studies

1016A AASLD ABSTRACTS HEPATOLOGY, October, 2015
1655
A Novel Entecavir Resistance Mutation RtA186T Causes
Viral Breakthrough in Chronic Hepatitis B Patients
Sanae Hayashi 1 , Shuko Murakami 1 , Katsumi Omagari 1 , Takeshi
Matsui 2 , Etsuko Iio 1 , Masanori Isogawa 1 , Tsunamasa Watanabe
1 , Yoshiyasu Karino 3 , Yuki Takamatsu 4 , Satoru Kohgo 5 , Kenji
Maeda 5 , Hiroaki Mitsuya 4,5 , Yasuhito Tanaka 1 ; 1 Department of
Virology & Liver unit, Nagoya City University Graduate School of
Medical Sciences, Nagoya, Japan; 2 Center for Gastroenterology,
Teine Keijinkai Hospital, Sapporo, Japan; 3 Department of Gastroenterology,
Sapporo Kosei General Hospital, Hokkaido, Japan;
4 Experimental Retrovirology Section, HIV and AIDS Malignancy
Branch, National Cancer Institute, National Institutes of Health,
Bethesda, MD; 5 National Center for Global Health and Medicine,
Tokyo, Japan
Background & Aim: Entecavir (ETV) is approved for the first-line
treatment of chronic hepatitis B virus (HBV) infections due to a
high genetic barrier. The aim of this study is to characterize
two novel reverse transcriptase (RT) mutations associated with
viral breakthrough (VBT) in an ETV-refractory patient. Methods:
Serum HBV from an ETV refractory patient was sequenced
before ETV treatment and after VBT. The clones with candidate
ETV resistance (ETVr) mutations (rtI163T and rtA186T)
were analyzed for replication efficacy and susceptibility to
ETV, Adefovir (ADV), Tenofovir Disoproxil Fumarate (TDF), and
novel nucleoside analogues (NAs) (4’-C-cyano-2-amino-2’-deoxyadenosine
(CAdA), 4’-C-cyano-2’-deoxyguanosine (CdG))
with similar efficacy to ETV in vitro. Moreover, chimeric mice
with human hepatocytes were inoculated with the patient’s
serum at VBT, and monitored for viral mutation pattern by
next-generation sequencing. To confirm the prevalence of the
novel mutations, 21 ETV-refractory patients were also examined.
Results: The novel mutations, rtI163V and rtA186T, were
detected together with LAMr at VBT, but not before ETV treatment.
RtA186T significantly reduced viral replication efficacy,
while rtI163V had no impact on it. RtA186T plus LAMr reduced
susceptibility to ETV as strongly as previously reported ETVr
mutations rtS202G plus LAMr, resulting in more than 111.1-
fold resistance to ETV compared with the wild-type clone. In
contrast, rtI163V plus LAMr resulted in a 20.4-fold resistance
to ETV, suggesting that rt186T was mainly responsible for the
VBT. The novel ETVr mutations did not confer cross-resistance
to ADV, TDF, as well as novel NAs, CAdA and CdG. The viral
mutation pattern in the chimeric mice indicated dominant proliferation
of a clone containing rtI163V and rtA186T mutations
plus LAMr under ETV treatment. In silico docking simulation
indicated that rtA186T reduced the RT binding ability to ETV.
In clinical practice, rtA186T, but not rtI163V, was detected
in another ETV-refractory patient with VBT, suggesting that
rtA186T could confer ETV resistance independently of rtI163V.
Conclusion: A novel ETV resistance mutation rtA186T causes
VBT, and should be closely monitored when chronic hepatitis B
patients exhibit VBT during prolonged ETV treatment.
