Rationale for Different Virological Targets in...
Transcript of Rationale for Different Virological Targets in...
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
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
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”
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?
HBV Lifecycle Showing Novel
Approaches for Viral Targets
Kapoor R & Kottilil S. 2014. Future Virol;9:565-585
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)
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
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.
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
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
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
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]
Stages of the HBV Life Cycle
Regulated by HBc Protein
Diab, A et al 2018. Antiviral Res;149:211–220
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
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)
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
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
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
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
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
• 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
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)
• 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
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
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
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
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
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.
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?