Rapid decrease in hepatitis C viremia by direct acting antivirals improves the natural killer cell response to IFNα

Abstract

Objective: Chronic HCV infection is characterised by innate immune activation with increased interferon-stimulated genes (ISG) expression and by an altered phenotype of interferon-responsive natural killer (NK) cells. Here, we asked whether a rapid reduction in viremia by daclatasvir (DCV) and asunaprevir (ASV) improves the response to pegylated interferon (PegIFN) in patients who had previously failed a standard course of PegIFN/ribavirin (RBV) therapy.

Design: Twenty-two HCV-infected non-responders to previous PegIFN/RBV therapy were studied for IFN-responsiveness of NK cells during quadruple (QUAD) therapy with DCV, ASV, PegIFN and RBV. A direct comparison of early NK cell responses in PegIFN/RBV therapy and QUAD therapy was performed for seven patients using paired cryopreserved peripheral blood mononuclear cells (PBMC) from both treatment courses. As a validation cohort, nine DCV/ASV-treated patients were studied for their NK cell response to in vitro stimulation with IFNα.

Results: The 24 h virological response to QUAD therapy correlated with an increase in signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (pSTAT1) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in NK cells, and the STAT1/pSTAT1/TRAIL induction was greater during QUAD therapy than during previous PegIFN/RBV therapy. Successful QUAD therapy as well as successful IFN-free DCV/ASV regimen resulted in an improved functional NK cell response (degranulation and TRAIL expression) to in vitro stimulation with IFNα.

Conclusions: IFN-responsiveness can be improved by inhibiting HCV replication and reducing the HCV-induced activation of the innate immune response. This may provide a rationale for clinical trials of a brief period of direct acting antiviral therapy followed by PegIFN/RBV therapy to reduce the overall treatment costs in low-income and middle-income countries.

Trial registration numbers: NCT01888900 and NCT00718172.

Keywords: HEPATITIS C; IMMUNE RESPONSE; IMMUNOLOGY; IMMUNOLOGY IN HEPATOLOGY.

Conflict of interest statement

COMPETING INTERESTS

The authors do not have a commercial or other association that might pose a conflict of interest.

Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Figures

Figure 1.. The rapid decrease in viremia during QUAD therapy is associated with a decrease in intrahepatic inflammation.

(A) Serum HCV RNA levels at the time of the pretreatment liver biopsy (pre) and at the start (day 0) and during the first 4 weeks of QUAD therapy. Filled squares represent data from patients who developed a virological breakthrough at week 8 (n=1) or relapsed after the end of therapy (n=1). L.l.o.q., lower level of quantitation; t.n.d., target not detected. (B) Serum ALT levels prior to and at week 4 of QUAD therapy. (C-D) CXCL10 protein concentration in the serum (C) and CXCL10 relative mRNA level in the liver (D) prior to and at week 4 of therapy. ‘Pre’ indicates the time point of the pre-treatment liver biopsy (up to 4 weeks prior to therapy). Statistical analysis: non-parametric paired Wilcoxon-signed-rank test.

Figure 2.. Detection of STAT1, TRAIL and pSTAT1-expressing NK cells of uninfected subjects and HCV-infected patients by flow cytometry.

(A)Flow cytometry gating strategy. Dot plots from left to right show gating on single cells in forward scatter (FSC) area versus FSC height plots, gating on lymphocytes in FSC versus side scatter (SSC) plots, exclusion of EMA+ dead cells, CD14+ monocytes and CD19+ B cells, and gating on CD56+CD3- negative NK cells. Percentages indicate the frequency of events in the red gate relative to total number of events in the respective plot. (B)Frequency of STAT1+ cells in the peripheral blood CD3-CD56+ NK cell population of a representative uninfected subject and a representative HCV-infected patient on day 0 and day 1 of QUAD therapy. FMO, fluorescence minus one control using all antibodies in the panel except for the anti-STAT1 A647 antibody to define the threshold of positivity. A647, alexa647. (C)Frequency of TRAIL+ cells in the peripheral blood CD3-CD56+ NK cell population of a representative uninfected subject and a representative HCV-infected patient on day 0 and day 1 of QUAD therapy. FMO, fluorescence minus one control using all antibodies in the panel except for the anti-TRAIL PE antibody to define the threshold of positivity. PE, phycoerythrin. (D)Frequency of pSTAT1+ cells in the peripheral blood CD3-CD56+ NK cell population of a representative uninfected subject and a representative HCV-infected patient on day 0 and day 1 of QUAD therapy. FMO, fluorescence minus one control using all antibodies in the panel except for the anti-pSTAT1 A488 antibody to define the threshold of positivity. A488, alexa488. Note that the CD56 dot plots in panels B/D (co-staining for STAT1/pSTAT1) differ from those in panel C (staining for TRAIL) because two different CD56 antibodies with different fluorochromes were used, and because of differential processing of the PBMC (fixation, permeabilisation and intranuclear staining for panels B/D; cell surface staining in panel C).

Figure 3.. NK cells express increased STAT1, TRAIL and pSTAT1 levels in HCV infection with further increase during QUAD therapy.

