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Case Reports
. 2015 May;89(10):5478-90.
doi: 10.1128/JVI.03717-14. Epub 2015 Mar 4.

Transmitted/Founder Viruses Rapidly Escape from CD8+ T Cell Responses in Acute Hepatitis C Virus Infection

Rowena A Bull  1 , Preston Leung  1 , Silvana Gaudieri  2 , Pooja Deshpande  3 , Barbara Cameron  1 , Melanie Walker  1 , Abha Chopra  4 , Andrew R Lloyd  1 , Fabio Luciani  5
Affiliations
Case Reports

Transmitted/Founder Viruses Rapidly Escape from CD8+ T Cell Responses in Acute Hepatitis C Virus Infection

Rowena A Bull et al. J Virol. 2015 May.

Abstract

The interaction between hepatitis C virus (HCV) and cellular immune responses during very early infection is critical for disease outcome. To date, the impact of antigen-specific cellular immune responses on the evolution of the viral population establishing infection and on potential escape has not been studied. Understanding these early host-virus dynamics is important for the development of a preventative vaccine. Three subjects who were followed longitudinally from the detection of viremia preseroconversion until disease outcome were analyzed. The evolution of transmitted/founder (T/F) viruses was undertaken using deep sequencing. CD8(+) T cell responses were measured via enzyme-linked immunosorbent spot (ELISpot) assay using HLA class I-restricted T/F epitopes. T/F viruses were rapidly extinguished in all subjects associated with either viral clearance (n = 1) or replacement with viral variants leading to establishment of chronic infection (n = 2). CD8(+) T cell responses against 11 T/F epitopes were detectable by 33 to 44 days postinfection, and 5 of these epitopes had not previously been reported. These responses declined rapidly in those who became chronically infected and were maintained in the subject who cleared infection. Higher-magnitude CD8(+) T cell responses were associated with rapid development of immune escape variants at a rate of up to 0.1 per day. Rapid escape from CD8(+) T cell responses has been quantified for the first time in the early phase of primary HCV infection. These rapid escape dynamics were associated with higher-magnitude CD8(+) T cell responses. These findings raise questions regarding optimal selection of immunogens for HCV vaccine development and suggest that detailed analysis of individual epitopes may be required.

Importance: A major limitation in our detailed understanding of the role of immune response in HCV clearance has been the lack of data on very early primary infection when the transmitted viral variants successfully establish the acute infection. This study was made possible through the availability of specimens from a unique cohort of asymptomatic primary infection cases in whom the first available viremic samples were collected approximately 3 weeks postinfection and at regular intervals thereafter. The study included detailed examination of both the evolution of the viral population and the host cellular immune responses against the T/F viruses. The findings here provide the first evidence of host cellular responses targeting T/F variants and imposing a strong selective force toward viral escape. The results of this study provide useful insight on how virus escapes the host response and consequently on future analysis of vaccine-induced immunity.

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Figures

FIG 1
FIG 1
Longitudinal course of viral load and antibody levels. Two subjects were chronic progressors (Ch_23 and Ch_240), and one cleared the infection (Cl_360). Gray shading indicates viral load, with open circles indicating viremic time points and filled circles indicating time points from which viral samples were deep sequenced. Black lines indicate antibody level, with black squares indicating data points. OD, optical density.
FIG 2
FIG 2
CD8+ T cell ELISpot responses against T/F variants. Each panel shows the time course of viral load (shaded gray), and ELISpot responses against T/F epitopes (colored lines). Legends indicate the epitope sequence with corresponding color, and red letters indicate mutated residues that become fixed during the course of the infection. The earliest CD8+ responses were detected preseroconversion (antibody positive [Ab +ve], vertical dotted line). The decline in viremia preceded the peak of HCV-specific CD8+ T cell responses. In the two chronic progressors (Ch_23 and Ch_240), recrudescent viremia coincided with a decline and temporary loss of detectable HCV-specific CD8+ T cell responses around 100 to 150 days p.i. In a subject that cleared infection (Cl_360), the T/F-specific CD8+ T cell responses remained stable after viral clearance.
FIG 3
FIG 3
CD8+ T cell escape from T/F virus in chronic subject Ch_23. (A) Viral load (shaded gray) and CD8+ T cell response measured via ELISpot assay against epitope 852RAEAQLHAW860. No immune responses were detected against mutants 852RTEAQLHAW860 and 852RAEAHLHAW860 (only one time point was tested), with the latter reaching fixation at 197 days p.i. A detailed list of evolving epitope variants along with the time of detection and frequency of occurrence (%) in the viral population is reported. (B) Viral load (shaded gray) and CD8+ T cell responses measured via ELISpot assay against epitope 2629KSKRTPMGF2637 and escape variant 2629NSKRTPMGF2637. A detailed list of evolving epitope variants is reported below. The high-resolution analysis of these epitope regions indicated significant residue toggling across the epitope at low frequency before preferential selection of a variant that coincided with recrudescent viremia. The color coding of the residues in the text correlates with the epitope variant colors in the graphs. DPI, days p.i.
FIG 4
FIG 4
CD8+ T cell escape from T/F virus in chronic subject Ch_240. (A) Viral load (shaded gray) and CD8+ T cell response measured via ELISpot assay against epitope 1633RLGPVQNEV1641. A moderate immune response was detected against escape variant 1633RLGPVQNEI1641 at 310 days p.i. (no further time points were tested), which reached fixation at 538 days p.i. A detailed list of evolving epitope variants along with the time of detection and frequency of occurrence (%) in the viral population is reported. (B) Viral load (shaded gray) and CD8+ T cell response against epitope 1602RAQAPPPSW1610. No immune responses were tested against escape variants. The variant 1602RAQALPPSW1610 rapidly reached fixation at 140 days p.i. A detailed list of evolving epitope variants is reported below. The high-resolution analysis of these epitope regions indicated significant residue toggling across the epitope at low frequency before preferential selection of a variant that coincided with recrudescent viremia. The color coding of the residues in the text correlates with the epitope variant colors in the graphs.
FIG 5
FIG 5
The rate of immune escape from T/F-specific CD8+ T cell responses. (A) In subject Ch_23, two T/F virus epitopes (in the legend) underwent escape mutations, the first (K2629N in black) at a rate of 0.122 per day and the second (Q856H in red) at a rate of 0.072 per day. (B) In subject Ch_240 two epitopes underwent escape mutations, the first (P1606L in black) at a rate of 0.102 per day and the second (V1641I in red) at a rate of 0.026 per day. Symbols represent the cumulative frequency of the escape variants within the epitope sequence. The lines represents the fit of the data with a mathematical model describing the escape dynamics under CD8+ T cell immune pressure (38).
FIG 6
FIG 6
Magnitude of CD8+ T cell response associated with escape. The CD8+ T cell responses measured via ELISpot assay for each T/F epitope during the first 101 days p.i. was compared between epitopes that underwent escape (n = 2 in Ch_23 and n = 2 in Ch_240, for a total of 8 immune responses) and those that did not (n = 9). The conserved epitopes were also further divided based on disease outcome. The epitopes that elicited greater IFN-γ ELISpot responses were more likely to have escape variants emerge (P = 0.03). Statistical comparisons are Mann-Whitney tests. Scatter plots represent means ± 1 standard deviation.
FIG 7
FIG 7
Mean Shannon entropy (SE) per site in epitope regions (E) and nonepitope regions (NE) calculated over time for the three subjects. Following a decline in the Shannon entropy per site within the first 100 days p.i. for all three subjects, the diversity index increased in the chronic phase of the infection. The mean Shannon entropy per site was generally higher within the epitope regions.
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