fitness and infectivity of drug- resistant and cross-resistant hbv mutants: why and how studying it?...

Fitness and infectivity of drug-resistant and cross-resistant

HBV mutants:why and how studying it?

David Durantel 5th of June 2009, Atlanta

Molecular physiopathology and new treatment for viral hepatitis - INSERM U871 (Pr Fabien Zoulim)

HBV life cycle in liver

Blood or inter-hepatocyte

spaces

From Seeger and Mason, Microbiol. Mol. Biol. Rev. 2000

Zoulim, Antiviral Research 2004

Concepts around treatment failure

Allen et al. Hepatology 1998; Gish et al. J Hepatol 2005; Qi et al. J Hepatol 2004; Tenney et al. AAC 2004; Lai et al. Gastroenterology 2005; Sheldon et al. Antivir Ther 2005; Delaney et al. AAC 2006 ; Schildgen et al NEJM 2006 ; Curtis et al JID 2007.

RNaseH

845 a.a.

Terminal protein Spacer Pol/RTPol/RT

AA BB CC EED D

349 692

YMDD

V173L

L180M M204I/V

I---V------LA---T

I(G)I(G) II(F) II(F)

(rt1) (rt 344)

LAM/ FTC

ETV I169T L180M S202G/I/C M250VT184S/Q/I/L/G/C/M

ADV A181V/T N236TI233V ?

LdT M204ITDF A194T ? ?

M204I/V

HBV drug resistance mutations

* In association wtih rtL180M+rtM204V (to be confirmed)

Impact of mutations in polymerase on S

TerminalProtein

spacer Pol/RT RNaseH

Surface

Polymerase

PreS1 PreS2

N2

36

T

L1

80

M

S

V17

3L

E1

64

D

A18

1V

/T

M2

04

V/I

W1

72*/

L1

73

F

I195

M/

W1

96*

Antiviral Drug-Associated HBsAg ChangesDrug Grouping Resistance Mutations HBsAg Corresponding Changes

L-Nucleosides

(LMV & LdT)

(* LdT)

rtL180M No Change

rtM204V sI195M

rtM204I * sW196*/S/L

Acyclic Phosphonates

(ADV & TFV)

(* LdT) (# LMV)

*#Also Affected

rtA181T *# sW172*

rtA181T *# sW172L

rtA181V * sL173F

rtN236T After End of HBsAg

Cyclopenta(e)ne

(ETV)

rtI169T sF161H/L

rtT184A No Change

rtT184C sL175F + sL176V

rtT184I No Change

rtT184G sL176V

rtT184S sL175F

rtT184M sL176*

rtT184L sL175F

rtS202C No Change / sS193F

rtS202I sV194F/S

rtS202G No Change / sS193L

rtM250I After End of HBsAg

rtM250V After End of HBsAg

Locarnini, Zoulim, submitted to Gastroenterology

Why studying viral fitness

Understand mechanism of emergence of drug-resistant strain

Determine whether a strain can propagate

Better monitoring and prediction of emergence

Define strategy for therapy: combination of appropriate antivirals to decrease viral fitness

Definition of viral fitness

A parameter that quantifies the adaptation of an organism or a virus to a given environment

For a virus, ability to produce infectious progeny relative to a reference viral clone, in a defined environment

Analysis of fitness is a complex matter because of the importance of model used to analyze it

Esteban Domingo, Fields Virology 2007

Treatment begins

Drug-resistant variant

Drug-susceptible virus

Naturally—occurring viral variants

Incomplete suppression Inadequate potency Inadequate drug levels Inadequate adherence Pre-existing HBV DR mt

Time

HB

V r

epli

cati

on

Kinetics of HBV drug resistance emergence

Si Ahmed et al., Hepatology, 2000; Zoulim, AVCC, 2001; Yuen et al., Hepatology, 2001; Locarnini et al ., Antiviral Therapy , 2004

Kinetics of spread and emergence of drug resistant virus during antiviral therapy

• Free liver space

• Mutant fitness

AntiviralAntiviral

II IIII IIIIII IVIV

wtwt mtmt

nini

INHIBITION OF WILD TYPE VIRUS REPLICATIONINHIBITION OF WILD TYPE VIRUS REPLICATION DELAYED EMERGENCE DELAYED EMERGENCE OF OF DRUG RESISTANT VIRUSDRUG RESISTANT VIRUS

XX

XX

XX

XX

XX

XXXX

Zhou et al ., AAC, 1999

Clearance of viral infection versus selection of escape mutants

The most important factors to consider:

The rate of immune killing of infected hepatocytes

The rate of replication and spread of mutant virus in the

chronically infected liver (i.e. fitness of the virus: the rate

of spread to uninfected hepatocytes)

Small changes in these factors may have profound effect

on whether treatment response is durable or subject to

rapid rebound

These factors may be subject to therapeutic intervention

Litwin et al, J Clin Virol 2005

Viral fitness as driving force for the emergence of drug resistant mutants?

