A novel cDNA‐uPA/SCID/Rag2−/−/Jak3−/− mouse model for hepatitis virus infection and reconstruction of human immune system

Although human hepatocyte‐transplanted immunodeficient mice support infection with hepatitis viruses, these mice fail to develop viral hepatitis due to the lack of an adaptive immune system. In this study, we generated new immunodeficiency cDNA‐urokinase‐type plasminogen activator (uPA)/SCID/Rag2−/−/Jak3−/− mice and established a mouse model with both a humanized liver and immune system. Transplantation of human hepatocytes with human leukocyte antigen (HLA)‐A24 resulted in establishment of a highly replaced liver in cDNA‐uPA/SCID/Rag2−/−/Jak3−/− mice. These mice were successfully infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) for a prolonged period and facilitate analysis of the effect of anti‐HCV drugs. Administration of peripheral blood mononuclear cells (PBMCs) obtained from an HLA‐A24 donor resulted in establishment of 22.6%–81.3% human CD45‐positive mononuclear cell chimerism in liver‐infiltrating cells without causing graft‐versus‐host disease in cDNA‐uPA/SCID/Rag2−/−/Jak3−/− mice without human hepatocyte transplantation. When mice were transplanted with human hepatocytes and then administered HLA‐A24‐positive human PBMCs, an alloimmune response between transplanted human hepatocytes and PBMCs occurred, with production of transplanted hepatocyte‐specific anti‐HLA antibody. In conclusion, we succeeded in establishing a humanized liver/immune system characterized by an allo‐reaction between transplanted human immune cells and human liver using a novel cDNA‐uPA/SCID/Rag2−/−/Jak3−/− mouse. This mouse model can be used to generate a chronic hepatitis mouse model with a human immune system with application not only to hepatitis virus virology but also to investigation of the pathology of post‐transplantation liver rejection.


| INTRODUC TI ON
Hepatitis B virus (HBV) and hepatitis C virus (HCV) impose a worldwide health burden involving acute hepatitis, chronic hepatitis, liver cirrhosis, and potentially death due to liver failure and hepatocellular carcinoma. The species tropism of these viruses is restricted to chimpanzee and human; thus, investigation of their pathology was long hampered by the lack of a small animal model. Immunodeficient mice in which mouse hepatocytes are replaced with human hepatocytes, such as urokinase-type plasminogen activator (uPA), 1,2 herpes simplex virus type-1 thymidine kinase (HSVtk) 3 transgenic mice, or a targeted disruption of the murine fumaryl acetoacetate hydrolase (FAH) 4,5 support infection and replication of hepatitis viruses and are commonly used for the study of viral hepatitis. [6][7][8] We previously reported that TK-NOG and cDNA-uPA/ SCID mice were useful animal models for the study of HBV and HCV virology as well as the analysis of the effect of antiviral drugs. 9,10 Although the main pathophysiology of viral hepatitis is the immune response to virus-infected hepatocytes, it is difficult to assess the immune response to hepatitis viruses because human hepatocyte chimeric mice are generated using immunodeficient mouse strains.
Establishment of an HBV infection model having a human immune system is of great interest to reveal the mechanism of viral hepatitis and to develop therapeutic approaches via the immune system.
Adaptive transfer of human immune cells is one approach to assessing the immune response to hepatitis virus infection. [11][12][13] The generation of double chimeric systems still represents a major challenge, and both the source of human cells and the mouse genetic background massively influence the outcome of cell engraftment and maturation. In this study, we generated novel cDNA-uPA/SCID/Rag2 −/− / Jak3 −/− mice with humanized livers that were almost completely repopulated with human hepatocytes. We were able to successfully infect these mice with both HBV and HCV. In addition, we attempted to reconstitute elements of the human immune system in this novel mouse model by administration of human peripheral blood mononuclear cells (PBMCs).

| Animal care and use for the survival study
All animal protocols described in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals When mice were transplanted with human hepatocytes and then administered HLA-A24-positive human PBMCs, an alloimmune response between transplanted human hepatocytes and PBMCs occurred, with production of transplanted hepatocytespecific anti-HLA antibody. In conclusion, we succeeded in establishing a humanized liver/immune system characterized by an allo-reaction between transplanted human immune cells and human liver using a novel cDNA-uPA/SCID/Rag2 −/− /Jak3 −/− mouse.
This mouse model can be used to generate a chronic hepatitis mouse model with a human immune system with application not only to hepatitis virus virology but also to investigation of the pathology of post-transplantation liver rejection.

K E Y W O R D S
HBV, HCV, human hepatocyte chimeric mice, human leukocyte antigen, humanized mice (https://grants.nih.gov/grant s/olaw/guide-for-the-care-and-use-oflabor atory-anima ls.pdf) and the local committee for animal experiments, and the experimental protocol was approved by the Ethics

| Measurement of the replacement index and human serum albumin levels
The replacement index of human hepatocytes was calculated as the ratio of the area occupied by hCK8/18-positive human hepatocytes relative to the entire area examined on immunohistochemical sections from 7 lobes, as described previously. 15 Cryosections prepared from the liver (5 μm thick) were incubated with anti-human cytoker- in mouse blood was measured by immunonephelometry in a JEOL BM6050 autoanalyser (JEOL, Tokyo, Japan) using using LX Reagent Eiken Alb II (Eiken Chemical, Tokyo, Japan).

