Hepatitis C Virus: Basics & Antiviral Therapy Ralf - - PowerPoint PPT Presentation

Ralf Bartenschlager AREVIR-GenaFor-Meeting Bonn, May 5-6, 2011

Hepatitis C Virus: Basics & Antiviral Therapy

Hepatitis C Virus Replication

• 3. Topology of HCV proteins
• 2. Genome organization
• 1. Entry
• 4. „Membranous Web“
• 5. RNA replication
• 6. Virus assembly and release

SOC therapy of hepatitis C

Poynard T et al. Lancet 2003;362:2095-2100

Virus

IFN-

dsRNA vRNA

IFN-

TLR3

TRIF RIG-I MAVS

IFN-

adapted from Haller et al., Virology, 2005 ISG ISRE

IFNAR

JAK / Stat ISGF-3

dsRNA

antiviral genes (~ 400)

The type 1 interferon system

ISGs impairing HCV replication

Schoggins et al., Nature 2011; Han et al., 2002; Gale et al., 1997; Helbig et al., 2005; Taylor et al., 2005

RIG-I MDA-5 IRF-1 IRF-2 IRF-7 MAP3K14 OASL RNaseL PKR RNA translation IFI44L GTPase NT5C3 nukleoside dephosph.? Viperin LDs? membrane curv? ADAR RNA editing DDIT4 ? signal transduction RNA degradation

Multiple attack strategies against HCV: little/no escape from effector mechanisms little/no inhibition of IFN-induces signalling (cell culture)

SOC-based treatment of HCV infection: correlation with IL28B polymorphism

*Ge et al., Nature 2009; Suppiah et al., Nat Gen 2009; Tanaka et al., Nat Gen 2009 Thomas et al., Nature 2009;

High positive correlation with therapy outcome High correlation with outcome of acute therapy A special role of IFN-lambda to combat HCV?

Mode-of-Action of Ribavirin

• broad spectrum virostatic
• reduces relapse

in combination with peg-IFN in combination with NS3 protease inhibitors

• little/no direct antiviral effect
• possible mechanism:

inhibition of IMPDH (no effect of direct IMPDH inhibitors) mutagenesis (‚error catastrophe‘) Th2 Th1 shift enhancement of IFN-response

NS3 protease NS5B polymerase NS3 helicase NS2-3 protease

Lorenz et al., Nature 2006 Kim et al., Cell 1996 Kim et al., Structure 1998 Tellinghuisen et al., Bresanelli et al.,PNAS, 1999 Nature 2005

NS5A Cyclophilin A Host factors:

HCV-specific targets

5B 5A 4B

4A

3 2

p7

E2 E1 C

Kwong et al., Gastroenterology 2011

HCV-specific DAAs

The NS3/4A protease

• chymotrypsin-like enzyme
• activation by NS4A
• shallow and exposed substrate binding pocket
• cleavage of viral and cellular proteins

polyprotein processing inhibition of innate immunity (TLR3, RIG-I)

• substrate specificity D/E - X4 - C/T S/A - X3
• end product inhibition

peptidomimetics

Development of NS3/4A protease inhibitors

Ac-Asp-Glu-Met-Glu-Glu-Cys-OH

Ingallinella et al., Biochemistry 1998

P6-P5-P4-P3-P2-P1--P1'-P2'-P3'-P4' P6-P5-P4-P3-P2-P1 P1'-P2'-P3'-P4'

Vx950

1 (+) RNA > 1.000 polyproteins RV LD

Translation Assembly

Mode-of-action of NS3/4A protease inhibitors

blockage of polyprotein processing disrupt formation of replication vesicles no effect on already established replication vesicles Restoration of signal transduction in innate immunity pathways RIG-I TLR3

ISG ISRE

IFNAR

JAK / Stat ISGF-3

dsRNA

antiviral genes (~ 400)

virus

IFN-

dsRNA vRNA

IFN-

TLR3

TRIF RIG-I MAVS

IFN-

adapted from Haller et al., Virology, 2005

Inhibition of IFN-production by NS3/4A protease

NS3/4A cleavage also in vivo?

Yes for MAVS ? for TRIF Reconstitution of immune response?

