Background
Hepatitis B virus (HBV) infection is one of the major threats to public health worldwide and more than 240 million people are currently infected. Approximately 25% of the HBV infected individuals develop HBV-associated diseases afterwards, including liver failure, cirrhosis and hepatocellular carcinoma (HCC) [
16].
An immunologically defined and reproducible small animal model for HBV infection remains unavailable due to the strict host specificity of HBV infection, which greatly hampers HBV related research. The laboratory mouse is genetically and immunologically well defined, and a large genetically modified animals are available for scientific research. However, mice could not be infected with HBV. Several lines of transgenic mice with replication competent HBV genomes have been established and showed powerful application value for HBV related research [
6]. Nevertheless, transgenic mice have integrated HBV genome and HBV replication existed in all the hepatocytes. The presence of HBV genomes in these mouse lines inevitably induces host immune tolerance against HBV antigens, which is different from that occurs during a natural infection [
1,
6,
9,
15]. In addition, the capability of production of HBV transgenic mouse line is not easy in ordinary laboratory conditions. Moreover, human liver transplanted mouse models were established and used for different studies [
3,
5,
8]. However, The transplant models are based on immunodeficient mouse strains and difficult to operate in majority of laboratory.
Hydrodynamic injection (HI) of replication-competent HBV clone into the tail veins of mice can establish HBV replication in the liver of mouse [
7,
18]. In 40% of C57/BL6 mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, the persistence of HBV surface antigenemia (HBsAg) was more than 6 months. The tolerance against HBsAg in this model was due to the insufficient cellular immunity against HBV core antigen, as has been documented in humans [
7]. The HBV HI mouse model is a highly interesting model for testing vaccination strategies and the mechanisms of viral persistence [
4,
10,
20‐
22]. This model also could be used to evaluate replication competence of HBV constructs [
10] as well as HBV related antiviral research [
17]. Therefore, increasing the percentage of HBV persistent mice is very important to optimize the application of HBV HI mouse model.
In this study, we tested the impact of the dose of injected HBV plasmid DNA on HBV persistence in both C57/BL6 and BALB/c mice. In previous study, Huang et al. showed that there were 40% of the C57/BL6 mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 6 months and none of the BALB/c mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 4 weeks [
7]. However, in our study, we found that 80% of the C57/BL6 mice receiving 5 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg persisted more than 6 months. The HBV persistent rate was 2-fold increase compared with the results shown in previous study. Furthermore, we found that 60% of BALB/c mice receiving 5 μg of pAAV-HBV1.2 plasmid DNA, serum HBsAg persisted more than 3 months which showed a dramatic improvement compared with the results in previous study. On the contrary, C57/BL6 mice injected with 100 μg high dose of pAAV-HBV1.2 plasmid DNA had the highest HBsAg and HBeAg expression at the beginning after HI whereas rapid clearance of them afterwards. In addition, 1 μg low dose of pAAV-HBV1.2 plasmid DNA showed a slight impact on HBV persistence in BALB/c mice.
To find the possible mechanism of HBV persistence in this study, we further detected ALT levels in serum of the mice and the expression of interferon regulatory factor (IRF3), interferon stimulate genes (ISGs) including ISG15, 2′-5′-oligoadenylate synthase (OAS) and Proteinkinase R (PKR) as well as several immune factors including Interferon gamma (IFNγ), Tumor necrosis factor α (TNFα), Transforming growth factor β (TGFβ), Interleukin 6 (IL-6), IL-10, Programmed cell death ligand 1 (PDL1) in liver of the mice at 3 dpi. The elevated serum ALT levels were detected in all groups of HBV plasmid DNA injected mice at first day after HI, which was due to the liver damage caused by HI method. Furthermore, no significant difference in the mRNA levels of IRF3 and ISGs was detectable between the PBS injected control group and different concentrations of HBV plasmid DNA injected groups. However, we found that the antiviral factor IFNγ was significant up-regulated in liver of the mice injected with 1 μg or 100 μg pAAV-HBV1.2 plasmid DNA. TNFα was up-regulated significantly in liver of the mice injected with 100 μg pAAV-HBV1.2 plasmid DNA. Those results suggest that IFNγ and TNFα still play important roles in controlling virus infection in this study. Moreover, we found that the immune negative regulatory factor PDL1 was up-regulated significantly in liver of the mice injected with 5 μg pAAV-HBV1.2 plasmid DNA.
