Oral Presentations

Session Title: Parallel Session: HEPATITIS B
Presentation Date: 01 APR, 2011

ADMINISTRATION OF THE ENTRY INHIBITOR MYRCLUDEX-B AFTER ESTABLISHMENT OF HEPATITIS B VIRUS INFECTION PREVENTS VIRAL SPREADING AMONG HUMAN HEPATOCYTES IN UPA MICE

M. Ben M´ Barek¹*, T. Volz¹, M. Lutgehetmann^1,2, T. Bornscheuer¹, L. Allweiss¹, A.W. Lohse¹, A. Alexandrov³, S. Urban⁴, J. Petersen⁵, M. Dandri^1
1Department of Internal Medicine, ²Department of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, ³Vision 7 GMBH, Frankfurt, ⁴University of Heidelberg, Heidelberg, ⁵IFI Institute for Interdisciplinary Medicine at Asklepios Clinic St. Georg, Hamburg, Germany. *m.benmbarek@t-online.de

Currently approved antiviral treatments based on interferon or inhibitors of the hepatitis B virus (HBV) polymerase are generally not curative and additional therapeutic strategies interfering with other HBV replication steps are needed. We previously demonstrated prevention of de novo HBV infection by pre-treating uPA mice with peptides derived from HBV envelope proteins (Petersen, Nat. Biotech.2008).
Aim: of this study was to investigate the ability of the entry inhibitor Myrcludex-B to block viral spreading in HBV-infected uPA/SCID mice, at a time when only few cells are infected and intrahepatic HBV replication is ongoing.
Experimental design: 15 human repopulated uPA/SCID mice were injected with HBV-infectious serum (5x10E7 HBV DNA copies/mouse). Three days post HBV-inoculation, 8/15 mice received daily subcutaneous injection of Myrcludex-B (2 mg/Kg). After 6 weeks, 3 treated and 3 sham-treated animals were sacrificed, whereas the remaining mice were monitored for additional 4 weeks to determine the occurrence of viral rebound. Mouse livers were analyzed by immunohistochemistry (human CK-18, HBcAg, HBsAg), while serological and intrahepatic measurements (HBV-DNA, cccDNA,) were performed by quantitative RT-PCR.
Results: In untreated mice viremia ranged from 3x10E5 to 4x10E8 HBV-DNA/ml 45 days post HBV-infection and the majority of human hepatocytes stained HBcAg-positive (1.2cccDNA copies/human cell), demonstrating efficient spreading of infection among human hepatocytes. However, very few isolated cells stained HBcAg/HBsAg-positive in Myrcludex-B treated mice at end of treatment, median viremia was 2.6x10E4 and cccDNA was still below detection (< 1/1,000 cells) (N=3). Two and four weeks after drug removal, median viral titres increased 6-fold and 38-fold, respectively, while in control mice median titres rose from 3x10E6 to 4x10E7 (14-fold) between week 6 and 10 post-infection. Notably, clusters of human hepatocytes became HBcAg-positive in the weeks following Myrcludex-B withdrawal, suggesting that slow spreading of HBV infection occurred preferentially among neighbouring human hepatocytes.
Conclusions: Application of Myrcludex-B post HBV-infection showed strong capacities to prevent spreading of HBV from initially infected cells, suggesting that its use in combination with current HBV-drugs may improve patients' outcome. Furthermore, the use of potent entry inhibitors in humanized mice may provide new insights about the kinetics and mechanisms of viral spreading occurring in vivo.