A - V

Report
Temporal evolution and application of
molecular diagnosis in the clinical
management of
patients with chronic viral hepatitis
P. Karayiannis
Imperial College London
Introduction of Molecular Based Tests for the Detection of
Hepatitis Viruses, Genotyping and Monitoring of Antiviral Therapy
Cloning
HBV
HDV
HAV
1970
Genotyping
Nucleic
acid
Detection
HEV
HCV
1980
1990
HBV
Dot-blot
HDV
PCR
2000
HCV
SemiqPCR
Real-time
qPCR
2010
GENOMIC
ORGANISATION
OF HEPATITIS B
VIRUS
Hunt et al, 2000
HEPATITIS C VIRUS GENOME ORGANISATION
ssRNA (9.6kb)
SIGNAL PEPTIDE PEPTIDASE
SIGNAL PEPTIDASE
VIRALLY ENCODED PROTEASES
GLYCOSYLATION SITES
Moradpour et al, Nature Rev Microbiol 2007;5:453-463
Cloned HBV-DNA
Hepatocyte DNA+
Restriction Enzymes
Liver HBV DNA
DETECTION OF HBV-DNA IN BODILY SECRETIONS
BY DOT-BLOT-HYBRIDISATION AND LIVER
BY SOUTHERN BLOT
Kb
- 2.37
HBsAg-ve
HBeAg+ve
anti-HBe
+ve
SALIVA –
- 7.7
URINE –
- 6.6
SEMINAL FLUID-
- 4.3
SPERM -
- 3.2
SERUM -
- 2.2
- 2.1
Karayiannis et al, Br Med J 1985;290:1853-1855.
MONITORING OF INTERFERON TREATMENT
EFFICACY IN CHRONIC HBV AND HCV
INFECTED PATIENTS
M 1
2
3
4
5
RESPONDER
NONRESPONDER
Dot-blot of HBeAg +ve patient
RT-PCR, 5’ NCR
QUANTITATION OF NUCLEIC ACIDS BY REAL-TIME PCR
Quantitative HCV RNA Assays
Method
Dynamic Range, IU/mL
(LLOQ-ULOQ)
LLOD, IU/mL
LLOQ = LLOD?
FDA Approved
Manual RT-PCR
600-500,000
N/A
N/A
Yes
Semiautomated RT-PCR
600-500,000
600
Yes
Yes
Semiautomated bDNA
signal amplification
615-7,700,000
615
Yes
Yes
LCx HCV RNA-Quantitative Assay
(Abbott Diagnostics)
Semiautomated RT-PCR
25-2,630,000
23
No
No
SuperQuant
(National Genetics Institute)
Semiautomated RT-PCR
30-1,470,000
30
Yes
No
Cobas TaqMan HCV Test
(Roche Molecular Systems)
Semiautomated
RT-PCR
43-69,000,000
18
No
Yes
COBAS TaqMan HCV Test v2.0
for use with High Pure System
(Roche Molecular Systems)
Semiautomated
RT-PCR
25-300,000,000
15
No
Yes
Semiautomated RT-PCR
12-100,000,000
12
Yes
Yes
Assay (Manufacturer)[1]
Amplicor HCV Monitor
(Roche Molecular Systems)
Cobas Amplicor HCV Monitor V2.0
(Roche Molecular Systems)
Versant HCV RNA 3.0 Assay (bDNA)
(Siemens Health Care Diagnostics)
Abbott RealTime HCV Assay
(Abbott Diagnostics)

Note that quantitative assays may have differing or identical LLOQ and LLOD levels

Phase III registration trials for both BOC and TVR used COBAS TaqMan HCV Test v2.0 for use with High
Pure System (1.3% false-positive rate)
Ghany MG, et al. Hepatology. 2009;49:1355-1374.
Clinical Care Options, Kuo P
Naeger LK, et al. Intl Workshop on Clinical Pharmacology of Hepatitis Therapy 2011. Abstract R-8.
