1. This program is supported by educational grants from Raffaele Bruno, MD Department of Infectious Diseases, Hepatology Outpatients Unit University of Pavia Fondazione IRCCS Policlinico San Matteo, Pavia, Italy HCV: clinica e virologia a confronto Francesca Ceccherini-Silberstein, PhD Associate Professor Chair of Virology Department of Experimental Medicine and Surgery University of Rome Tor Vergata

2. Changes in standard of care for HCV, and improvements in numbers of sustained virological responses

3. Who does (not) care about HCV drug resistance?

4. Treatment of HCV in 2015 (including protease, NS5B, and NS5A inhibitors that are approved or are about to be approved) *NS5A inhibitor. †NS5B inhibitor. ‡Protease inhibitor

5. Impact of baseline RAVs on response What is the role of the resistance test in the context of a SVR> 90%?

6. Differently from HIV, where natural resistance segregates only within minority species, in HCV, major species with decreased sensitivity to direct-acting antivirals (DAAs) can be observed in DAA-naive patients prior to treatment Kunzen, Hepatology 2008 5.5% in G1a and 1.4% in G1b dominant NS3 PI resistance mutation Possible fitness changes are adjusted by the genetic background (compensatory mutations) No impact of NS3 PI resistance mutations on viral load Johnson and Geretti JAC 2010 Background HIV-1 variants containing mutations associated with drug resistance

7. VF rates at M12 were 6.0% (95% confidence interval [CI]: 5.5; 6.5), 6.3% (4.2; 9.3) and 16.2% (13.0; 20.1) for no TDR group, TDR and fully active group and TDR and resistant group, respectively. Wittkop et al Lancet 2011 HIV-1 transmitted drug resistance is associated with a poorer virological response when patients received cART containing ≥1 drug not fully active

8. Jacobson I, et al. Lancet 2014 GT 1aGT 1b SMV + P/R P/R 105/11729/56105/14736/74 71 49 90 52 100 60 20 0 SVR12 (%) 80 40 Virologic failure during simeprevir treatment was more common in treatment-naive patients with genotype 1a with Q80K No differences between 1b and 1a without Q80K Differences in SVR12 by Subgroup (95% CIs) GT 1a/other HCV - With baseline Q80K vs Pbo - Without baseline Q80K vs Pbo GT 1b HCV 28.2 (13.4-42.9) 4.7 (-14.6 to 24.1) 40.3 (25.8-54.8) 42.1 (26.5-57.6) 147 60 86 117 74 74 74 56 SMV (n) Pbo (n) Favors Placebo Favors SMV -100-50050100 SVR12 with baseline Q80K: 16/30 (53%) SVR12 without baseline Q80K: 73/86 (85%)

9. SMV + SOF (12 weeks) SVR12 rates according to Q80K presence at baseline in G1a cirrhotic patients: OPTIMIST-2 Eligible patients were 18–70 years with chronic HCV G1 infection; HCV RNA >10 000 IU/mL at screening, platelets >50 000/mm 3, albumin >3 g/dL and presence of cirrhosis determined by any of: FibroScan with kPa >12.5 within 6 months of screening; FibroTest score of >0.75 and AST to platelet ratio >2 at screening; liver biopsy documenting cirrhosis Treatment-experienced = IFN-intolerant, prior relapsers, prior nonresponders, other Brackets = 95% CI; AST = aspartate aminotransferase Lawitz E, et al. EASL 2015. Poster LP04 60/7225/3435/38 83 (74.0,92.6) 74 (57.2,89.8) 92 (82.2,100) Asselah T, EASL 2015 SYMPOSIUM

10. Treatment options for treatment-naive patients with HCV genotype 1a who are initiating therapy Daily sofosbuvir (400 mg) plus simeprevir (150 mg) with or without weight-based RBV for 12 weeks (no cirrhosis) or 24 weeks (cirrhosis) for patients with a negative test result for the Q80K variant using commercially available resistance assays. In patients with HCV genotype 1a and cirrhosis who have the Q80K variant, one of the other regimens for cirrhosis detailed above is recommended. (IIa-B) AASLD GUIDELINES HEPATOLOGY UPDATE JUNE 2015 AASLD guidelines recommends Q80K testing in GT-1a patients candidate to a SOF+SMV regimen

11. The prevalence of pre-treatment NS5A RAVs in GT-1 is different across different countries, ranging from 6% to 25%, and different according to subtype….. The analysis of >3000 GT-1 NS5A sequences form 14 countries showed a high prevalence of baseline Y93H mutation (associated with resistance to daclatasvir 100 fold) in GT -1 b infected patients, ranging from 7% to 15%. Svarovskaia E.S., EASL 2015