Virus related

Genome replication capacity of a strain Transcription Packaging and RT

Virion production of a strain

Virion stability outside cells

Infectivity of a strain Entry process Post-entry

Immune-related

Cellular antiviral innate response to infection

Clearance of infected hepatocytes by immune cells

Clearance of virion in the circulation

Therapeutic interventionImmunosupression

Studying viral fitness in vitro

No ideal model to study viral fitness HepaRG and PHH Virus derived from patient versus virus produced in vitro

Disconnection between genome replication, virus assembly, and infectivity

Replication assays (with or without antiviral treatment)

Transfection or tranduction of hepatoma cells Stably transformed cell lines (HepG2.2.15 etc….)

Assembly assays Infectivity assays

Method to study HBV replication capacity

Durantel et al., Hepatology, 2004Durantel et al., J. Clin. Virol. , 2005

Evaluation of drug susceptibility in vitro

Durantel et al., Hepatology, 2004Durantel et al., J. Clin. Virol. , 2005

Longitudinal studies: replication capacity and drug suceptibility of lamivudine-resistant strains

Durantel et al., Hepatology, 2004

Yang et al., Antiviral Res, 2004

Other phenotypic assay

0

20

40

60

80

100

WT NT LMMV LMMVNT

mo

ck

WT

WT NT LMMV LMMVNT

3

2

4

1,5

1,2

kB

4330

7

100

rép

lica

tion

du

VH

B (%

)

NT

LM

MV

VHB EC50 (µM) EC90 (µM)facteur de résistance EC50 (µM) EC90 (µM)

facteur de résistance

WT 0,3 ± 0,09 2,3 ± 1,9 1 5,5 ± 1 52,5 ± 2,5 1NT 0,12 ± 0,03 1,8 ± 1 0,4 9,2 ± 0,8 > 75 1,7LMMV >10 > 10 >33 7,35 ± 1 51,5 ± 8,5 1,3LMMVNT >10 > 10 >33 43 ± 3 > 75 7,8

Lamivudine (LAM) Adéfovir (ADV)

Phenotypic studies with Bac-HBV

Replication capacity

Drug susceptibility

and cross resistance

MockW

TLMMV

NT LMMNNT

Encapsidated intracellular HBV DNA

Lucifora, Durantel et al., JGV, 2008

Relative Replication Yield (RC) of HBV Mutants

R. Edwards, T. Shaw, V. Sozzi & S. Locarnini. 2005. Global Antiviral Journal;1(Suppl2):77

0 1 2 3 4 5 6 7 8 9 10

L180M+A181V+N236T

A181V+N236TL180M

L180M+T184G+S202I+M204V*

A181V

M250V*M204I

L180M+A181V+M204V

I169T+V173L+L180M+M204V+M250V*N236T

L180M+M204V*

WTA181T+N236T

L180M+A181V

A181T

I169T+V173L+L180M+M204V*V173L+L180M+M204V*

Replication capacity varies with cell type

Brunelle et al, Hepatology, 2005

1 3 6 9 12 days

32

4

1,5

0,5

kB

32

4

1,5

0,5

mo

ck

HepG2

proliferativeHepaRG

3

21,5

dif ferenciatedHepaRG

Lucifora et al, submitted

Antiviral drug potency depends on hepatoma cell type

Seignères et al, Hepatology 2002

Impact of HBV genome background on antiviral drug efficacy and cross-resistance

Villet et al, J Hepatol 2008

Transfected or tranduced cells

Stably-transformed cell lines

HDV particles

HBV pseudoparticles

Models for the production of HBV particles used for assemby and infectivity assays

Transiently transfected versus stably transformed cell line

Transiently-transfected with plasmids or PCR-amplicons

Easier and flexible Cell line can be selected on

choice Less producing cells More non-enveloped

capsids? Lower specific infectivity

Stably-transformed

Work intensive and less flexible

All cells produce Less non-enveloped

capsids? Higher specific infectivity

Production of significant

amount of non-enveloped capsid

Transfection or transduction

Problem of production of non enveloped capsids in transfected/transduced cells

Problem of standardisation

of input for infectivity assay

Interest of stably transformed cell lines

Hepatology , 2008

HDV life cycle

Courtesy from Dr Camille Sureau

Production of HDV and HBV

Huh7

Transfection

Production and secretion of HDV and

HBV

Target Cell

Fixation and internalisation via HBV receptor(s)

