Employing human adipose-derived stem cells to propagate serum-derived hepatitis C virus and use thereof
Inventors
Assignees
Frontier Bio-Drug Development Ltd
Publication Number
US-10273461-B2
Publication Date
2019-04-30
Expiration Date
2035-01-07
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Abstract
Hepatitis C virus replication at extrahepatic sites has been suggested; however, complete viral replication has only been confirmed in hepatocytes. Here we show that human adipogenic DLK-1+ stem cells (hADSC) freshly isolated from HCV-infected individuals contained viral transcripts, replication intermediates and viral antigens in vivo, and viral transcripts increased in supernatants upon prolonged ex vivo culture. Furthermore, naive hADSC isolated from HCV (−) individuals support complete replication of clinical isolates in vitro, and the infection is donor-nonspecific for cells and cross-genotypic for viruses. Viral infection/replication is mediated through CD81, LDL-R, SR-B1, EGFR, Apolipoprotein E, occludin, claudin-1, NPC1L1 and diacylglycerol acetyltransferase-1, and can be inhibited by anti-viral drugs. In addition, the physical properties of hADSC-propagated viral particles resemble clinical isolates more than JFH1/HCVcc, and viruses propagated by in vitro infected hADSC are infectious to primary human hepatocytes. Therefore, hADSC are an in vivo HCV reservoir and represent a novel venue of clinical virus-host interaction. hADSC can also be exploited as a physiologically relevant primary cell culture system to propagate clinical isolates.
Core Innovation
The invention discloses systems and methods employing human adipose-derived DLK-1+ stem cells (hADSCs) for propagating hepatitis C virus (HCV). It reveals that hADSCs, freshly isolated from HCV-infected individuals, contain viral transcripts, replication intermediates, and viral antigens in vivo, and that naive hADSCs from HCV-negative individuals support complete replication of clinical HCV isolates in vitro. The infection is donor-nonspecific for cells and cross-genotypic for viruses, mediated through host factors such as CD81, LDL-R, SR-B1, EGFR, Apolipoprotein E, occludin, claudin-1, NPC1L1, and diacylglycerol acetyltransferase-1 and can be inhibited by antiviral drugs.
The problem being solved addresses the limitations of existing in vitro systems for studying extrahepatic HCV replication. Current models employ primarily hepatocytes, which are difficult to maintain and exhibit donor-to-donor variability, or use molecular clones not reflective of natural virus. There is a lack of physiologically relevant primary cell culture systems allowing propagation of serum-borne clinical HCV isolates, particularly at extrahepatic sites. The invention provides an alternative, physiologically relevant cell culture system using hADSCs that supports complete replication of HCV, enabling better clinical virus-host interaction studies and anti-viral screening.
Claims Coverage
The patent contains four independent claims covering systems, methods for propagating HCV using hADSCs, methods for diagnosing HCV infection using hADSCs, and methods for screening anti-HCV compounds using hADSCs.
System for propagating hepatitis C virus using human adipose-derived stem cells
A system comprising hADSCs, culture medium suitable for culturing hADSCs, and HCV; wherein hADSCs are used to propagate HCV under conditions suitable for viral replication.
Method for propagating hepatitis C virus using hADSCs
A method comprising contacting hADSCs with HCV in suitable culture medium and conditions that support complete viral replication.
Method for diagnosing hepatitis C virus infection using hADSCs
A method including providing hADSCs, incubating them with a biological sample from a subject in suitable culture medium, culturing to permit HCV replication, and detecting the level of HCV replication as an indication of infection.
Method for screening anti-hepatitis C virus compounds using hADSCs
A method comprising contacting hADSCs with HCV in absence and presence of a candidate compound, determining HCV levels in both conditions, comparing them, and identifying a compound as anti-HCV if it reduces HCV level.
The claims collectively cover systems and methods for propagation, diagnosis, and therapeutic screening of HCV using hADSCs, emphasizing the capability of hADSCs to support complete replication of clinical HCV isolates in vitro.
Stated Advantages
hADSCs represent a novel in vivo reservoir for HCV, providing new insight into virus-host interactions at extrahepatic sites.
The hADSC-based system supports complete viral replication of clinical HCV isolates, overcoming limitations of current in vitro models that use hepatocytes or molecular clones.
The system enables physiologically relevant studies of HCV life cycle and facilitates the screening of antiviral compounds using primary human cells.
Documented Applications
Propagating clinical isolates of hepatitis C virus in vitro using human adipose-derived stem cells.
Conducting hepatitis C virus life cycle analyses in a physiologically relevant primary cell culture system.
Diagnosing hepatitis C virus infections by incubating hADSCs with biological samples and detecting viral replication.
Screening candidate anti-viral compounds for efficacy against hepatitis C virus using hADSC-based infection models.
Characterizing hepatitis C virus genotypes and infection status in subjects using hADSC cultures.
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