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Rationale for
Different Virological
Targets in HBV
Professor Stephen Locarnini WHO Regional Reference Laboratory for Hepatitis B
Victorian Infectious Diseases Reference Laboratory,
Doherty Institute
Melbourne, Victoria 3000, AUSTRALIA
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Disclosure
Clear-B
Therapeutics
Gilead
Sciences
Inc
Arrowhead
Research
Corp
Spring Bank
Pharmaceuticals,
Inc.
Roche
Molecular
AusBio
Ltd
Janssen
(J&J)
AbbVie
Consulting Fees
(eg. Advisory
Boards)
YES YES YES YES YES YES YES
Contract
Research (grant)YES YES
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Outline of Presentation
1. Overview of Current Virological
Targets
2. Possible New Directions
3. Opportunities for Integration into
Existing and New Therapies
“Combine or Perish”
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Current Efforts
1. Entry (Myrcludex B)
2. Viral Transcriptome (RNAi)
3. Capsid Assembly – Disassembly
(CpAM)
Q. How many targets need to be
blocked to achieve cure?
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HBV Lifecycle Showing Novel
Approaches for Viral Targets
Kapoor R & Kottilil S. 2014. Future Virol;9:565-585
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Li, J et al 2016. Hepatol;63(1):11-13
HBV Entry into Hepatocytes
• Internalization via Clathrin-Mediated Endocytosis
• IMPORTANT CONCEPT EMERGING: PROTECT CELLS FROM
MULTIPLE ROUNDS OF VIRAL RE-INFECTION (Urban, S)
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Wang, J et al 2016. J Hepatol;65:700-710
Entry & Re-Entry and HBV
• HBV WT and HBV DSL DNA
• HBV SPLICE VARIANTS
• HBV FL RNA
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HBx RNA Was Detected Very Early
After HBV Infection
Beran, R et al 2017. EASL
*Mock infection. LHB, large HBV surface protein; d, day; h, hour; MHB, middle
HBV surface protein; ORF, open reading frame; SHB, small HBV surface protein.
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HBsAg
HBV DNA levels decline during Myrcludex B treatment in 4/8 patients (consistent with HBV trial).
More pronounced decline of HBV DNA in the Myrcludex B/PEG-IFNa group (5/8 patients).
No significant changes (except patient 1027) in HBsAg levels .
HBV DNA
(B) HBV Serum DNA- and HBsAg Levels During Myr B and MyrB/IFNa Treatment
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HBV Genome and
Proteome
• 3.2kb DNA virus
• Hepadnaviridae, Orthohepadnavirus
• 10 well described genotypes (A-J)
• rcDNA (+strand incomplete)
• 4 overlapping genes (P, S, X, C)
• HBV proteins (HBsAg, HBeAg, HBcAg, HBx, Pol)
• Multiple splice variants
Molecular Variants Generated:
• G → A hypermutation (RT)
• APOBEC-3
• Recombination
• Splice Variants
Kann, M. 2008. In “Hepatitis B Virus”. Ed: Lai, CL and Locarnini, S. International Medical press. Page 2.4.
p17
HBx
p90
Pol
p21
HBcAg
core
p17HBeAg
corePre-core
p25/p22
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Viral Transcriptome: Mechanism of RNA Interference
(RNAi)
Natural Process of
RNAi
cleaved mRNA
Selective GeneSilencing
mRNAdegradation
dicerdicer
dsRNA
siRNAs
cleavage
RISC
strand separation
cleavage
Therapeutic Gene Silencing complementary pairing
mRNA(A)n
SyntheticsiRNAs
11
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Mean Decline in HBsAg by Cohort
with HBV-ARO
Will this result in restoration of immune responses?
Gane, E et al 2018. AASLD Late Breaker [LB-25]
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Stages of the HBV Life Cycle
Regulated by HBc Protein
Diab, A et al 2018. Antiviral Res;149:211–220
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Phase 1b Clinical Trial:CpAM NVR 3-778 Reduces Serum HBV
DNA and RNAEfficacy shown in hepatocyte culture and chimeric mouse models of HBV.