V36 A/M T54 A R155 K/T V36 A/M +R155K/T A156 S/T/V A36A/M+A156V/T V36 A/L/M F43 C/S V55A T 54 A/S V36M+T54S R155 K/T/P V36A+R155K T54S/A+R155K A156 S T54S+A156S V170A/T/L

Telaprevir Boceprevir

clinic

Telaprevir Boceprevir

Replicon

T54 A/T R155 R/K A 156 A/S/T/V T 54 A/S R155 K A156 S/T V170A

• R. de Francesco, INGM

A156 R155 T54 V36 D168 V170 F43 NS4A

Resistance against NS3/4A protease inhibitors

• broader spectrum of mutations in vivo vs. in vitro

limited viral diversity in vitro

• double mutations in vivo

R155K+V36M: most frequently associated with ‘break through’ compensatory mutations necessary to restore fitness

NS3 protease NS5B polymerase NS3 helicase NS2-3 protease

Lorenz et al., Nature 2006 Kim et al., Cell 1996 Kim et al., Structure 1998 Tellinghuisen et al., Bresanelli et al.,PNAS, 1999 Nature 2005

NS5A Cyclophilin A Host factors:

HCV-specific targets

5B 5A 4B

4A

3 2

p7

E2 E1 C

The NS5B RNA-dependent RNA polymerase

right-hand shape closed active site

thumb palm fingers

Nucleosidic and non-nucleosidic NS5B inhibitors

De Francesco & Migliaccio, Nature 2005

NM283 2'-C-methyl cytidine 7-deaza-2'-C-methyl-adenosine

Nucleoside analogs Non-nucleosidic inhibitors

Benzimidazole derivative Thiophene derivative Thiadiazine derivative

1 (+) RNA > 1.000 polyproteins RV LD

Translation Assembly

Block of RNA synthesis affect already established replication vesicles direct inhibition of NS5B (NNI) block of elongation (NI)

Mode-of-action of HCV NS5B polymerase inhibitors

• R7128: 10x and 15x IC50 eliminate HCV replicon within 3 weeks

no resistance detected

• HCV-796: 10x and 15x IC50 do NOT eliminate HCV replicon

C316Y and S365S/A

• Telaprevier: 10x and 15x IC50 do NOT eliminate HCV replicon

A156T/S and T54T/A

Telaprevir

1X IC50 10X IC50 15X IC50

HCV-796

1X IC50 10X IC50 15X IC50

NS5B NI NS5B NNI NS3/4A PI

R7128 Active Moiety (PSI-6130)

Higher genetic barrier of nucleoside analogues as compared to non-nucleosidic inhibitors and protease inhibitors

Untreated 1X IC50 10X IC50 15X IC50

Mutations conferring NS5B NNI resistance

• ABT-333; ANA598

Cys 316; Met 414; Tyr 448; Gly 554; Asp 559

• VX-222; VX-759; Filibuvir

Leu 419; Met 423; Ile 482; Val 494

• BI 207127; MK-3281

Pro 495; Pro 496; Val 499

• HCV-796

Leu 314; Cys 316; Ile 363; Ser 365; Met 414

• GS-9190

Cys 445; Tyr 448; Tyr 452

A B D E C

• cross resistance against different NNI classes
• R. de Francesco, INGM

NS3 protease NS5B polymerase NS3 helicase NS2-3 protease

Lorenz et al., Nature 2006 Kim et al., Cell 1996 Kim et al., Structure 1998 Tellinghuisen et al., Bresanelli et al.,PNAS, 1999 Nature 2005

NS5A Cyclophilin A Host factors:

HCV-specific targets

5B 5A 4B

4A

3 2

p7

E2 E1 C

• discovered via high-throughput screen with subgenomic HCV replicons
• active against genotype 1 – 6 replicons (chimeras)

M Gao et al. Nature 465, 96-100 (2010)

HCV subtype 1a 1b 2a 3a 4a 5a Replicon EC50 (pM) 50 9 12-63 127 12 33

BMS-79005: The most potant DAA

Mode-of-action of NS5A inhibitors

(polyU) Basic groove

• F. Penin
• F. Penin

NS5A inhibitors are dominant negative (1 inhibitor per 100 – 1.000 NS5A molecules) block of 5A oligomerization? block NS5A recruitment to LDs? block NS5A hyperphosphorylation?

