a polypeptide segment of amino acid 1-183 of the HBV core antigen.

6. A kit for predicting the therapeutic effect of alpha interferon in chronic hepatitis B patients, comprising an HBV core antigen polypeptide segment and an IFNα inducer, and an agent for detecting markers OAS2 and USP18 levels.

7. The kit of item 6, wherein the HBV core antigen polypeptide segment is a polypeptide segment of amino acid 1-183 of the HBV core antigen.

8. Use of a reagent for detecting a marker level of OAS2 and/or USP18 for the preparation of a diagnostic agent for predicting the therapeutic effect of alpha interferon in a chronic hepatitis B patient.

9. Use of a reagent for detecting a marker level of OAS2 and/or USP18 in the preparation of a kit for predicting the therapeutic effect of alpha interferon in a chronic hepatitis B patient.

10. Use of the markers OAS2 and/or USP18 for predicting the therapeutic effect of alpha interferon in chronic hepatitis B patients.

11. The use of any of items 8-10, wherein the detecting of the markers OAS2 and/or USP18 comprises quantifying the mRNA levels of the markers OAS2 and/or USP18.

12. The use of any of items 8-10, wherein the mRNA levels of the markers OAS2 and/or USP18 are quantitatively detected by fluorescent PCR.

13. The use of any of items 8-10, wherein the mRNA for detecting the markers OAS2 and/or USP18 is used The sequence of the flat primers and probes is:

Advantageous effects of the invention

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 IFNα treatment and those who do not respond to IFNα treatment, thereby establishing a simple, A method for predicting the therapeutic effect of IFNα on a subject having chronic hepatitis B or the subject's response to IFNα treatment is convenient, rapid, and highly accurate and specific.

Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

(1) It was first discovered that the expression level of OAS2 in PBMC of patients with chronic hepatitis B without or after in vitro stimulation can be used to predict the outcome of IFNα treatment in patients with CHB;

(2) It was first discovered that the expression level of USP18 in PBMC of patients with chronic hepatitis B after in vitro stimulation can be used to predict the outcome of IFNα treatment in CHB patients;

(3) The operation involved is simple and convenient: for example, by collecting whole blood, separating PBMC, substituting it into a cell culture plate and adding IFNα, and then culturing it in a constant temperature incubator; after a certain period of time, performing mRNA or Detection of protein levels;

(4) The requirements for experimental conditions, technical capabilities, equipment and environment of personnel are not high;

(5) The prediction accuracy of the response to IFNα treatment is high;

(6) The cost is not high, it is easy to promote, and the scope of use is wide.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The various objects and advantageous aspects of the invention will be apparent to those skilled in the <

DRAWINGS

Figure 1 shows the significant difference in OAS2 mRNA levels of PBMCs in CHB patients between the IFNα treatment response group (Rs) and the non-response group (NRs) under different treatment conditions. Among them, 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 IFNα 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 IFNα treatment response group (Rs) and the non-response group (NRs) under different treatment conditions. Among them, 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 IFNα in vitro; Figure 2b shows the relative expression level of USP18 mRNA in the response group after PBMC was induced by IFNα 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 IFNα 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 IFNα therapy.

Sequence information

The sequence information referred to in the present invention is provided in Table 1 below.

Table 1: Sequence information

detailed description

The invention is described with reference to the following examples which are intended to illustrate, but not limit the invention. Unless otherwise indicated, it is carried out essentially according to conventional methods well known in the art and described in various references. The experiments and methods described in the examples (for example, molecular biology experimental methods and immunoassays). See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992). ), all of which are incorporated herein by reference. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially. The invention is described by way of example, and is not intended to limit the scope of the invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1. Peripheral blood PBMC isolation and in vitro induced culture

1.1 Peripheral blood PBMC separation

(1) Whole blood was centrifuged at 4000 rpm for 10 min.

(2) Aspirate the serum and add the same amount of PBS to dilute the remaining whole blood.

