2016年EASL：REP2139摘要2

StephenW

THU-204
NUCLEIC ACID-BASED POLYMERS EFFECTIVE AGAINST HEPATITIS B
VIRUS INFECTION IN PATIENTS DO NOT HARBOUR IMMUNE
STIMULATORY PROPERTIES IN PRIMARY ISOLATED BLOOD OR
LIVER CELLS
C. Real1, M. Werner1, A. Paul2, G. Gerken1, J. Schlaak1,3, A. Vaillant4,
R. Broering1. 1Deptartment of Gastroenterology and Hepatology;
2Deptartment of General-, Visceral- and Transplantation Surgery,
University Duisburg-Essen, Medical Faculty, Essen; 3Evangelisches
Klinikum Niederrhein GmbH, Duisburg, Germany; 4Replicor Inc.,
Montreal, Quebec, Canada
E-mail: [email protected]
Background and Aims: Nucleic acid polymers (NAPs) block the
release of HBsAg from infected hepatocytes in vivo. Although this
mechanism is likely responsible for the activity of NAPs against
hepatitis B virus (HBV) in patients, the role of potential
immunostimulatory effects have not been explored. In this study,
the immune stimulatory properties of NAPs were examined in
primary isolated human blood and parenchymal and nonparenchymal
liver cells.
Methods: Human peripheral blood mononuclear cells (PBMCs) and
primary isolated hepatocytes (PHH) and Kupffer cells (KC) were
treated with the following NAPs: REP 2006, the prototypic
degenerate NAP (dN)40, contains residual CpG (TLR-9 stimulatory)
content, REP 2055 is clinically active having a sequence (dAdC)20
devoid of CpG content and REP 2139 (also clinically active) and REP
2165 are REP 2055 analogues further rendered immunologically
inactive by replacing cytidine with 5-methylcytidine replaces and 2′-
O methylation of riboses. Immune responsiveness was confirmed
with known ligands for TLR-7 (poly I:C), TLR-3 (ssRNA) or TLR-9 (CpG
ODN2216). Total RNA was isolated and quantitative RT-PCR was
performed to analyse gene expression indicating antiviral (IFNA4,
IFNB1, IFNG, IFNL2) and inflammatory (TNF, IL6, and IL10) effects. The
intracellular uptake of CY3-labelled NAPs was confirmed using
fluorescence microscopy.
Results: REP 2006, REP 2139 and REP 2165 induced proinflammatory
responses in PBMCs but displayed no significant
antiviral activity. In PHH, no significant inflammatory or antiviral
responses were detected for any NAP. In KC, pro-inflammatory
activity (restricted to TNF) was observed with REP 2006 and REP
2055, whereas a weak but significant induction of interferon genes
(INFA and IFNL2) was only observed with REP 2006 at the highest
concentration. These signals were comparable to those induced by
ODN2216 stimulation.
Conclusions: The data presented here suggest that NAPs optimized
to treat hepatitis B virus infection in patients do not induce antiviral
responses in primary isolated blood or parenchymal and nonparenchymal
liver cells. We therefore hypothesize that the antiviral
activity of NAPs against HBV infection cannot be explained by direct
induction of innate antiviral responses.

StephenW

THU-204
核酸基于聚合物的有效的抗乙肝
病毒感染的患者并不港免疫
刺激性质的一次分离血液或
肝细胞
C. Real1，M. Werner1，A Paul2，G Gerken1，J. Schlaak1,3，A Vaillant4，
R. Broering1。胃肠病学和肝病学1Deptartment;
常规 - ，Visceral-和移植外科2Deptartment，
杜伊斯堡 - 埃森大学，医学院，埃森; 3Evangelisches
KLINIKUM莱茵有限公司，德国杜伊斯堡; 4Replicor公司，
蒙特利尔，魁北克，加拿大
电子信箱：[email protected]
背景和目的：核酸聚合物（国家行动计划）阻断
从体内感染的肝细胞的HBsAg的释放。虽然这
机制是国家行动方案的反对活动有可能负责
乙型肝炎病毒（HBV）的患者，潜在的作用
免疫刺激作用没有得到探讨。在这个研究中，
国家行动方案的免疫刺激性质进行了检查
一次分离人体血液和实质和非实质
肝细胞。
方法：人外周血单核细胞（PBMC）和
初级分离肝细胞（PHH）和枯否细胞（KC）是
2006年REP的原型：与下列处理行动方案
退化NAP（DN）40，含有残留CPG（TLR-9刺激）
内容REP 2055是具有序列（DADC）20临床活动
缺乏CpG含量和REP 2139（临床上也有源）和REP的
2165是REP 2055类似物进一步呈现免疫
通过用5-甲基胞苷替换胞苷非活性取代并2'-
核糖的O-甲基化。免疫反应被证实
与TLR-7已知的配体（聚I：C）中，TLR-3（单链RNA），或TLR-9（CpG的
ODN2216）。分离总RNA和定量RT-PCR是
进行分析基因表达表明抗病毒（IFNA4，
IFNB1，IFNG，IFNL2）和炎症（TNF，IL-6，和IL10）的影响。该
CY3标记的国家行动方案的细胞内吸收使用确认
荧光显微镜。
结果：2006年REP，REP 2139和2165 REP诱导促炎
在外周血单个核细胞，但反应未显示显著
抗病毒活性。在PHH，没有显著炎性或抗病毒
任何NAP检测反应。在KC，促炎
活动（仅限于TNF）与2006年REP和REP观察
2055，而干扰素基因的微弱但显著感应
（INFA和IFNL2）与2006年REP最高仅见
浓度。这些信号是与那些由感应
ODN2216刺激。
结论：这里介绍的数据显示，国家行动方案进行了优化
治疗乙肝病毒感染的患者不要诱导抗病毒
在一次分离血液或实质和非实质回应
肝细胞。因此，我们推测这种抗病毒
预防HBV感染的国家行动方案活动不能被直接解释
感应先天抗病毒反应。