In conclusion, this study illustrated that HI with 5 μg medium dose of pAAV-HBV1.2 plasmid DNA significant increased the rates of HBV persistence in both C57/BL6 and BALB/c HI mouse model. The IRF3 and ISGs do not account for HBV clearance in our study. However, the antiviral factors IFNγ, TNFα as well as immune negative regulatory factor PDL1 play important roles on HBV persistence. In sum, this study will help us to further understand the mechanism of HBV persistence and better apply this model to explore new treatments against chronic HBV infection.
Methods
HI experiments
The pAAV-HBV1.2 plasmid contains an 1.2 fold over-length HBV genotype A genome (nt 1400-3182-1987) [
7], was kindly provided by Prof. Pei-Jer Chen. The persistence of HBV in vivo was tested by HI method with different concentrations of HBV plasmid DNA in both C57/BL6 mice (male, 6–8 weeks old, from the breeding colonies of the experimental animal center in Shanghai, China) and BALB/c (male, 6–8 weeks old, from the breeding colonies of the experimental animal center in Hubei Province, China). HI experiments were carried out as described previously [
7], 5 μg, 10 μg or 100 μg pAAV-HBV1.2 plasmid DNA were injected into the tail veins of C57/BL6 mice, separately and 1 μg, 5 μg or 10 μg pAAV-HBV1.2 plasmid DNA were injected into the tail veins of BALB/c mice, respectively, with 30 mice per group. Furthermore, to find the possible mechanism of the HBV persistence, 1 μg, 5 μg, 10 μg, or 100 μg pAAV-HBV1.2 plasmid DNA were injected into the tail veins of BALB/c mice, respectively, with five mice per group. The pAAV-HBV1.2 plasmid DNA were in a volume of 0.9% NaCl, equivalent to 8% of the mouse body weight. The total volume was delivered within 5–8 s. The control mice were injected only with the same volume of 0.9% NaCl, equivalent to 8% of the mouse body weight.
The animal experiments were carried out in concordance with the guidelines established by the Institutional Animal Care and Use Committee at the Huazhong University of Science and Technique and the Tongji Hospital of Tongji Medical College. The mice used in this study were anesthetized with ketamine and xylazine.
Detection of ALT, HBsAg, HBeAg, HBcAb and HBsAb in mouse sera
Mouse sera from both C57/BL6 and BALB/c were collected at the 1d, 3d, 7d, 10d, 2w, 3w, 4w, 5w, 6w, 7w, 8w, 9w, 10w… after being injected with different concentrations of pAAV-HBV1.2 plasmid DNA. Alanine aminotransferase (ALT) levels of the BALB/c mice injected with 1 μg, 5 μg, 10 μg, or 100 μg pAAV-HBV1.2 plasmid DNA were measured on full automated biochemistry analyzer (7600 Series; Hitachi, Tokyo, Japan) by using ALT reagent (Denka Seiken, Tokyo, Japan).
The HBsAg, HBeAg, HBcAb and HBsAb were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Kehua, Shanghai, China). Ten-fold diluted serum samples were used for detection.
Isolation and analysis of HBV DNA in mouse sera
HBV DNA was extracted from 40 μl of mouse serum samples. The protocol was described previously [
17], 40 μl of samples were treated with 10 μg DNase I for 16 h at 37 °C. 100 μl of lysis buffer (20 mM Tris-HCl, 20 mM EDTA, 50 mM NaCl and 0.5% SDS) containing 50 μg proteinase K were added. After incubation at 65 °C for 3 h, viral DNA was isolated by phenol/chloroform extraction and ethanol precipitation. The DNA pellet was rinsed with 70% ethanol and resuspended in 10 μl of ddH
2O.