(3) A 15 mL centrifuge tube was prepared, and a lymphocyte separation solution (Ficoll-page Tm PLUS, GE Helathcare Life Sciences) equivalent to whole blood was added.

(4) The diluted whole blood is slowly added to the lymphocyte separation liquid along the tube wall to maintain a layered state.

(5) Centrifuge the above liquid at 1000 g for 22 min, and adjust the rate of the rate of increase and decrease to a minimum.

(6) Carefully pipette the white cloud-like lymphocyte layer into another centrifuge tube, add PBS and dilute it, centrifuge, 600g×7min, discard the supernatant. Further, it was diluted with PBS, centrifuged for 400 g × 7 min, and the supernatant was discarded. The cells were resuspended in 1 ml of culture medium.

(7) A 1:5 cell dilution was prepared by adding 10 μl of the prepared sample cell suspension and 40 μl of 0.4% trypan blue to a 1.5 ml centrifuge tube. 10 μl of trypan blue/cell mixed suspension was added to the blood cell counting plate and subjected to viable cell counting, wherein the number of cells per ml = cell count × dilution factor × 10 ⁴ .

1.2 In vitro induction culture of PBMC by IFNα and/or stimulator

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 IFNα and HBcAg polypeptide library) labeled as N, and the second hole is added with IFNα (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 μg/) was added to the third well. Strips/mL, a total of 35), the fourth well was added to the above IFNα and HBcAg polypeptide libraries. After the culture plate was placed in a carbon dioxide incubator at 37 ° C for 24 hours, cells were collected.

Example 2. Detection of mRNA levels of genes by real-time PCR

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 β-actin. Quantitative PCR detection system.

2.1 Extraction of RNA from PBMC

(1) The PBMC cells obtained in Example 1 were collected by centrifugation, and the supernatant was discarded; 250 μl of RPMI1640 medium and 750 μl of Tripure were added to each well and mixed well; 200 μl of chloroform was added per 1 ml of cells, mixed upside down, and allowed to stand at 4 ° C. Set for 10min.

(2) 12,000 rpm, 4 ° C, centrifugation for 15 min.

(3) After the above treatment, the sample in each tube is divided into three layers, and the uppermost layer is taken (note that the protein layer is not absorbed) and transferred to a new EPPC treated with DEPC, and an equal volume of isopropanol is added. Mix upside down and let stand at -20 °C for 15 min.

(4) 12,000 rpm, 4 ° C, centrifugation for 10 min, discard the supernatant.

(5) Two times with 1 mL of 75% ethanol, 12,000 rpm, 4 ° C, and centrifuged for 5 min.

(6) Discard the ethanol, air dry for 5-10 min, dissolve the RNA with 35 μL of DEPC water preheated at 55-60 ° C, and extract the extracted RNA and store it in a refrigerator at -80 ° C.

2.2 RNA reverse transcription

The concentration and purity of the RNA samples were determined using a spectrophotometer. 2μL RNA sample was used to measure the absorbance of A260 and A280 in a 2μL 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.

Reverse transcription system:

Reverse transcription was carried out for 30 min at 42 ° C by the above reverse transcription system. The obtained cDNA is a template in a fluorescent quantitative PCR detection system and stored at -20 °C.

2.3 Establishment of a real-time PCR system

(1) RT-PCR system:

Amplification reaction process:

95 ° C 3 min, then 95 ° C 10 sec, 55 ° C 45 sec, a total of 39 cycles.

Primers and probes for RT-PCR were designed using primer software and synthesized by Yingjun Biotechnology Co., Ltd. The specific sequence is shown in Table 2 below:

Table 2. Primers and probes for fluorescent quantitative PCR reactions

(2) Primer pre-experiment

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 β-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. To efficient amplification, it is further shown that 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.

Table 3. Pre-experiment of primers in a real-time PCR assay system

Example 3. Differential expression of OAS2, USP18 in CHB patients who responded to IFNα therapy and did not respond

The invention has systematically tracked 150 chronic hepatitis B specimens, among which 66 patients received IFNα treatment and 24 weeks of treatment results; among them, 47 patients had no response to IFNα treatment and 19 patients had IFNα 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.