HCV RNA Assays:
LLOD Is Distinct From LLOQ
• LLOQ
– Lowest HCV RNA concentration within linear range of assay
• ie, smallest amount of HCV RNA that can be not only detected but
also accurately quantified
• LLOD
– Lowest amount of HCV RNA concentration that can be detected
with 95% probability to determine presence or absence
• Beneath LLOD threshold, HCV RNA is “undetectable,” also known
as “target not detected”
• Commercially available quantitative assays may
have differing LLOQ and LLOD levels
Clinical Care Options, Kuo P
HCV RNA Levels and Relationship to
LLOD and LLOQ
HCV Treatment
Log10 Viral Titer
8
HCV RNA Assay Result:
Detectable/
quantifiable
6
Reported as quantitative
number within performance
range of assay
4
Reported as detected
2
Detectable/not quantifiable
Not quantifiable/
not detectable
1
0
0
LLOQ
LLOD
Reported as undetected or
target not detected
Time
SVR
Adapted from Naeger LK, et al. Intl Workshop on Clinical Pharmacology of Hepatitis Therapy
2011. Abstract R-8. Clinical Care Options
DETERMINATION OR RESPONSE BASED ON
HCV-RNA DETECTION AT SET POINTS DURING
ANTIVIRAL TREATMENT
HCV RNA (log10 IU/mL)[1]
7
Null response
6
5
Partial response
4
3
Relapse
40% chance
of SVR with
pegIFN/RBV[2]
2
1
Undetectable
RVR
0
-8
-4
-2
0
4
EVR
8
12
EOT
16
20 24
32
Wks After Start of Therapy
1. Ghany MG, et al. Hepatology. 2009;49:1335-1374.
2. McHutchison JG, et al. N Engl J Med. 2009;361:580-593.
40
48 52 60
SVR
72
Cloning and Sequencing
Studies
Manual
Automated
• Genotyping
• Quasispecies
• Viral variants
– Pre-core
– Basal Core promoter
– Vaccine escape
– Polymerase
PHYLOGENETIC TREE
BASED
ON 100 COMPLETE HBV
GENOME SEQUENCES:
GENOTYPES A TO H
GEOGRAPHIC DISTRIBUTION OF HBV GENOTYPES
C
99
A
99
100
A,D
A
B
D
100
0.016
E
G
A
100
100
E
100
100
G
F
100
100
10
H
A
F
D
96
C,B
100
5
Arauz-Ruiz et al, J Gen Virol 2002;83:2059-73
Woolly Monkey
%Nucleotide
0 Divergence
D
B
PHYLOGENETIC TREE OF COMPLETE HBV GENOME SEQUENCES:
GENOTYPES AND SUBGENOTYPES
B1-5
C1-5
F1-4
H
G
E
Schaefer, World J Gastroenterol 2007;7:14-21
A1-5
D1-5
GENOTYPES AND SUBTYPES:
GEOGRAPHIC DISTRIBUTION
Simmunds P. J Gen
Virol 2001;82:693−712,
www.hopkins-gi.org/GDL_Disease.aspx?CurrentUD
HEPATITIS C VIRUS PREVALENCE
>10%
2.5-9.9%
>1.2.4%
0-0.9%
Commercially Available HCV Genotype
Assays
Genotype assay
Manufacturer
Method
Siemens
Direct sequence analysis of the
5' noncoding region
Innogenetics
Reverse hybridization analysis using genotype-specific
oligonucleotide
probes located in the 5' noncoding region
Versant HCV Genotyping
Assay 2.0
Siemens
Reverse hybridization analysis using genotype-specific
oligonucleotide
probes located in the 5' noncoding region
Abbott RealTime HCV
Genotype II
Abbott
Genotype-specific real-time PCR of the
5' noncoding region and NS5b
Trugene 5'NC HCV Genotyping
kit
INNO-LiPa HCV II
 Incorrect typing among major genotypes rare (< 3%)
Ghany MG, et al. Hepatology. 2009;49:1335-1374.
Clinical Care Options, Kuo P
Genotypes: Natural History and Response to
Antiviral Treatment
• HBV:
– Gen B milder disease and
more frequent loss of
HBeAg
– Gen C more severe chronic
disease and HCC
– Gen A and B better
response to IFN treatment
– Gen C and D respond less
well to IFN
• HCV:
– Gen 1 & 4 difficult to treat
– Gen 2 & 3 easy to treat
– Gen 1 associated with more
severe disease
– Gen 3 associated more
often with steatosis
Natural course of chronic HBV infection
Chu et al, Hepatology 1985;5:431-34
Kwon, H. & Lok, A. S. (2011) Hepatitis B therapy
Nat. Rev. Gastroenterol. Hepatol.