Frequency of (A) STAT1+, (B) TRAIL+ and (C) pSTAT1+ NK cells and mean fluorescence intensity (MFI) of these markers per NK cell prior to (pre, day 0) and during (day 1, week 4) of QUAD therapy (filled circles). Immunological read-outs are shown for the first 4 weeks of QUAD therapy, because HCV is already undetectable at week 4, and values of the immunological read-outs plateau with continued weekly PegIFN injections. NK cells of uninfected subjects are shown for comparison (open squares). Statistical analysis: non-parametric paired Wilcoxon-signed-rank test or unpaired Mann-Whitney test. Median and IQR are shown.

Figure 4.. The first-phase virological response correlates with the increase in STAT1 and TRAIL levels in NK cells during QUAD therapy.

(A, B) Linear regression analysis of the decrease in viremia during the first 6h of QUAD therapy and the 24h-increase in STAT1 (A) and TRAIL (B) mean fluorescence intensity (MFI) on NK cells. (C, D) Linear regression analysis of the decrease in viremia during the first 24h of QUAD therapy and the increase in STAT1 (C) and TRAIL (D) MFI on NK cells during the same period. R2: Coefficient of determination. Filled squares represent data from patients who developed a virological breakthrough at week 8 (n=1) or relapsed after the end of therapy (n=1).

Figure 5.. IFN-induced STAT1 and pSTAT expression in NK cells is greater during QUAD therapy than during PegIFN/RBV therapy.

(A)Comparison of the decrease in HCV RNA levels during the first 24 h of therapy in 7 patients who were non-responders to a full course of PegIFN/RBV therapy but had an SVR during a subsequent course of QUAD therapy. (B, C) Comparison of changes in the expression level of STAT1+ (B) and pSTAT1 (C) NK cells during the first 24 h of therapy in patients who were non-responders to a full course of PegIFN/RBV therapy but had an SVR during a subsequent course of QUAD therapy. (D) Comparison of changes in the percentage of pSTAT1+ NK cells during the first 24 h of therapy in patients who were non-responders to a full course of PegIFN/RBV therapy but had an SVR during a subsequent course of QUAD therapy. Statistical analysis: non-parametric paired Wilcoxon-signed-rank test. Median and IQR are shown in the left graphs in panels A-D.

Figure 6.. IFN-induced TRAIL expression in NK cells is greater during QUAD therapy than during PegIFN/RBV therapy.

(A, B) Comparison of changes in the expression level (A) and frequency (B) of TRAIL+ NK cells during the first 24 h of therapy in patients who were non-responders to a full course of PegIFN/RBV therapy but had an SVR during a subsequent course of QUAD therapy. (A, B) Comparison of changes in the expression level (A) and frequency (B) of TRAIL+ CD56dim NK cells during the first 24 h of therapy in patients who were non-responders to a full course of PegIFN/RBV therapy but had an SVR during a subsequent course of QUAD therapy. Statistical analysis: non-parametric paired Wilcoxon-signed-rank test. Median and IQR are shown in the left graphs in panels A-D.

Figure 7.. Successful QUAD therapy normalizes NK cell response to in vitro IFNα stimulation for at least 24 weeks after the end of therapy.

(A, B) The ex vivo frequency of CD107a+ total NK cells (A) and CD107a+ CD56dim NK cells (B) was determined in uninfected controls and in HCV patients prior to and at week 24 after the end of QUAD therapy. Data in the left graphs in panels A and B are normally distributed. Mean and SEM are indicated and parametric paired and unpaired t-tests were used. Data in the right graphs in panels A and B are not normally distributed. Median and IQR are indicated and non-parametric paired Wilcoxon-signed-rank test and Mann Whitney unpaired t-test are used. (C) Linear regression analysis of the decrease in the frequency of CD107a+ CD56dim NK cells from prior to QUAD therapy to week 24 after the end of QUAD therapy and the strength of the NK cell response (CD107a expression) to in vitro stimulation with IFNα at week 24 after the end of QUAD therapy. R2: Coefficient of determination. One HCV patient was excluded from the analysis in figure 7, because the sole pretreatment blood sample of this patient that was available for the CD107 assays was obtained more than 2 years prior to QUAD therapy.

Figure 8.. IFN-free therapy with DCV/ASV improves IFNα-signaling in NK cells.

(A) Decrease in HCV RNA levels during the first 24h of IFN-free DCV/ASV therapy in 8 patients with an SVR. (B, C) Fold-increase in the frequency (C) and mean fluorescence intensity (D) of pSTAT1+ NK cells in response to in vitro stimulation of PBMC with IFNα. The in vitro response to IFNα was assessed at day 0 and day 1 of DCV/ASV therapy. Statistical analysis: non-parametric paired Wilcoxon-signed-rank test. (D, E) Fold-increase in the frequency (D) and mean fluorescence intensity (E) of pSTAT1+ NK cells in response to in vitro stimulation of PBMC with IFNα. The in vitro response to IFNα was assessed at day 0 and week 24 of DCV/ASV therapy. Statistical analysis: non-parametric paired Wilcoxon-signed-rank test.

Figure 9.. IFN-free therapy with DCV/ASV improves the effector response of NK cells.

PBMC were stimulated in vitro with the indicated concentration of IFNα and the percentage of TRAIL+ cells in the CD56bright NK cell population was determined. For each of the indicated patients (patient numbers refer to Table 1), the NK cell response in PBMC sample prior to DCV/ASV therapy (open circles, dotted line) was compared to the NK cell response in a PBMC sample at the end of successful DCV/ASV therapy (filled circles, continuous line). EC50, IFNα concentration resulting in 50% of the maximal TRAIL response of CD56bright NK cells.