HBV or HDV replication quantified by qRT-PCR orSB/NB

HDV

Replication of HDV and HBV genomes &

glycoproteinssynthesis

CMV HDV genome (3x) polyA

Sureau, Curr Top Microbiol Immunol, 2006

Sph I Swa I

Sal I/Xho I

Baculovirus sequences Baculovirus sequencesCMVie enh

Actin promoter

Nsi I

T7terminator

Rabbit globinterminator

HBV 1.1 genome unit

Not I Nco I

C X

Pol

S

Sph I Swa I

Sal I/Xho I

Baculovirus sequences Baculovirus sequencesCMVie enh

Nsi I

T7terminator

Rabbit globinterminator

HBV 1.3 genome unit

Nco I

C X

P

S

Nco I

Pre-C

Pre-C

Pre-CX

EN1 EN2

EN1 EN2DR1

DR1DR2

DR1DR2EN1 EN2 DR1

DR2

TYPE I

TYPE II

HBV

Production of HDV particles without production of HBV

Huh7

CMV HBV large polyA

Transfection

Production and secretion

of HDV

Target Cell

Fixation and internalisation via HBV receptor(s)

HDV replication quantified by qRT-PCR

or Northern blot

HDV

Expression of HBV glycoproteins

Replication of HDV genome

CMV HBV medium polyA

CMV HBV small polyA

CMV HDV genome (3x) polyA

Sureau, Curr Top Microbiol Immunol, 2006

Production of retroviral particles pseudotyped with HBV/DHBV

glycoproteins?

293FT

CMVGag

PolpolyA

CMV CMVU5R

U3 U5R

3’ LTR5’ LTR

CMV HBV large polyA

Transfection

Production and secretion

of HBVpp

Target Cell

Fixation and internalisation

Reporter expression measurement

Biochemical and antigenic studies

HBVpp

Expression of HBV glycoproteins

Luciferase

CMV HBV medium polyA

CMV HBV small polyA

Chai et al., J Virol, 2007 Saha, J Virol, 2005

Primary human hepatocytes

HepaRG cells

Other cell culture models

Models used for infectivity assays

Infection of PHH with HBV

Gripon et al., J Virol, 1988

Model difficult to master Variability from batch to batch

Infection of HepaRG by HBV

Easier to manipulate than PHH Only 10-20 % cells infected Type-I IFN may be responsible for low HBV replication

Gripon et al., J Virol, 1988 Hantz et al., J Gen Virol, 2009 Maire te al., BBRC 2008

1 3 6 9 12 jours

32

4

1,5

0,5

kB

32

4

1,5

0,5

mo

ck

HepG2

HepaRGp

3

21,5

HepaRGd


Type-I IFN response restrict HBV replication launched by Bac-HBV in proliferative HepaRG cells

INFR1

Gus β

polyIC : - - + + -

Hep

aR

G

Hep

aR

GshIF

NR

1

Hep

aR

G

Hep

aR

GshIF

NR

1

H20

Encapsidated intracellular HBV DNA RT-PCR


Lucifora, Durantel et al., submitted

1 3 6 9 12

0Bac-HBV-WT :

joursmo

ck

1 3 6 9 12

0 + 3

mo

ck

1 3 6 9 12

0

mo

ck

1 3 6 9 12

0 + 3

mo

ck

jours

3

2

4

1,5

kB

HepaRG HepaRG shIFNR1

M J1 J3 J6 M J1 J3 J6Transduction Infection

M J1 J3 J6 J9 J1 J3 J6 J9

Ac anti-IFNß

+-

M J1 J3 J6 J9 M J1 J3 J6 J9

- +

Immunofluorescence

Other cell systems ?

Total DNAEncaps DNA

RT-PCR

Animal model to study viral fitness

Kremsdorf and Brezillon, WJG, 2007

Chimpanzee to study viral fitness ??

Hepatology , 2009

Study of viral fitness in vitro to get insight on the emergence of mutants in vivo: an example

Villet et al., Gastroenterology, 2006 and 2009

Genotypic and viral quasispecies studies

Longitudinal studies: replication capacity and drug suceptibility of multiresistant strains

WT mut-3 mut-2 mut-1 mut-4

The selected mutant replicates better in presence of drugs

Replication capacity and magnitude of

resistance to AV are not the only factors

In vivo selected mutant is better produced in vitro

HBV transfection HBV/HDV transfection

In vivo selected mutant is more infectious in vitro

Conclusions

Among the four resistance mutants, the mutant that presented: • The strongest resistance to antivirals

• The best secretion of HBsAg and Dane particles

• The highest infectivity

…was finally selected and became dominant

Acknowledgements

Liver department

F ZoulimC. TrépoPh. MerleF. Bailly

P. MiailhesM. MaynardC. FournierS. Si Ahmed

INSERM U871

S. VilletC. PichoudF. Lavocat

R. EgounletyG. BillioudJ. Lucifora

M. A GoughoulteP. DenyO. Hantz

D. DurantelS. Durantel

MN Brunelle

Collaborations

K. Lacombe, ParisJ.P. Villeneuve, Montreal

P. Lampertico, MilanS. Locarnini, Australia

H. Wedemeyer, HannoverJ. Petersen, HamburgT. Santantonio, Bari

J. Neyts, LeuvenM. Levrero, RomaM. Buti, Barcelona

fitness and infectivity of drug- resistant and cross-resistant hbv mutants: why and how studying it?...

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