SERUM HBV-DNA AND LIVER HBcAg DETECTION IN anti-HBe
POSITIVE PATIENTS
HRP staining for HBcAg
Race
HBeAg+
Anti-HBe+
HBVDNA+ (%)
HBVDNA- (%)
HBVDNA+ (%)
HBVDNA- (%)
N. Europe
98
2
6
94
S. Europe
94
6
21
79
Africa
78
22
13
86
Far East
100
0
58
41
Karayiannis et al, J Hepatol 1985;1:99-106
PRE-CORE STOP CODON MUTATION ABROGATING
HBeAg PRODUCTION
1814
a) ATGCAACTTTTTCACCTCTGCCTAATCATCTTTTGTTCATGTCCTAC
b) ----------------------------------------------c) ----------------------------------------------1896
1899
1903
TGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATG
----------------------------------TAG---------------------------------------TAG-A----
Carman et al, Lancet 1989;2:588-591
ACUTE EXACERBATIONS IN AN ANTI-HBe POSITIVE PATIENT
800
IU/L
700
600
IFN
500
IFN
A1
ALT
A4
400
300
A6
200
A2
100
A5
A3
A7
Time (Days)
0
0
200
400
600
800
1000
1200
1400
1600
1800
HBV QUASISPECIES IN AN ANTI-HBe POSITIVE PATIENT
Core
Pre-C
Nt position
A1 direct seq
Codon
Amino-acid
1896
A
28
stop
1907
A
3
I
1913
C
5
L
1934
A
12
T
1938
A
13
E
1983
G
28
R
1985
G
29
D
2239
A
113
E
A1-1 (2 cls)
A1-2
A1-3
A1-4
A1-5 (3 cls)
A1-6
A1-7
A1-8 (2cls)
A1-9
A1-13
A1-14
W
W
-
L
L
L
L
-
A
A
A
A
-
S
S
-
V
V
V
V
V
V
V
V
-
E
E
-
H
H
-
-
A4-1 (9cls)
A4-6
A4-7 (5cls)
-
L
A
-
-
V
-
-
-
-
A5-1
A5-2 (2cls)
A5-3 (4cls)
A5-4 (3cls)
A5-6
A5-10
A5-11 (3cls)
-
L
-
A
A
-
S
S
-
V
V
V
-
-
D
Alexopoulou et al,
J Hepatol 1997;26:
748-753
ANTIGENICALLY VARIABLE DOMAINS IN THE ENVELOPE
GLYCOPROTEINS
aa 1
5’
192
384
E1
809
E2 / NS1
C
VARIABLE REGION aa
HCV-1
HCV-BK
HCV-J1
HCV-J
HCV-J4
HCV-JH
HCV-H
HCV-UK
PK - 95
(1a)
(1b)
(1a)
(1b)
(1b)
(1b)
(1a)
(1a)
H
Y
Y
V
T
-
T
S
-
246-275
G
-
G
-
S
A
Q
R
A
V
N
A
A
Q
V
Q
-
G
A
A
A
S
A
H
K
R
S
R
K
T
A
S
V
G
V
T
M
T
T
T
T
T
S
N
Q
A
-
386-411
G
R
S
T
T
T
F
L
L
L
L
L
L
-
V
A
T
A
S L L A
- M F- - F T
W - SQ
- - F S
- - FR
G - - T
G - F -
456-482
PG
S - - - - - - -
A
P
P
S
K
S
S
S
S
R
Q
R
-
N V
K I
- I
K I
- I
K I
- I
- I
HCV QUASISPECIES: CHANGES IN THE
HYPERVARIABLE REGION 1 OF THE E2/NS1
WITH TIME IN A CHRONIC CARRIER
Time 0
STRVTGGQQGRAVHGI ASLFSLGASQK
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q- F- - - - R- - - - - E
8 months
N
G
-
-
- - - H-
S
-
- - - -A
R
R
L
-
-
-
14 months
- - Q-M - - - - - - - Q-M - - - - - - - Q-M - - - - - -
-
A -S
A -S
A -S
AY-
LTLTLT- T-
F
L
- P- PT- -H-
- - - N
- - EN
- - -N
P - - -
- AYSL - - - L- P- - N- - AYSL - - - LGP- - - - - AYSL - - - L- P- - - - -
Kato et al, 1992
PK - 95
Interference of HCV with
the Innate
Immune Defence Pathways
Doehle & Gale. In “Nucleic Acid Sensors and Antiviral Immunity”. Edited by: S Sambhara and T Fujita.