The quantification of HBV DNA was performed by using a routine real time PCR (qPCR), described previously [
11‐
14,
19,
23]. The HBV copy numbers were determined by SYBR Green Real time PCR Master Mix (commercially available assay kit, TOYOBO, Osaka, Japan). Melt curve analysis and agarose gel electrophoresis were used to verify the specificity of the qPCR. The following primers were used: forward primer: 5′-CTG CAT CCT GCT GCT ATG-3′ (nt 408-425), reverse primer: 5′-CAC TGA ACA AAT GGC AC-3′ (nt 685-701) according to the reference sequence with Genbank accession number (AY220698.1). Serum containing a known concentration of HBV DNA was used as a positive control.
Immunohistochemistry
The liver tissue was taken from the mice injected with different concentrations of pAAV-HBV1.2 plasmid DNA at 1, 3, 5, 9, 12 and 24 weeks post injection (wpi), and used for immunohistochemical staining of the HBcAg in hepatocytes. The liver tissue of the mice received 0.9% NaCl was used as negative control. Liver tissue was collected from the mice and embedded in paraffin. Intrahepatic HBcAg was visualized by immunohistochemical staining with rabbit anti-HBc (Dako) of the liver tissue sections. The liver tissue sections were also stained with hematoxylin.
Purification of RNA from mouse liver tissue and real-time PCR detection
The BALB/c mice were killed at 3 day after being injected with 1 μg, 5 μg, 10 μg, or 100 μg pAAV-HBV1.2 plasmid DNA, respectively and receiving perfusion via the Hepatic portal vein. Total RNA was isolated from the liver tissue at 3 day post injection (dpi) by tissue RNA extraction kit (OMEGA, Norcross, USA) and used for analyzing the copy number of mouse IRF3, ISG15, OAS, PKR, IFNγ, TNFα, TGFβ, IL-6, IL-10 and PDL1 mRNA by using SYBR Green one-Step reverse trscription quantitive PCR (qRT-PCR) Kit (Takara, Dalian, China). Primers for qRT-PCR detection were provided by Qiagen Company (Qiagen, Hilden, Germany). β-actin was used as housekeeping gene to normalize qRT-PCR results.
Statistical analysis
The data were evaluated by Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. The statistical significance of the data was considered at p < 0.05.
Discussion
Establishment of a stable as well as genetically and immunologically defined mouse model is very important to accelerate HBV related basic and clinical research process. HBV HI mouse model is a HBV replication-competent mouse model which showed usual definition of persistent HBV infection in humans. It is a very valuable model that will help to further understand the mechanism of HBV tolerance and also provide a useful tool for HBV related antiviral research. In previous study, they found that both vector construction and mouse genetic background determined HBV persistence in this model. The tolerance toward HBV surface antigen in HI mouse model was shown to be due to an insufficient cellular immunity against HBcAg, as was documented in humans [
7].
As we know, HBV persistent mouse model is needed for HBV related antiviral research. However, the previous study showed that only 40% of the C57/BL6 mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 6 months and none of the BALB/c mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 4 weeks [
7]. Therefore, optimization of the existing HBV HI mouse model is necessary to accelerate the widespread application of this model. The previous study showed that the size of the viral inoculum contributes to the outcome of HBV infection in adult chimpanzees. They found that CD4
+ T-cell depletion before inoculation of a normally rapidly controlled inoculum precluded T-cell priming and caused persistent infection with minimal immunopathology, suggesting that the relationship between the kinetics of viral spread and CD4
+ T-cell priming determines the outcome of HBV infection [
2]. However, it should be noted that Asabe et al. used adult chimpanzees in their study so that the age, sex and genetic diversity of the chimpanzees probably have an impact on the outcome of HBV infection. However, in our study, 5 weeks old genetically and immunologically defined laboratory mice were used so that the dose of the HBV plasmid DNA should be the unique factor which account for the outcome of HBV invasion.
