Temporal dynamics of viral fitness and the adaptive immune response in HCV infection
- Viral Immunology Systems Program, The Kirby Institute, Australia
- School of Biomedical Sciences, Faculty of Medicine, The University of New South Wales, Australia
Figures
Figure 1 with 1 supplement
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 (Bull et al., 2011; Cai et al., 2022). Escape variants are shown with a clear symbol of the original epitope found in the transmitted/founder (see key). IFN-γ ELISpot values were generated from multiple biological samples. Peptides were pooled and tested in technical duplicate; positive responses were confirmed by testing the individual peptide in a follow-up IFN-γ ELISpot assay. (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 >90 DPI (purple) (E) and <90 DPI (green) (F) are also shown. p-Values (P) and Pearson’s correlation coefficient (R) are shown in the top left corner of each panel.
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Figure 1—source data 1
Source data of IFN-γ response, viral diversity and the rate of immune escape in subjects chronically infected with HCV.
- https://cdn.elifesciences.org/articles/102232/elife-102232-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
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. The 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.
Figure 2 with 1 supplement
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. Gray shade indicates viral load and is measured in IU/ml on the right y-axis. Colored 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.
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Figure 2—source data 1
Source data containing Days post infection, viral load and NS2, NS3, NS5B fitness estimate data.
- https://cdn.elifesciences.org/articles/102232/elife-102232-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
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 (Bull et al., 2011; Walker et al., 2019; Bull et al., 2015; Cai et al., 2022).
Figure 3
The relationship between fitness and the magnitude of the immune response (SE and IFN-γ ELISPOT) and the rate of escape at <90 DPI and >90 DPI.
The relationship of population fitness against (A) average IFN-γ ELISPOT, (B) average Shannon entropy (SE), and (C) rate of escape at <90 DPI (green) was 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 >90 DPI (purple) was 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.
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Figure 3—source data 1
Source data containing SE, IFN-y ELISPOT and the rate of escape data at >90DPI and <90DPI.
- https://cdn.elifesciences.org/articles/102232/elife-102232-fig3-data1-v1.xlsx
Figure 4
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 labeled by frequency of occurrence (%) and days post infection (DPI). Specific amino acid changes are shown in Tables 3 and 4.
Figure 5 with 1 supplement
HCV neutralizing antibody (nAb), CD8 +T cell responses, and viral fitness.
The timing (Days post-infection) of CD8 +T cell and nAbs is 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. (C-E) show three representative subjects who developed chronic HCV infection. The blue line represents the IFN-γ ELISPOT (SFU/million). The maroon line represents HCV nAb ID50 titer 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) is shown. Black arrows represent increases in average relative fitness. Neutralization results were generated from multiple biological samples with each sample assayed in technical quadruplicates across two independent experiments.
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Figure 5—source data 1
Source data containing viral load, CD8 T cell, neutralisation and fitness data.
- https://cdn.elifesciences.org/articles/102232/elife-102232-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
HCV neutralizing antibody (nAb) and CD8 +T cell responses and viral fitness.
(A-C) show 3 subjects who cleared HCV infection and (D-F) show 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 titer with squares representing timepoints tested on autologous virus and circles representing timepoints tested on heterologous virus. The green line illustrates average relative fitness values. (A-C) The shaded area represents the longitudinal HCV RNA levels (IU/ml). (D-F) Population average relative fitness estimates of regions are shown (see key).
Tables
Table 1
Subject characteristics and time point analysis.