Clinical studies:
Serum HBV DNA: mean 1.7 log reduction (600 mg BID)
Serum HBV RNA: mean 0.86 log reduction (600 mg BID)
Yuen M-F, et al. AASLD 2015, San Francisco. #LB-10Lam A, et al. AASLD 2015, San Francisco. #33
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New Understanding of HBV Life- Cycles
and Potential Antiviral Opportunities
1. HBeAg-Pos vs HBeAg –Neg HBV
2. Chromatinisation (HBx)/Epigenetic
Regulation Minichromosome (cccDNA)
3. HBV DNA Integration
4. RNA and RNA Splicing
5. Packaging pgRNA and Reverse
Transcription (Protein
Priming/Translocation)
6. Entry and Re-Entry (HBx)
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HBV Replication: Pre ARC-520 Era
(HBeAg-Positive HBV)
Uncoating
ER
Mature
Nucleocapsid
Immature
Nucleocapsid
Nuclear
Transport
RC-DNA
Transcription
viralRNA
Core
Polymerase
Surface
HBeAgSpherical &
Filamentous HBsAg
GOLGI
Translation
Precore
Mature HBV
virion
Intracellular Conversion PathwayRC-DNA
Reverse
Transcription
RC-DNA
• cccDNA/minichromosome based replication
• “minimal” integration
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HBV Replication: ARC-520 Era
(HBeAg-Negative HBV)
Uncoating
HBsAg
Pre-S truncation
ER
Mature
Nucleocapsid
Immature
Nucleocapsid
Nuclear
Transport
RC-DNA
Transcription
Viral RNA
Core
Polymerase
Reverse
Transcription
Surface
Viral Integration
HBeAg
Spherical & Filamentous
HBsAg
GOLGI
Translation
Intracellular Conversion PathwayRC-DNA
DSL- DNA
Precore
Mature HBV virion
NEG
• HBsAg derived predominately from integrated HBV DNA
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Bock, T. et al 2001. JMB;307:183
High Replication
PhenotypeTranscriptionally Active
High Viraemia
Low Replication
PhenotypeQuiescent or active
Medium to Low or
No Viraemia
The HB Core Protein is a Component of
the Minichromosome
Chromatinization mediated by Smc5/6 Complex: involved in DNA repair, chromosome topology and organization
Fully chromatinized MC are transcriptionally SILENT
• HBcAg binds to CpG Island II (Guo, YH et al 2011. Epigenetics;6:720)Newbold, J. et al 1995.
J.Virol;69:3350
• cccDNA = 21 Topoisomers [NOT a single entity]
• Reflects Differences in Transcriptional Activities Newbold, J. et al 1995. J.Virol;69:3350
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HBx Promotes Degradation of the Smc5/6 Complex
to Prevent Silencing of HBV cccDNA
cccDNA, covalently closed circular DNA; Cul4, Cullin 4; DDB1, damage-specific DNA-binding protein 1;
HBV, hepatitis B virus; HBx, HBV X protein; ND10, nuclear domain 10; Smc5/6, structural maintenance of
chromosome 5/6 complex
HBx-minus or HBx variant HBV strains essentially transcriptionally SILENT
Niu, C et al PLoS One. 2017 ;12(1):e0169648
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Addressing the HBV DNA
Integration Issue• Integration occurs early when infection is first established
(Tu, T et al 2018. J Virol;92(11):e02007-17)
• integrated HBV DNA is an important source of HBsAg
• in the context of natural history, 1-2% of patients do lose
HBsAg
• mechanism involved double-stranded linear (DSL) HBV
DNA as major precursor
• ? role in clonal expansion of “resistant” hepatocytes (Mason, WS et al 2016. Gastroenterol;151(5):986-998)
• PCR Assay available for HBV DSL DNA in serum (Zhao, X-L et al 2016. GUT;65:502-511)
• can be successfully targeted with molecular based
therapeutics
– RNAi: ARO-HBV
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• Splicing of HBV is a common event during chronic infection, occurring
in over 80% of patients
– HBV splicing levels change dramatically over time, suggesting a highly dynamic
process
– Higher HBV viral load results in increased HBV splicing
– Splicing increases each year prior to the development of HCC (Bayliss, J et al 2013. J Hepatol;59:1022)
– Asian HBV genotypes (B and C) have significantly greater levels of HBV splicing
than European genotypes (A and D)
• Up to 10% of the virion DNA populations contain spliced genomes
• splice mutants are replication defective due to intron removal
generating truncated viral proteins (Sommer et al Virology, 1997;239:402)
• three neoproteins translated from spRNA: HBSP, HBDSP, P-S FP
• secreted splice RNA recently identified in patient serum (Espiritu..Lam, AASLD, 2016)
• Clinical, virological and pathological significance now emerging
HBV Splicing and Natural History of CHB
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HBV Splicing
Allain and Candotti. 2012. Biologicals;40(3):180-186.
