Fitness and infectivity of drug-resistant and cross-resistant HBV mutants : why and how studying it?

1. 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)

2. HBV life cycle in liver From Seeger and Mason, Microbiol. Mol. Biol. Rev. 2000 Blood or inter-hepatocyte spaces

3. Concepts around treatment failure Zoulim, Antiviral Research 2004

4. HBV drug resistance mutations Pol/RT Spacer RNaseH Terminal protein 349 692 845 a.a. (rt 344) (rt1) I---V------LA---T YMDD I(G) II(F) A B C D E V173L L180M M204I/V LAM/ FTC A181V/T N236T I233V ? ADV M204I/V I169T L180M S202G/I/C M250V T184S/Q/I/L/G/C/M ETV M204I LdT A194T ? ? TDF * In association wtih rtL180M+rtM204V (to be confirmed) 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.

5. A181V/T M204V/I V173L L180M N236T I195M/ W196* W172*/ L173F E164D Impact of mutations in polymerase on S Pol/RT spacer RNaseH Polymerase Terminal Protein Pre S2 Surface PreS1 S

6. Antiviral Drug-Associated HBsAg Changes Locarnini, Zoulim, submitted to Gastroenterology

7. 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

8. 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

9. Drug-susceptible virus Naturally—occurring viral variants Drug-resistant variant Kinetics of HBV drug resistance emergence Treatment begins • Incomplete suppression • Inadequate potency • Inadequate drug levels • Inadequate adherence • Pre-existing HBV DR mt HBV replication Time Si Ahmed et al., Hepatology, 2000; Zoulim, AVCC, 2001; Yuen et al., Hepatology, 2001; Locarnini et al ., Antiviral Therapy , 2004

10. Antiviral wt mt X X X X X X X ni I II III IV DELAYED EMERGENCE OF DRUG RESISTANT VIRUS INHIBITION OF WILD TYPE VIRUS REPLICATION Kinetics of spread and emergence of drug resistant virus during antiviral therapy • Free liverspace • Mutant fitness Zhou et al ., AAC, 1999

11. 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

12. Viral fitness as driving force for the emergence of drug resistant mutants? • Immune-related • Cellular antiviral innateresponse to infection • Clearance of infectedhepatocytes by immune cells • Clearance of virion in the circulation 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 Therapeutic intervention Immunosupression

13. Studying viral fitness in vitro • No ideal model to study viral fitness • HepaRG and PHH • Virus derived from patient versusvirus 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

14. Method to study HBV replication capacity Durantel et al., Hepatology, 2004 Durantel et al., J. Clin. Virol. , 2005

15. Evaluation of drug susceptibility in vitro Durantel et al., Hepatology, 2004 Durantel et al., J. Clin. Virol. , 2005

16. Longitudinal studies: replication capacity and drug suceptibility of lamivudine-resistant strains Durantel et al., Hepatology, 2004

17. Other phenotypic assay Yang et al., Antiviral Res, 2004

18. Phenotypic studies with Bac-HBV LMMNNT Mock LMMV NT WT Replication capacity Encapsidated intracellular HBV DNA Drug susceptibility and cross resistance Lucifora, Durantel et al., JGV, 2008

19. Relative Replication Yield (RC) of HBV Mutants R. Edwards, T. Shaw, V. Sozzi & S. Locarnini. 2005. Global Antiviral Journal;1(Suppl2):77

20. Replication capacity varies with cell type Lucifora et al, submitted Brunelle et al, Hepatology, 2005

21. Antiviral drug potency depends on hepatoma cell type Seignères et al, Hepatology 2002

22. Impact of HBV genome background on antiviral drug efficacy and cross-resistance Villet et al, J Hepatol 2008

23. Studying viral fitness in vitro • No ideal model to study viral fitness • HepaRG and PHH • Virus derived from patient versusvirus 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

24. Models for the production of HBV particles used for assemby and infectivity assays Transfected or tranduced cells Stably-transformed cell lines HDV particles HBV pseudoparticles

