What are the pros and cons of HBsAg

loss as the endpoint for treatment?

Jordan J Feld MD MPH

Toronto Centre for Liver Disease

Sandra Rotman Centre for Global Health

University of Toronto

Disclosures

• Consulting: Abbvie, Contravir, Enanta, Gilead, Merck

• Research: Abbvie, Gilead, Janssen, Merck, Wako

HBsAg – the optimal marker of cure?Pros

• Well documented excellent

prognosis after HBsAg loss –

naturally or with therapy

• Durable endpoint – sero-reversion

relatively rare even with

immunosuppression

• Regulators may demand it

Cons

• High bar – current therapies rarely

achieve this

• May not mean what we think it

means

– False positives: HBsAg loss without

benefit

– False negatives: Persistent HBsAg

loss but with all the benefits

• Sensitivity of the assay may be an

issue…threshold for HBsAg loss…

What are the goals of therapy?

Learning from natural history

0

100

80

60

40

20

0 5 10 15 20 25

Su

rviv

al p

rob

ab

ilit

y (

%) Inactive CHB

HBeAg-/HBV DNA+

or HBeAg reversion

HBeAg+ persistence

Time (years)

• Very inactive disease and ideally HBsAg loss associated with excellent long-term and cancer-free survival

• A good goal for therapy

Fattocvich Gut 2008, Yang NEJM 2002

sAg + /eAg +

sAg + /eAg -

sAg - /eAg -Cu

mu

lati

ve H

CC

In

cid

ence

(%

)

Survival HCC

Spontaneous HBsAg clearance rate

Yeo Gastro 2018

Pooled annual rate 1.02%...associated with lower DNA, e-neg disease

34 studies – 42,588 patients, 303,754 patient years of f/u

5 year 10 year 15 year

HBsAg loss is a high bar…

TDF+PegIFN x 24w

TDF+PegIFN x 16w +

TDFx12w

TDFx120wPegIFNx48w

9.1%

Hard to achieve with monotherapy (even long-term) or current combos,

particularly for HBeAg- patients

Composite of numerous trials, not head-to-head, Marcellin Gastro 2016

2.8%

0%

Caveats with sAg loss

CCC DNA

HBV RNATranscripts

PregenomicRNA

EnvelopeProteins (sAg)

S, M, L

e Ag

PolymeraseProtein

Core Protein

1. Loss of sAg = loss of sAg transcription → silent/absent cccDNA = our goal

Caveats with sAg loss

HBV RNATranscripts

PregenomicRNA

EnvelopeProteins (sAg)

S, M, L

e Ag

PolymeraseProtein

Core Protein

1. Loss of sAg = loss of sAg transcription → silent/absent cccDNA = our goal

Elimination

Caveats with sAg loss

CCC DNA

HBV RNATranscripts

PregenomicRNA

EnvelopeProteins (sAg)

S, M, L

e Ag

PolymeraseProtein

Core Protein

x x

1. Loss of sAg = loss of sAg transcription → silent/absent cccDNA = our goal Transcriptional

Silencing

Caveats with sAg loss

CCC DNA

HBV RNATranscripts

PregenomicRNA

EnvelopeProteins (sAg)

S, M, L

e Ag

PolymeraseProtein

Core Protein

xx

1. Loss of sAg = loss of sAg transcription → silent/absent cccDNA = our goal

2. Loss of sAg = loss of sAg translation →siRNA…unclear what this means…may still be very helpful but unknown if the same as 1 (our usual sAg loss)

TranslationalSilencing

(siRNA)

Caveats with sAg loss

CCC DNA

EnvelopeProteins (sAg)

S, M, L

HBV RNATranscripts

PregenomicRNA

e Ag

PolymeraseProtein

Core Protein

sAgx x

1. Loss of sAg = loss of sAg transcription → silent/absent cccDNA = our goal

2. Loss of sAg = loss of sAg translation →siRNA…unclear what this means…may still be very helpful but unknown if the same as 1 (our usual sAg loss)

3. sAg may still be made from integrated HBV DNA –makes 1 and 2 hard to confirm!

Integration

Control without sAg loss may be enough…

Cu

mu

lati

ve li

feti

me

inci

de

nce

of

HC

C

Age (years)

Persistently eAg +ve

eAg loss but persistent DNA +ve

eAg –ve but persistent DNA +ve

DNA –ve, sAg loss

aHR p-valueDNA –ve, sAg loss 1.0 DNA –ve, no sAg loss 1.53 0.37eAg –ve, DNA +ve 3.99 <0.001eAg loss, DNA +ve 15.1 <0.001eAg+ 20.4 <0.001

Liu Gut 2014

Follow-up of the REVEAL Study

DNA –ve, no sAg loss

Have they

cleared

sAgcccDNA?

Clinical Data with RNAi

sAg

crAg

eAg

Placebo

HBV Protein reduction with ARC520 sAg decline eAg + vs -

• Effective knockdown of all HBV proteins

• Much more effective in HBeAg + than HBeAg -

• e+ - initial 1.6 log to 2.9 log → cccDNA?

• e- - initial 0.5 log to 1.2 log → integrated?

