Is Host Genome Integrated HBV

Relevant?

The CON Perspective

Professor Stephen Locarnini WHO Regional Reference Laboratory for Hepatitis BVictorian Infectious Diseases Reference Laboratory,

Doherty InstituteMelbourne, Victoria 3000, AUSTRALIA

Types of Chronic HBV Control and Cure

Peters, MG & Locarnini, S. 2016. Gastroenterol and Hepatol;13(6):348-356.

Inactive StateSustained, off drug:• No inflammation: normal ALT and liver biopsy• HBV DNA low or undetectable• HBsAg-positiveFunctional Cure (Clinical Resolution)Sustained, off drug:• No inflammation: normal ALT and liver biopsy• HBsAg loss• Anti-HBs gainComplete Cure (Virologic Cure)• All of the above plus• Loss of cccDNA in the liver

What Would HBV Funcational Cure Look Like?

In the blood: HBV DNA/HBsAg both undetectableanti-HBs positive

In the liver: no HBV cccDNAno pgRNA/no precore mRNAno HBV RC/DSL DNAHBcAg staining negative± HBsAg (occasional)

[reflecting integrated HBV DNA]

Background

“Relevant” in Which Context?A.Natural History and Viral Replication [Impact of Therapy]B.Host Survival DisadvantageC.Host Survival AdvantageD.Ability to Achieve Functional Cure

TREAT EARLY

A. Natural History and Viral Replication: #1• HBV DNA integrations are frequent, occur early on in infection and

steadily increase over the 4 (5) phases of CHB• in Woodchuck (and duck), hepadnaviridae integration occurs within 6

days (Summers, J and Mason, WS. 2004. Proc Natl Acad Sci (USA);101(2):638-640)

• in cell culture, HBV DNA integration occurs within 3 days (Tu, T and colleagues Personal Communication. 2017)

• following perinatal transmission, significant integration of HBV DNA can be found by the teenage years (Mason, W and colleagues, 2016. Gastroenterol;151:986)

• following acute infection integrated HBV DNA can be detected (Kimbi, GP and Kramvis, A. 2005. World J Gastroenterol;11:6416)

YET……• HCC only occurs much later (40-60 years later) suggesting integration

may not be directly causative• over the natural history of CHB, not every patient develops liver

cirrhosis or HCC, and 80% of patients with CHB, die from other causes• many patients are in the “inactive phase” of the disease and are in

general good health

A. Natural History and Viral Replication #2: VIROME Perspective

• up to 10% of the human genome is a consequence of multiple viral integrations over millennia – VIROME

• cells are actually quite robust against insertional mutagenesis and have developed multiple strategies/host-restriction mechanisms including Nuclear Domain 10 Smc 5/6 to cope and manage incoming viral and transposable elements

• integration rates have been shown to be low with around 1 integration detected per 104 cells (Summers, J and Mason, WS. 2004. Proc Natl Acad Sci (USA);101(2):638-640)

Is that really enough to drive the HCC process?

• splicing rather than HBV integrations may in fact be at least another major mechanism by which HBV is oncogenic (Bayliss J et al, 2013. J Hepatol;59(5):1022-8)

How Might HBV Splicing be Contributing to HCC #2 ?

• Alexander [PLC/PRF/5] cells derived from HCC encode many integrated splice variants

• Secreted splice variants associated with advanced liver disease (Soussan , P et al 2008. J Infect Dis;198(2):218-25)

• HBSP (Soussan P et al 2000. J Clin Invest;105:55-60) associated with increased cell proliferation and liver disease (Redelsperger, F et al 2012. Virology;431(1-2):21-8)

• Recently shown that splicing generates oncogenic chimeric integrants in setting of hepatocellular carcinoma (Chiu, YT et al 2016. Jhepatol; 64(6):1256-64)

Sommer and Heise. Frontiers in Bioscience, 2008

Multiple splice variants have been identifiedMultiple Splice Variants Have Been Identified

The most common transcript (Sp1) is a 2.2kb molecule that encodes precore/core, X and a novel protein in the hepatitis B splice protein (HBSP) (Soussan et al 2008)

Hepatitis B Splice Protein (HBSP)

AAA

ACCEPTOR SITEDONOR SITE

(2447) (489)

AUG CAPSID AUG POL STOP POLSTOP CAPSID

INTRON3.5kb

STOP CAPSID

AUG POL-HBSP STOP HBSPAUG CAPSID2.2kb AAA

40AA 40AANEW ORFPOL ORF

10.4kDa PROTEINChronic Hepatitis B

• 40% of patients have anti-HBSP• associated with HBeAg-positivity and high VL• associated with liver fibrosis• induces apoptosis without cell cycle block in vitro

INTRON

Soussan P et al 2000. J Clin Invest;105(1):55-60

How Might HBV Splicing be Contributing to HCC?

