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?