Optimization Of A Hepatitis C Virus Mouse Model With Reporter Gene And Its Application In Vaccine Development

Hepatitis C virus (HCV) is a hepatotropic pathogen of vital importance to public health. Chronic HCV infection frequently results in serious liver disease, such as cirrhosis and hepatocellular carcinoma. For lack of suitable animal models, no vaccine against HCV infection is currently available and the interferon-based treatments in use tend to be ineffective and have significant side effects. A new suitable animal model will hold the promise for new routes for the study of HCV pathogenesis, as well as drug and vaccine development.Previously, we have reported an HCV model system based on hydrodynamic injection andφC31 integrase. The HCV expression cassette was introduced into mice livers by hydrodynamic injection and integrated permanently into mouse chromosome by theφC31 integrase to achieve long-term expression. But the recombination efficiency ofφC31 remained limited. And the repeatability and stability of this model have not been validated. To improve the recombination efficiency, the recombination efficiency ofφC31 andφC31o integrase in mice livers was compared. Our study confirmed thatφC31o integrase improved recombination efficiency in mouse livers and could be a feasible alternative method to create genetically modified laboratory mouse models. Then, the HCV expression cassette was introduced into mice livers by hydrodynamic injection and integrated permanently into mouse chromosome by theφC31o integrase. Expression of HCV in mouse livers was greatly improved. The repeatability and stability of this model was evaluated by repeated experiments. The use of luciferase as a reporter for HCV made possible the real-time, noninvasive, whole-body imaging of HCV expression under a variety of physiologic and phathological conditions. Finally, the inhibitory effect of NS3/4A DNA vaccine against HCV was evaluated by this model through bioluminescence imaging.The main research content and results are as follows:1. Analysis ofφC31 andφC31o integrase activity in mouse livers.To assess the recombination efficiency ofφC31o integrase in mouse livers,φC31 integrase expression vector pCMV-int orφC31o integrase expression vector Pphic31φalong with vector pAA-Fluc were cotransfected into mouse livers by hydrodynamic injection. Bioluminescence imaging revealed that mice coinjected with Pphic31φall displayed high luciferase activity for over 60 days. In marked contrast, mice coinjected with pCMV-int displayed either no or low luciferase activity. This indicated thatφC31o integrase improved recombination efficiency in mice livers.?2. Sustained expression of HCV in mouse model.ΦC31o integrase was then utilized to prolong HCV expression in the liver. pAA-Fluc-IRES-HCV together with Pphic31φwas hydrodynamically injected into mice tail veins. Levels of luciferase, monitored over time, remained at high levels for over 180 days, indicating that recombination had occurred. Expression of the reporter and HCV proteins mediated by Pphic31φin mouse livers was validated by Western blot and RT-PCR. Nest PCR confirmed the integration mediated byφC31o integrase. The repeatability and stability of this model was validated by repeated experiments.?3. Application of this mouse model in vaccine development.To compare the transfection efficiency between hydrodynamic limb vein and electroporation, plasmid pGL3-CMV containing Fluc reporter gene was transfected into different mouse limb by hydrodynamic limb vein injection (HLV) and? electroporation, respectively. Bioluminescence imaging was used to real time detect the Fluc expression. Our experiment confirmed that Fluc expression was higher and longer in HLV-transfected group.Then, BALB/c mice were immunized with pVAX-NS3/4A or pVAX by HLV. The plasmid pAA-Fluc-IRES-HCV was cotransfected with plasmid Pphic31φinto mice by hydrodynamics-based procedure 14 days post last immunization. Bioluminescence imaging revealed that Fluc activity decreased significantly 9 days after injection in the group immunized with pVAX-NS3/4A, indicating that this model has potential in vaccine development.In conclusion,this study described an HCV model system based on hydrodynamic injection andφC31o integrase. The HCV expression cassette was introduced into mice livers by hydrodynamic injection and integrated permanently into mouse chromosome by theφC31o integrase to achieve long-term expression. The repeatability and stability of this model was validated by repeated experiments. Also, the potential of this mouse model in vaccine development was validated.