studies

294A AASLD ABSTRACTS HEPATOLOGY, October, 2015
167
Characterisation of the immune profile in Chronic Hepatitis
B, with CyTOF, to identify biomarkers of immune
control following NUC therapy discontinuation
Upkar S. Gill 1 , Laura Rivino 2 , Nina Le Bert 3 , Kamini Kunasegaran
2 , Damien Tan 3 , Sarene Koh 3 , Machteld Van Den Berg 2 , Yang
Cheng 4 , Navjyot K. Hansi 1 , Graham R. Foster 1 , Evan W. Newell
4 , Antonio Bertoletti 2,3 , Patrick T. Kennedy 1 ; 1 Hepatology Unit,
Centre for Immunobiology, Blizard Institute, Barts and The London,
School of Medicine & Dentistry, QMUL, London, United Kingdom;
2 Program Emerging Viral Diseases, Duke-NUS Graduate Medical
School, Singapore, Singapore; 3 Infection & Immunity Program,
Singapore Institute for Clinical Sciences, Agency for Science, Technology
& Research (A*STAR), Singapore, Singapore; 4 Singapore
Immunology Network, Singapore Agency for Science, Technology
& Research (A*STAR), Singapore, Singapore
INTRODUCTION: The absence/functional exhaustion of
HBV-specific T cells is the hallmark of chronic HBV infection
(CHB); conversely a robust functional response of these cells
is associated with viral control. Current therapies are limited
in their ability to restore the functional HBV-specific repertoire.
Consequently, Nucleot(s)ide analogue (NUC) therapy remains
indefinite in the majority. Clinical parameters alone cannot
distinguish in which patients NUC therapy can be safely discontinued,
with durable immune control from those who will
relapse and develop hepatic flares (HF). We studied a cohort
of virally suppressed patients on potent NUC therapy prior to
and after treatment discontinuation to characterise the immune
profile associated with viral control. PATIENTS & METHODS:
PBMC were analysed at 4-weekly intervals prior to and after
NUC discontinuation. The frequency of HBV-specific T cells
was assessed by IFNγ ELISPOT after 10 day expansion in the
presence of HBV peptides spanning the entire viral proteome.
Phenotypic and functional characteristics of T & NK cells were
studied with an in-depth longitudinal analysis of the expression
of >35 markers involved in cell activation, differentiation and
exhaustion, by cytometry time of flight (CyTOF), a novel mass
cytometry technology. Serum cytokine and chemokine levels
(IL-1β, IL-6, TNF-α, IL-10, CXCL-8, CXCL-10) were also measured
using Luminex. RESULTS: Prior to NUC discontinuation,
patients could be divided into 2 groups based on the presence/absence
of HBV-specific T cells. Upon NUC discontinuation,
those patients with detectable frequencies of circulating
HBV-specific T cells were able to control HBV replication and
did not demonstrate HF’s. In these patients HBV-specific T cells
preferentially target HBV polymerase followed by core proteins.
In patients without viral control who developed HF, we
noted a significant increase in serum CXCL10 and IL10 levels,
correlating with ALT. Patients without HF’s were characterised
by increased frequencies of a CD8+ T cell subset expressing
CD56, CD107, CCR5, CD127, CD28, 2B4, KLRG1, IFNγ,
TNFα, MIP1β & IL2. NK cell profiles did not differ depending
on viral rebound/HF upon NUC discontinuation, but all CHB
patients expressed significantly higher levels of the exhaustion
marker, KLRG1 on NK cells, compared with healthy controls.
CONCLUSIONS: Our data suggest that HBV-polymerase
specific T cells represent a potential biomarker that can be
used to predict those patients who will control HBV after NUC
discontinuation. These results further support the concept that
HBV-specific T cells are associated with viral control rather than
contributing to immunopathology.
Disclosures:
Graham R. Foster - Advisory Committees or Review Panels: GlaxoSmithKline,
Novartis, Boehringer Ingelheim, Tibotec, Chughai, Gilead, Janssen, Idenix,
GlaxoSmithKline, Novartis, Roche, Tibotec, Chughai, Gilead, Merck, Janssen,
Idenix, BMS; Board Membership: Boehringer Ingelheim; Grant/Research Support:
Chughai, Roche, Chughai; Speaking and Teaching: Roche, Gilead, Tibotec,
Merck, BMS, Boehringer Ingelheim, Gilead, Janssen
Patrick T. Kennedy - Grant/Research Support: Roche, Gilead; Speaking and
Teaching: BMS, Roche, Gilead
The following authors have nothing to disclose: Upkar S. Gill, Laura Rivino, Nina
Le Bert, Kamini Kunasegaran, Damien Tan, Sarene Koh, Machteld Van Den Berg,
Yang Cheng, Navjyot K. Hansi, Evan W. Newell, Antonio Bertoletti
168
Hepatitis B virus X protein stimulates HBV replication by
regulating transcription factors associated with DNA or
histone methylation
Naoki Oishi 1,2 , Xuyang Wang 2 , Kazunori Kawaguchi 1,2 , Masao
Honda 1,2 , Seishi Murakami 2 , Shuichi Kaneko 1,2 ; 1 Department of
Gastroenterology, Kanazawa University Hospital, Kanazawa,
Japan; 2 Department of Disease Control and Homeostasis,
Kanazawa University Hospital, Kanazawa, Japan
Background: Hepatitis B virus (HBV), a small enveloped DNA
virus, chronically infects more than 350 million people worldwide
and causes liver diseases, from hepatitis to cirrhosis and
liver cancer. HBx is a multifunctional protein encoded by the
HBV genome that stimulates HBV replication. Previously, we
found that HBx has an important role in stimulating HBV transcription
and replication and that the transcriptional transactivation
function of HBx may be critical for its augmentation
effect on HBV replication. However, the molecular mechanism
of HBx in transcriptional coactivation remains unclear. In this
study, we provide a new insight into the coactivator mechanism
and the possibility of a new treatment target for HBV replication
and HBV-related hepatocarcinogenesis. Methods: We
used a retroviral vector to introduce wild-type HBx (HBxwt) or
empty vector (EV) into HepG2 cells. Gene expression profiling
was performed using Affymetrix GeneChip Human U133A2.0
ver.2.0 arrays according to the manufacturer’s protocol. Unsupervised
hierarchical clustering analysis and class comparison
analysis were performed with BRB-Array Tools software version
4.2.2. Transcription factor analysis was performed using
Ingenuity Pathway Analysis (IPA; Ingenuity Systems) to identify
potential upstream transcription factors (TFs). We used array
data from 244 chronic hepatitis B patients for analyzing clinical
features. Results: Unsupervised hierarchical clustering analysis
of HepG2-HBxwt or HepG2-EV cells revealed that the gene
expression profiles of these cell lines were significantly different.
Class comparison analysis between both cell lines identified
803 HBx-related genes. Six HBx-related activated TFs that
affected HBV replication were identified by IPA, small interfering
RNA, and inhibitor analyses. Three of these six TFs control
HBV replication by modifying DNA or histone methylation. By
analysis of 244 hepatitis B patients, these three TFs were found
to be expressed at a significantly highly level in HBeAg(+)
HBeAb(-) cases than in HBeAg(-)HBeAb(+) cases. Moreover,
these TFs up-regulated the expression of stemness markers
(EpCAM, AFP, and SOX9) and epithelial-mesenchymal transition
markers (ZEB1, ZEB2, and VIM) and were associated with
a poor prognosis of HCC. Conclusions: HBx upregulated three
TFs associated with DNA or histone methylation. Moreover,
HBx stimulates HBV replication and hepatocarcinogenesis by
modifying DNA or histone methylation. Our study suggests that
TFs activated by HBx will be an important therapeutic target
against both virus replication and hepatocarcinogenesis aimed
at the prevention of HCC.