12. In vitro potency, genotypic coverage and resistance profiles of DCV and LDV Gao M. Curr Opin Virol. 2013;3:514 In vitro HCV replicon potency and RAV sensitivity to DCV vs LDV: GT 1 DCV and LDV display similar potencies against GT 1a and GT 1b wild type replicons in vitro DCV is superior in GT 1a and 1b against Y93H resistant variant (≈10% in baseline) GT 1bGT 1a LDV = ledipasvir; RAV = resistance-associated variant; WT = wild-type

13. Zeuzem S et al., Ann Intern Med. 2015

15. Kwo P, et al. EASL 2015 Grazoprevir/elbasvir +/- RBV for 12 or 16 weeks in patients with HCV G1, G4 or G6 infection who previously failed peg IFN/RBV: C-EDGE treatment-experienced

16. Krishnan P et al., AAC 2015

17. HCV-1a HCV-1b Prevalence of baseline polymorphisms in GT-1a and GT-1b in the Aviator trial M28V confers 58-fold resistance to ombitasvir in the GT1a replicon, while S556G confers 30-fold resistance to dasabuvir. Y93H confers 77-fold resistance to ombitasvir in the GT1b replicon, while the combination of variants C316N plus S556G confers 38-fold resistance to dasabuvir. Krishnan P et al., AAC 2015

18. Although the number of patients with baseline variants other than Q80K was small, there was no difference in SVR24 rates among GT1a-infected patients with any of these variants at baseline as compared to patients with the reference amino acid residue at the corresponding position Observed data SVR24 rate a in the presence of baseline variants Krishnan P et al., AAC 2015

19. Prevalence and Impact of NS3 Baseline Variants on Efficacy in Hepatitis C genotype 1 infected patients treated with Grazoprevir+Elbasvir±Ribavirin: the C-WORTHY Study Black S. et al., EASL 2015

20. Overall, 97% SVR12 after 12 weeks of DCV + SOF QD and 76% SVR12 in patients treated for 8 weeks in HIV/HCV coinfected patients In 12-week group, 97% in GT 1; 100% in GT 2, 3, and 4 97% in treatment-naive and 98% in treatment-experienced patients Wyles DL et al. CROI 2015

21. The new NS5B RAV L159F associated with virological failure to sofosbuvir can be found as natural polymorphism in GT-1b Donaldson, Hepatology 2014 Svarovskaia E.S., EASL 2015 In a Russian study, L159F showed a lower SVR12 rate in treatment-naive GT-1b patients with 16 weeks of SOF + RBV therapy. In a recent, worldwide analysis of >7800 GT-1 NS5B sequences from 22 countries, the L159F variant was mostly found in GT-1b, with the highest prevalence in Russia (34%), followed by Spain (32%), and the lowest prevalence in Japan and Taiwan (1% and 0%, respectively). In Italy the prevalence was around 20%. Zhdanov K., APASL 2015

22. Should we be worried about baseline RAVs? SVR rates to NS3 protease inhibitor plus NS5A inhibitor combination regimens in HCV genotype 1 infected patients according to the presence of baseline RAVs. SVR rates for NS3 protease inhibitor plus nucleos(t)ide NS5B inhibitor combination regimens in HCV genotype 1 infected patients according to the presence of baseline RAVs. SVR rates NS5A inhibitor plus nucleos(t)ide NS5B inhibitor DAA combination regimens in HCV genotype 1 infected patients according to the presence of baseline RAVs. Sarrazin C et al., J Hepatol 2015

23. Chevaliez, PLoS ONE 2010

24. How to genotype/subtype HCV?

26. Messina et al., Hepatology 2015 HCV genotypes show different geographical distribution

27. Genotype 1 is by far the most frequent genotype in chronically infected patients worldwide as well as in Europe Esteban JI et al J Hepatol 2008 3a 1b 2 1a 3a 2 2 1b 1a 3a 1a 3a 1a 3a 1a 3a 1a 3a 1a 3a 1a 3a 4 4 4 1a 2 4 3a

28. 37.5% HCV-1 30% HCV-3 8.5 % HCV-4 Distribution of HCV genotypes (G) was: G1a, 439 (30%); G1b, 178 (12%); G2, 35 (2%); G3a, 339 (23%); G4, 147 (10%). 92% were currently receiving ART. M Shanyinde et al ICAR 2015 1,462 PLHIV-HCV patients

