Dynamic Changes of Post-Translationally Modified Forms of CXCL10 and Soluble DPP4 in HCV Subjects Receiving Interferon-Free Therapy

Abstract

Serum levels of the interferon (IFN)-stimulated chemokine CXCL10 are increased during chronic HCV infection and associate with outcome of IFN-based therapy. Elevated levels of NH2-terminal truncated CXCL10 (3-77aa), produced by DPP4 cleavage, negatively associate with spontaneous clearance of acute HCV infection and sustained virological response (SVR) with IFN-based therapy for chronic infection. The association of different CXCL10 forms and DPP4 with outcome during IFN-free HCV therapy has not been examined. Using novel Simoa assays, plasma was analyzed from HCV genotype-1 (GT1) subjects who relapsed (n = 11) or achieved SVR (n = 10) after sofosbuvir and ribavirin (SOF/RBV) treatment, and from SOF/RBV relapsers who achieved SVR with a subsequent SOF/ledipasvir regimen (n = 9). While the NH2-truncated form of CXCL10 was elevated in HCV infection relative to healthy controls, pre-treatment plasma concentrations of CXCL10 forms failed to stratify subjects based on treatment outcome to IFN-free regimens. However, a trend (statistically non-significant) towards elevated higher levels of total and long CXCL10 was observed pre-treatment in subjects who relapsed. All forms of CXCL10 decreased rapidly following treatment initiation and were again elevated in subjects who experienced HCV relapse, indicating that CXCL10 production may be associated with active viral replication. While soluble DPP4 (sDPP4) and NH2-truncated CXCL10 concentrations were highly correlated, on-treatment sDPP4 levels and activity declined more slowly than CXCL10, suggesting differential regulation. These data suggest post-translationally modified forms of CXCL10 will not support the prediction of treatment outcome in HCV GT1 subjects treated with SOF/RBV.

Fig 1. CXCL10 forms do not differ pre-treatment in patients who achieve SVR vs. relapse after SOF/RBV treatment.

Plasma collected pre-treatment from 21 subjects (SVR = 10, relapse = 11) treated with SOF/RBV in the SPARE trial and 8 healthy controls was analyzed for total, long, and short forms of CXCL10 using Simoa. Analysis is by Kruskal-Wallis with a multiple test correction. Shown are individual values and medians. NS = not significant (p>0.05), SVR = sustained virologic response, Rel = relapser.

Fig 2. CXCL10 forms decline rapidly during IFN-free therapy with SOF/RBV.

Plasma collected pre-treatment, day 6–11 of treatment, and at week 20 from 21 subjects (SVR = 10, relapse = 11) treated with SOF/RBV in the SPARE trial was analyzed for total, long, and short forms of CXCL10. For SPARE relapsers, 9 subjects who were treated with SOF/ledipasvir (SYNERGY trial) were assayed pre-treatment and at week 8. Longitudinal changes in total (A), long (B), and short (C) forms are displayed for subjects achieving SVR (black circles) and for relapsers (red circles). Statistical analysis is by Kruskal-Wallis with a multiple test correction considering data from all subjects (SPARE data before dotted line) or Wilcoxon paired test (SYNERGY data after dotted line). Assays were run in technical triplicates. Shown are individual values and medians.

Fig 3. sDPP4 activity and levels decline during treatment with slower kinetics than CXCL10 forms.

sDPP4 levels (A) and activity (B) were determined longitudinally on samples from subjects as described in Fig 2. Data from subjects achieving SVR (black circles) and those who relapsed (red circles) on SPARE are shown. Statistical analysis is by Kruskal-Wallis with a multiple test correction considering data from all subjects (SPARE data before dotted line) or Wilcoxon paired test (SYNERGY data after dotted line). Assays were run in technical triplicates. Shown are individual values and medians.

Fig 4. Total and short CXCL10, but not long CXCL10, correlate with sDPP4 activity.

(A) Correlation between DPP4 levels and activity for all subjects and all time points tested (n = 80). (B-D) Correlation of total, long, and short CXCL10 forms with DPP4 activity. For long CXCL10, only data from samples with detectable long CXCL10 (above LOD of 1 pg/ml) were used for analysis (n = 26). Spearman correlation (r_s) was determined for each analysis.