Table 4. Characteristics of selected study specimens

Peripheral blood was collected from chronic hepatitis B patients who had not received IFNα treatment, PBMC was extracted according to the method of Example 1 above, and PBMC was treated and the method of Example 2 was used to extract RNA, and real-time PCR was performed to detect and calculate 17 genes to be tested ( mRNA levels of USP18, OAS2, LGP1, LAP3, GIP3, CEB1, ATF5, GIP2, OAS3, RPLP2, STXBP5, Viperin, RPS28, PI3KAP1, MX1, DUSP1, ETEF1), the mRNA levels were determined by relative quantification (Comparative Ct method) Calculated by calculating the Ct value of each target gene minus the Ct value of each internal reference β-actin, and obtaining the ΔCt value of each target gene (ΔCt=Ct _{target gene-} Ct _{internal reference gene} ) Calculating a 2- ^ΔCt value, which is a multiple of the expression level of the gene in the sample to be tested relative to the expression level of the reference gene (ie, the normalized mRNA level of the gene relative to the reference gene); The ΔCt value of the gene in the sample to be tested is subtracted _{from the} ΔCt value of the gene in the control sample to obtain a ΔΔCt value (ΔΔCt=ΔCt _{sample to be tested-} ΔCt _{control sample} ), and finally a 2- ^ΔΔCt value is calculated, which represents Measuring the gene Expression relative to the control sample fold change expression levels of the gene (i.e., the gene in the test sample relative to the control sample gene mRNA relative expression level).

By comparing the differences in mRNA levels of the above respective genes in the response group and the non-response group, it was found that the mRNA levels of OAS2 and USP18 were significantly different between the response group and the non-response group. The results are shown in Fig. 1 and Fig. 2, in which the PASMC cells of the OAS2N group were not induced in vitro, and the group represented the normalized mRNA level of OAS2 relative to the reference gene in the sample to be tested; OAS2 (IFNa+NN) The in vitro induction conditions of PBMC in the group, USP18 (IFNa+NN) group and USP18 (IFNa+HBcAg-HBcAg) group are shown in Table 5. 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.

Table 5. In vitro induction conditions of samples to be tested and control samples in different groups

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 IFNα 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 IFNα 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 IFNα 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 IFNα and HBcAg peptide libraries (containing HBcAg antigenic fragments shown in SEQ ID NO: 3-37) were accepted. In the PBMC samples before IFNα treatment, the relative expression level of USP18 mRNA in the response group was significantly lower than that in the non-responder group.

The above results indicate that OAS2 levels in pre-treatment PBMC samples in patients treated with IFNα are significantly higher than those in patients not responding to IFNα treatment; meanwhile, pre-treatment PBMC samples in IFNα-treated patients are induced in vitro by IFNα 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 4. ROC curve analysis of OAS2N detection method and OAS2 (IFNa+N-N) detection method

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. In this example, 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. Then use SPSS 17.0 software to normalize the mRNA level of OAS2 and the relative expression level of OAS2 mRNA as diagnostic indicators, and plot the ROC curve to test the use of OAS2N assay or OAS2 (IFNa+NN) assay alone to predict IFNα to CHB Performance of the treatment effect or response of CHB patients to IFNα treatment. The ROC curve analysis results are shown in Fig. 3 and Table 7. The results show that the area under the ROC curve (AUC) of the OAS2N test and the OAS2 (IFNa+NN) test method are 0.774 and 0.814, respectively; the sensitivity is 72% and 71%, respectively. The specificities were 80% and 83%, respectively.

The above results indicate that both methods have high sensitivity and specificity. OAS2N assay or OAS2 (IFNa+NN) assay can be used to predict the therapeutic effect of IFNα on CHB patients or CHB patients on IFNα therapy. Response, with high sensitivity and specificity.