29. Daclatasvir is highly potent and pan- genotypic: Useful for combination therapy Potent NS5A inhibitor with pan-genotypic coverage (EC50: 9 to 146 pM in vitro) 1. Gao M, et al. Nature. 2010;465:96; 2. Fridell RA, et al. Hepatology. 2011;54:1924; 3. Fridell RA, et al. J Virol. 2011;85:7312; 4. Wang C, et al. Antimicrob Agents Chemother. 2013;57:611; 5. Wang C, et al. Antimicrob Agents Chemother. 2012;56:1588. GTEC 50 1a (H77)50 ± 13 pM 1b (Con1)9 ± 4 pM 2a (JFH)71 ± 17 pM 3a 146 ± 34 pM 4a12 ± 4 pM 5a33 ± 10 pM Fold resistance GT 1a 1 GT 4 5 GT 2 3 GT 1b 2 GT 3 4 In vitro resistance profileIn vitro potency 1 EC = effective concentration

30. In vitro potency, genotypic coverage and resistance profiles of DCV and LDV Gao M. Curr Opin Virol. 2013;3:514 GT 2aGT 3aGT 4aGT 5aGT 6a In vitro HCV replicon potency and RAV sensitivity to DCV vs LDV: GT 2, 3, 4, 5, 6 DCV has significantly higher potency in GT 2a and GT 3a in vitro compared to LDV In vitro potency, genotypic coverage and resistance profiles of DCV and LDV

31. NS3 NS5A NS5B By phylogenetic analysis of NS3, NS5A and NS5B, genotype assignments were 100% each other concordant (Ceccherini-Silberstein et al., Hepatology 2015 in press) The best method to assess genotype and subtype is the direct sequencing followed by phylogenetic analysis

32.  Trugene HCV Genotyping assay Direct sequencing  INNO-LiPA HCV 1.0 Reverse hybridization  INNO-LiPA HCV 2.0 Reverse hybridization  Abbott RealTime HCV Genotype II assay Real time PCR  Target Regions: HCV 5’ UTR, CORE & NS5B region  5’ UTR3’ UTR All assays target the 5’NCR gene for genotypes 1-6, in addition, the 2 assays more used in diagnostics, Abbott and INNO- LiPA-HCV-2.0, target also the NS5B and the core gene, respectively, providing additional information also in subtyping: for genotype 1 (1a/1b, both), and for all genotypes (only Innolipa). Several commercial assays are available for determining genotype/subtype

33.  Trugene HCV Genotyping assay Direct sequencing  INNO-LiPA HCV 1.0 Reverse hybridization  INNO-LiPA HCV 2.0 Reverse hybridization  Abbott RealTime HCV Genotype II assay Real time PCR  Target Regions: HCV 5’ UTR, CORE & NS5B region  5’ UTR3’ UTR However, not all genotypes can be resolved, with results being reported as: ‘indeterminate’, ‘mixed’, ‘genotype X reactivity with Y’, or just the major genotype 1 alone. A correct determination of HCV-genotype is relevant prior to treatment initiation

34. We reanalyzed genotype data by phylogenetic analysis of 601 HCV-infected patients candidate to DAA-treatment, who performed a genotypic-resistance-test between 2011 and 2015. To confirm the appropriate genotype allocation, HCV-sequencing was performed by home-made protocols, specific for each genotype, on NS3-protease (95% samples), together with/in alternative to NS5A (9%) and/or NS5B (14%). Notably, all patients with a previous result of ‘mixed’ or ‘indeterminate’ HCV-genotype or subtype by commercial-assays were instead precisely resolved by HCV-sequencing. Update from Ceccherini-Silberstein F. et al, Hepatology 2015 Patients (N)Patients (%) Genotype/subtype confirmed54590.7% Genotype 1 with no subtype142.3% Discordant genotypes81.4% Genotype 1 with discordant subtype172.8% Mixed genotypes/indeterminate/unknown172.8% Total601100% HCV-sequencing and commercial-assays were concordant in 90.7% of cases analysed

35. We reanalyzed genotype data by phylogenetic analysis of 601 HCV-infected patients candidate to DAA-treatment, who performed a genotypic-resistance-test between 2011 and 2015. To confirm the appropriate genotype allocation, HCV-sequencing was performed by home-made protocols, specific for each genotype, on NS3-protease (95% samples), together with/in alternative to NS5A (9%) and/or NS5B (14%). Notably, all patients with a previous result of ‘mixed’ or ‘indeterminate’ HCV-genotype or subtype by commercial-assays were instead precisely resolved by HCV-sequencing. Update from Ceccherini-Silberstein F. et al, Hepatology 2015 Patients (N)Patients (%) Genotype/subtype confirmed54590.7% Genotype 1 with no subtype142.3% Discordant genotypes81.4% Genotype 1 with discordant subtype172.8% Mixed genotypes/indeterminate/unknown172.8% Total601100% HCV-sequencing and commercial-assays were concordant in 90.7% of cases analysed Overall, 25 out of 601 (4.6%) HCV infected patients candidate to start a treatment containing a DAA showed a discordant genotype or subtype according to the sequencing