Disclosures:
Yoshiyasu Karino - Speaking and Teaching: BMS KK
Yasuhito Tanaka - Grant/Research Support: Chugai Pharmaceutical CO., LTD.,
MSD, Bristol-Myers Squibb; Speaking and Teaching: janssen pharma, Bristol-Myers
Squibb
The following authors have nothing to disclose: Sanae Hayashi, Shuko Murakami,
Katsumi Omagari, Takeshi Matsui, Etsuko Iio, Masanori Isogawa, Tsunamasa
Watanabe, Yuki Takamatsu, Satoru Kohgo, Kenji Maeda, Hiroaki Mitsuya
1656
Hepatitis B Virus Surface Protein-induced hPIAS1 Transcription
Requires TAL1, E47, MYOG, NFI, andMAPK
Signal Pathways
Hongyan Wang, Di Wu, Xiaofeng Wang, Guang Chen, Yuanya
Zhang, Weiming Yan, Meifang Han, Qin Ning; Department and
institute of Infectious Disease,, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan,
China
The protein inhibitor of activated STAT1 (PIAS1) is ubiquitous
in mammals and fulfills key functions in cell proliferation and
inflammation responses. Despite the importance of PIAS1 in
regulating virus-induced or IFN-stimulated chronic hepatitis, little
is known about the molecular mechanisms of activation in
response to hepatic virus infection. Here, we report that the
hepatitis B virus surface protein (HBs) enhancedhPIAS1 promoter
activity in luciferase reporter assays. A strong regulatory
region from −712 to −507 (relative to the transcriptional start
site) was responsible forhPIAS1 gene transcription in response
to HBs. TAL1, E47, myogenin (MYOG), and NFI were highly
expressed and localized to the nucleus in response to HBs.
Binding of TAL1, E47, MYOG, and NFI to a cis-regulatory
element in the hPIAS1 promoter was confirmed by electrophoretic
mobility shift assays and chromatin immunoprecipitation.
siRNA knockdown of TAL1, E47, MYOG, and NFI
expression inhibited hPIAS1transcription in response to HBs.
Increased ERK and p38MAPK phosphorylation was observed
in HBs-transfected HepG2 cells, and treatment with the ERK
inhibitor PD098059 or p38MAPK inhibitor SB203580 abolished
increase in nuclear TAL1, E47, MYOG, and NFI levels
and hPIAS1 induction. Peripheral blood mononuclear cells from
patients withhigh HBsAg levels (also withhigh levels of HBV
DNA ) displayed increased ERK and p38MAPK phosphorylation
and high levels of TAL1, E47, MYOG, and NFI, compared
to those with low HBsAg levels or healthy controls. Increased
hPIAS1 expression was detected in liver biopsies from CHB
patients with high HBV replication compared to those of undetectable
for HBV or healthy controls.These findings suggest that
the HBs protein enhances hPIAS1transcription through activities
of TAL1, E47, MYOG, and NFI that are dependent on MAPK
signaling pathways.
Disclosures:
Qin Ning - Advisory Committees or Review Panels: ROCHE, NOVARTIS, BMS,
MSD, GSK; Consulting: ROCHE, NOVARTIS, BMS, MSD, GSK; Grant/Research
Support: ROCHE, NOVARTIS, BMS; Speaking and Teaching: ROCHE, NOVAR-
TIS, BMS, MSD, GSK
The following authors have nothing to disclose: Hongyan Wang, Di Wu,
Xiaofeng Wang, Guang Chen, Yuanya Zhang, Weiming Yan, Meifang Han
1657
The activating receptor NKP46 is essential for the development
of chronic hepatitis B
Wanyu Li, Xiuzhu Gao, Ruqi Mei, Jinglan Jin, Junqi Niu; the first
hospital of jilin university, Changchun, China
BACKGROUND & AIMS: Our pervious study indicated that
NKp46 expression regulated NK cell cytolytic function. NKp46
may moderate NK cell activity during HBV replication suppression
and HBV-associated liver damage in peripheral blood.
Here we studied the functions of NKP46 during hepatitis B
virus (HBV) infection in vivo and in the liver of patients. METH-
ODS: Intrahepatic NKP46 was investigated in chronic hepatitis
B patients(n=25) and healthy controls(n=15) by immunohistochemistry.We
examined the effects of blocking antibodies
against NKP46(50ug) in immunocompetent mice that express
HBV from a pAAV/HBV1.2 plasmid(10ug) and are positive