Table 6. Main steps of the OAS2N assay and the OAS2 (IFNa+N-N) assay

Table 7. ROC analysis of OAS2N assay and OAS2 (IFNa+N-N) assay

Example 5. Analysis of OAS2 (IFNa+N-N) detection method, USP18 (IFNa+N-N) detection method and ROC curve analysis of combined detection

According to the experimental conditions of the OAS2 (IFNa+NN) group and the USP18 (IFNa+NN) group in Example 3, the corresponding OAS2 (IFNa+NN) detection method and USP18 (IFNa+NN) detection method were respectively established, and Table 8 records The main steps of the above two methods. In this example, 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 IFNα by OAS2 (IFNa+NN) assay or USP18 (IFNa+NN) assay alone. The performance of CHB patients or the response of CHB patients to IFNα treatment; and the above indicators as a joint detection index, through the logistic regression model, the statistical analysis values were obtained, and the ROC curve was again drawn with this value, and the joint detection of the two was evaluated. Predict performance. ROC curve analysis results As shown in Fig. 4 and Table 9, the results showed that the area under the ROC curve (AUC) of the two methods was 0.814 and 0.823, respectively; the sensitivity was 71% and 60%, respectively; the specificities were 83% and 95%, respectively. The AUC of the two methods combined detection can reach 0.861, and the sensitivity and specificity are 74% and 89%, respectively. The above results indicate that the OAS2 (IFNa+NN) assay and the USP18 (IFNa+NN) assay and the combined assay have good sensitivity and specificity, further indicating that the OAS2N assay or the OAS2 (IFNa+NN) assay is used alone. Or combined detection of both, have a good sensitivity and specificity for predicting the therapeutic effect of IFNα on CHB patients or the response of CHB patients to IFNα therapy.

Table 8. Main steps of the OAS2 (IFNa+N-N) assay and the USP18 (IFNa+N-N) assay

Table 9. ROAS analysis of OAS2 (IFNa+N-N) assay and USP18 (IFNa+N-N) assay and their combined detection

While the invention has been described in detail, it will be understood by the . The invention is generally divided into the appended claims and any equivalents thereof.

Claims (9)

1. A kit comprising a first reagent capable of detecting the expression level of a marker, optionally further comprising IFNα and/or a stimulator; wherein the marker is selected from OAS2 or USP18, the stimulator is selected from the group consisting of HBV antigen , an antigenic fragment of the HBV antigen, or any combination thereof;

   Preferably, the HBV antigen is HBcAg; more preferably, the HBcAg has an amino acid sequence as shown in SEQ ID NO: 38; preferably, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NO: 3-37;

   Preferably, the stimulant comprises an antigenic fragment having an amino acid sequence as shown in SEQ ID NOs: 3-37, respectively;

   Preferably, the first agent is an agent capable of detecting the mRNA level or protein level of the marker, for example, an agent capable of detecting the mRNA level of the marker.
2. The kit of claim 1 comprising a first agent capable of detecting the level of expression of OAS2, optionally further comprising IFNα and/or a stimulator.
3. The kit of claim 1 comprising a first agent and IFN[alpha] capable of detecting USP18 expression levels, optionally further comprising an irritant.
4. The kit of any one of claims 1 to 3, further comprising 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 diluting the sample (for example, phosphate buffer or physiological saline); an anticoagulant (for example, heparin) for preventing blood coagulation; an agent for maintaining PBMC activity (for example, a medium or a culture solution); An agent for isolating PBMC (for example, a lymphocyte separation solution such as a sucrose-diammonia solution); more preferably, the second reagent includes an agent for maintaining PBMC activity (for example, culture medium or culture) Liquid), and/or reagents for isolating PBMC (eg, lymphocyte separation fluid, such as a sucrose-diammonia solution);

   Preferably, the kit 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).
5. The use of an agent capable of detecting the expression level of a marker in the preparation of a kit for predicting the therapeutic effect of IFNα on a subject having chronic hepatitis B or the response of the subject to IFNα treatment; The marker is selected from OAS2 or USP18;

   Preferably, the kit further comprises IFNα and/or a stimulator selected from the group consisting of an HBV antigen, an antigenic fragment of an HBV antigen, or any combination thereof;

