Signatures of protective memory immune responses during hepatitis C virus reinfection

Abstract

Background & aims: Development of a vaccine against hepatitis C virus (HCV) has been hindered by our limited understanding of immune correlates of protection during real-life exposure to the virus. We studied the immune response during HCV reinfection.

Methods: We analyzed blood samples from participants in the Montreal Acute Hepatitis C Injection Drug User Cohort Study who were reinfected with HCV from 2009 to 2012. Five patients spontaneously resolved their second infection and 4 developed chronic infections. We monitored the phenotypic and functional dynamics of HCV-specific memory T cell responses in all subjects during natural re-exposure and re-infection.

Results: Populations of CD4(+) and CD8(+) T cells with HCV-specific polyfunctional memory were expanded in all 5 individuals who resolved 2 successive HCV infections. We detected CD127(hi) HCV-specific memory CD8(+) T cells before reinfection regardless of a subject's ability to clear subsequent infections. Protection against viral persistence was associated with the expansion of a CD127(neg), PD1(lo) effector memory T cells at the peak of the response. We also observed broadening of T-cell response, indicating generation of de novo T-cell responses. The 4 individuals who failed to clear their subsequent infection had limited expansion of HCV-specific CD4(+) and CD8(+) memory T cells and expressed variable levels of the exhaustion marker PD1 on HCV-specific CD8(+) T cells. Dominant epitope regions of HCV strains isolated from patients with persistent reinfection had sequence variations that were not recognized by the pre-existing memory T cells.

Conclusions: Protection from persistent HCV reinfection depends on the magnitude, breadth, and quality of the HCV-specific memory T-cell response. Sequence homology among viruses and ability of T cells to recognize multiple strains of HCV are critical determinants of protective memory.

Keywords: Cytokines; Immune Regulation; Protective Immunity; Viral Infection.

Figure 1. Higher magnitude and breadth of the HCV-specific T cell response in the SR/SR as compared to SR/CI patients

PBMCs from the indicated time points were tested in an IFNγ ELISPOT assay against overlapping peptide pools representing the HCV (H77) polyprotein. The frequencies of IFNγ spot forming cells (SFC) per million PBMCs from four SR/SR patients (A) and four SR/CI patients (B) are shown. The dashed lines delineate the different infection episodes. (C) Frequency of cells targeting the structural versus the non-structural proteins at the earliest time point (mean 8 weeks) during reinfection in the SR/SR (open symbols) and the SR/CI (solid symbols) patients.

Figure 2. Higher proliferative capacity of HCV-specific CD4 and CD8 T cells from SR/SR patients as compared to SR/CI patients

Proliferation of HCV-specific T cells in a 6-day CFSE proliferation assay against overlapping peptide pools spanning the HCV proteins indicated. Representative proliferation data for CD4 T cells from two SR/SR (A) and two SR/CI (B) patients. Representative proliferation data for CD8 T cells from two SR/SR (C) and two SR/CI (D) patients. Proliferating cells were identified as percent viable CFSE^lo, CD4+ or CD8+, CD3+ T cells and presented as stimulation index (SI).

Figure 3. Higher production of cytokines by HCV-specific CD4 T cells from SR/SR patients as compared to SR/CI patients during reinfection

PBMCs were stimulated with overlapping peptide pools representing HCV (NS3 and NS5B) and pools showing the highest response in the IFNγ ELISPOT then ICS was performed to measure the production of IFNγ, IL-2 and TNFα. Representative results from two SR/SR patients (A, B) and two SR/CI patients (C and D), gated on CD4+ CD3+ viable T lymphocytes. Peptide pools are indicated between brackets and represent the following regions: NS3-1 (NS3 aa: 1016–1341), NS5B-1 (NS5B; aa: 2416–2720) and NS5B-2 (NS5B; aa: 2710–3014). For polyfunctionality, orange represents triple functional cells, green represents double functional and yellow represents monofunctional cells of any combination.

Figure 4. Higher polyfunctionality of HCV-specific CD8 T cells from SR/SR patients in comparison to SR/CI patients during reinfection

PBMCs were stimulated overnight with overlapping peptide pools representing HCV (NS3 and NS5B) and HCV pools showing the highest response in the IFNγ ELISPOT. ICS was performed to measure the production of IFNγ, IL-2 and TNFα and expression of CD107a. Representative results from two SR/SR patients (A, B) and two SR/CI patients (C and D), gated on CD8+ CD3+ viable T lymphocytes. Peptide pools are indicated between brackets and represent the following regions: NS3-1 (NS3 aa: 1016–1341), NS5A-2 (NS5A; aa: 2192–2426) and NS5B-1 (NS5B; aa: 2416–2720). For polyfunctionality, orange represents triple functional cells, green represents double functional and yellow represents monofunctional cells of any combination.

Figure 5. Higher polyfunctionality of HCV-specific CD8 T cells targeting minimal epitopes in SR/SR patients in comparison to SR/CI patients during reinfection

PBMCs from the indicated time points were stimulated overnight with the minimum cognate peptide and ICS/CD107a staining was performed. Representative results from two SR/SR patients (A B) and two SR/CI patients (C and D), gated on viable CD3+CD8+ T lymphocytes. Peptides used are: 1073 = A2 restricted NS3-1073 and 2594 = A2 restricted NS5B-2594.

Figure 6. Expansion of HCV-specific tetramer+ CD8 T cells upon reinfection and generation of a CD127^lo effector T cell population in SR/SR patients

HCV tetramer staining and phenotyping were performed directly ex vivo on PBMCs from the indicated time points. Representative dot plots for longitudinal tetramer staining (upper panels) and phenotyping (lower panels) for (A) patient SR/SR-1 with A2/NS3-1073 tetramer and (B) patient SR/CI-1 with A1/NS3-1436 tetramer. Top panels are gated on viable CD3+ T lymphocytes. Lower panels are gated on viable CD3+CD8+ T lymphocytes. Tetramer frequency is represented as percent Tetramer+ CD8+ CD3+ T cells. In lower panels, black dots represent the tetramer+ HCV-specific CD8+ T cells overlaid on grey contour plots of CD8+ CD3+ T cells. Numbers in each quadrant represent percent expression on tetramer+ CD8+ CD3+ T cells. The dashed lines delineate the different infection episodes. (C–E) Tetramer expansion and phenotyping data from all SR/SR and SR/CI patients; (C) Frequency of HCV tetramer+ CD8+ CD3+ T cells, (D) Frequency of CD127+ tetramer+ CD8+CD3+ T cells, (E) Frequency of PD1+ tetramer+ CD8+CD3+ T cells.

Figure 7. Sequence variation in targeted epitope and reduced recognition by CD8 T cells in SR/CI patients

(A) Sequences of the epitopes targeted by tetramer+ CD8 T cells in SR/CI patients at early and late time points during reinfection. (B) Responses of total PBMCs collected during reinfection in a dose-response IFN-γ ELISPOT assay to the reference sequences versus variant autologous sequences for two SR/SR patients in response to the NS3-1073 epitope (left panels), two SR/CI patients in response to the NS3-1073 epitope (middle panels) and two SR/CI patients in response to the NS3-1436 epitope (right panels).