Research ArticleThe entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus
Introduction
The hepatitis B virus (HBV) still represents a major health threat with about 350 million chronically infected individuals worldwide. HBV infection leads to a wide spectrum of liver disease ranging from acute to chronic viral hepatitis, which is often associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). Despite the availability of an effective prophylactic vaccine, about 1 million people die each year due to HBV-associated liver pathologies [1]. Currently approved antiviral treatments based on interferon alpha or nucleos(t)ide analogues that inhibit the viral reverse transcriptase can induce long-term responses, defined as HBsAg loss or seroconversion, only in a minority of patients. Therefore the development of novel therapeutic strategies interfering with other steps of the viral life cycle is needed to improve the treatment outcome.
Viral entry inhibition may represent a rather new and attractive therapeutic concept to combat viral infections both in the acute or chronic phase. Indeed, maintenance of chronic HBV infection is thought to depend on a dynamic turnover of infected hepatocytes that are cleared by the immune system and cells that become newly infected. Moreover, independent reports have shown that cccDNA intrahepatic loads are often below 1 cccDNA copy per cell in human liver biopsies (median 0.1–1 cccDNA copy/cell) [8], [12], [16], [17], [18], [19], indicating that the liver of HBV-chronically infected patients often harbors a significant fraction of uninfected hepatocytes. Furthermore, drug resistant variants may eventually emerge under antiviral pressure using nucleos(t)ide analogs. Thus, the use of drugs able to prevent infection of the hepatocytes and spreading of drug resistant variants may represent an important new antiviral concept in the setting of chronic HBV infection.
We have previously shown that Myrcludex-B, which is the GMP version of a synthetic lipopeptide derived from the preS1 domain of the HBV envelope protein, specifically targets the hepatocytes and efficiently blocks de novo HBV infection both in vitro [5], [15] and in vivo, after pretreatment of human chimeric uPA/SCID mice [13]. To investigate whether Myrcludex-B administration may also prevent intrahepatic viral spreading in HBV-infected uPA/SCID mice, at a time when only a minority of the human hepatocytes are infected, mice repopulated with primary human hepatocytes were first injected with HBV to permit an initial infection establishment and than treated with the HBV entry inhibitor. We show here that 6 weeks of Myrcludex-B administration, initiated either 3 days or 3 weeks post HBV-injection, efficiently blocked HBV dissemination and cccDNA amplification in vivo.
Section snippets
Generation of human chimeric mice and infection with HBV
UPA/SCID mice were maintained under specific pathogen free conditions in accordance with institutional guidelines, under approved protocols [13]. Humanized homozygous uPA/SCID mice were generated as previously described [10]. All animal experiments were conducted in accordance with the European Communities Council Directive (86/EEC) and approved by the Ethical Committee of the city and state of Hamburg and according to the principles of the Declaration of Helsinki. Human chimeric animals
Administration of Myrcludex-B starting 3 days post HBV infection prevents viral spreading in humanized uPA mice
To investigate the capacity of the entry inhibitor Myrcludex-B to inhibit infection of the human hepatocytes in vivo, by treating mice after the initial infection establishment, 15 naïve humanized uPA/SCID mice were inoculated with 5 × 107 copies of HBV genome equivalents to permit viral entry. As depicted in Fig. 1A, three days after infection, mice received either Myrcludex-B treatment (n = 8; 2 μg/g body weight/day) or saline buffer as control (n = 7) for 6 weeks. Two weeks after viral injection,
Discussion
The limited availability of animal models of HBV infection and difficulties encountered by cultivating HBV in vitro have hindered investigation of the molecular mechanisms adopted by HBV to infect the hepatocytes and disseminate the liver. Upon viral entry and release of the genome into the cell nucleus, the rcDNA is converted into the cccDNA, which after being associated with histone and non-histone proteins, is incorporated into the host chromatin [9]. Thus, establishment of productive HBV
Financial support
The study was supported in part by an unrestricted grant from the MYR GmbH. MD is supported by the Deutsche Forschungsgemeinschaft (SFB 841, Da 1063/2-1). ML is supported by the Deutsche Forschungsgemeinschaft (Lü 1707/1-1). SU and MD received funding from the Deutsche Zentrum für Infektionsforschung (TP1-2011-2012). SU received financial support by the Bundesministerium für Bildung und Forschung (BMBF), “Innovative Therapieverfahren”, Grant No. 01GU0702.
Conflict of interest
Alexander Alexandrov is Medical Director/COO of the company MYR GmbH, which is manufacturing the compound used in this study. All the other authors have no conflict of interest to disclose.
Acknowledgements
We thank R. Reusch and A. Groth for their excellent assistance with the mouse colony and G. Apitzsch and C. Dettmer for great technical help. We are grateful to J. Bierwolf for her support during human liver cell isolation.
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