   Preferably, the HBV antigen is HBcAg; more preferably, the HBcAg has an amino acid sequence as shown in SEQ ID NO: 38; preferably, the antigenic fragment has an amino acid sequence selected from the group consisting of SEQ ID NO: 3-37;

   Preferably, the stimulant comprises an antigenic fragment having an amino acid sequence as shown in SEQ ID NOs: 3-37, respectively;

   Preferably, the reagent capable of detecting the expression level of the marker is an agent capable of determining the mRNA level or protein level of the marker, for example, an agent capable of determining the mRNA level of the marker; more preferably, the capable An agent for detecting the expression level of the marker quantitatively determines the mRNA level of the marker by PCR;

   Preferably, the kit further comprises one or more reagents or devices selected from 1) to 6):

   1) a diluent for diluting the sample, such as phosphate buffer or physiological saline;

   2) an anticoagulant for preventing blood coagulation, such as heparin;

   3) an agent for maintaining the activity of PBMC, such as a medium or a culture solution;

   4) an agent for isolating PBMC, such as a lymphocyte separation solution, such as a sucrose-diammonia solution;

   5) a blood collection device, such as a pyrogen-free vacuum blood collection tube;

   6) a PBMC separation device, such as a PBMC cell separation tube;

   Preferably, the kit includes an agent (for example, a medium or a culture solution) for maintaining PBMC activity, and/or an agent for isolating PBMC (for example, a lymphocyte separation solution such as polysucrose-diatrizoate) Solution).
6. The use according to claim 5, wherein the kit predicts the therapeutic effect of IFNα on a subject having chronic hepatitis B or the response of the subject to IFNα treatment by a method comprising the following steps:

   (1) determining the expression level of said marker in a sample from said subject using an agent capable of detecting the expression level of the marker, wherein said marker is OAS2;

   (2) comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject will IFNα Treatment produces a response;

   Wherein 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;

   Preferably, in step (2), the expression level is statistically analyzed using a logistic regression model;

   Preferably, prior to step (1), the method further comprises one or more of the following steps: (a) obtaining a sample from the subject using a blood collection device; (b) treating the blood collection device with an anticoagulant Or a sample from the subject; (c) treating the sample from the subject with an agent for maintaining PBMC activity; (d) diluting the sample from the subject with a diluent; and (e PBMC were isolated from samples from the subject using reagents or PBMC separation devices for isolating PBMC.
7. The use according to claim 5, wherein the kit predicts the therapeutic effect of IFNα on a subject having chronic hepatitis B or the response of the subject to IFNα treatment by a method comprising the following steps:

   (1) stimulating at least one sample from the subject as a test sample using IFNα, and using a sample that is not stimulated by IFNα as a control sample; or co-stimulation with the IFNα and the stimulator from the subject At least one sample is used as a sample to be tested, and a sample stimulated by the stimulator but not stimulated by IFNα is used as a control sample; wherein the stimulator is selected from the group consisting of an HBV antigen, an antigenic fragment of an HBV antigen, or any combination thereof;

   (2) determining the expression level of the marker in each sample in the step (1) using an agent capable of detecting the expression level of the marker, and obtaining the expression level of the marker in the sample to be tested compared to the control sample. a fold change in expression level of the marker, the fold change being used as a relative expression level of the marker in the sample to be tested, wherein the marker is selected from OAS2 or USP18;

   (3) comparing the relative expression level with a reference value, or performing statistical analysis on the relative expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject is Will respond to IFNα treatment;

   Wherein 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;

   Preferably, in step (2), a change ratio of the mRNA level of the marker in the sample to be tested compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the test is to be tested. The relative expression level of the mRNA of the marker in the sample;

   Preferably, in step (3), the relative expression level is statistically analyzed using a logistic regression model. analysis;

