Maureen J. Donlin Saint Louis University School of Medicine

1. HCV Genetic and Phenotypic Variation in the Context of Antiviral Therapy and Pathology Maureen J. Donlin Saint Louis University School of Medicine

2. Genotype • Six genotypes • >28% divergence • Subtype • ~15-20% divergence • Isolate • ~5-10% divergence • Intra-patient • ~1-3% divergence • Quasispecies Alignment of 70 HCV subtype 1a sequences with variations highlighted HCV genetic variation

3. Pre-therapy HCV genetic associations with the outcome of therapy

4. The Virahep-C viral genetics study Question: Are there HCV genetic patterns associated with outcome of therapy at the viral isolate level? • Approach: • Sequence the full viral ORF from genotype 1 patients undergoing interferon α/ribavirin treatment • Identify genetic patterns associated with outcome of therapy.

5. Virahep-C genetics cohort • The viral genetic patterns were compared between sequences stratified by either Day 28 response or by treatment outcome. • Genotype 1a and 1b sequences were analyzed separately.

6. Amino acid differences associated with response to therapy Positions at which marked and poor alignments have different amino acids conserved at > 60% • Very few amino acid positions differed consistently by response • The amino acid variations at these sites were conservative Donlin et al. J. Virol. 81:8211 and unpublished

7. Association of pre-therapy genetic diversity with treatment outcome • Marked sequences are more diverse than poor sequences • This difference is not uniformly distributed, NS3 and NS5A in 1a; Core and NS3 in genotype 1b. • Associations of genetic variability with treatment outcome are similar to associations with day 28 response Donlin et al. PLoS One 5:e9032

8. Other relevant observations: Core, NS3, and NS5A can counteract the type 1 interferon response in cultured cells Only those HCV isolates that were most fit survived the strong interferon response induced by therapy Interpretation High viral diversity is associated with successfull therapy because there are only a few ways to optimize protein function, but many ways to impair it

9. HCV genetic associations rate of progression of liver disease

10. The HALT-C viral genetics study Question: Are there HCV genetic patterns associated with an increased rate of disease progression? • Approach: • Sequence the full viral ORF from 30 genotype 1a patients undergoing long-term interferon α treatment at two intervals, 3 years apart • Identify genetic patterns associated with rate of disease progression.

11. HALT-C viral genetics cohort • Inclusion criteria: • HCV 1a • Fibrosis score = 3 or 4 • Failed standard IFN+RV or PegIFN+RBV therapy • Exclusion criteria: • HBV or HIV co-infection • Rapid progressors: • Have primary HALT-C outcome or 2-point increase in Ishak Fibrosis score • Slow progressors: • Matched based on pre-IFN therapy titre, Ishak score, age and gender

12. Amino acid identity differences at only a few positions correlate with rate of disease progression • Amino acid skewing at most positions was very modest and involved similar amino acids—Probably noise • The most interesting observation was the cluster of 3 significant differences in NS4B • 2 of the 3 were visually apparent in the alignments • We have never seen a correlation in NS4B before

13. Viral diversity differences are not associated with disease progression All variations Unique variations • Diversity differences associated with rate of disease progression were not seen in any gene • This is very different from the strong association of high diversity with sensitivity to interferon-based therapy Unique variations Association with response to therapy

14. Viral genetic diversity is stable over a three year interval • Genetic analyses: • Average pair-wise protein distance • Number of variations from population reference • Number of variations from time point 1 to time point 2 within same patient • Results: • We saw no significant differences between the time points by any of the genetic measures

15. HALT-C versus Virahep-C cohort All variations Question: Are there genetic differences in HCV associated with advancing disease? • HALT-C sequences are more similar to the Poor than to the Marked VHC sequences • The exception is that NS5B is more diverse in HALT-C compared to either Marked or Poor sequences • Patterns remain consistent in the variations unique to HALT-C or VHC sequences p = 0.002 P < 0.001 Unique variations

16. HCV genetic associations with development of hepatocellular carcinoma

17. The HCC viral genetics study Question: Are there HCV genetic patterns associated with development of hepatocellular carcinoma? • Approach: • Sequence the full viral ORF from genotype 1b patients diagnosed with HCC and from age- and gender-matched cirrhotic controls • Identify genetic diversity patterns associated with the development of HCC

