AASLD2014:GS9620细胞因子通过Toll样受体7激动剂有效抑制乙肝病

StephenW

1879
Cytokines Induced by a Toll-Like Receptor 7 Agonist Potently Inhibit HBV RNA, DNA and Antigen Levels in Primary Human Hepatocytes
Congrong Niu, Stephane Daffis, Mei Yu, Guofeng Cheng, William E. Delaney, Simon P. Fletcher;
Gilead Sciences, Foster City, CA

Background & Aims: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in phase 2 trials for the treatment of chronic hepatitis B (CHB). Infrequent dosing of GS-9620 (e.g. once a week) induced prolonged suppression of serum viral DNA and antigens in animal models of CHB. Here we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 by evaluating the antiviral activity of TLR7 agonists in vitro. Methods: Primary human hepatocytes (PHH) were infected with HBV and ≥3 days later were treated with a TLR7 agonist (compound A), with media from human PBMCs treated with the TLR7 agonist (TLR7 conditioned media; TLR7-CM) or with recombinant cytokines. Antiviral activity was evaluated by quantifying extracellular HBV DNA (qPCR), HBeAg and HBsAg (ELISA), as well as intracellular HBV RNA (qRT-PCR). Results: The TLR7 agonist compound A (a close analog of GS-9620), had no direct antiviral activity in HBV-infected PHH, consistent with the lack of functional TLR7 in hepatocytes. In contrast, sustained exposure of HBV-infected PHH to TLR7-CM strongly reduced the levels of HBV DNA and HBeAg (>90%), as well as HBsAg and HBV RNA (>75%), without detectable toxicity. Reductions in viral parameters were observed when PHH were treated with TLR7-CM early (3 days) or late (13 days) post-infection, but were not induced by media from control DMSO-treated PBMCs. We next compared the durability of short (3 day) and long (10 day) treatment of HBV-infected PHH with TLR7-CM. Short duration treatment (days 3-6 post-infection) only transiently reduced HBeAg. In contrast, prolonged treatment (days 3-13 post-infection) induced more sustained suppression of HBeAg, with rebound still incomplete 7 days after removal of TLR7-CM (day 20). A survey of cytokines in TLR7-CM identified recombinant IFN-α2a as a potent antiviral cytokine (EC50 ≤ 10 IU/mL for HBV DNA and HBeAg) in HBV-infected PHH. Recombinant TNF-α, IFN-γ and IFN-λ1 also strongly reduced HBV DNA, RNA and antigen levels, whereas IL-6 had only weak antiviral activity. Since TLR7 agonists induced substantially more IFN-α and IL-6 in human PBMCs than other cytokines, these data indicated that IFN-α was likely the principal mediator of TLR7-CM antiviral activity in HBV-infected PHH. Conclusion: Sustained exposure to antiviral cytokines directly induced by TLR7 activation, such as IFN-α, potently inhibited HBV in PHH in vitro. However, since short duration exposure had only a transient antiviral effect, additional components of the TLR7-induced immune response may also play an important role in the antiviral response to GS-9620 in vivo.

Disclosures:

Congrong Niu - Employment: Gilead Science Stephane Daffis - Employment: Gilead Sciences

Guofeng Cheng - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

William E. Delaney - Employment: Gilead Sciences; Patent Held/Filed: Gilead Sciences; Stock Shareholder: Gilead Sciences

Simon P. Fletcher - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

The following people have nothing to disclose: Mei Yu
1880

StephenW

1879
细胞因子通过Toll样受体7激动剂有效抑制乙肝病毒的RNA诱导，在人原代肝DNA和抗原水平
从戎牛，斯特凡Daffis，余梅，程国丰，威廉·E·德莱尼，西蒙体育弗莱彻;
吉利德科学公司，加州福斯特城

背景与目的：GS-9620，Toll样受体7（TLR7）的口服激动剂，在第2阶段临床试验用于治疗慢性乙型肝炎（CHB）的治疗。 GS-9620的不频繁给药（如每周一次）引起的血清病毒DNA和乙肝动物模型抗原的长期抑制。在这里，我们调查了有助于通过评估TLR7激动剂的抗病毒活性在体外对GS-9620的抗病毒应答的分子机制。方法：原代人肝细胞（PHH）感染了乙肝病毒，并≥3天后被用TLR7激动剂（化合物A）对待，从与TLR7激动剂（TLR7条件培养基; TLR7-CM）处理的人外周血单个核细胞介质或重组细胞因子。抗病毒活性通过定量细胞外HBV DNA（定量PCR），HBeAg和HBsAg的（ELISA），以及细胞内的HBV RNA（RNA的qRT-PCR）进行评估。结果：TLR7激动剂化合物A（GS-9620的亲密模拟），并没有直接的抗病毒活性，在HBV感染PHH，在肝细胞的功能缺失TLR7一致。相反，持续HBV感染PHH暴露于TLR7-CM大大减少HBV DNA和HBeAg的（> 90％）的水平，以及HBsAg和HBV RNA（> 75％），没有可检出的毒性。在病毒参数的减少观察时PHH分别与TLR7-CM早期（3天）或晚期（13天），感染后治疗，但没有引起从控制DMSO处理的PBMC中的介质。我们接下来比较短（第3天）和长期（10天）治疗HBV感染PHH与TLR7-CM的耐久性。短的治疗时间（3-6天感染后）只有短暂降低大三阳。相比之下，e抗原诱导更多的持续抑制，随着反弹长期治疗（3-13天感染后）仍不能完全除去TLR7-CM后7天（20天）。细胞因子在TLR7-CM的调查确定重组IFN-α2A作为一种强效的抗病毒细胞因子（EC≤10国际单位/毫升的血清HBV DNA和HBeAg），乙肝病毒感染者PHH。重组TNF-α，IFN-γ和IFN-λ1也大大减少HBV DNA，RNA和抗原水平，而IL-6只有弱的抗病毒活性。因为TLR7激动剂诱导的基本上更多的IFN-α和IL-6在人PBMC比其它细胞因子，这些数据表明，IFN-α是可能的TLR7-CM的抗病毒活性在HBV感染PHH的主要介体。结论：持续暴露于直接诱导TLR7激活抗病毒细胞因子，如干扰素-α，有效抑制HBV在PHH体外。然而，由于时间短曝光只有短暂的抗病毒效果，TLR7-诱导的免疫应答的附加组分也可起到在体内对GS-9620的抗病毒反应中起重要作用。

披露：

从戎牛 - 就业：吉利德科学斯特凡Daffis - 就业：Gilead Sciences公司

国丰诚 - 就业：Gilead Sciences公司;股股东：Gilead Sciences公司

威廉·德莱尼 - 就业：Gilead Sciences公司;专利已挂起/归档：Gilead Sciences公司;股股东：Gilead Sciences公司

西蒙·P.弗莱彻 - 就业：Gilead Sciences公司;股股东：Gilead Sciences公司

下面的人都没有透露：美宇