   Preferably, prior to step (1), the method further comprises one or more of the following steps: (a) obtaining a sample from the subject using a blood collection device; (b) treating the blood collection device with an anticoagulant Or a sample from the subject; (c) treating the sample from the subject with an agent for maintaining PBMC activity; (d) diluting the sample from the subject with a diluent; and (e PBMC were isolated from samples from the subject using reagents or PBMC separation devices for isolating PBMC.
8. A method for predicting a therapeutic effect of IFNα on a subject having chronic hepatitis B or a response of the subject to IFNα treatment, or a method of obtaining a result of a diagnostic analysis, wherein the diagnostic assay is for predicting IFNα The therapeutic effect of a subject with chronic hepatitis B or the subject's response to IFNα treatment includes the following steps:

   (1) providing a sample from the subject;

   (2) determining the expression level of the marker in the sample, wherein the marker is OAS2;

   (3) comparing the expression level with a reference value, or performing statistical analysis on the expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject will IFNα treatment produces a response;

   Wherein 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;

   Preferably, in step (2), the mRNA level or protein level of the marker is determined, for example, determining the mRNA level of the marker; more preferably, in step (2), the marker is quantitatively determined by PCR. mRNA level of the substance;

   Preferably, in step (3), the expression level is statistically analyzed using a logistic regression model;

   Preferably, prior to step (1), further comprising one or more of the following steps: (a) obtaining a sample from the subject; (b) adding anticoagulation to a sample from the subject a reagent such as heparin; (c) obtaining PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer) from a sample from the subject; (d) adding to a sample from the subject Culture medium or medium; and, (e) diluting the sample from the subject.
9. A method for predicting a therapeutic effect of IFNα on a subject having chronic hepatitis B or a response of the subject to IFNα treatment, or a method of obtaining a result of a diagnostic analysis, wherein the diagnostic assay is for predicting an IFNα pair The therapeutic effect of a subject with chronic hepatitis B or the subject's response to IFNα treatment includes the following steps:

   (1) providing at least two samples from the subject;

   (2) stimulating at least one sample as a test sample using IFNα, and using a sample not stimulated by IFNα as a control sample; or co-stiming at least one sample from the subject using IFNα and a stimulator as a test sample a sample, and a sample stimulated by the stimulator but not stimulated by IFNα is used as a control sample; wherein the stimulator is selected from the group consisting of an HBV antigen, an antigenic fragment of an HBV antigen, or any combination thereof;

   (3) determining the expression level of the marker in each sample in the step (2), and calculating 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;

   (4) comparing the relative expression level with a reference value, or performing statistical analysis on the relative expression level to obtain a statistical analysis value, comparing the statistical analysis value with a reference value, and determining whether the subject is Will respond to IFNα treatment;

   Wherein 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;

   Preferably, in the step (2), the HBV antigen is HBVcAg; more preferably, the HBVcAg has an amino acid sequence as shown in SEQ ID NO: 38; preferably, the antigenic fragment has a selected from the group consisting of Amino acid sequence: SEQ ID NO: 3-37; more preferably, the stimulant comprises an antigenic fragment having an amino acid sequence as set forth in SEQ ID NOs: 3-37, respectively;

   Preferably, in step (3), the mRNA level or protein level of the marker is determined; for example, determining the mRNA level of the marker; more preferably, in step (3), the marker is quantitatively determined by PCR mRNA level of the substance;

   Preferably, in step (3), a change ratio of the mRNA level of the marker in the sample to be tested compared to the mRNA level of the marker in the control sample is obtained by comparing the Ct method, that is, the test is to be tested. The relative expression level of the mRNA of the marker in the sample;

   Preferably, in step (4), the relative expression level is statistically analyzed using a logistic regression model;

   Preferably, prior to step (1), further comprising one or more of the following steps: (a) obtaining a sample from the subject; (b) adding anticoagulation to a sample from the subject Agent, such as heparin; (c) from Obtaining PBMC or a blood component containing PBMC (for example, a peripheral blood leukocyte layer) in the sample of the subject; (d) adding a culture solution or a medium to the sample from the subject; and, (e Diluting the sample from the subject.

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