18. HCC viral genetics patient cohort • Inclusion criteria: • HCV 1b • Cirrhotic • Adequate clinical monitoring to detect cancer if it existed • Exclusion criteria: • HBV or HIV co-infection • Heavy alcohol use • Other liver diseases including NASH, abnormal α1-antitrypsin levels, or hemochromatosis

19. Amino acid identity differences at only a few positions correlate with HCC • Amino acid skewing at most positions was modest and involved similar amino acids • The most interesting differences were in P7 (residues 762, 767), NS3 (1323, 1329), NS5A (2356), and 2543 (NS5B) • 3 of the 63 residues in the P7 ion channel were significantly different in HCC and cirrhotic sequences

20. Only very small diversity differences are associated with HCC All variations Unique variations • Diversity differences associated with HCC were not seen in any gene when all variations were considered • Weak differences were seen in E1 and p7 when only unique variations were examined • This is similar to the weak association of diversity differences with HALT-C Unique variations Association with response to therapy

21. Genetic variability within the HCC patients compared to Virahep-C cohort All variations • The genetic diversity of the HCC sequences are similar to VHC Poor sequences for Core, E2 and NS2 • The HCC diversity patterns are more similar to VHC Marked sequences for NS3 and NS5A Unique variations

22. Summary • Virahep-C study: • Very few amino acids differed consistently by response to interferon-based therapy, and these differences were conservative. • Responder sequences were more diverse than non-responder sequences. • HALT-C study: • HCV sequences from patients with rapidly advancing disease were very similar to those with stable disease. • HCV sequences from patients with advanced liver disease were stable over a three-year interval. • HCV sequences from patients with advanced liver disease are similar to primary interferon non-responder sequences. • HCV sequences from patients with advanced liver disease have increased NS5B variability compared to the Virahep-C Marked or Poor sequences. • HCC study: • HCV sequences from patients with HCC were similar to those from cirrhotic patients without cancer. • The HCV sequences from patients with HCC or advanced cirrhosis did not consistently resemble either the VHC Marked or Poor sequences.

23. Conclusions • Viral survival during interferon-based therapy requires robust suppression of interferon responses. • HCV’s suppressive activities can be impaired by a variety of genetic variations. • The obvious genetic diversity differences associated with HCV’s sensitivity to interferon α are not seen with rapid disease progression or HCC. • Differential sensitivity to endogenous antiviral interferon responses does not appear to key to whether a patient will have severe disease. • We have not yet found strong genetic evidence for differential virulence among HCV genotype 1 strains. • Previous studies demonstrated a correlation between NS5B enzymatic activity and higher ALT levels. • The higher level of variability in NS5B seen in the genotype 1a for HALT-C suggests there may be an association of NS5B genetic variability with pathology.

24. Acknowledgements Personnel: • Dr. John Tavis • Dr. Adrian Di Bisceglie • Dr. Nathan Cannon • Dr. Feng Cao • Dr. Alexi Kiss • Dr. Elena Lomonosova • Xiaohong Cheng • Julia Gray • Virahep-C Study Group • Funding: • NIH grants U01DK060345, R21CA125321, U54AI057160, R01CA126807, and R01DK074515 • Saint Louis University President’s Research Fund Seed Grant • Friends of the Saint Louis University Liver Center Grant-in-Aid

25. Extra slides

26. Effect of the HCV’s high genetic variation on the phenotype of its proteins Example analyses

27. SVR Relapser Variant RdRp Phenotypic variation among HCV RNA polymerases matches their genetic variability In vitro poly-G and poly-U synthesis by purified variant HCV RdRps from the Virahep-C cohort • RNA polymerase activity varied by >300 fold among the variant enzymes. • Nucleotide use preference was not uniform among the RdRps. • Major variation was found in all 5 biochemical parameters we assessed. Cao et al., J. Viral Hepatitis, 18:349

28. Effect of NS3/4A variants on stimulation of the IFN-β promoter poly(IC) HCV-1a responder patient isolates (Duplicate samples) HCV-1a non-responder patient isolates (Duplicate samples) HCV-1a reference isolate (Technical repetitions) Normalized promoter activity • Over 80% of the subtype 1a NS3/4A variantssuppressed induction of IFN-β promoter activity by >20% • Suppressive activity varied 7-fold among the variants, but suppression by responders and non-responder genes was equivalent • Variation in suppression of dsRNA sensing by NS3/4A is not a primary determinant of differential response to therapy

29. Higher RdRp activity is weakly correlated with elevated ALT levels in patients Correlation of pre-therapy ALT levels with RNA synthesis activity of pre-therapy RdRps