• Splicing removes cis-acting sequence (hM) which promotes circular DNA, leading to double stranded linear DNA (Abraham et al. 2008)

• Double stranded linear DNA integrates into the host genome

Lewellyn, EB and Loeb, DD 2007. J Virol;81(12):6207-6215

HBV Lifecycle

• Double-stranded linear (DSL) DNA is the dominant template for Integration into the host genome

• As well as splicing, DSL DNA is also generated by in-situ priming of (+) DNA following failure of 18 nt capped RNA oligomer to prime

2.4kb S

HBV Transcripts Differ Between HBeAg+ and HBeAg-Chimps PacBio Single Molecule Real-Time (SMRT) Sequencing

DR1

DR2 HBV Poly(A) signal

2.1kb S

HBeAg-(88A010)

HBeAg+(A2A004)

HBeAg-• Majority of S transcripts are

fused at the 3’ end to chimp sequence

• Fusion points typically between DR2 and DR1as expected if transcripts arose from integrated HBV dslDNA

HBeAg+• Most S transcripts terminate

near HBV poly(A) signal as expected

HBV-aligning

HBV non-aligning

S ORFs

S transcripts in HBeAg- chimps often lack target sites for ARC-520

ARC-520 siRNAs

Wooddell, C et al 2017. Sci Transl Med;9(409):pii: eaan0241

ORFs of HBV dslDNA Form

Tu, T et al 2017. Viruses; 9(4). pii: E75.

• Result: HBsAg Expressed from Integrants

• Is expression of HBsAg needed for HBV persistence?• Will clearance of HBsAg from the liver result in immune

recovery?• Why does HBsAg loss <50 yo reduce HCC risk?

The REVEAL Study• Risk Evaluation of Viral Load Elevation and

Associated Liver Disease/Cancer-Hepatitis B Virus (REVEAL-HBV) study.

• Taiwanese study of 3653 male and female participants who were seropositive for HBsAg and seronegative for anti-HCV.

• The REVEAL study showed that the greatest risk factors for liver cancer were:– High viral load– HBeAg positivity– HBV genotype C

Chen, CJ et al 2007. Clin Liver Dis;11(4):797-816

Are secreted splice variants predictive of HCC in this large Asian cohort?

• Most HCC patients who had elevated spHBV levels occurred up to 5 years prior to HCC diagnosis. The adjusted odds ratio of developing HCC within 5 years for spHBV ≥20% was 32.8 (P<.0001)

Black dots = HCC patientsGrey dots = non HCC patients

Splice Variants Were Strongly Associated With Liver Cancer

Yang, H-I and Revill, P (2017). Personal Communication

A. Natural History and Viral Replication: #3

• HBV DNA integrations are not necessary for viral replication as they do not result or are needed for a productive infection, compared to retroviruses

• in HBeAg-positive CHB, 90% of intrahepatic HBV DNA is episomal (minichromosomal DNA) whilst HBeAg-negative CHB, <10% of intrahepatic HBV DNA is episomal, rest is integrated (Wooddell, C and colleagues 2017. Sci Transl Med;9(409):pii: eaan0241)

YET……..

• Both phases respond well to the current approved therapies of interferon (immune based) and NUCs (direct acting antivirals)

B. Host Survival Disadvantage• the major viral product produced following HBV DNA integration

is HBsAg, usually as sub-viral particles, secreted in vast excess over virions (>103 – 104 fold) and circulate in blood at concentrations 100-400 µg/ml (at least 1% of total serum proteins)

• HBsAg plays a key role in HBV persistence, suppresses both innate [TLR-2, TLR-9 and IFN-α] as well as adaptive (m DC) and B-cell responses to infection (Wang, 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)

• persistence of HBsAg is associated with increased risk HCC (Yuen MF et al 2008. Gastroenterology;135:1192)

• HOWEVER……– Loss of HBsAg <50 years age: ↓↓ risk HCC– Loss of HBsAg >50 years age: HCC risk same as if still HBsAg-

positive– ? Impact of underlying cirrhosis

C. Host Survival Advantage: Clonal Expansion

• Evidence from both human and animal studies show that clonal hepatocyte expansion is a major risk factor for HCC and HBV DNA integration is a potential initiating event for HCC

• this is prevalent not just in the later stages of CHB, but also in the IT phase

• Tu, T et al. Personal Communication, 2017: demonstrated that virus integration and hepatocyte expansion are present in the immune tolerant (IT) phase

• Mason, W et al 2016. Gastroenterol;151:986: using inverse PCR; HBV DNA integration and clonal hepatocyte expansion were similar in patients in the IT phase to those that have HBeAg-positive, immune active, CHB

• Argument: Clonal expansion of hepatocyte CHB provides a selective advantage in that these mutated cells are “resistant” to elimination as a consequence of host cell immune attack

D. Ability to Achieve Functional Cure

• Is reversal of “immune exhaustion” sufficient?

