Hepatitis B Pathogenesis: The Importance of cccDNA and the Precore Protein

1
Hepatitis B Pathogenesis The Importance of
cccDNA and the Precore Protein

• Professor Stephen Locarnini
• Victorian Infectious Diseases Reference
  Laboratory,
• North Melbourne, Victoria 3051,
• AUSTRALIA
• www.vidrl.org.au/publications/hep_updates.htm

2
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.

3
HBV Replication cccDNA Generation
Nucleus
ER/Golgi
Cytosol
4
HBV Replication Recycling or Release
transport to cell
nucleus
CCC DNA
Attachment and Penetration
DNA
uncoating
repair
MINICHROMOSOME
Nucleus
Re-entry
pregenomic RNA
HBV RNA
Golgi
transcripts
complex
HBV polymerase
protein

envelope proteins S, M, L
?
Precore protein
core proteins (HBcAg)
Release
HBV Virion
HBeAg
Secretory Pathway
HBV DNA SYNTHESIS
HBsAg
5
EVASION STRATEGY 1cccDNA in Chronic Hepatitis B

• cccDNA is produced by repair of double stranded
  DNA and incorporated in the nucleosome as a
  stable, episomal minichromosome
• The source of cccDNA is incoming virus and the
  recycling of nucleocapsids from the hepatocyte
  cytoplasm
• cccDNA serves as the transcriptional template for
  viral mRNAs
• (that is pregenomic RNA and mRNA for surface,
  core, and polymerase)

6
Role of HBV cccDNA

• Viral persistence is due to maintenance of the
  HBV cccDNA pool in the nuclei of infected cells
• HBV cccDNA does not self-replicate, so is not
  directly affected by NA-based therapy
• Stability related to long half-life of
  hepatocytes
• cccDNA represents an genetic archive for a rapid
  reselection of resistant mutants
• Little is known of the significance of HBV cccDNA
  levels during
• acute infection
• progression to chronic infection
• resolution of acute or chronic infection
• antiviral drug therapy

Werle-Lapostolle et al (2004) Gastroenterology
1261750
7
Selective PCR for HBV cccDNA
Werle-Lapostolle et al (2004) Gastroenterology
1261750
8
Hepatic HBV cccDNA Levels in Different Patient
Populations
Quantitative real-time PCR used to measure cccDNA
from liver biopsies
Werle, Petersen, Locarnini, Zoulim
Gastroenterology 2004
9
cccDNA Levels as a Predictor of Reactivation
After Chemotherapy(Dr George Lau, QMHK, Hong
Kong)

• HBsAg positive lymphoma (patients 22)
• Treated with intense chemotherapy
• Evaluated pre-chemotherapy serum HBV load,
  intrahepatic HBV DNA and cccDNA as predictors of
  reactivation

Hui CK et al (2005). Blood 105 2616.
10
Optimal Cut-Off of cccDNA to Predict Reactivation
Hui CK et al (2005). Blood 105 2616.
11
Is cccDNA Clearance Achievable?

• Possibly, in a small percentage of patients using
  antiviral therapies that are more potent and have
  a negligible rate of resistance for long
  durations
• Since cccDNA can be detected in inactive carriers
  and people with resolved infections, long-term
  suppression to low concentrations may be
  sufficient in preventing progressive liver damage
  
• Need to Activate Host Immune Responses against HBV

12
A Lok 2005
13
HBV Minichromosomes and Chromatin Modelling

• Structure of Chromatin
• Altered by post-translational modification of
  histones
• Acetylation, phosphorylation, methylation and
  ubiquitylation
• Two groups of enzymes
• Histone deacetylases (HDACs) and histone
  acetyltransferases (HATs) determine the
  acetylation status of histones

14
Acetylation Status of HBV cccDNA Bound H3 and H4
From Pollicino, T. et al 2006. Gastroenterology13
0823
15
Comparison Between Amounts of Intrahepatic and
Serum Markers in CH-B
  HBeAg Anti-HBe Occult HBV P-value
Liver Total HBV DNA (copies/ml) 651 300 0.06 lt0.001
cccDNA (copies/ml) 11.4 0.58 0.02 lt0.001
pgRNA (copies/cell) 248 3 0.001 lt0.001
Serum HBV DNA (copies/ml) 6 x 108 4 x 106 14 lt0.001
HBsAg (IU/ml) 12,748 8,113 ND lt0.001
Pollicino, T. et al 2007.
16
Conclusion

• Markers of HBV replication reflect the natural
  history phase of CH-B.
• No correlation was found between cccDNA amounts
  and serum HBeAg levels in any group of patients.
• HBV cccDNA appears to be hypoacetylated and
  methylated in occult infection compared to CH-B,
  suggesting that epigenetic silencing might have a
  role in suppressing viral activity.