Mutations in the Reverse Transcriptase Region of HBV
Polymerase Conferring Resistance to Nucleos(t)ide
Analogues
Kwon, H. & Lok, A. S. (2011) Nat. Rev. Gastroenterol.
Hepatol.
MUTATIONS CONFERING RESISTANCE TO BOTH LAMIVUDINE AND ADEFOVIR
B
C
D
E
AYW SHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHLESLFTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGCYG
ADW ------V----------------------------------------------------------------------------------------------------------------Y----------------------------------------------------------------I---SW-M1
M7
------------------------------------------T----------------------------------------------------------------------------Y--------------------------------------------------------------------SW-------------------------------------------T----------------------------------------------------------------------------Y-----------------------------------------------------D-------------SW--
S1
S5
------------------------------------------V----------------------------------------------------------------------------------------------------------------------------- ------H-------------------------------------------------------------T----------------------------------------------------------------------------------------------------------T-----------------------H--------------------
J1
------------------------------------------V----------------------------------------------------------------------------- Y---------------------------------------------------------------------------
B1
B5
B8
-------M---------------------------------T-----------------------------------------------------------------------------Y---------------------------T----------------------------------------------------M---------------------------------V-----------------------------------------------------------------------------Y---------------------------------------------------------------------------------M---------------------------------T-----------------------------------------------------------------------------Y---------------------------T----------------------------------------------
181
236
LMV
ADV
TDF
A181T
10
3.2
2.8
A181V
7.7
7.8
2.4
A181T+N236T
35
>10
6.8
A181V+N236T
43
4.5
1.2
Villet et al, J Hepatol 2008;48:747-55
FoldResistance
Evolution of Approved HBV and HCV Therapy
Over Time
PegIFN a-2a
Entecavir
Lamivudine
HBV
1990
1998
IFN a-2a
HCV
1990
2002
Adefovir
2005
Tenofovir
2006
Telbivudine
Ribavirin
PegIFN a-2a
1998
2005
IFN a-2a
In combination with:
2008
2011
Telaprevir
Boceprevir
IFN a-2a
Ribavirin
IFNa-2a+Ribavirin
How Hepatitis C Drugs in Development May Affect Practice Today and Tomorrow
clinicaloptions.com/hepatitis
Each Drug Class Has Unique Features
NS3/4A
Protease
Inhibitors
 High efficacy
 Low genetic
barrier to
resistance
 Macrocyclic or
linear
 Phase III:
BI 201335,
TMC435
NS5B Polymerase Inhibitors
Nucleos(t)ide
Analogue
Non-nucleos(t)ide
 Mimic natural
substrates of the
polymerase
 Bind to several
different allosteric
enzyme sites;
results in
conformational
change
 Incorporated into
RNA chain causing
chain termination
 Broad genotypic
coverage
 High genetic barrier
to resistance
 Phase III:
PSI-7977
 Resistance more
frequent than
nucs
 Several agents in
phase II
NS5A Inhibitors
Cyclophilin A
Inhibitors
 NS5A has role in
assembly of
replication
complex
 Supports HCVspecific RNA
replication,
protein
expression
 Mechanism of
inhibition under
study
 Phase III:
Daclatasvir
(BMS-790052)
 Interacts with
NS2, NS5A,
NS5B
 May regulate
polypeptide
processing, viral
assembly
 Phase III:
Alisporivir
Phase III trials with telaprevir in interferon-naive (ADVANCE and ILLUMINATE)
and interferon-experienced patients (REALIZE)
Jacobson et al, NEJM 2011;364:2405-16
Sherman et al, NEJM 2011;365:2417-28
Zeuzem et al, NEJM 2011;364:2417-28
Soriano V et al. J. Antimicrob. Chemother. 2011;66:1673-1686
Phase III trials with boceprevir in interferon-naive (SPRINT-2) and interferonexperienced patients (RESPOND-2).
