Maureen J. DonlinSaint Louis University

School of Medicine

HCV Genetic and Phenotypic Variation in the Context of Antiviral

Therapy and Pathology

HCV genetic variation• 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

Pre-therapy HCV genetic associations with the outcome of therapy

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.

The Virahep-C viral genetics study

Virahep-C genetics cohortDay 28 versus Treatment outcome

Genotype 1a Genotype 1b

SVR NR SVR NR

Total 22 25 26 21

Marked 15 1 13 2

Intermediate 5 10 10 6

Poor 2 14 3 13

• 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.

Amino acid differences associated with response to therapy

Subtype Gene Position Amino Acids

1a NS4A 29 V (marked), I (poor)

1bCore 70 R (marked), Q (poor)

E1 139 T (marked), A (poor)

• Very few amino acid positions differed consistently by response

• The amino acid variations at these sites were conservative

Positions at which marked and poor alignments have different amino acids conserved at > 60%

Donlin et al. J. Virol. 81:8211 and unpublished

Positions with statistically significant skewing of amino acid distributions between

HCV 1b SVR and NR patientsPosition P value Protein

75 0.001 Core147 0.002 E1221 0.049 E1240 0.040 E1310 0.021 E1397 0.006 E2766 0.034 P7790 0.026 P7837 0.038 NS2

1158 0.045 NS31370 0.002 NS31409 0.046 NS32016 0.035 NS5A2033 0.049 NS5A2270 0.035 NS5A2377 0.035 NS5A2465 0.023 NS5B2517 0.017 NS5B2673 0.038 NS5B

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 responseDonlin et al. PLoS One 5:e9032

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

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

HCV genetic associations rate of progression of liver disease

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.

HALT-C viral genetics cohort

Rate Time point 1 Time point 2

Rapid 30 28

Slow 30 29

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

Amino acid identity differences at only a few positions correlate with rate of disease

progression

Positions with statistically significant skewing of amino acid distributions between rapid and slow progressors

Position P value Protein308 0.040 E1333 0.040 E1453 0.031 E2464 0.028 E2490 0.050 E2766 0.038 P7827 0.050 NS2883 0.043 NS2

1723 0.006 NS4B1746 0.046 NS4B1747 0.010 NS4B2181 0.040 NS5A2185 0.010 NS5A2361 0.040 NS5A2482 0.012 NS5B

• 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

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

Association with response to therapy

Unique variations

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

HALT-C versus Virahep-C cohort

Question: Are there genetic differences in HCV associated with advancing disease? p

= 0.

002

• 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 <

0.00

1

All variations

Unique variations

HCV genetic associations with development of hepatocellular carcinoma

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

HCC viral genetics patient cohort

Controls HCC P

N 25 22 --

Age 57.5 ± 10 62 ± 8.3 0.13

Sex (M) 16 (73%) 19 (76%) 0.80

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

Amino acid identity differences at only a few positions correlate with HCC

Positions with statistically significant skewing of amino

acid distributions between HCC and Cirrhotic patients

Position P value Protein75 0.038 Core

274 0.015 E1330 0.033 E1438 0.020 E2476 0.025 E2496 0.028 E2524 0.026 E2538 0.046 E2699 0.013 E2741 0.044 E2759 0.019 p7762 0.018 p7767 0.028 p7938 0.046 NS2

1290 0.016 NS31323 0.018 NS31329 0.008 NS31536 0.012 NS31694 0.038 NS4A2016 0.029 NS5A2278 0.031 NS5A2356 0.046 NS5A2385 0.028 NS5A2543 0.001 NS5B2650 0.014 NS5B

• 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

Only very small diversity differences are associated with HCC

Unique variations

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

Association with response to therapy

Genetic variability within the HCC patients compared to Virahep-C cohort

• 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

All variations

Unique variations

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.

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.

AcknowledgementsPersonnel:• 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

Extra slides

Effect of the HCV’s high genetic variation on the phenotype of its proteins

Example analyses

Phenotypic variation among HCV RNA polymerases matches their genetic variability

• 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.

In vitro poly-G and poly-U synthesis by purified variant HCV RdRps from the Virahep-C cohort

Cao et al., J. Viral Hepatitis, 18:349

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Poly-G synthesisPoly-U synthesis

Variant RdRpSVR Relapser

Effect of NS3/4A variants on stimulation of the IFN-β promoter poly(IC)

pcDNA

H77-Exp73

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HCV-1a reference isolate(Technical repetitions)

HCV-1a non-responder patient isolates(Duplicate samples)

HCV-1a responder patient isolates(Duplicate samples)

• Over 80% of the subtype 1a NS3/4A variants suppressed 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

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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