| Subject ID* | Age | Sex | Disease outcome | GT† | HLA-A | HLA-B | First sampling point (DPI‡) | Time to clearance | Initial viral load | No. samples sequenced§ | No. T/F¶ viruses | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 300023 | 22 | M | Chronic | 1 a | 02:01 | 02:01 | 44:02:00 | 57:01:00 | 36 | - | 19,234,348 | 6 | 2 |
| 300240 | 21 | M | Chronic | 3 a | 02:01 | 02:01 | 15:01 | 57:01:00 | 44 | - | 54,887 | 5 | 1 |
| 300256 | 31 | M | Chronic | 1 a | 03:01 | 24:02:00 | 07:02 | 35:01:00 | 44 | - | 34,149,824 | 5 | 1 |
| HOKD0485FX | 26 | F | Chronic | 1b | 01:01 | 30:01:00 | 08:01 | 13:02 | 30 | - | 733,849 | 8 | 1 |
| THDS1086MX | 25 | M | Chronic | 1 a | 02:01 | 32:01:00 | 14:02 | 27:05:00 | 16 | - | 235,662 | 6 | 1 |
| THGS0684MX | 28 | M | Chronic | 1 a | 02:01 | 32:01:00 | 27:02:00 | 40:01:00 | 2 | - | 140,200 | 5 | 1 |
| 300360 | 29 | M | Clearer | 3 a | 68:02:00 | 32:01:00 | 14:02 | 40:01:00 | 30 | 178 | 5,648,631 | 3 | 1 |
| 300277 | 25 | M | Clearer | 3 a | 02:01 | 11:01 | 44:02:00 | 44:02:00 | 39 | 69 | 5,482,503 | 3 | 1 |
| MCRL0786FX | 25 | F | Clearer | 1 a | 01:01 | 29:02:00 | 44:02:00 | 44:02:00 | 80 | 115 | 1846 | 1 | 1 |
| 400087 | 32 | F | Clearer | 1b | 24:02:00 | 30:04:00 | 14:02 | 15:06 | 45 | 139 | 13,118,082 | 2 | 1 |
| 300364 | 29 | M | Clearer | 1 a | 01:01 | 03:01 | 07:02 | 57:01:00 | 337 | 352 | 1932 | 1 | 1 |
| 300231 | 22 | M | Clearer | 3 a | 01:01 | 01:01 | 07:02 | 57:01:00 | 6 | 148 | 2,242,163 | 1 | 1 |
| 300089 | 26 | M | Clearer | 1b | 01:01 | 30:01:00 | 07:02 | 57:01:00 | 181 | 357 | 70,737 | 1 | 1 |
| 300164 | 22 | F | Clearer | 3 a | 24:02:00 | 32:01:00 | 07:02 | 40:01:00 | 71 | 204 | 684,028 | 1 | 1 |
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*
Identification.
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†
Genotype.
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‡
Days post infection.
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§
Next generation sequencing.
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¶
Transmitted/Founder.
Table 2
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.
| Subject ID | GT | Epitope (MT)*,† | Epitope (WT)† | Start aa | End aa | Region | ELISPOT‡ | DPI§ | Matching Subject Allele | Predicted Rank (WT) | Rank Change | Epsilon Estimate¶ | p-Value | Fitness Estimated |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 300023 | 1a | RAEAHLHAW | RAEAQLHAW | 852 | 860 | NS2 | 91 | 60 | HLA-B*57:01 | 0.3 | 0 | 0.0625 | 0.0001 | Yes |
| NSKRTPMGF | KSKRTPMGF | 2629 | 2637 | NS5B | 484 | 60 | HLA-B*57:01 | 0.45 | –2.2 | 0.1243 | 0.0036 | Yes | ||
| 300240 | 3a | RAQALPPSW | RAQAPPPSW | 1602 | 1610 | NS3 | 55 | 44 | HLA-B*57:01 | 0.2 | 0 | 0.1025 | 0.0236 | Yes |
| RLGPVQNEI | RLGPVQNEV | 1633 | 1641 | NS3 | 300 | 71 | HLA-A*02:01 | 1.6 | –2.1 | 0.0303 | 0.0001 | Yes | ||
| 300256 | 1a | DYPYRLWHY | HYPYRLWHY | 610 | 618 | E2 | 750 | 58 | HLA-A*24:02 | 1.45 | –0.5 | 0.4813 | 0.0242 | N/A |
| GPKMGVRAT | GPRLGVRAT | 41 | 49 | Core | 25 | 58 | HLA-B*07:02 | 0.4 | –1.4 | 0.0532 | 0.0413 | N/A | ||
| HPSIEEVAL | HPNIEEVAL | 1359 | 1367 | NS3 | 150 | 58 | HLA-B*35:01 | 0.5 | 0 | 0.0565 | 0.0001 | Yes | ||
| HOKD0485FX | 1b | HSRRKCDEL | HSKKKCDEL | 1395 | 1403 | NS3 | 55 | 79 | HLA-B*08:01 | 3.3 | 0.5 | 0.2685 | 0.972 | Yes |
| THDS1086MX | 1a | KLVAMGLNAV | KLVAMGINAV | 1406 | 1415 | NS3 | 85 | 72 | HLA-A*02:01 | 0.75 | 0.5 | 0.5098 | 0.0005 | Yes |
| TLSPYYKRHI | TLSPYYKRYI | 830 | 839 | NS2 | 30 | 72 | HLA-A*02:01 | 3.35 | –6.05 | 0.0689 | 0.0003 | Yes | ||
| VRMVMMTHF | ARMVMMTHF | 2841 | 2849 | NS5B | 25 | 72 | HLA-B*27:05 | 0.2 | –0.1 | 0.2582 | 0.0034 | Yes | ||
| THGS0684MX | 1a | TSILGIGTV | TSILGIGTA | 1324 | 1332 | NS3 | 230 | 58 | HLA-A*02:01 | 32 | 17 | 0.2861 | 0.0145 | Yes |
| SILGIGTVL | SILGIGTAL | 1325 | 1333 | NS3 | 405 | 58 | HLA-A*02:01 | 5.6 | –1.2 | 0.2861 | 0.0145 | Yes | ||
| AWETARYTPV | AWETARHTPV | 2816 | 2825 | NS5B | 50 | 58 | HLA-A*02:01 | 36.5 | 3.5 | 0.1082 | 0.0484 | Yes |
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*
Final most dominant escape variant is shown (i.e. frequency of occurrence >70%).