Therefore, to test whether the dose of the HBV plasmid DNA influence the course and outcome of HBV infection in HI mouse model, we start with injection of different concentrations of pAAV-HBV1.2 plasmid DNA in the C57/BL6 mice. The results demonstrated that there were 80% of the C57/BL6 mice showing HBsAg positive more than 6 months after being injected with 5 μg pAAV-HBV1.2 plasmid DNA. It was two fold increase compared with the mice injected with 10 μg pAAV-HBV1.2 plasmid DNA in previous study. Whereas, the viruses in the mice injected with 100 μg higher dose of pAAV-HBV1.2 plasmid DNA were cleared quickly with declined viral kinetics even if high HBsAg and HBeAg levels appeared at the first day after HI. To further confirm the results and investigate whether the lower inocula could promote HBV persistence, BALB/c mice were injected with 1 μg, 5 μg or 10 μg pAAV-HBV1.2 plasmid DNA, separately. We found that there were 60% of the BALB/c mice showing HBsAg positive more than 3 months after being injected with 5 μg pAAV-HBV1.2 plasmid DNA. This was a significant improvement compared with the previous results which illuminated that none of the BALB/c mice showing HBsAg positive more than 4 weeks after being injected with 10 μg pAAV-HBV1.2 plasmid DNA. In addition, we found that injection of 1 μg lower dose of pAAV-HBV1.2 plasmid DNA just exert a slight influence on the course and outcome of HBV invasion in this model. The inability to produce anti-HBs after injection of 5 μg pAAV-HBV1.2 plasmid DNA into both C57/BL6 and BALB/c mice (As shown in Table
1) suggesting that the tolerance against HBsAg was generated in vivo.
To investigate the possible mechanism of HBV persistence in this study, we detected the ALT levels in serum and the expression of IRF3, ISGs as well as several immune factors in liver of the mice at 3 day after the BALB/c mice being injected with 1 μg, 5 μg, 10 μg, or 100 μg pAAV-HBV1.2 plasmid DNA. We found that the elevated serum ALT levels in the mice of all the groups at first day after HI were due to the liver damage caused by HI method. Furthermore, no significant difference in the mRNA levels of IRF3 and ISGs was detectable between the PBS injected control group and different concentrations of pAAV-HBV1.2 plasmid DNA injected groups. However, we found that the antiviral factor IFNγ was significantly up-regulated in liver of the mice injected with 1 μg or 100 μg pAAV-HBV1.2 plasmid DNA. TNFα was up-regulated significantly in liver of the mice injected with 100 μg pAAV-HBV1.2 plasmid DNA. Moreover, the immune negative regulatory factor PDL1 was up-regulated significantly in liver of the 5 μg pAAV-HBV1.2 plasmid DNA injected mice. Taken together, the results highlight that the IRF3 and ISGs do not account for HBV clearance but IFNγ, TNFα as well as immune negative regulatory factor PDL1 play important roles on HBV persistence in HI mouse model.
In a word, our study demonstrated the significant increases of HBV persistent rates in both C57/BL6 and BALB/c HI mouse model after being injected with 5 μg pAAV-HBV1.2 plasmid DNA, which will greatly improve the application of this model. As we know, a lot of HBV related antiviral researches are based on HBV chronic infection mouse model. Therefore, increasing the percentage of HBsAg positive mice in HBV HI mouse model will greatly reduce the number of experimental animals. In addition, our study also provide an opportunity to better understand the mechanism of HBV persistence in HI mouse model, which is very important to disclose the host immune response against HBV invasion. Taking togethr, here we provide a valuable tool which will contribute a lot to the HBV related basic research and also useful for the evaluation of anti-HBV therapy.
Conclusion
In this paper we demonstrated that, in the HBV HI mouse model, the different concentrations of the injected pAAV-HBV1.2 plasmid DNA contribute to the diverse kinetics of HBsAg and HBeAg levels in the serum as well as HBcAg expression in the liver, which then determined the HBV persistence. In addition, we found that the antiviral factors IFNγ, TNFα as well as immune negative regulatory factor PDL1 play important roles on HBV persistence. In brief, here we optimized the HBV HI mouse model which will greatly improve the application of this model for HBV related research.