Soluble Inflammatory Mediators Identify HCV Patients Who May Be Cured With Four Weeks of Antiviral Treatment

Tanvi Khera; Lone Wulff Madsen; Yanqin Du; Søren Thue Lillevang; Peer Brehm Christensen; Heiner Wedemeyer

Disclosures

J Viral Hepat. 2022;29(6):447-454. 

In This Article

Abstract and Introduction

Abstract

Soluble inflammatory mediators (SIM) can be predictive of treatment outcome in antiviral treatment of chronic hepatitis C. Recently, it was shown that a subgroup of patients can be cured with four weeks of therapy. We here profiled patients for 70 SIM before and during treatment of hepatitis C with glecaprevir/pibrentasvir (GLE/PIB) +/− ribavirin. Proximity extension assay was performed in a total of 32 patients. Pre-treatment SIM profiles did not distinguish patients achieving an SVR (n = 21) from patients experiencing antiviral relapse (n = 11). However, after 4 weeks of therapy, eight markers were identified that could distinguish patients with SVR from the relapsed group, namely MMP-10, CCL20, CXCL11, FGF-23, TNF, MCP-2, IL-18R1 and CXCL10. Thus, this study shows that a distinct on-treatment immune profile is associated with cure of HCV infection after ultrashort treatment.

Introduction

The estimated global prevalence of chronic hepatitis C is about 1%.[1] In the absence of a prophylactic vaccine for hepatitis C virus (HCV) infection, direct acting antivirals (DAA) have played a major role in revolutionizing HCV therapy with an overall effectiveness of 98%. International guidelines for treatment of chronic HCV recommend 8–12 weeks of treatment with DAA. However, emerging evidence suggests that short duration treatment regimens given for 6 weeks or less can be sufficient for distinct subgroups of patients with chronic hepatitis C.[2–4] Hence, shortening therapy could improve real world access especially among active drug users where compliance is a major concern. Nonetheless, the target population for short duration treatment is still not clear and determining which patients could benefit from short duration treatment would be a significant progress. Soluble mediators together with cellular components play an essential role in regulating the immune network. It is well established that persistent infection with HCV causes profound alterations in the inflammatory milieu and limited data suggests that these imprints are not completely restored after clearance.[5,6] Soluble inflammatory mediators (SIM) could serve as biomarkers for prediction of disease severity or treatment outcome. We have previously shown that based on the expression of SIMs, patients with SVR clearly differed from the ones that relapsed before the start of DAA therapy in chronic hepatitis C patients.[5]

The overall aim of this study was to analyse whether distinct SIMs differ between patients who achieve SVR and patients with treatment failure for patients who were treated with 4 weeks of glecaprevir/pibrentasvir with or without ribavirin.[7,8] The underlying hypothesis was that SIMs as predictive biomarkers could help to distinguish the SVR from the relapsed group after an ultrashort course of antiviral therapy.

Study Population

In this study, a total of 32 patients were evaluated. The patients were randomized to glecaprevir/pibrentasvir (GLE/PIB) +/− ribavirin for four weeks. Study details have recently been reported.[8] In total, 17 received GLE/PIB and 15 patients received GLE/PIB plus ribavirin. Overall, 21 patients achieved SVR and 11 experienced treatment failure. Blood plasma was collected from (EDTA) anticoagulated peripheral blood samples, taken at two distinct time points at baseline (BL) and by the end of treatment (EOT) and stored at −20°C until analysis.

Protein Analysis

Plasma samples were thawed, and 20μl of each sample was analysed by Olink where proximity extension analysis (PEA, Olink AB) was performed. A pre-defined panel simultaneously measuring 92 inflammation-related proteins in plasma was used (Inflammation panel). The panel included cytokines, chemokines, growth factors and ligands according to established SOPs.[6,9] For more information regarding limit of detection, reproducibility, and validation, see the supplementary materials and methods section. Of these, 70 markers showed expression above limit of detection and did pass the internal quality control for the assay (Table 2). Briefly, in PEA, a pair of oligonucleotide-labelled antibodies, Proseek probes, bind to the target protein in the plasma sample. When the 2 Proseek probes are in close proximity, a new polymerase chain reaction target sequence is formed by a proximity-dependent DNA polymerization event. This complex is subsequently detected and quantified using standard real-time qPCR. The generated normalized protein expression unit is on a log2 scale, where a larger number represents a higher protein level in the sample. The quantification cycle (Cq) values from a DNA extension control are subtracted from the measurement of the Cq value, an interpolate control, is corrected for and finally a correction factor is subtracted to yield a normalized protein expression (NPX) value which is log2 transformed. For more information regarding the limit of detection, reproducibility, and validation, please see the manufacturer's homepage (www.olink.com).

Statistics

Data were analysed using Graph Pad Prism v8 (Graph Pad Software,). In general, quantitative comparisons were performed using parametric student t test for normally distributed values. Principal component analysis (PCA) was performed using Qlucore Omics Explorer v3.2 (Qlucore, Lund, Sweden). Repetitive t testing was used in PCA to compare groups to each other. For analysis, p values were set to 0.05 and a Q value of <0.2. Correction for multiple t testing was performed using the false discovery rate (FDR) approach using the Benjamini–Hochberg method.

Ethics

Patients gave informed written consent for the study. The study was approved by The Danish health Medicines Authorities, Eudra CT no: 2017–005179–21, and the Regional Committees on Health Research Ethics for Southern Denmark ID S-20180013.

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