25. 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

26. Problem of production of non enveloped capsids in transfected/transduced cells Production of significant amount of non-enveloped capsid Transfection or transduction Problem of standardisation of input for infectivity assay Interest of stably transformed cell lines

27. Hepatology , 2008

28. Studying viral fitness in vitro • No ideal model to study viral fitness • HepaRG and PHH • Virus derived from patient versusvirus 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

29. HDV life cycle Courtesy from Dr Camille Sureau

30. Production of HDV and HBV polyA HDV genome (3x) CMV Fixation and internalisation via HBV receptor(s) Transfection Target Cell HDV Huh7 HBV or HDV replication quantified by qRT-PCR orSB/NB Replication of HDV and HBV genomes & glycoproteins synthesis Production and secretion of HDV and HBV HBV Sureau, Curr Top Microbiol Immunol, 2006

31. Production of HDV particles without production of HBV polyA HDV genome (3x) CMV polyA HBV large CMV polyA HBV medium CMV Fixation and internalisation via HBV receptor(s) polyA HBV small CMV Transfection Target Cell HDV Huh7 HDV replication quantified by qRT-PCR or Northern blot Production and secretion of HDV Expression of HBV glycoproteins Replication of HDV genome Sureau, Curr Top Microbiol Immunol, 2006

32. DY Pol polyA CMV Gag Production of retroviral particles pseudotyped with HBV/DHBV glycoproteins? 5’ LTR 3’ LTR Y R Luciferase U3 U5 CMV U5 R CMV polyA HBV large CMV Biochemical and antigenic studies polyA HBV medium CMV polyA HBV small CMV Fixation and internalisation Transfection Target Cell HBVpp 293FT Reporter expression measurement Production and secretion of HBVpp Expression of HBV glycoproteins Chai et al., J Virol, 2007 Saha, J Virol, 2005

33. Studying viral fitness in vitro • No ideal model to study viral fitness • HepaRG and PHH • Virus derived from patient versusvirus 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

34. Modelsused for infectivityassays Primary human hepatocytes HepaRG cells Other cell culture models

35. Infection of PHH with HBV • Model difficult to master • Variability from batch to batch Gripon et al., J Virol, 1988

36. 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

37. Type-I IFN response restrict HBV replication launched by Bac-HBV in proliferative HepaRG cells RT-PCR Encapsidated intracellular HBV DNA Encapsidated intracellular HBV DNA Encapsidated intracellular HBV DNA Lucifora, Durantel et al., submitted

38. Other cell systems ? Ac anti-IFNß - + Transduction Infection M J1 J3 J6 J9 J1 J3 J6 J9 M J1 J3 J6 M J1 J3 J6 M J1 J3 J6 J9 M J1 J3 J6 J9 Total DNA Encaps DNA - + Immunofluorescence RT-PCR

39. Animal model to study viral fitness Kremsdorf and Brezillon, WJG, 2007

40. Chimpanzee to study viral fitness ?? Hepatology , 2009

41. 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

42. 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

43. Genotypic and viral quasispecies studies

44. Longitudinal studies: replicationcapacity and drugsuceptibility of multiresistantstrains WT mut-3 mut-2 mut-1 mut-4

45. The selected mutant replicatesbetter in presence of drugs Replication capacity and magnitude of resistance to AV are not the only factors

46. In vivo selectedmutant isbetterproduced in vitro HBV transfection HBV/HDV transfection

47. In vivo selectedmutant is more infectious in vitro

48. 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

49. 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

50. Acknowledgements Collaborations K. Lacombe, Paris J.P. Villeneuve, Montreal P. Lampertico, Milan S. Locarnini, Australia H. Wedemeyer, Hannover J. Petersen, Hamburg T. Santantonio, Bari J. Neyts, Leuven M. Levrero, Roma M. Buti, Barcelona INSERM U871 S. Villet C. Pichoud F. Lavocat R. Egounlety G. Billioud J. Lucifora M. A Goughoulte P. Deny O. Hantz D. Durantel S. Durantel MN Brunelle Liver department F Zoulim C. Trépo Ph. Merle F. Bailly P. Miailhes M. Maynard C. Fournier S. Si Ahmed