1105 LONG-TERM FOLLOW-UP OF OCCULT HBV QUASISPECIES DIVERSITY AND LYMPHOTROPISM IN HIV-1-INFECTED PATIENTS REACTIVE FOR ANTIBODY TO HBV CORE ANTIGEN (ANTI-HBC)

Abstract

the cells. Changes in mitochondrial calcium flow and blocking cytosolic calcium signalling were shown to impact HBV replication. Furthermore, HBsAg was shown to interact with cyclophilin A in liver cells in vitro. In the present study we employed a panel of liver cell lines to investigate the ability of cyclophilin inhibitors DEB025 (Alisporivir) and NIM811 to affect HBV replication and viral particle secretion from cells. Material and Methods: Stably transfected HepG2215 cells, with full virion and HBsAg production, and the parent cells (HepG2) were cultured for 7 days (baseline) and subsequently treated with 0.25/1.0/5.0 ug/ml of NIM811 or DEB025. Cells and supernatants were harvested at baseline; 6, 24, 48 and 72 hours after addition of NIM811/DEB025. Intracellular and secreted HBV levels were quantitated by Taqman qPCR (ABI7500). Western blot and ELISA were used to assess intracellular and secreted HBsAg, respectively. Transiently transfected Huh-7 cells (with full length HBVDNA plasmid) were treated similarly with NIM811/DEB025. PLC/PRF/5 cells, expressing only HBsAg, were also tested. To determine the roles of individual cyclophilins in HBV replication, HepG2215 and Huh-7 cells were transfected with siRNA for cyclophilins A, B, C, D, F. Cell death was evaluated by Annexin V expression (flow cytometry) and LDH/ALT quantitation (ELISA) in supernatants. Results: Both cyclophilin inhibitors significantly reduced cytoplasmic core-particle associated HBVDNA levels in the cells, between 2 and 10-fold, lower than the control cells. The most pronounced reduction of intracellular HBVDNA levels (by10-fold at 72 hours) was observed with DEB025 (5ug/ml), which was greater than the reduction observed with NIM811. Similarly, DEB025 (both at 1 and 5ug/ml) showed a greater impact in reducing HBV virion secretion in the supernatants, compared with NIM811. Both compounds significantly reduced HBsAg secretion from cells by 50% vs controls. Conclusion: Cyclophilin inhibition interferes with HBV replication within liver cell lines, and in addition, it significantly reduces the secretion of virions and HBsAg particles from the cells. A combination of cyclophilin inhibitor and nucleos(t)ide anti-HBV agent may be useful for treatment of chronic hepatitis B.