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Immune-mediated effects targeting hepatitis C virus in a syngeneic replicon cell transplantation mouse model.

ABSTRACT: OBJECTIVE:HCV is characterised by its ability to establish chronic infection in hepatocytes and to replicate in the presence of an inflammation. We mimicked this situation in vivo in immune-competent mice by syngeneic transplantation of HCV replicon-containing mouse hepatoma cells. DESIGN:A total of 5?million H-2b positive Hep56.1D cells, carrying a subgenomic genotype (gt) 2a replicon (HCV replicon cells) or stably expressing comparable levels of the HCV NS3/4A protease/helicase complex (NS3/4A hepatoma cells), were injected subcutaneously into syngeneic H-2b-restricted mice. Kinetics of tumour growth, HCV RNA replication levels and HCV-specific immune responses were monitored. For immune monitoring, new H-2b-restricted cytotoxic T cell epitopes within the gt2a NS3/4A region were mapped. Immune mice were generated by DNA-based vaccination. RESULTS:HCV replicon and NS3/4A hepatoma cells generated solid tumours in vivo. Similar to what is seen in human HCV infection did HCV RNA replicate in the presence of inflammation. NS3/4A-specific CD8+ T cells seemed to transiently reduce HCV RNA levels. Both CD4+ and CD8+ T cells were required for protection against tumour growth. Vaccine-induced NS3/4A(gt2a)-specific T cells protected against HCV replicon tumours in wild-type, but not in HCV NS3/4A(gt1a)-transgenic mice with dysfunctional HCV-specific T cells. Importantly, as in human HCV infection, HCV replicon cells neither primed nor boosted a strong NS3/4A-specific T cell response. CONCLUSION:Syngeneic transplantation of mouse HCV replicon cells into immune-competent animals mirrors many in vivo events in humans. This system is versatile and can be applied to any genetically modified H-2b-restricted mouse strain.

SUBMITTER: Levander S

PROVIDER: S-EPMC6204962 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Immune-mediated effects targeting hepatitis C virus in a syngeneic replicon cell transplantation mouse model.

Levander Sepideh S Holmström Fredrik F Frelin Lars L Ahlén Gustaf G Rupp Daniel D Long Gang G Bartenschlager Ralf R Sällberg Matti M

Gut 20170623 8

<h4>Objective</h4>HCV is characterised by its ability to establish chronic infection in hepatocytes and to replicate in the presence of an inflammation. We mimicked this situation in vivo in immune-competent mice by syngeneic transplantation of HCV replicon-containing mouse hepatoma cells.<h4>Design</h4>A total of 5 million H-2<sup>b</sup> positive Hep56.1D cells, carrying a subgenomic genotype (gt) 2a replicon (HCV replicon cells) or stably expressing comparable levels of the HCV NS3/4A proteas ...[more]

PMID: 28646094

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Project description:To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA.The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 10(8) ± 2.747 × 10(7) vs 5.368 × 10(7) ± 1.016 × 10(7), P < 0.05; 1.049 × 10(8) ± 2.747 × 10(7) vs 5.243 × 10(7) ± 1.194 × 10(7), P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells.Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.

Dataset Information

Immune-mediated effects targeting hepatitis C virus in a syngeneic replicon cell transplantation mouse model.

Publications

Immune-mediated effects targeting hepatitis C virus in a syngeneic replicon cell transplantation mouse model.

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