Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment (original) (raw)
Robust repopulation with human hepatocytes. To undertake infections with hepatotropic human viruses, we first devised novel strategies to increase the extent of human hepatocyte reconstitution in Fah–/–Rag2–/–Il2rg–/– mice. This involved primarily increasing the number of transplanted human hepatocytes (see Methods for details). Briefly, we transplanted 50 animals with 3 × 106 to 5 × 106 human hepatocytes from 12 different donors (4–67 years; Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI40094DS1) and with no pretreatment other than NTBC. Table 1 shows that in 50 reconstituted mice, an average human albumin level of 7.6 mg/ml was detected in the mouse serum. Based on quantitative morphometry of FAH-positive, human hepatocytes in the mouse liver, this corresponds to a chimerism of 42% (see also Figure 1F). These levels were about 10–100 times higher than previously reported (16). Figure 1, A–D, shows a highly repopulated mouse with near complete humanization of the mouse liver as evidenced by immunostaining for FAH and human-specific cytokeratin 18 antibodies. It thus appears that high human chimerism can be achieved in our Fah–/–Rag2–/–Il2rg–/– mouse strain by simply transplanting more human hepatocytes.
Nearly complete repopulation of the Fah–/–Rag2–/–Il2rg–/– mouse liver with human hepatocytes. (A–E) Immunostaining of the same liver for FAH (A and B), human-specific cytokeratin-18 (C and D), and no primary antibody (control, E). (B and D) Higher-magnification views of the boxed areas in A and C, respectively. Scale bars: 1 mm (A, C, and E), 50 μm (B and D). (F) Correlation of human albumin level in the murine serum and repopulation as assessed by morphometry of FAH immunostaining. A, C, and E are composite images.
Repopulation rates of 50 human liver chimeric mice
Since human albumin is a convenient, noninvasive marker for human hepatocyte repopulation, we analyzed the correlation between human albumin levels in the murine serum and immunostaining for FAH. Figure 1F shows a good correlation by regression analysis (r2 = 0.88). Therefore, only a few microliters of murine blood are needed for estimating human hepatocyte repopulation.
HBV infection in human liver chimeric mice. We inoculated 4 highly repopulated mice with HBV (see Methods). After 6 weeks, maximal serum titers of up to 8.15 × 108 genome equivalents (GE)/ml were observed. Analysis of liver tissue at 2 and 7 weeks after inoculation confirmed intrahepatic propagation and revealed a commensurate increase in HBV cccDNA (from 1.2 to 98 copies/ng human liver DNA), HBV RNA (from 206 to 5,550 copies/ng human liver RNA), and HBV DNA replicative intermediates (from 1,001 to 58,000 copies/ng human liver DNA). Figure 2A shows a highly repopulated chimeric mouse liver (~80% chimerism) that was inoculated with HBV. Costaining for HBcAg and FAH revealed extensive infection of human hepatocytes (Figure 2, B–D). Within a given cluster, 50%–80% of human hepatocytes stained for HBcAg with the typical strong nuclear and faint cytoplasmic distribution. While mouse tissue was subject to tyrosine-related toxicity and hence cell death, we did not observe any cytopathic effect related to HBV infection in the human counterpart (Supplemental Figure 1).
HBV-infected mouse with high human chimerism. (A) Immunostaining for FAH and counterstaining with hematoxylin shows that 80% of the liver consists of human hepatocytes. (B) Immunostaining for HBcAg (red) and counterstaining with DAPI (blue). (C and D) Fluorescent costaining for FAH (green) and HBcAg (red). The image in D is a higher-magnification view of the boxed area in C. B and C show the same human hepatocyte cluster. Scale bars: 1 mm (A), 50 μm (B–D). A is a composite image.
Interestingly, 4 additional animals with very low human albumin levels (0.04–0.16 mg/ml) could also be infected with HBV. The presence of HBV intermediates, cccDNA, as well as HBV RNA in liver tissue confirmed viral propagation. The sparse human hepatocytes in the murine liver stained positive for HBcAg (Supplemental Figure 2).
HCV infection in mice with high human chimerism. We inoculated 5 highly repopulated chimeric mice with 2 × 104 focus-forming units (ffu) of HCV genotype 2a (JFH-1 strain). HCV RNA could be detected 24 hours later and increased after 1 week in inoculated chimeric mice (Figure 3A). Interestingly, there was no further increase in titer thereafter. Six weeks after inoculation, we harvested the livers and analyzed them for HCV RNA. Figure 3C shows that HCV RNA could be detected in all of the examined livers.
HCV infection of mice with high human chimerism. HCV RNA in mouse serum after inoculation with HCV genotype 2a (A) or clinical isolate of HCV genotype 1a or chimeric HCV genotypes (1a/2a and 1b/2a) (B). (C) HCV RNA intermediates detected in chimeric livers and human albumin levels in the murine serum of HCV-infected mice. HCV RNA was normalized to human GAPDH levels and expressed as GE per μg total RNA. (D) Antiviral treatment with peg-IFN alone, peg-IFN and ribavirin, and peg-IFN and Debio 025 for 2 weeks. Results are shown as normalized mean ± SD. (E–G) Fluorescent costaining of an HCV-infected chimeric liver for FAH (green) and HCV NS5a (red); nuclear counterstaining with DAPI (blue). (E) FAH and nuclear staining. (F) NS5a and nuclear staining. (G) Merge of E and F showing colocalization of HCV and human hepatocytes in yellow. (H) Control mouse liver repopulated with human hepatocytes but not inoculated with HCV. Costaining for FAH (green) and HCV NS5a (red); nuclear counterstaining with DAPI (blue). Scale bars: 50 μm.