Targett-Adams et al., JVi 2011

X X NS5A inhibitors are dominant negative (1 inhibitor per 100 – 1.000 NS5A molecules) block of 5A oligomerization? block NS5A recruitment to LDs? block NS5A hyperphosphorylation?

Mode-of-action of NS5A inhibitors

IRBM/ Merck Genelabs Bristol Myers Squibb A92V Y93H R157W L28V L31V P58L Y93H M28T (1a); L28V (1b) Q30H/R L31V/F/M Y93H/C/W

Resistance against NS5A inhibitors in replicon studies

HCV RNA

Schmitz Recent Pat Antiinfect Drug Discov. 2008

Gao et al. Nature 465, 96-100 (2010)

Resistance against NS5A inhibitors in replicon studies

HCV and therapy resistance: a few remarks in advance

• HCV persistence requires continuous replication

no latency, no integration, no cccDNA continuously vulnerable to DAAs

• Resistant virus variants are not archived

most often impaired fitness require compensatory mutations non-adapted variants are out-competed

HCV and therapy resistance: a few remarks in advance

How much of a concern will DAA resistance be in the future? Preexisting resistance DAAs combined with SOC IFN-free therapy

Pre-existing PI-resistant HCV variants: of clinical relevance? Boceprevir + IFN/R

Deep sequencing of NS3 protease at baseline and during treatment (n=29, SPRINT-1

• Resistant variants detectable also with SVR patients
• SVR upon triple therapy depends on sensitivity to IFN/R and type of PI

Courtesy C. Sarrazin, Ffm

0.6 Probability of being WT Time after failure (months)

1b: 54% (64/119) 1a: 84% (225/269)

0.0 0.1 0.2 0.3 0.4 0.5 0.7 0.8 0.9 1.0 2 4 6 8 10 12 14 16 18

median

Persistence of resistance? Telaprevir + IFN/R

Population-based sequencing, treatment failures, pooled phase 3 studies

• Reduction of frequency of resistant variants after cessation of therapy

Sullivan et al., EASL 2011 Courtesy C. Sarrazin, Ffm

HCV and therapy resistance: a few remarks in advance

How much of a concern will DAA resistance be in the future? Preexisting resistance clinically probably little relevance DAAs combined with SOC IFN-free therapy

High potency of DAA combination therapy with/without SOC

Genotype 1 NULL RESPONDERS BMS-790052 (NS5A) and BMS-650032 (PI) for 24 weeks +/- IFN/R 5A and PI alone: 4/11 achieved SVR12 and SVR24 6/11 had breakthrough 4/6 had SVR after addition of IFN/R HCV infection can be cured w/o IFN/R quadruple (5A, PI, IFN, R): 10/10 achieved SVR12 9/10 achieved SVR24 high rate of cure in this difficult to treat population

• A. Lok et al., EASL 2011

Open Questions

• Does suppression of viremia restore immunological competence?

DAAs plus IFN/R increases SVR even in null responsers

• Is there an (extra-hepatic) reservoir of HCV persistence?

Relapse even a few months after cessation of therapy

• Will resistance become a (major) issue?

Role of genotypic resistance assays? Role of phenotypic resistance assays?

Approach for Phenotypic Resistance Assay

Tripathi et al., Antiviral Res. 2007

Phenotypic (replicon-based) assays

Kwong et al., Gastroenterology 2011

1b (Con1) 1a (H77) 1b (N)

Comparison of in vitro vs. in vivo fitness measurements for variants resistant to a PI

Kwong et al., Gastroenterology 2011

Phenotypic assays

Infection with primary isolates too inefficient too variable requires primary cell cultures Replicons cover complete replicase, but lack core to NS2 authentic replication, predicting major clinical resistance mutations limited to some HCV isolates (1a, 1b, 2a) replicon vector backbone required for each geno-/subtype incompatibility between replicon fragments Infectious cell culture grown HCV covers all steps of HCV replication cycle limited to only one isolate (JFH-1) BSL3 lab required most compounds target replicase