What are the pros and cons of HBsAg loss as the endpoint...
Transcript of What are the pros and cons of HBsAg loss as the endpoint...
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