Poordad et al, NEJM 2011;364:1195-206
Bacon et al, NEJM 2011;364:1207-17
Soriano V et al. J. Antimicrob. Chemother.
2011;66:1673-1686
REPLICATION MACHINERY: MEMBRANOUS WEB
Alisporivir
Bode et al, Biol. Chem., Vol. 390, pp. 1013–1032
Replication strategy
of HCV
www.postech.ac.kr/dept/life/mv1/hcv.html
Membranous
web
Moradpour et al, Nat Rev
Microbiol 2007:5;453
DRUGS FOR FUTURE TREATMENT
OF CHRONIC HCV INFECTION
RIBBON MODEL OF THE HCV POLYMERASE
Active site with bound inhibitor and non-nucleoside
inhibitor (NNI) sites 1 to 4. Palm, thumb and fingers
coloured red, green and blue, respectively.
DAAs and HTAs IN CLINICAL TRIALS
Welsch et al, Gut 2012;61:36-46.
RNA
binding
INHIBITORS: POTENTIAL NS3 SERINE
PROTEASE/HELICASE
TARGETS AND RESISTANCE
MUTATIONS
HELICASE
NTPase site
PROTEASE
NH2
Membrane
binding
domain
In vivo
Zinc
In vitro
Inhibitor
Catalytic site
BILN-20611,2
Telaprevir2,3,7
(VX-950)
SCH-64
Boceprevir (SCH5030345)
ITMN-1916
A156V/T
R155Q
D168A/V/Y
A156S/V/T
A156V/T
R109K
A156S/T
T54A
V170A
A156S/V
Q41R, F43S,
S138T, D168A,
S489L,
V23A (NS4A)
NA
V36A/M
T54A
R155K/T
A156S/V/T
NA
V36A/M, T54A,
R155K/Q/T/M,
A156S, V170A/T,
F43C/S
NA
Direct-Acting Antiviral Resistance Mutations (Compiled In Vitro
and In Vivo Data)
Schaefer et al, Gastroenterology 2012;142:1340-50.
Genomic overview of the region of
19q13.13 surrounding the genomewide significant determinant of
response to treatment and
including the IL28B gene
Single Nucleotide
polymorphism
The SNPs that show genome-wide significant association with
SVR are marked in red. The polymorphism rs12979860 (red arrow)
is 3 kb upstream to the gene encoding IFN--3 (IL28B, blue arrow).
Ge et al Nature 2009;461:399-401
Genomics of HCV
1. SNPs in proximity to IL28B
• rs12979860 (“The Duke SNP”; CC favourable, TT not)
• rs8099917 (“Triple nine”; TT favourable, GG not)
2. Inosine Triphosphatase (ITPA) Gene Variants
• Deficiency leads to increased ITP
• Protect against ribavirin induced anaemia
• Rs1127354 (P32T in exon 2) and rs7270101 (intron 2)
3. Genes affecting Hepatic Steatosis and Fibrosis in HCV
• Palatin-like phospholipase (PNPLA 3)
• Cyclooxygenase-2 promoter (COX-2)
• RNF7, MERTK and TULP1
Percentage of SVR by IL28B genotypes of rs12979860 and
Geographic Distribution
Ge et al, Nature 2009:461;399-401.
Thomas et al, Nature
2009:461:798-801
Temporal evolution and application of
molecular diagnosis in the clinical
management of
patients with chronic viral hepatitis
by
P. Karayiannis
Imperial College
London
Phylogenetic Tree of all Known Subtypes
and Genotypes of HCV
2
1
4
6
5
3
Simmonds et al, J Gen Virol 2004:85;3173-3188
NATURAL HISTORY OF CHRONIC HBV INFECTION
HBeAg+
Immune
Clearance
Non-Replicative
Re-Activation
ALT
HBV-DNA
Immune
Tolerance
HBeAg-/anti-HB+
Time (Years)
Chu et al, Hepatology 1985;5:431-34

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