Pollicino, T. et al 2007
17
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.

18
EVASION STRATEGY 2Precore 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

19
Precore/core Protein Production and Processing
Pregenomic mRNA translates p21 (core)
20
Virus Detection by I.D.
21
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.

22
Precore (PC) G1896A
HBV Variants Reduce HBeAg Production
Translational stop codon - PC mRNA transcribed -
no HBeAg translated
23
HBV Variants Reduce HBeAg Production
Basal Core Promoter (BCP) A1762T/G1764A(genotype
s A,F)
Reduced transcription - reduced PC mRNA -
reduced HBeAg
24
Clinical relevance

• HBeAg seroconversion a clinical endpoint for
  therapy
• Predicts
• Normalization of ALT / Profound drop in VL /
  Remission of histology
• Drop in cccDNA
• (Werle-Lapostolle, Gastr, 2004)
• Preliminary data from the pegylated -interferon
  registration trials suggests a potential role for
  quantitative HBeAg titres in
• Predicting seroconversion baseline level of
  186.3IU/ml - 41
• Predicting treatment failure level gt 100IU/ml at
  6/12 - 4
• (Cooksley, EASL, 2005 (abs 71) Fried, AASLD,
  2005, (abs 182))

25
Assay validation

• Architect immunoassay
• Optimal linear range
• High throughput commercial
• Cost per sample 5 AUD
• Accurate and reproducible
• 1) Intra-assay variation
• the intra-assay variation was less than 5 (0 -
  6.1)
• Again, the higher variation was seen at lower
  HBeAg levels
• 2) Inter-assay variation
• the percentage coefficient of variation ranged
  from 2.7 - 9.8
• the higher coefficient of variation was seen at
  lower HBeAg levels.

1 10
100 PE IU/ml
26
Population Distribution of HBeAg Titre
In 40 the HBeAg titre lt 1000 PE IU/ml
Number of cases
(n 85)
27
Population Distribution of HBeAg Titre
Most patients have a titre lt 100 PE IU/ml
Number of cases
28
HBV variants and HBeAg titre in vivo
PC/BCP variants reduce HBeAg titre
Wildtype
BCP
PC
Mann-Whitney U-Test p lt 0.05
29
Natural History HBeAg-POS
(Adapted from Anna Lok 1996)
30
Natural History HBeAg-NEG
(Adapted from Anna Lok 1996)
31
Discussion Future work

• With such refinement there is exciting potential
  for the use of qHBeAg titres as a clinical tool
• in guiding patient selection
• in monitoring antiviral therapy
• We are currently analyzing a nucleoside analogue
  treatment cohort to define the kinetics of HBeAg
  decline in relationship to viral load with view
  to an algorithm for predicting seroconversion

32
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

33
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
34
MyD88 Dependent Pathway
35
MyD88 Independent Pathway
TLR
TRIF
TBK1
IRF3
Interferon a/ß
36
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
37
TLR2 Expression in HBeAg-POS and HBeAg-NEG CH-B
Patients
Visvanathan, K. et al 2007. Hepatology45102
38
TOLL 2 Precore Interaction
39
TOLL 2 Precore Interaction
40
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
41
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
42
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
43
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
44
Effect on HBeAg Secretion
Relative HBeAg secretion
No reduction in secreted HBeAg despite DNA
inhibition.
45
TOLL 2 HBV Interaction
TLR2 EFFECT IS POST-TRANSCRIPTIONAL
46
Thompson A., Visvanathan K. and Locarnini S.
Gastro1131031.
47
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
• 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

48
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.