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†
Red shows the positions which undergo amino acid change.
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‡
IFN-γ ELISPOT test of autologous PBMC measured in SFU per million PBMC against wild type epitope at earliest sample time point with a positive assay (shown in DPI column). WT - Wild Type and MT - Mutant Mutant epitopes are sourced from the final available deep sequenced data point.
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§
Days post infection.
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¶
The epsilon estimate is the rate of escape given in per day.
Table 3
Co-occurring mutations, epitope escape mutants, and the associated frequency of occurrence and relative fitness of the NS3 region of subject THDS1086MX.
| Time | Viral load (IU/ml) | Frequency | Relative fitness | 1406KLVAMGLNAV1415mutations | Co-occurring mutations† | ||
|---|---|---|---|---|---|---|---|
| 16DPI | 235662 | 61.70% | 1.000 | ||||
| 18.10% | 1.000 | ||||||
| 6.00% | 1.000 | ||||||
| 4.80% | 0.364 | V1408A | |||||
| 4.60% | 1.000 | ||||||
| 1.70% | 1.000 | ||||||
| 1.60% | 0.364 | V1408A | |||||
| 1.40% | 1.000 | ||||||
| 72DPI | 176550 | 48.50% | 0.453 | H1115T | |||
| 24.70% | 0.453 | H1115T | |||||
| 6.20% | 0.453 | H1115T | |||||
| 6.20% | 0.453 | H1115T | |||||
| 3.70% | 0.084 | H1115T | C1318Y | ||||
| 3.60% | 0.453 | H1115T | |||||
| 3.30% | 0.453 | H1115T | |||||
| 2.10% | 0.084 | H1115T | |||||
| 1.80% | 0.069 | H1115T | C1318Y | V1458A | |||
| 109DPI | 108737 | 35.80% | 0.223 | I1412V | H1115S | R1118H | |
| 19.10% | 0.384 | I1412L | H1115S | ||||
| 8.60% | 0.910 | I1412L | V1112A | H1115S | |||
| 7.70% | 0.360 | I1412L | V1112I | H1115S | |||
| 6.30% | 0.061 | I1412L | H1115G | ||||
| 5.70% | 0.247 | I1412L | H1115T | ||||
| 3.70% | 0.657 | I1412L | V1112T | H1115S | |||
| 2.60% | 0.145 | I1412L | V1112A | H1115T | |||
| 2.30% | 0.062 | I1412L | V1112I | H1115G | |||
| 2.20% | 0.229 | I1412L | V1112I | H1115T | |||
| 2.20% | 0.573 | I1412L | V1112A | H1115T | |||
| 2.10% | 0.624 | I1412V | H1115S | ||||
| 1.80% | 0.309 | I1412L | H1115A | ||||
| 198DPI | 681389 | 73.60% | 0.360 | I1412L | V1112I | H1115S | |
| 12.60% | 2.149 | I1412L* | V1112I | H1115S | A1593V | ||
| 4.70% | 1.683 | A1409T | V1112P | H1115S | |||
| 2.90% | 0.360 | I1412L | V1112I | H1115S | |||
| 1.80% | 0.360 | I1412L | V1112I | H1115S | |||
| 1.70% | 1.683 | A1409T | V1112P | H1115S | |||
| 1.60% | 0.066 | I1412L | V1112I | H1115S | M1268I | ||
| 1.20% | 0.360 | I1412L | V1112I | H1115S | |||
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*
Bold - indicate escape variants with fitness estimate ≥1.
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†
Only non-synonymous mutations are shown.