36. EASL 2015

38. Do DAA resistance mutations “disappear” following discontinuation of therapy?

39. Long-term follow-up data on a cohort of 314 BOC- failing patients indicated that NS3 RAVs reverted to wild-type in 73% of cases within 3 years post-therapy From a different perspective, these data indicate that 27% of patients still presented dominant resistant (and presumably fit) variants after 3 years since boceprevir discontinuation. Furthermore, the remaining 73% can still harbor minority residual resistant strains, since reversion was evaluated by population sequencing, able of detecting variants at a frequency >20%. Howe, Antivir Res 2014

40. In the majority of patients PR RAVs disappear…. Lentz O, et al. EASL 2014 Simeprevir Krishnan P et al. EASL 2015 Paritaprevir/r

41. The absence (by Sanger sequencing) or low prevalence of RAVs does not always means low amount of circulating resistant virus WT R155 (65.5%) WT= wild-type PT 13PT 11 WT R155 (96.3%) R155K (35.5%) R155K (3.7%) Mutational Load of R155K: 77,966 IU/ml Mutational Load of R155K: 594,022 IU/ml HCV-RNA : 1,671,926 IU/mlHCV-RNA: 2,090,903 IU/ml Mutational Load of R155K: 44,167 IU/ml WT R155 (96.0%) R155K (4.0%) HCV-RNA: 1,104,189 IU/ml At 100 weeks of therapy interruption At 62 weeks of therapy interruption PT 211 Three HCV-1a infected patients, all failing TVR-containing regimen with RAVs, still showed the R155K after 60-100 weeks of therapy interruption. Di Maio VC, EASL 2015

42. Majority of RAVs Detected After 96 Weeks (> 1% of Population) Registry Study 62/6358/5842/4345/4552/5550/58 Almost all patients developed NS5A RAVs at treatment failure Patients with NS5A RAVs Patients without NS5A RAVs Long-Term Persistence of HCV NS5A Variants After Treatment With LDV NS5A RAVs in patients who failed HCV treatment with ledipasvir (LDV) in the absence SOF Positions 24, 28, 30, 31, 32, 58, 93 that confer >2.5- fold reduced susceptibility to LDV in vitro were included Wyles et al. Abstract O059, EASL 2015

43. Retreatment of Patients Who Failed 8 or 12 Weeks of Ledipasvir/Sofosbuvir-Based Regimens With Ledipasvir/Sofosbuvir for 24 Weeks SVR12 by baseline RAVs shows that the presence of baseline NS5A RAV(s) is associated with virological failure. Lawitz E. et al., EASL 2015 Hadas Dvory-Sobol IWDR Berlin, June 2014 Prior to re-treat no NS5B RAVs (S282T, L159F, V321A) At second virologic failure 4 of 12 (33%) patients had NS5B RAVs: S282T (n=2) L159F (n=1) S282T + L159F (n=1)

44. Hepatology 2015

45. Sofosbuvir + simeprevir (± ribavirin) Sofosbuvir + daclatasvir (± ribavirin) When is the Role of Ribavirin Unclear? Treatment Options for HCV Genotype 1 Sofosbuvir + Ledipasvir (± ribavirin) Sofosbuvir + daclatasvir (± ribavirin) Treatment Options for HCV Genotype 3

46. Can Ribavirin protect against the emergence of resistance?

47. 23/2686/9132/34164/16917/20113/11314/1444/44 12 Weeks 24 Weeks Sarrazin et al., EASL 2015 LDV/SOF ± RBV: SVR12 in GT 1 Treatment-naïve Patients With Cirrhosis ± Baseline NS5A RAVs 18% (94/511) cirrhotic patients had BL RAVs; Need for RBV?

48. LDV/SOF ± RBV: SVR12 in GT 1 Treatment-naïve Patients With Cirrhosis ± Baseline NS5A RAVs Sarrazin et al., EASL 2015 Different impact according to HCV-1 subtype……

49. Treatment should be individualized Sarrazin C et a., J Hepatol 2015 Both in terms of treatment duration and number of effective drugs

50. ACKNOWLEDGEMENTS