The Outcome of Acute Hepatitis C Predicted by the Evolution of the

Viral QuasispeciesFarci et al. (2000) Science 288, 339-344.

Georg Gerber

HST.120

December 15, 2006

HCV overview

• HCV sequenced in 1989 – major cause of non-A, non-B viral hepatitis

• Approximately 170 million people infected worldwide• > 38,000 new cases in U.S. annually• Chronic infection in ≈70% untreated people (≈10% if

treated)• Cirrhosis in ≈20% and hepatocellular carcinoma in

≈2.5% of chronically infected• No vaccine• Treatment effective in only 50-60% of patients and has

significant toxicity

EnvelopeEnvelope

CoreCore

EnvelopeEnvelopeGlycoproteinsGlycoproteins

Viral RNA Viral RNA (9400 (9400 nucleotides)nucleotides)

55-65 nm55-65 nm

Hepatitis C Virus

HCV Genotypes

• Viral polymerase is highly error-prone (≈1 nucleotide change per replication cycle)

• At least 6 viral genotypes and >90 subtypes• Genotypes determined by highly conserved 5’NC

sequence• In the U.S.:

– 75% genotype 1– 15% genotype 2– 10% genotype 3

• Difference in sustained virological response (SVR) w/ Rx:– genotype 1 - 42-46% SVR– genotypes 2 and 3 – 76-80% (shorter Rx effective)

HCV Genotype Distribution

1a, 1b 2a, 2b,

3a

1b 2a, 2b, 2c,

3a

4

5a

1b

1b, 6

1b, 3a

1b, 3a

3b

2a4

Concept of Viral Quasispecies

• Low-fidelity viral polymerase produces an “ensemble” of related sequences

• View evolutionary pressure as acting on the viral population, not individual variants

• Extremely high mutations rates bad for the virus, but too low rates make non-adaptive

• Recent work by Vignuzzi et al. (Nature 2006):– Poliovirus mutant with high fidelity polymerase– Achieves WT replication rates, but loses neurotropism and

pathogenicity– Chemically induced mutagenesis → restored neurotropism and

pathogenicity– Demonstrate strains that complement each other (strain that was

pathogenic but couldn’t cross BBB complemented by “opposite” phenotype)

Host Response

• Clearance of acute infection doesn’t protect against re-infection, but does protect against chronic state

• Role of naturally acquired antibodies unclear, because don’t protect against re-infection and viral clearance can occur w/o anti-HCV antibodies in both humans and chimps

• Unclear why some become chronically infected; many proposed mechanisms for viral modulation of host defenses

“The Outcome of Acute Hepatitis C Predicted by

the Evolution of the Viral Quasispecies”

• Investigated relationship between viral genetic diversity in patients and infection outcome (e.g., resolving, chronic, fulminant)

• Opportunity to study natural history of HCV infection because:– Post-transfusion infections (before universal

blood screen) = can pinpoint date of infection– Prior to current treatment protocols

Patients

• Twelve patients categorized as:– Fulminant hepatitis (FH)– Resolving hepatitis– Chronic, slowly progressors (mild and stable

for more than 20 years)– Chronic, rapid progressors (liver-related death

within 5 years of the onset of infection)

• Three patients in each group

Viral Population Analysis

• For patients w/o FH, 3-4 viral infection time-point samples:

1. The first HCV PCR-positive sample (2-5 weeks post-transfusion)

2. Prior to antibody seroconversion (before or at the time of ALT peak)

3. One to two add’l after seroconversion

• FH patients: 2 time-points (one before and one after seroconversion)

• DNA amplified from E1/E2 genes (558 nucleotides) and cloned

• Mean of 10.6 clones sequenced from each sample (total of 414 sequences)

Viral Genetic Diversity and Number of Viral Variants

• Genetic diversity = mean Hamming distance between a.a. sequences w/in or outside 31 a.a.’s hypervariable region 1 (HVR1) of E2

• Number of viral variants = number of unique a.a. sequences w/in or outside 31 a.a.’s HVR1 of E2

Viral Genetic Diversity and Number of Viral Variants Analysis Conclusions

• After seroconversion, patients w/ chronic infection had large increase in viral genetic diversity w/in HVR1; patients w/ resolving disease showed a marked decrease in the same measure (both changes statistically significant)

• Genetic diversity and the # of viral variants outside the HVR1 region was consistently lower than w/in HVR1 and showed little temporal change

• Patients w/ FH had the lowest levels of viral genetic diversity

Analysis for positive selection

• Analyzed the # of synonymous (silent) versus non-synonymous (a.a. replacements) changes

• For each patient, derived time-point 1 consensus sequence and compared to all sequences from the last time-point

• Mean # of non-syn. substitutions per site per week in HVR1 higher in patients w/ progressing hepatitis than in patients w/ either resolving or FH (statistically significant)

• Non-syn. substitutions consistently lower outside HVR1 w/ no significant differences among patient populations

• No significant differences in syn. substitutions either inside or outside HVR1 among patient populations

• Syn. substitutions slightly higher both inside and outside the HVR1 in patients w/ FH (not statistically signif.)

Study conclusions

• Provides evidence that outcome of HCV infection may be related to viral genetic diversity that emerges w/in first few months of infection

• Patients w/ chronic infection had large increase in viral genetic diversity w/in HVR1 and significantly more non-synonymous substitutions than in other patient populations

• Patients w/ resolving disease had decrease in viral genetic diversity w/in HVR1

• Genetic diversity outside HVR1 was consistently lower than w/in HVR1, showed little temporal change, and low rates of both syn. and non-syn. substitutions across all patient populations

Criticism

• Small patient population → limited statistical power

• Limited statistical analysis:– Correction for multiple hypothesis testing? – Significance of phylogenetic findings?

• Lack of explicit biological mechanism doesn’t allow inference of causality

• Thus, conclusions really only suggestive of associations, patterns or trends

Criticism (cont.)

• Are these findings counterintuitive? Shouldn’t an effective immune response exert selective pressure → viral diversification?

• Authors speculate that patients who clear HCV are mounting an effective response that eliminates many viral variants, and note a similar trend in patients responding well to interferon therapy

• 2006 study by same authors (similar methodology) of children perinatally infected w/ HCV:– Children w/ highest ALT levels (worst liver damage) harbored

the most homogeneous viral populations– Authors speculate due to differences in strength of the cytotoxic

T-cell response versus B-cell mediated response

Viral tropism

• All necessary host receptors not yet elucidated• E2 binds to CD81 (present on many cells

including hepatocytes); necessary but not sufficient

• Many other receptors identified: low-density lipoprotein receptor, scavenger receptor class-B type-I (SR-BI), L-SIGN and DC-SIGN

• Some evidence can infect non-hepatocytes

Treatment and prophylaxis

• Pegylated IFN-alpha and ribavirin for 24 or 48 weeks

• Sustained virological response (SVR) = no detectable HCV RNA during treatment and for >6 months after stopping therapy:– genotype 1 - 42-46% SVR– genotypes 2 and 3 – 76-80% (shorter Rx effective)

• Concerns about drug resistance and relatively low SVRs, so much work on developing new drugs and a vaccine