Table 4
Co-occurring mutations, epitope escape mutants, and the associated frequency of occurrence and relative fitness of NS3 region of subject THGS0684MX.
| Time | Viral load (IU/ml) | Frequency | Relative fitness | 1325SILGIGTAL1333Mutations | Co-occurring mutations* | |
|---|---|---|---|---|---|---|
| 2DPI | 140,200 | 87.9% | 1.000 | |||
| 4.8% | 0.148 | G1076W | ||||
| 3.2% | 1.000 | |||||
| 3% | 0.155 | G1076R | ||||
| 1.1% | 0.022 | G1076W | Y1521C | |||
| 58DPI | 19,932 | 74.2% | 1.000 | |||
| 10.8% | 1.000 | |||||
| 8.4% | 0.322 | |||||
| 5.5% | 0.162 | M1649T | ||||
| 1% | 0.322 | S1289G | ||||
| 184DPI | 221,964 | 90.9% | 1.042 | A1332V† | P1496S | |
| 9.1% | 0.157 | A1332V | D1316G | P1496S | ||
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*
Only non-synonymous mutations are shown.
-
†
Bold - indicate escape variants with fitness estimate ≥1.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | Lenti-X 293T Cell Line | Takara, Mountain View, CA, USA | Cat# 632180 | Cell line maintained in High Glucose Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum |
| Cell line (Homo sapiens) | Huh7.5 | Apath, New York, NY, USA | Cell line maintained in High Glucose Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum | |
| Transfected construct (Photinus pyralis) | pTG126 MLV luciferase | Prof. Francois-Loic Cosset; Bartosch et al., 2003 | Vector to produce HCVpp | |
| Transfected construct (murine leukemia virus) | phCMV-5349 MLV gag/pol | Prof. Francois-Loic Cosset | ||
| Peptide, recombinant protein | CD8 T-cell (9-10mers) | Mimotopes | ||
| Commercial assay or kit | Mammalian Calphos transfection kit | Macherey-Nagel | Cat#631312 | For HCVpp transfection |
| Software, algorithm | ShoRAH | ShoRAH | RRID:SCR_005211 | |
| Software, algorithm | LoFreq | LoFreq | RRID:SCR_013054 | |
| Software, algorithm | Geneious | Geneious | RRID:SCR_010519 | |
| Software, algorithm | QuasiRecomb | QuasiRecomb | RRID:SCR_008812 | |
| Software, algorithm | GraphPad | GraphPad | RRID:SCR_000306 | |
| Software, algorithm | Immune Epitope Database and Analysis Resource (IEDB) | Immune Epitope Database and Analysis Resource (IEDB) | RRID:SCR_006604 | |
| Software, algorithm | Python | Python | RRID:SCR_008394 | |
| Software, algorithm | RStudio | RStudio | RRID:SCR_000432 | |
| Software, algorithm | ShoRAH | ShoRAH | RRID:SCR_005211 | |
| Software, algorithm | LoFreq | LoFreq | RRID:SCR_013054 | |
| Software, algorithm | Geneious | Geneious | RRID:SCR_010519 | |
| Software, algorithm | QuasiRecomb | QuasiRecomb | RRID:SCR_008812 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/102232/elife-102232-mdarchecklist1-v1.pdf
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Supplementary file 1
Summary of epitope selection and (IFN-γ) ELISPOT assay responses in subjects who cleared infection.
- https://cdn.elifesciences.org/articles/102232/elife-102232-supp1-v1.docx
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Supplementary file 2
Summary of epitope selection and positive (IFN-γ) ELISPOT assay responses in subjects who developed chronic infection.
- https://cdn.elifesciences.org/articles/102232/elife-102232-supp2-v1.docx
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Supplementary file 3
Subject 300023 relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.
- https://cdn.elifesciences.org/articles/102232/elife-102232-supp3-v1.docx
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Supplementary file 4
Subject 300240 relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.
- https://cdn.elifesciences.org/articles/102232/elife-102232-supp4-v1.docx
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Supplementary file 5
Subject 300256 relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.
- https://cdn.elifesciences.org/articles/102232/elife-102232-supp5-v1.docx
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Supplementary file 6
Subject HOKD0485FX relative fitness estimate, co-occurring mutations and frequency of occurrence for each reconstructed haplotype.
- https://cdn.elifesciences.org/articles/102232/elife-102232-supp6-v1.docx
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Temporal dynamics of viral fitness and the adaptive immune response in HCV infection
eLife 13:RP102232.
https://doi.org/10.7554/eLife.102232.3
