Abstract
BackgroundHepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, causing a major risk of liver disease and hepatocellular carcinoma (HCC). Many mouse models have been tried to establish HBV infection through injection with various HBV-containing plasmids. However, it is not well understood that different plasmids, all of which contain the similar HBV genome, even the same plasmids with different dose, results in opposite immune responses toward HBV.MethodsIn this study, we investigated the role of HBV-containing plasmid backbones and the HBcAg in determining the HBV persistence. C57BL/6 mice were injected hydrodynamically with 6 μg or 20 μg of WT pAAV/HBV1.2 plasmid, e/core-null pAAV/HBV1.2 plasmid, or none-HBV genome pAAV/control plasmid. Serum levels of HBV-related markers were measured by quantitative immunoradiometric assay (IRMA). Liver HBcAg expression was detected by immunohistochemical staining. The mRNA levels of cytokines and Th1-related immune factors were quantified by qRT-PCR.ResultsAll mice injected with 6 μg of the pAAV/HBV1.2 plasmid shows HBsAg positive at week 6 after hydrodynamic injection (AHI) as previously investigated. However, the mice injected with 20 μg pAAV/HBV1.2 or 6μgpAAV/HBV1.2 plus 14μgpAAV/control plasmid results in HBV clearance within 4 weeks AHI, indicating the anti-HBV activity is induced by 20 μg plasmid DNA, but not by the inserted viral genome. This anti-HBV activity is independent of HBcAg and Toll like receptor (TLR) signaling pathway, since the lack of HBcAg in pAAV/HBV1.2 plasmid or stimulation with TLRs agonists does not influence the kinetics of serum HBsAg in mice. The mRNA levels of t-bet and cxcr3 were dramatically up-regulated in the liver of the mice injected with 20 μg plasmid DNA.ConclusionOur studies demonstrate that plasmid backbones are responsible for modulating immune responses to determine HBV persistence or clearance in our HBV mouse model by hydrodynamic injection of HBV-containing plasmid, and Th1 cells play key roles on HBV clearance.
Highlights
Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, causing a major risk of liver disease and hepatocellular carcinoma (HCC)
80% of the mice injected with 20 μg pAAV/HBV1.2 plasmids produced anti-HBs antibody in the serum, while only 25% of those receiving 6 μg pAAV/HBV1.2 plasmid were anti-HBs positive at week 11 after hydrodynamic injection (AHI) (Table 1)
Our results have shown that HBV clearance still occurs in mice injected with20 μg of the e/core-null pAAV/HBV1.2 plasmids (Fig. 3c-d), and HBV persistence induced by 6 μg pAAV/HBV1.2 plasmid is broken by mixing with 14 μg of the control plasmid or with even other irrelevant plasmids at injection time (Fig. 4 and Fig. 5c)
Summary
Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, causing a major risk of liver disease and hepatocellular carcinoma (HCC). The appearance of HBV-transgenic mice has promoted HBV related research [6], it is not a good model to investigate the immunological mechanisms of HBV because of the central tolerance to HBV-related antigens To overcome this challenge, many researchers created HBV persistence models in immunocompetent mice through hydrodynamic injection of HBV genome-containing plasmids, by which the injected plasmids could mainly target hepatocytes [7]. Many researchers created HBV persistence models in immunocompetent mice through hydrodynamic injection of HBV genome-containing plasmids, by which the injected plasmids could mainly target hepatocytes [7] In these HBV persistent mice, viral replication intermediates, transcripts, and all HBV related proteins can be detected in the liver tissues for several months. Hydrodynamic injection of different plasmids, in spite of containing same HBV genome, or the same plasmids at different doses induce totally different immune responses toward HBV and subsequently result in HBV persistence or HBV clearance [8–11]. This opposite results suggest that plasmid backbones other than HBV genome are involved in triggering innate immunity [12], which eventually influence the status of HBV infection
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