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441 Maximal Boosting of Innate Immunity During Pegylated Interferon-Aplha Therapy is Reached at 48 Weeks in E-Antigen Positive Chronic Hepatitis B

2012, Journal of Hepatology

POSTERS increased over time relative to GS-9620 exposure, and this was noted as early as Day 7. After repeated QD and QOD administration of 0.15 mg/kg, the ratio of serum IFN-a to GS-9620 Cmax was 8- to 19-fold higher than on Day 1. In comparison, with QW administration of 0.15 mg/kg, serum IFN-a to GS-9620 Cmax ratio was ≤2-fold higher as compared to Day 1. At higher doses, GS-9620 administered QOD or QW resulted in high IFN-a responses that did not increase over time relative to GS-9620 exposure. A select number of additional cytokines, such as IL-6, MCP-1, MIG (CXCL9) and MIP-1b, were induced by GS-9620; the magnitude and number of which increased with increasing GS-9620 dose. Graph 1. Mean IFNa following QD, QOD, or QW administration of 0.15 mg/kg GS-9620. Dose/regimen Duration of dosing Serum IFN-a:GS-9620 Cmax ratio 0.15 mg/kg QD QOD QW 0.5 mg/kg QOD 1.5 mg/kg QOD QW 1 4 7 8 15 22 29 − 184 − 336 8270 − − − 3385 2405 − − − − − 516 − 1916 − − − − − 570 − − 7974 − 4 weeks 13133 − − − − − 1752 4 weeks 4 weeks 692 1896 − − − − − − 963 − − 3413 162 − 1 2 4 4 week weeks weeks weeks Ratio not calculated on day 1 for animals given 0.15 mg/kg QD or QOD as values were below the limit of detection. Conclusions: Compared to daily or every-other-day dosing, weekly administration of GS-9620 in cynomolgus monkeys resulted in the most consistent type 1 IFN-a response. Phase 1 clinical development of GS-9620 is exploring low oral doses of GS-9620 administered once weekly. 441 MAXIMAL BOOSTING OF INNATE IMMUNITY DURING PEGYLATED INTERFERON-APLHA THERAPY IS REACHED AT 48 WEEKS IN E-ANTIGEN POSITIVE CHRONIC HEPATITIS B U.S. Gill1 , M. Papadaki1 , D. Peppa2 , L. Micco2 , L. Li1 , I. Ushiro-Lumb3 , G.R. Foster1 , M.K. Maini2 , P.T.F. Kennedy1 . 1 Department of Hepatology, Institute of Cell & Molecular Science, Barts and The London School of Medicine & Dentistry, 2 Division of Infection & Immunity, University College London (UCL), 3 Department of Virology, Barts and the London NHS Trust, London, UK E-mail: [email protected] Background and Aims: Stopping-rules now exist for Pegylated Interferon-Alpha (PEG-IFNa) treated Chronic Hepatitis B (CHB) patients. Despite the utility of such strategies, the immunological mechanisms that drive HBV DNA and HBsAg decline remain poorly understood. Recent data have identified changes in a subset of NK cells in HBeAg negative disease, which may determine treatment response. However, HBeAg positive disease responds more favourably to PEG-IFNa; here we report on a longitudinal analysis of changes in the immune profile in this cohort, to define the effects of PEG-IFNa on innate immunity. Methods: PBMCs from a cohort of 17 HBeAg positive patients followed longitudinally at 3 monthly intervals pre, during and post PEG-IFNa therapy were utilised. Phenotypic analysis of NK cells was performed by multicolour flow cytometry. Changes in the immune responses were correlated with simultaneous measurements of ALT, HBV DNA and quantitative HBsAg levels (Abbott ARCHITECT). Results: PEG-IFNa increased CD56bright NK cells by four-fold (mean fold change; MFC 3.7, p = 0.0001). This was paralleled by the activation and proliferation of this subset, as marked by HLADR and Ki67 expression respectively (MFC 1.5 and 2.3, p = 0.009 and p = 0.0001 respectively). This increase was more marked at 48 weeks treatment, correlating with a nadir of HBV DNA and HBsAg. The activating (NKG2C and NKp30) and inhibitory (NKG2A) receptors were also analysed in this population. A two-fold increase in NKp30 expression (MFC 2.27, p = 0.04) was seen which was maximal at 48 weeks, while no significant change was noted for NKG2A and NKG2C. There was a two fold up-regulation of TRAIL expression on CD56bright NK cells, which temporally correlated with ALT levels, (MFC 1.8, p = 0.0001), this effect was most dramatic at 24 weeks of therapy and sustained to 48 weeks. Conclusions: PEG-IFNa therapy in this cohort enhances and activates CD56bright NK cells. Similarly, TRAIL and NKp30 expression is augmented and sustained throughout treatment and all these effects are maximal at 48 weeks. The restorative innate immune changes begin early and increase throughout therapy in all patients. Thus, 48 weeks therapy may provide the optimal immunological conditions to introduce an oral-antiviral to achieve disease control in PEG-IFNa non-responders. 442 A CELLULAR SPHINGOLIPID IS A KEY ELEMENT FOR THE EARLY STEPS OF HEPATITIS B VIRUS INFECTION A. König, C.M. Bremer, N. Kott, D. Glebe. Institute of Medical Virology, Justus-Liebig University of Giessen, Giessen, Germany E-mail: [email protected] Background and Aims: Recently, we demonstrated that special viral lipids (e.g. cholesterol) within the viral envelope are crucial for infectivity of HBV. To determine the role of key lipids of the cellular plasma-membrane in HBV binding, uptake and infection, we selectively depleted cellular cholesterol and sphingolipids by specific inhibitors or selective enzymes. Methods: Binding and uptake as well as infection and control experiments were done with primary Tupaia hepatocytes in vitro using highly purified, human plasma-derived virions and subviral particles. Data obtained with HBV were directly compared with results from model viruses (Semliki Forest virus, SFV; Simian virus 40, SV40) with well-known binding and entry kinetics. Results: While SV40 was reported to be found in detergentresistant membranes (DRM) that are characterized as cholesterolrich membrane-domains; we found that HBV did not bind to DRMs for initiating infection. Consistent with that, depletion of cholesterol from the plasma-membrane of susceptible hepatocytes had no effect on HBV-binding and infection. Interestingly, selective depletion of the sphingolipid sphingomyelin from the extracellular leaflet of the plasma-membrane by specific sphingomyelinase drastically reduced HBV binding, subsequent uptake and infection but had no effect on the clathrin-dependent infection process of SFV, or caveolin-dependent infection process of SV40. Confocal analysis showed that HBV and subviral particles are colocalisized to cellular sphingomyelin micro-domains with several micrometers in diameter, suggesting that membrane sphingomyelin is essential for binding, membrane-clustering or uptake of HBV. Conclusions: In summary, we could show that HBV binding and infection is independent on cellular cholesterol but needs sphingomyelin within the cellular plasma-membrane for specific infection of susceptible hepatocytes. Journal of Hepatology 2012 vol. 56 | S71–S224 S175