Yuen AASLD 2015, Yuen EASL 2017

HBeAg -ve

HBeAg +ve

Important lessons from RNAiRNAi ARC520 IV qw to chimps – reduced HBsAg decline in HBeAg –ve

compared to HBeAg +ve chimps

% of cccDNA transcripts much higher in e+ vs e- chimps

Convincingly showed that much of HBsAg from HBeAg –ve patients is from integrated HBV DNA rather than cccDNA…important implications

Wooddell Sci Trans Med 2017

1. qPCR

HBeAg-

HBeAg+

siRNA target

3. mRNAseq2. Single molecule real-time seq

Fusion of HBV and chimp DNA in e- samples ie. intregration

Integration in the viral lifecycle

Nguyen J Hep 2010

Why does integration occur?

Staprans J Virol 1991, Tu Viruses 2018

pgRNA

Minus strand

DNA synthesis

RNase H

activity

DR1 DR1DR2

Translocation

then priming

rcDNA

No translocation

dslDNA

• Double stranded linear DNA (dslDNA) made by priming ‘error’

• Integration via host DNA repair mechanisms

• Can occur anywhere but predilection for site between DR1 & DR2

Integrated HBV DNA

Features of integrated DNA

- PreS1 and S2/S with promoters →

can make HBsAg

- Shorter than pgRNA – no

replication

- PreC/Core separate from promoter

(may be made by host promoter)

- X protein truncated but possibly still

functional as transactivator

Tu Viruses 2018

Is there a way to distinguish HBsAg from cccDNA vs integrated DNA?

Total HBsAg LHBs

P=0.07 P=0.051

* *

*p<0.01, **<0.001

** ** ****

HBsAg components helpful

• Inactive carriers best recognized by low LHBs – integrated may not make LHBs

• Other sAg species in IC may all be from integrated DNA ie sAg+ but active cccDNA-

Pfefferkorn Gut 2018 – slide courtesy of T. Berg

HDV can use integrated DNA

Hep3b

PLC/PRF/5

(Alexander cells)

Both support

HDV production

HDV

HDV

Functional PreS1, PreS2

& part of S

HDV

PHH

Infectious

Truncated PreS1 fused to

inverted Core/PC

HDV xNon-infectious

This means that integrated DNA can make functional LHBs (at least in vitro)

Freitas J Virol 2014

Integrant HBV

Integrant HBV

Clinical data on effect of integrated DNA

70 patients of whom 11 with detectable integrated HBV DNA

• Initial 2 years – similar reduction e+ vs e- ?cccDNA

• Latter 2 years – markedly reduced from baseline

• Intriguing…need more data

Hu J Gastro Hep 2018

Baseline to Year 2 Year 2 to Year 4

Baseline Year 2 Year 4

HB

sA

g L

evels

(lo

g I

U/m

L)

Red

ucti

on

in

HB

sA

g

Levels

(lo

g I

U/m

L)

Int No Int Int No Int

Integration

No integration

Quant sAg levels correlate with phase of disease

HBsAgint?

HBsAgcccDNA?

IT IC LR ENH

LR ENH

HBsAgint?

• If integration happens early, it’s a wonder anyone clears HBsAg!

• And if this is not true, why not?

Jaroszewicz J Hep 2010

Timing of integrationIT PatientsIT & IA Patients

• Integration events clearly happen early

• But clone size increased in age-matched HBeAg- CHB…hence HBsAgint more

relevant in HBeAg- CHB but unclear why this is true

Mason Gastro 2017

Estimated clone size with HBVint

Do we need more than HBsAg?HBV core-related Ag by phase

of CHB

IT IC ENH ENQ

HBV core-related Ag vs quant

HBsAg by phase of CHB

ENQENH

ICIT

HBsAgcccDNA

HBsAgint

Have they already achieved functional cure?

Additional measures of cccDNA transcription may be useful…more from Massimo

Massoumy Clin Micro & Inf 2015

Some key questions

• Can we distinguish HBsAgint vs HBsAgcccDNA? Using LHBs? Something else?

• If integration occurs early, why is it so much more relevant in HBeAg- CHB?

• How relevant is HBsAgint to disease pathogenesis? HCC? Other?

• Does HBsAgint have a ‘purpose’ for the virus?

• Are there therapeutic approaches to target HBsAgint? Or is it unnecessary?

• Can other markers of cccDNA transcription be used with HBsAg? Do they correlate with long-term outcome?

So should we use HBsAg?

• Yes for now…but…

• For HBeAg+ CHB – probably a good (and feasible) choice

• For HBeAg- CHB – false ‘negatives’ a real risk → give up a promising therapy!– Consider accepting:

1. HBsAg loss OR

2. HBsAg + with negative crAg OR

3. possibly HBsAg + low level LHBs

• Need alternative ways to predict reliable off-treatment good prognosis

Alternatives to HBsAgHBV RNA

• Marker of cccDNA transcription

• Biology poorly understood

• Measurement challenging – various

assays

• Undetectable in many HBeAg-

patients

• Cannot be produced from

integrated HBV DNA

HBV core-related Ag

• Correlates with cccDNA transcription

• Component of HBeAg, p22 and core

• Of limited value in HBeAg +ve CHB

• Stable, simple assay

Major weakness – clinical relevance vs ‘hard endpoints’ – survival/HCC

More evidence

Plasmid safe DNase digestion with greater

effect on e- chimps → digesting integrated

DNA b/c cccDNA resistant to PSD

With redesign of RNAi to target a site present

in integrated HBV DNA effective

Wooddell Sci Trans Med 2017

The process of integration

e and core ORFs separated from promotors

but may come from host promotor

Tu Viruses 2018