In order to determine whether these mice were susceptible to other HCV genotypes, we inoculated 2 chimeric mice with a clinical sample of HCV genotype 1a and 1 mouse each with 2 different genotypes — the chimeric HCV viruses 1a/2a and 1b/2a (H77/C3 and Con1/C3) — that are infectious in vitro (18). Figure 3B shows that all the different HCV genotypes could be propagated in chimeric mice. Intriguingly, there was a 1,000-fold difference in titers between the clinical sample (mouse 164 and 195) and the chimeric HCV genomes (mouse 205 and 206). The successful propagation of all 4 different HCV viruses could be confirmed by the detection of HCV RNA in total liver RNA (Figure 3C). Like HCV RNA levels in the serum, intrahepatic HCV RNA levels normalized for human GAPDH did not correlate with human serum albumin levels and hence repopulation rates. Liver tissue of chimeric mice was stained for HCV nonstructural protein 5A (NS5a) and FAH. Figure 3, E–G, shows that immunostaining for NS5a colocalized with human hepatocytes that were FAH positive. Histological examination did not reveal any cytopathic effect of HCV infection (data not shown). In a few chimeric livers, we observed hepatocellular carcinoma, exclusively originating from mouse tissue. Hence, these tumors reflect a known complication of tyrosinemia type I rather than virus-related tumor growth (19).
Antiviral therapy in human liver chimeric mice. To extend the utility of this animal model, we explored its suitability for evaluation of antiviral drug efficacy. The state-of-the-art therapy against HCV is the combination of pegylated interferon α 2a (peg-IFN) and the nucleoside analog ribavirin. We compared this therapy to a more experimental therapy in clinical evaluation (19), the cyclophilin inhibitor Debio 025. We set up 1 control group without treatment and 3 treatment groups (peg-IFN alone, peg-IFN and ribavirin, peg-IFN and Debio 025). The control group received saline injections and oral gavages of the vehicle only. All 12 animals (4 × 3 mice) had high human liver chimerism and were inoculated with a clinical isolate of HCV genotype 1a. Antiviral therapy was performed for 2 weeks, after animals had reached the plateau phase of HCV titer (day 21). All treated animals showed an approximately 3 log reduction in HCV RNA after 2 weeks of treatment (Figure 3D). We also inoculated 2 mice with a clinical isolate of HCV genotype 3a, which is known to be more sensitive to interferon. Indeed viral titers decreased even further than those of HCV genotype 1a upon peg-IFN treatment (Supplemental Figure 3). All treatments were well tolerated by all animals.
We investigated yet another class of antiviral compounds, this time in human liver chimeric mice infected with HBV. The nucleotide analog adefovir dipivoxil, which is used in the treatment of HBV patients, could lower the HBV titers in the murine serum (Supplemental Methods and Supplemental Figure 4).
Sustained viral propagation in human liver chimeric mice. To evaluate long-term infection, we inoculated 6 animals with HCV genotype 1 and monitored them for more than half a year (34 weeks). Three of the 6 mice had been treated with the peg-IFN and Debio 025 for 4 weeks. Five to 6 weeks after antiviral therapy was ceased, the mean HCV levels of the treatment group were again comparable to those of the nontreated ones and reached plateau phase for the remainder of the study (Figure 4A).
Long-term propagation and serial passage of HCV. (A) Mice were treated for 4 weeks with peg-IFN and Debio 025. The treatment was started 3 weeks after inoculation with HCV genotype 1a, when the viral titers reached the plateau phase. Serum HCV RNA levels are presented as mean ± range of treatment (blue, n = 3) and control (red, n = 3) groups. (B) Two mice were inoculated with a clinical isolate of HCV genotype 1a. (C) Serum from mouse 164 and 195 was used as inoculum for mouse 235 and 239 (see text for details).
Encouraged by this long-term propagation in human liver chimeric mice, we were interested to see whether the virus could also be passaged from one chimeric mouse to another. We inoculated 2 mice with a clinical sample of HCV genotype 1a (Figure 4B). After 4 weeks, we used their serum to inoculate 2 different mice by diluting the virus to match the titer of the clinical inoculum (Figure 4C). The HCV RNA levels of the 4 mice were comparable in terms of maximal viral titer and kinetics of early infection.
Some of the chimeric mice used for HBV and HCV infection were repopulated with hepatocytes from the same donor (Supplemental Tables 1 and 2) and therefore offered the possibility to compare viral infection under standardized host conditions. Supplemental Figure 5 shows HBV and HCV infection in chimeric mice of the same hepatocyte donor (44-year-old, mixed European descent) and exemplifies the differences in viral dynamics not only between HBV and HCV, but also between different HCV isolates.