Subject characteristics and time point analysis.

IFN-γ response and the relationship between viral diversity and the rate of immune escape in subjects chronically infected with HCV.

IFN-γ ELISpot values (SFU/million, right y-axis) and viral load (IU/mL, left y-axis) measured in subjects A. 300256, B. THDS1086MX and C. THGS0684MX for epitope-specific CD8+ T cell responses (see key). Subjects HOKD0485FX, 300023 and 300240 have been shown previously (10, 16). Escape variants are shown with a clear symbol of the original epitope found in the transmitted/founder (see key). D. Plot of average Shannon entropy (SE) against the rate of escape for each epitope in each protein region per subject. Plots of average SE against average IFN-γ ELISPOT response at >90DPI (purple) (E.) and <90DPI (green) (F.) are also shown. P values (P) and Pearson’s correlation coefficient (R) are shown in the top left corner of each panel.

Epitopes and escape rate of epitopes from chronic progressors with fixation events where positive IFN-γ ELISPOT response was detected on the T/F virus.

Co-occurring mutations, epitope escape mutants and the associated frequency of occurrence, and relative fitness of NS3 region of subject THDS1086MX.

Co-occurring mutations, epitope escape mutants and the associated frequency of occurrence and relative fitness of NS3 region of subject THGS0684MX.

Longitudinal fitness plots of subjects chronically infected with HCV.

Longitudinal fitness plots of subjects A. 300023, B. 300240, C. 300256, D. HOKD0485FX, E. THDS1086MX and F. THGS0684MX are shown. Grey shade indicates viral load and is measured in IU/ml on the right y-axis. Coloured lines indicate population average relative fitness estimate (right y-axis) for protein regions (see key). Vertical bars indicate standard deviation of population average relative fitness.

The relationship between fitness and the magnitude of the immune response (SE and IFN-γ ELISPOT) and the rate of escape at <90DPI and >90DPI.

The relationship of population fitness against A. average IFN-γ ELISPOT, B. average Shannon entropy (SE) and C. rate of escape at <90DPI (green) were measured by Pearson’s correlation. The relationship of population fitness against D. average IFN-γ ELISPOT, E. average Shannon entropy (SE) and F. rate of escape at >90DPI (purple) were also measured by Pearson’s correlation. P values (P) and Pearson’s correlation coefficient (R) are shown in the top left corner of each panel.

Longitudinal co-occurring mutations in the NS3 region for subjects THDS1086MX and THGS0684MX.

Highlighter plots (Geneious Prime 2023) derived from longitudinal sequencing from subjects THDS1086MX (top) and THGS0684MX (bottom) indicating co-occurring mutations relative to the transmitted/founder (TF) across the NS3 region. Numbers above highlighter plots denote the genomic amino acid number for the NS3 region. Sequences are labelled by frequency of occurrence (%) and days post infection (DPI). Specific amino acid changes are shown in Table 3 and Table 4.

HCV neutralizing antibody (nAb), CD8+ T cell responses and viral fitness.

The timing (Days post infection) of CD8+ T cell and nAbs are compared for clearer subjects (A) and chronic subjects (B). Statistical significance (Wilcoxon matched-pairs signed rank test) is represented by asterisks (p<0.05 (*)) and non-significance by NS. Panels C - E shows 3 representative subjects who developed chronic HCV infection. The blue line represents the IFN-γ ELISPOT (SFU/million). The maroon line represents HCV nAb ID50 titre with squares representing timepoints tested on autologous virus and circles representing timepoints tested on heterologous virus. Population average relative fitness estimate of regions NS5B (purple), NS3 (green) and NS2 (pink) are shown. Black arrows represent increases in average relative fitness.

Summary of epitope selection and (IFN-γ) ELISPOT assay responses in subjects who cleared infection.

Summary of epitope selection and positive (IFN-γ) ELISPOT assay responses in subjects who developed chronic infection.

Subject 300023 relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.

Subject 300240 relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.

Subject 300256 relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.

Subject HOKD0485FX relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.

Magnitude of CD8+ T cell responses associated with escape.

CD8+ T cell responses were tested by IFN-γ ELISPOT assays measuring spot-forming unit per million cells (SFU/million cell) of epitopes (vertical axis) across three populations. Horizontal axis from left to right shows the population of epitopes that undergo fixation events in subjects who became chronically infected by HCV (Ch), epitopes that had no mutations and epitopes from subjects that spontaneously cleared HCV infection (Cl). Statistical comparisons are Mann-Whitney tests. Scatter plots represent means and standard deviation.

Phylogenetic analysis of subject 300023 NS5B region.

Diamonds represent haplotypes with blue indicating consensus sequence and green diamonds representing reconstructed haplotypes at 36DPI. Purple diamonds indicate reconstructed haplotypes at 44DPI. The NS5B region shows diversity consistent with the presence of two T/F viruses as previously described by us (10, 11, 15, 16).

HCV neutralizing antibody (nAb) and CD8+ T cell responses and viral fitness.

Panels A - C shows 3 subjects who cleared HCV infection and Panels D-F shows three subjects who developed chronic HCV infection. The blue line represents the IFN-γ ELISPOT response (SFU/million). The maroon line represents HCV nAb ID50 titre with squares representing timepoints tested on autologous virus and circles representing timepoints tested on heterologous virus. For panels A-C, the shaded area represents the longitudinal HCV RNA levels (IU/ml). For panels D-F population average relative fitness estimate of regions are shown (see key).