Two AASLD Presentations in 2017 AND

One at EASL in 2017

Soluble PD-1 as a Novel Biomarker for HBsAg SC

Baseline sPD-1 (pg/ml)

Spontaneous HBsAg SC per 100,000 years

Rate Ratio (95% CI) of HBsAg SC Compared

to the >4,000 pg/ml group

>4,000 11.5 -536-3,999 61.7 5.7125-535 96.7 8.9

<125 151.00 14.6

Those patients with BL sPD-1 >4,000 pg/ml who reduce sPD-1 <125 pg/ml during follow-up had an OR (95% CI) of HBsAg SC of 68.6 compared to those who persistently had sPD-1 >125 pg/ml.

Conclusion: Reduced sPD-1 levels strongly predict greater chance of HBsAg decline and HBsAg SC in patients in inactive phase of CHB.

Hu, H et al 2017. AASLD Poster #175. Hepatology.

21

Immunological assessment of HBeAg-negative Chronic Hepatitis B patient

responses following anti-PD-1 treatment

Daniel Verdon1, Anna E. Brooks1, 4, Anuj Gaggar2, Jacky Woo2, Mani Subramanian2, Christian Schwabe3, Edward J. Gane3, Rod Dunbar1, 4

o 1University of Auckland, Auckland, New Zealand, 2Gilead Sciences, Foster City, CA, United States, o 3Auckland Clinical Studies, Auckland, New Zealand, 4Maurice Wilkins Centre for Molecular Biodiscovery,

Auckland, New ZealandEmail: EdGane@adhb.govt.nz

Study Design

Primary efficacy endpoint: Change in HBsAg log10 IU/mL levels 12 weeks following Nivolumab treatment

o Clinically approved dose 3mg/kg fortnightly (melanoma, NSCLC, RCC)

Results: HBsAg change from baseline

o 2/22 (9%) at Week 12 and 3/22 (14%) at Week 24 with a >0.5 log10 reduction in HBsAg• One patient with >1 log10 reduction in HBsAg at either timepoint• 19/22 patients treated with 0.3mg/kg showed some decline in serum HBsAg by

week 24.

Sentinel A (0.1mg/kg)Cohort A (0.3mg/kg)Cohort B (0.3mg/kg + GS-4774)

Results: Case Study

o Patient remains off-treatment for >9 months with sustained HBsAg loss and anti-HBs seroconversion

Results: Case Study

CD4+ T Cells CD8+ T Cells

On Treatment ALT Flares Associated with HBsAg Loss

• Wong, D et al 2017. J Hepatol;66(Suppl):p260; #162. EASL

• 16 genotype A patients lost HBsAg during TFV/ADV monotherapy [GS-US-147-0103]

• 9 flared; 7 no flare

• Multiple pathways to functional cure?

D. Ability to Achieve Functional Cure #2

Functional Cure Perspective• in the near future, it will be possible to target these

HCC/pre-neoplastic cells as nearly all still produce HBsAg,

• the next wave of DAA therapies will include novel therapeutic vaccines and chimeric antigen receptors (CAR) for T-cells used in adoptive T-cell receptor gene therapies as well as liver targeted RNAi based approaches, all of which will be able to clear these HBsAg-positive, host immune resistant pre-malignant and malignant cells from the liver

Conclusion

• There are multiple pathways to achiving functional cure in CHBCytolyticNon-cytolytic

• Either way, it is possible to achieve functional cure in CHB and this does not seem to be affected by HBV integration

Is HBV Genome Integrated HBV Relevant?NO, because

i.Spliced HBV DNA/RNA maybe more relevant as the oncogenic driver to HCCii.Does not affect response to current standard of care therapyiii.Persistent HBsAg positivity at least as importantiv.Clonal expansion protects host from hepatocyte destructionv.New molecular-based therapies will achieve functional cure and possibly even complete cure can be achieved

We need to: TREAT EARLY [Pre-Cirrhosis] SHOULD ACHIEVE SIGNIFICANT REDUCTION IN THE HCC RISK

Papatheodoridis, G et al 2014. J Hepatol; 60(1):62-8

Acknowledgements

• Dr Thomas Tu, University of Heidelberg, GermanyMany thought provoking discussions and providing excellent guidance

• Dr Daniel Verdon/Prof Ed Gane, University of Auckland, New Zealand