Regulation of HBV Pre-S/S mRNA and HBsAg Production by Spliced Pre-S/S mRNA(Hass, M et al 2005. Hepatol;42:93)
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• From a reactivation case of OBI, G458A mutation identified (affected the
5' splice site by disrupting RNA secondary structure/stem loop formation)
which inhibited Pre-S2/S splicing, resulting in a marked decrease in
unspliced Pre-S2/S transcript and HBsAg (Hass, M et al 2005. Hepatol;42:93)
• Candotti, D et al 2012. GUT;61:1744 reported on mutations in the 5' splice-
site 458 vicinity:– 25/55 (45%) OBIB vs 5/47 (11%) non-OBIB– 14/33 (42%) OIBC vs 5/48 (10%) non-OBIC
• Amongst OBI Variants: 44% OIBB and 36% OBIC splice donor region
mutations disrupted stem loop structure as described with the G458A
mutation
• None of the (few) substitutions found in non-OBI variants predictive of
affecting the RNA stem loop structure
IMPLICATIONS FOR HBsAg LOSS VIA Pre-S2/S 5' SS MUTATIONS
Lessons Learned:
HBV Splicing and OBI
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Key Step 2: Reverse Transcription
CORE-CAPSID PROTIEN DEPENDENT
Host Enzymes
Covalently Closed
Circular
(ccc) DNA
HBV RC Genomic
DNA
(-) (+)
HBV DNA
PolymeraseHost RNA
Polymerase II
HBV Reverse
Transcriptase
HBV Minus (-) DNA
(-)
Pregenomic HBV
(pg) RNA
AAAA[RNaseH]
Key Step 1: Conversion of RC DNA into cccDNA
1. Removal of RT
2. Removal of r
3. Ligation of (-) DNA
4. Completion of (+) DNA
5. Removal of capped RNA
6. Ligation of (+) DNA
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Assessment of Antiviral Effect of SB9200
Compared to Other HBV Inhibitors
Colledge, D et al. 2018 AASLD Poster # 383
• Antiviral effect involves inhibition of HBV replication at the level of reverse
transcription without affecting nucleocapsid assembly [novel MOA]
• ? Blocking priming or primer translocation within the capsid
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Patients: HBsAg <4 log: 16 HBeAg –ve, 10 HBeAg +ve
I N A L L C O H O RT S : B A S E L I N E H B s A g P R E D I C T S
R E S P O N S E O F B O T H D N A A N D R N A TO I N A R I G I V I R
1
-1
-2
0
-3
Subjects with
Baseline
HBsAg >4 log
Subjects with
Baseline
HBsAg <4 log
HBV DNA
p<0.001
Subjects with
Baseline
HBsAg >4 log
Subjects with
Baseline
HBsAg <4 log
2
-2
-4
0
-6
HBV RNA
p<0.007
HBsAg >4 log: 1 HBeAg –ve, 19 HBeAg +ve
Lo
g10 D
eclin
e H
BV
DN
A
Lo
g10 D
eclin
e H
BV
RN
A
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HBsAg Targeting Strategies
• HBsAg clearance an endpoint of therapy
• Decline in HBsAg levels may restore the
antiviral activity of exhausted T/B/NK
cells
• Several strategies in evaluation
– RNA interference (siRNA): « gene silencing »
– Anti-HBs antibodies
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Immune Regulation by HBsAg
• HBsAg secreted in vast excess
over virions (>103 fold)
• Circulate in blood 100-400 g/ml
(1% of total serum protein)
• Unique conformational structure
(8 cysteines and 8 prolines )
• Associated with increased risk of
HCC (Yuen, MF. et al 2008. Gastro;135:1192–1199)
• Plays a key role in HBV
persistence
• Suppress both innate (TLR-2,
TLR-9 and IFN-α) as well as
adaptive (mDC) responses to
infectionWang, S et al 2013. J Immunol;190:5142.; Xu,
Y et al 2009. Mol Immunol;46:2640.; Op den
Brouw, ML et al 2009. Immunol;126:280.
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What Might a HBV Curative Regimen Look Like?
Potent
NA
cccDNA
Inhibitor
HBV Antigen
Inhibitor
Immune
Activator
/
agent to prevent viral spread and cccDNA
re-amplification
safe and selective agent to reduce or
silence cccDNA
agent(s) to block/inhibit the HBV life-cycle
[entry, cell-spread, capsid assembly, HBx,
HBeAg, HBsAg]
agent(s) to activate specific antiviral
immune responses or relieve
repression/exhaustion of the system
• The medium-term aim for the field is to achieve “functional cure” − HBsAg seroconversion off treatment
How Many Targets to be Blocked to Achieve Functional Cure?