Exosome-mediated diagnosis of hepatitis virus infections and diseases

Abstract

A method for diagnosing hepatitis virus infection or a hepatitis disease condition in a subject based on hepatitis virus-associated biomarkers present on exosomes in a bodily fluid sample from the subject is disclosed. Also disclosed are a method for monitoring the course of a hepatitis virus infection or a hepatitis disease condition in a subject and a method for monitoring effectiveness of treatment to a subject with an anti-hepatitis virus agent based on hepatitis virus-associated biomarkers present on exosomes in bodily fluid samples from the subject, as well as a kit for diagnosing hepatitis virus infection and/or a hepatitis disease condition in a subject based on hepatitis virus-associated biomarkers on exosomes in bodily fluid samples from the subject.

What is claimed is: 
     
         1 . A method for diagnosing hepatitis virus infection or a hepatitis disease condition in a subject caused by a hepatitis virus, comprising:
 (a) preparation of an exosome(s) from a bodily fluid sample from a subject;   (b) contacting said exosome preparation with one or more hepatitis virus-associated biomarker binding agent(s) selective for a hepatitis virus and/or one or more detection reagent(s) suitable for detecting one or more hepatitis virus-associated biomarker(s); and   (c) determining whether the exosome preparation comprises at least one hepatitis virus biomarker,   wherein a determination of the presence of the at least one hepatitis virus-associated biomarker in step (c) is indicative of a hepatitis virus infection or hepatitis disease condition in the subject and wherein a determination of the absence of the at least one hepatitis virus-associated biomarker in step (c) is indicative of the absence of hepatitis virus infection or a hepatitis disease condition in the subject.   
     
     
         2 . The method of  claim 1 , wherein the bodily fluid is urine. 
     
     
         3 . The method of  claim 1 , wherein the exosome preparation comprises whole exosomes. 
     
     
         4 . The method of  claim 1 , wherein the exosome preparation comprises an exosome lysate. 
     
     
         5 . The method of  claim 1 , wherein the hepatitis-virus is selected from the group consisting of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV). 
     
     
         6 . The method of  claim 1 , wherein step (b) further comprises contacting each one of a plurality of exosome preparations with one or more different hepatitis virus-associated biomarker binding agent(s). 
     
     
         7 . The method of  claim 6 , wherein each hepatitis virus-associated biomarker binding agent is selective for a common hepatitis virus. 
     
     
         8 . The method of  claim 6 , wherein each hepatitis virus-associated biomarker binding agent is selective for a different hepatitis virus. 
     
     
         9 . The method of  claim 6 , wherein the biomarker binding agents are selective for HAV, HBV, HCV, HDV and HEV. 
     
     
         10 . The method of  claim 1 , wherein step (b) comprises contacting said exosome preparation with one or more detection reagents capable of detecting one or more hepatitis virus-associated biomarker(s), wherein said detection reagents are selected from the group consisting of one or more reagents for reverse transcription of RNAs, one or more PCR reagents for amplification and detection of genomic nucleic acids or mRNAs and one or more oligonucleotides for detecting miRNAs. 
     
     
         11 . The method of  claim 1 , wherein step (b) comprises contacting said exosome preparation with one or more hepatitis virus-associated biomarker binding agent(s) and one or more detection reagent(s) suitable for detecting one or more hepatitis virus-associated biomarkers, wherein said one or more detection reagents are suitable for detecting a miRNA selected from the group consisting of miR-92a, miR-122, miR-148a, miR-194, miR-155, miR-483-5p and miR-671-5p, miR-106b, miR-1274a, miR-130b, miR-140-3p, miR-151-3p, miR-181a, miR-19b, miR-21, miR-24, miR-375, miR-5481, miR-93, and miR-941, miRNA-1, miRNA-122, miR-584, miR-517c, miR-378, miR-520f, miR-142-5p, miR-451, miR-518d, miR-215, miR-376a, miR-133b and miR-367. 
     
     
         12 . The method of  claim 1 , wherein step (b) comprises contacting one or more exosome preparation with one or more agents capable of detecting liver damage and/or hepatocellular carcinoma selected from the group consisting of CD10, CD26, CD81, AST, ALT, α-fetoprotein (AFP) and its various isoforms, including AFP-L1, AFP-L2, AFP-L3, AFP-P4, AFP-P5 (E-PHA), and monosialylated AFP; des-carboxyprothrombin (DCP), α-1-fucosidase (AFU), γ-glutamyl transferase, glypican-3 (GPC-3), squamous cell carcinoma antigen (SCCA), golgi protein 73 (GP73) and mucin 1 (MUC-1), 14-3-3 gamma, alpha-1-fucosidase, gamma-glutamyl transferase, glypican-3, squamous cell carcinoma antigen, protein C (PROC), retinal binding protein 4 (RBP4), alpha-1-B glycoprotein (A1BG), alpha-1-acid glycoprotein (AGP), Mac-2-binding protein (M2BP), complement Factor H (CFH) and insulin-like growth factor binding protein acid labeled subunit (IGFALS). 
     
     
         13 . The method of  claim 1 , wherein said exosome preparation is isolated by differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, size-exclusion chromatography, ultracentrifugation, magnetic activated cell sorting (MACS) or a combination thereof. 
     
     
         14 . The method of  claim 1 , further comprising the step of administering a therapeutic drug for hepatitis. 
     
     
         15 . A method for monitoring the course of hepatitis virus infection or a hepatitis disease condition in a subject, comprising:
 (a) measuring the level of one or more hepatitis virus-associated biomarkers in a first exosome preparation representing a first time point from a subject;   (b) measuring the level of the one or more hepatitis virus-associated biomarkers in a second exosome preparation representing a first time point from a subject;   (c) comparing the level of the one or more hepatitis virus-associated biomarkers in the first exosome preparation to the level of the one or more hepatitis virus-associated biomarkers in the second exosome preparation; and   (d) determining the disease progression between the first time point and the second time point based on the result of step (c).   
     
     
         16 . A method for monitoring the effectiveness of treatment to a subject with an anti-hepatitis virus agent, comprising:
 (a) measuring the level of one or more hepatitis virus-associated biomarkers in a first exosome preparation representing a first time point from a subject;   (b) measuring the level of the one or more hepatitis virus-associated biomarkers in a second exosome preparation representing a first time point from a subject;   (c) comparing the level of the one or more hepatitis virus-associated biomarkers in the first exosome preparation to the level of the one or more hepatitis virus-associated biomarkers in the second exosome preparation; and   (d) determining the disease progression between the first time point and the second time point based on the result of step (c).   
     
     
         17 . A kit for diagnosing a hepatitis virus infection and/or a hepatitis disease condition in a subject, comprising:
 one or more reagents for preparing an exosome preparation;   one or more hepatitis virus-associated biomarker binding agents and detection reagents selective for a hepatitis virus,   and one or more hepatitis virus-associated biomarker standards.   
     
     
         18 . The kit of  claim 17 , further comprising one or more centrifuge filters for collecting exosomes in bodily fluid samples, wherein the one or more centrifuge filters have a molecular weight cutoff of 500 kd-50 kd. 
     
     
         19 . The kit of  claim 17 , wherein the bodily fluid is urine. 
     
     
         20 . The kit of  claim 17 , wherein said hepatitis virus is selected from the group consisting of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV).