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Title: Pathogenesis of Hepatitis B: New Insights From Studies of the Innate Immune Response


1
Pathogenesis of Hepatitis BNew Insights From
Studies of the Innate Immune Response
  • Professor Stephen Locarnini
  • Victorian Infectious Diseases Reference
    Laboratory,
  • North Melbourne, Victoria 3051,
  • AUSTRALIA
  • www.vidrl.org.au/publications/hep_updates.htm

2
Geographic Distribution of Chronic HBV Infection
WHO global estimate gt 350 million
chronically infected individuals
gt 2 million liver related deaths annually
3
WHO Global Program for Hepatitis B
ACUTE /CHRONIC HEPATITIS B
CIRRHOSIS ESLD
HEPATOCELLULAR CARCINOMA
HBV
PRIMARY PREVENTION
CHEMO PREVENTION
CANCER SCREENING
Antivirals Cytokines
Tumour Marker Ultrasound Transplantation
Vaccination
4

5
HBV Genome Map
6
HBV Genome Map
Circular ss/ds DNA virion, supra-genomic RNA
Four major overlapping ORFs Alternative start
sites Many regulatory elements
7
Late Events Assembly Release versus Recycling
  • Precore protein
  • inhibits core dimerization.
  • essential for persistent infection.
  • first protein made on infection

8
Pre-C/C ORF
9
Viral Evolution
  • With up to 1012 copies HBV DNA per ml
  • A virion half-life of less than one day
  • Replication via an RNA-dependent DNA polymerase
  • Single base changes will be generated every day
    for
  • all residues.
  • This genomic plasticity means that selective
    pressure will inevitably cause sequence
    evolution.
  • This can be seen during the life of HBV carriers
    but surprisingly is more pronounced in the
    anti-HBe carrier with lower replication than in
    the high replicating HBe carrier.

10
590 Full Length HBV Sequences Including 23 NCBI
Prototypes
http//www.vgb.ucl.ac.uk/starn.shtml
11
Orangutan
Asian
Gorilla
Great Apes
Human
African
Chimpanzee
Bonobo
9 8 7 6
5 4 3 2
1 Present Millions of years ago
12
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13
HBV Infection Pathogenesis
  • Represents the outcome of the interplay between
    the virus, the hepatocyte and the hosts immune
    response
  • Under normal circumstances, HBV is not
    cytopathic
  • In CHB, liver damage produced as a result of
    hosts cellular immune response to HBV-infected
    hepatocytes as part of the immune clearance
    phase

14
Natural History HBeAg-POS
(Adapted from Anna Lok 1996)
15
Natural History HBeAg-NEG
(Adapted from Anna Lok 1996)
16
Strategies Used by HBV to Ensure Persistence
  • 1. HBeAg
  • Soluble and secreted protein
  • Toleragen perinatal transmission and is
    essential for PERSISTENT infection
  • Possible immune regulatory function both INNATE
    and ADAPTIVE
  • dampens hosts immune response to
    virus-infected hepatocytes
  • Pre-core protein regulates level of HBV
    replication
  • Excess empty virus particles (decoy anti-Pre-S1)
  • 2. HBsAg
  • Excess production (decoy anti-HBs) of 22 nm
    particles and filaments
  • Diverts anti-HBs neutralization of virions (42nm
    forms)
  • 3. HBV cccDNA
  • major transcriptional template
  • heterogeneous topoisomer species
  • variable half-life
  • resistant to nucleoside analogue therapy
  • 4. HBV DNA Integration
  • HBV can be generated from spliced HBV mRNA
    transcripts
  • Role in latency and reactivation.

17
Background Precore Protein
  • a unique feature of the hepatitis B virus (HBV)
    is the production of a secreted, non-particulate
    form of the nucleoprotein of the virus, the
    hepatitis Be antigen (HBeAg) Milich et al.
    1998.
  • little is known about the function of HBeAg in
    the viral life-cycle, but it is not essential for
    infection or replication Chang et al. 1987
    Schlicht et al. 1997
  • the expression of the HBeAg is one of several
    strategies used by HBV to ensure persistence
  • the HBeAg may function as an immune toleragen in
    utero Milich et al. 1990 since soluble HBeAg
    can cross the placenta
  • HBeAg has been shown to regulate the hosts
    immune response in animal models Milich et al.
    1998.
  • Cellular processing of the precore protein is
    complex

18
HBeAg Processing
S K L C L G W L W G - M
(core)
19
Virus Detection by I.D.
20
Expression of HBeAg in HBV Infections Defines
State
21
Viral Prevention of HBeAg Expression in HBV
Infections G1896A Precore Stop Codon Mutation
note these changes follow after loss of HBeAg
which tells us something
22
Precore/core Protein Production and Processing
Pregenomic mRNA translates p21 (core)
23
Precore Protein Collaborative Group
  • Locarnini laboratory (VIDRL)
  • Revill laboratory (VIDRL)
  • Visvanathan laboratory (Monash)
  • Mansell laboratory (Monash)
  • Hudson/Nuttal laboratory (CSIRO)
  • Beard laboratory (Adelaide University)
  • Lemon/Li laboratory (UTMB, Texas)
  • Kuiper laboratory (VPAC)

Clinical Collaborators
  • Professor Paul Desmond Dr Alex Thompson (St
    Vincents)
  • Professor Bill Sievert (Monash)
  • Professor Sharon Lewin (Alfred)
  • Professor Peter Angus (ARMC)
  • Dr Joe Sasadeusz (VIDS)

24
Goals of Collaborative Group
  • To try to understand the biology of the precore
    protein!!!!!!!

25
Cellular Response to Conditional Expression of
the Hepatitis B Virus Precore and Core Proteins
in Cultured Hepatoma (Huh-7) Cells
  • S Locarnini1, J Dean1, T Shaw1, D Colledge1, K
    Li2, S Lemon2, G Lau3, P Bhat1, M Beard2
  • 1 VIDRL, North Melbourne, Victoria, Australia,
  • 2 UTMB, Galveston, Texas, USA
  • 3 University of Hong Kong, SAR, China

AIM To determine the effect of the HBV precore
protein on the cellular transcriptome
Locarnini S., et al. 2005. J. Clin
Virol32113-121
26
pTRE-HBV Producing Cell Lines
  • Cloning
  • HBV genotype D in pBluescript II SK (Stratgene)
    was kindly provided by Prof H. Schaller
  • PCR products were generated using primers
    incorporating the core and precore region and the
    products were cloned into pTRE2 (Clontech)
  • plasmids co-transfected into Huh-7 cells with
    Tet-off plasmid (Clontech) and Huh-7 pTRE-insert
    clones selected with G418
  • three cell lines generated 1.
    Precore-producing
  • 2. Core-producing and
  • 3. Control cell line

27
Affymetrix Analysis (Hu95a)
  • Cell Line
  • Precore (45) Core (5) Control (8)
  • Functional Grouping Down Up Down Up
    Down Up
  • 1. Regulators of Nucleotide
  • Metabolism and Transcription 12 - 1 - 1 -
  • 2. Signal Transducers,
  • Membrane-associated and
  • Cell Trafficking Proteins 11 - 3 - 2 -
  • 3. Regulators of Cell Cycle
  • and Apoptosis 6 - - - --
  • 4. Structural and Metabolic
  • Proteins 3 1 - - 1 1
  • 5. Cytokines Other
  • Secreted Proteins 4 - - 1 - 1
  • 6. Unknown 7 1 - - 2 -
  • TOTAL 43 2 4 1 6 2

28
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29
Conclusions
  • Expression of HBV precore protein has a much
    greater effect on cellular gene expression in
    comparison to the core protein expressing or
    control cell line.
  • Genes affected during precore protein induction
    included those involved in cell-signalling, RNA
    transport and processing, cytosol-nuclear
    trafficking and innate immune responses
  • Precore protein is behaving like a tumour
    suppressor protein with anti-apoptotic activity
  • Effect on transcriptome is significant
  • Several putative innate immune response genes
    downregulated

30
Precore Protein Effect on Secretion
From Scott Preiss 2006
31
Suggests that Pre-core reduces efficiency
reporter expression translationally or
transcriptionally.
From Scott Preiss 2007
32
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33
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34
Electron Microscopy and Crystallisation of
Core-Precore Protein
  • Dr Renae Walsh
  • 2006 / 2007
  • (CSIRO and VIDRL Group)

35
Constructs
36
HBeAg (N-terminal GST)
Coomassie
Western Blot
37
Crystallisation
  • Set up
  • HBeAg .Some success?
  • IgNAR (Help 6) binder
  • Co-crystallisation of HBeAg and IgNAR.

HBeAg IgNAR (Help 6)
HBeAg
HBeAg IgNAR (Help 6)
38
EM - HBeAg HBcAg Salt Gradient (0.15M NaCl)
HBeAg (0.15M NaCl) HBcAg (0.15M
NaCl)
Physiological salt induces capsid formation,
more pronounced for HBeAg
HBeAg (0.75M NaCl) HBcAg (0.75M
NaCl)
Highest level of HBeAg capsids. HBcAg capsids
more likely to congregate, may only appear like
less capsids that eAg?
39
EM - HBeAg HBcAg Salt Gradient
Overall Comments HBcAg capsid seem more uniform
and robust in structure than HBeAg capsids HBeAg
capsids appear to require lower NaCl to induce
capsid formation than HBcAg capsids HBcAg capsids
seem to clump together more than HBeAg capsids,
which affects counting
40
EM - pCI Precore Grid Immuno (18/4) Dako Ab
Lysate None
s/n None
Fraction18 Maybe?
Fraction4 Numerous capsids
41
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42
The Innate Immune System and Hepatitis B
  • Kumar Visvanathan1 and Stephen Locarnini2
  • 1 Medicine, Monash Medical Centre, Monash
    University, Clayton, Australia and 2Victorian
    Infectious Diseases Reference Laboratory, North
    Melbourne, Australia

43
TLR2/1 TLR2/6 TLR4
TLR effector pathways
Nucleus
Penetration Endosomal compartment
mRNA transcripts
Translation / proteolytic processing
Uncoating
Viral proteins
Attachment
Genomic Replication
  • RNA / DNA
  • Intermediates
  • single-stranded
  • double-stranded

TLR3 TLR7/8 TLR9
TLR effector pathways
RLH effector pathways
RIG-I MDA5
Release
Viral Assembly
Cytoplasm
44
MyD88 Dependent Pathway
45
MyD88 Independent Pathway
TLR
TRIF
TBK1
IRF3
Interferon a/ß
46
TLR-2 and 4 Expression in Chronic Hepatitis B
  • Liver biopsies and whole blood (PBMC) on 21
    patients 9 HBeAg positive and 12 HBeAg negative
    CH-B
  • 5 disease controls (steatosis)
  • Liver biopsy processed to yield approx 6x104
    hepatocytes (no collagenase, DNase or trypsin
    used)
  • Liver biopsies and PBMCs stained with
  • CD14- Per CP (Becton-Dickinson)
  • TLR-2-FITC, and TLR-4-PE (eBiosciences)
  • Appropriate isotype controls
  • Cells gated according to CD14 expression CD14
    high or CD14 low

Visvanathan. K. et al. 2007. Hepatology45102-110
47
TLR2 Expression in HBeAg-POS and HBeAg-NEG CH-B
Patients
Visvanathan, K. et al 2007. Hepatology45102
48
IL-6 Production in HBeAg-Pos and HBeAg-Neg
Patients
HBeAg-Pos
HBeAg-Neg
Visvanathan K and colleagues. 2007. Unpublished
data.
49
Hepatocyte Interleukin-1 and Toll-Like Receptor
Signalling Effects on Hepatocyte Biology and
HBV Replication in vitro
  • Alex Thompson, S Preiss, P Revill, S Rodgers, PV
    Desmond, K Visvanathan, SA Locarnini
  • Hepatology Fellow
  • PhD student

VIDRL
St. Vincents Hospital Melbourne
50
Aims
  • To develop a cell culture model allowing for
    examination of the relationship between innate
    immune signaling and HBV
  • To define TLR signaling pathways in HepG2 cells,
    a hepatoblastoma cell-line permissive for HBV
    replication in vitro
  • To test for an antiviral effect of TLR signaling
    on HBV in this cell culture system, both
    HBeAg-positive and negative strains

51
Methods
  • ATCC HepG2 cells
  • TLR-2,3,4,5,7,9 ligands (Invivogen)
  • IL-1? (Roche) positive control
  • TLR expression
  • mRNA expression - qPCR (ABI Prism 7000)
  • Taqman probes - RPLPO, TLR 1- 9, TNF?, IFN?/?
  • TLR signaling
  • NFKB-Luciferase reporter (M. Gale)
  • Induction of cytokine mRNA - TNF?, IFN?
  • ELISA - TNF? (BD Biosciences)

1)
2), 3)
(Akira and Takeda, 2004)
52
Methods
  • Recombinant HBV-baculovirus transduction model in
    HepG-2 cells
  • HBeAg-POS (WT)
  • HBeAg-NEG (G1896A)
  • HBV Replication
  • Intracellular core-associated HBV DNA
  • Southern blot
  • Radio-labelled P32 / phospho-imaging /
    densitometry
  • HBV nucleocapsid assay
  • Immunoblot on agarose
  • Quantitative HBeAg
  • Architect assay (Abbott, IL)
  • Paul-Ehrlich International Units

Delaney, W and Isom, H. Hepatology, 1998
Melegari,M. et al. JV, 2005
Perrillo,R. et al. Hepatology, 1993
53
Do HepG2 Cells Express TLR?
Non-synchronized, harvested d1 post-passage
Not expressed
5,8
Low
1,4,7,9
Intermediate
2,3,6
mRNA expression detectable for most TLR
TLR-2,3,6 gt 1,4,7,9 gt 5,8
54
PAMP Ligand Screen
IL-1
IL-1R
Pam2Cys
TLR2/6
Pam3Cys
TLR2/1
IL-1 and TLR-2 induce TNF mRNA predictably
following stimulation Maximal stimulation
observed at 60 min
55
PAMP Ligand Screen
HepG2 cells harvested 1 hr post stimulation for
qPCR
TNF?
IFN?
Fold induction cytokine mRNA
TLR2
TLR4
TLR5
TLR3
TLR7
TLR9
RIG-I / MDA5
Minimal TNF / IFN-? mRNA induction observed
56
Are Functional IL1R, TLR2 Present?
P2C / IL-1 stimulation
Relative luciferase activity

IL-1 and TLR-2 ligand induce NFKB reporter
activity - dose response
57
Are Functional IL1R, TLR2 Present?
P2C / IL-1 stimulation ELISA

P2C
IL-1

TNF pg/ml

TNF protein - secretion peaks at 6 hrs post
stimulation by ELISA
p lt 0.05, MWU test
58
Methods 2
Recombinant HBV Baculovirus Transduction Ligand
Stimulation Protocol
  • D minus 1 - seed
  • D 0 - transduce rBCV construct
  • D 2 - media change mock / TLR ligand
  • D 3 - media change - mock / TLR ligand
  • D 4 - harvest

59
IL-1 Stimulation Inhibits HBV Replication
Wildtype rHBV - IL-1


rc
dsl
ss

LMV 0.01 uM
IL-1 5 ng/ml
Mock
p lt 0.05, MWU test
60
TLR2 Stimulation Inhibits HBV Replication WT
(HBeAg-Positive) HBV

rc

dsl


ss
LMV 0.01 uM
TNF 10 ng/ml
  • P2C
  • 10 100 1000
  • ng/ml

Mock
p lt 0.05, MWU test
61
TLR2 Stimulation Inhibits HBV ReplicationPC
Variant (HBeAg-Negative) HBV
rc
dsl
ss
LMV 0.01 uM
TNF 10 ng/ml
  • P2C
  • 10 100 1000
  • ng/ml

Mock
p lt 0.05, MWU test
62
Effect on HBeAg Secretion
Relative HBeAg secretion
No reduction in secreted HBeAg despite DNA
inhibition.
63
TOLL 2 HBV Interaction
64
TOLL 2 HBV Interaction
TLR2 EFFECT IS POST-TRANSCRIPTIONAL
65
TOLL 2 Precore Interaction
66
TOLL 2 Precore Interaction
67
NF-KB Output of Cells Transfected with Increasing
Amounts of HBV Pre-Core
Ashley Mansell Sally Rodgers, 2007
68
Summary and Conclusion
  • TLRs expressed on hepatocytes in vivo and in
    vitro
  • Precore protein/HBeAg downregulates TLR-2
    expression and functional signaling
  • Absence of Precore protein/HBeAg upregulates
    TLR-2 expression and functional signaling
  • Stimulation of TLR-2 can result in blocking
    core-associated HBV DNA replication
  • HBsAg clearance associated with increased TLR-7
    activity
  • Identification of a potentially important
    interaction of the precore protein/HBeAg and
    HBsAg with the innate immune response to HBV
  • Pathogenic and therapeutic implications in the
    management of HBeAg-Pos and HBeAg-Neg CH-B are
    significant

69
Goals of Collaborative Group
  • To try to understand the biology of the precore
    protein!!!!!!!

70
END
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