| Hepatitis virus infection in human liver chimeric mice
Twelve weeks after human hepatocyte transplantation, cDNA-uPA/ SCID/Rag2 −/− /Jak3 −/− mice were injected intravenously with serum containing either HBV or HCV. HBV serum was obtained from an HBeAg-positive patient with chronic genotype C HBV infection who had a high virus titre (8.2 log IU/ml). HCV serum was obtained from a patient with chronic genotype 1b HCV infection with high virus titre (6.3 log IU/ml). After serum inoculation, mouse blood samples were obtained serially, and serum viral titres and HSA levels were measured. Human serum samples were obtained from patients who provided written informed consent using a form that was made in accordance with the Declaration of Helsinki and was approved by the ethical committee of Hiroshima University. The individual serum samples were divided into small aliquots and stored separately in liquid nitrogen until use.

| Quantification of HBV DNA and HCV RNA
Serum HBV DNA and HCV RNA levels were measured using the COBAS TaqMan test (Roche Diagnostics). The lower detection limits of the assay for HBV DNA and HCV RNA are 4.4 and 3.5 log copies/ ml, respectively.

| Preparation of human PBMCs and transplantation into mice
Characteristics of human PBMCs used in this study are shown in Table 1. PBMCs were isolated using Ficoll-Paque density gradient centrifugation according to the manufacturer instructions. 5 × 10 6 of PBMCs were transplanted into cDNA-uPA/SCID/Rag2 −/− /Jak −/− mice by peritoneal injection.

| Flow cytometry
We collected mouse liver-infiltrating lymphocytes flowing through the portal vein after hepatectomy. 16

| Histochemical analysis of mouse liver
Histochemical analysis and immunohistochemical staining using antibodies against human albumin (Bethyl Laboratories Inc., Montgomery, TX) were performed as described previously. 10,17 Immunoreactive materials were visualized using a streptavidinbiotin staining kit (Histofine SABPO kit; Nichirei, Tokyo, Japan) and diaminobenzidine.

| Detection of anti-HLA antibody in mice
Anti-human leukocyte antigen (HLA) antibody was detected using WAKFlow class I antibody specificity identification reagent Changes in the body weight and serum HSA levels of 13-week-old mice are shown in Figure 1 and Table S1. The HSA levels increased steadily after transplantation of human hepatocytes and reached a steady state at around 11 weeks ( Figure 1A). Mouse serum HSA levels were higher in cDNA-uPA homozygote chimeric mice compared to hemizygote chimeric mice from 6 weeks old (3 weeks after transplantation) to 13 weeks old, indicating that engraftment of human hepatocytes was superior in cDNA-uPA homozygote host mice. Body weights were higher in cDNA-uPA hemizygote mice than in homozygote mice ( Figure 1A). Mean HSA levels in males were slightly higher than those in females of both cDNA-uPA homozygote and hemizygote human hepatocyte-replaced mice. The correlation between HSA levels at 17 weeks old and the human hepatocyte replacement index, estimated by immunohistochemistry in 16 cDNA-uPA homozygote mice, is shown in Figure 1B. HSA levels were exponentially correlated with replacement index in cDNA- The formula indicated that a level of more than 6.2 mg/ml HSA represents a replacement index exceeding 70% in human hepatocyte-  Figure 4D). Therefore, the symptoms following human PBMC administration seemed to be caused by alloimmunity between administered human PBMCs and transplanted human hepatocytes. Next, we tested PBMC administration into HBV-infected cDNA-uPA/SCID/Rag2 −/− /Jak3 −/− mice. Human hepatocyte-transplanted mice were infected with HBV and administered with PBMCs obtained from Donor 1. We also tested PBMCs obtained from a patient who had recovered from an episode of acute severe hepatitis B (Donor 2) and a patient with chronic HBV infection (Donor 3) ( Table 1). Mice experienced weight loss, and mouse serum HSA level decreased 2 weeks after PBMC administration, similar to results using uninfected mice ( Figure 5). Although HBV DNA levels decreased in these mice in response to the reduction in HSA level, HBV-specific cytotoxic T lymphocytes (CTLs) were not detected in liver-infiltrating cells obtained from these mice (data not shown),

| Hepatitis virus infection of human
indicating that the reduction of HSA levels was not due to a specific response to HBV-infected hepatocytes.

| Alloimmune response with production of transplanted hepatocyte-specific anti-HLA antibody
Because no reduction of HSA after PBMC transplantation was ob- Meanwhile, the immune response between graft and host is a serious problem when constructing in vivo immune system models and is mainly thought to be caused by MHC class1/2 molecules. 25 Various xeno-immunity mouse models are reported using immunodeficiency mouse strains. 26 However, no alloimmunity mouse model has been reported so far. According to a previous report, allo-specific anti-HLA antibody was difficult to detect in   and JP22fk0310513). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.

CO N FLI C T O F I NTE R E S T
Michio Imamura has received research funding from AbbVie.
Hiroshi Aikata has received honoraria from Eisai and Bayer. Kazuaki

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
All animal protocols described in this study were performed in accordance with the Guide for the Care and Use of F I G U R E 5 PBMC administration into HBV-infected cDNA-uPA/SCID/ Rag2 −/− /Jak3 −/− mice. Human hepatocytetransplanted cDNA-uPA/SCID/Rag2 −/− / Jak3 −/− mice were infected with HBV and administered human peripheral blood mononuclear cells (PBMCs) obtained from a healthy volunteer (Doner 1), a patient recovered from an episode of acute hepatitis B (Donor 2) or a chronic HBV-infected patient (Donor 3). Changes in body weight, serum HBV DNA level, and human serum albumin concentration in each mouse are shown.