Synergy of entry inhibitors with direct-acting antivirals uncovers novel combinations for prevention and treatment of hepatitis C

Abstract

Objective: Although direct-acting antiviral agents (DAAs) have markedly improved the outcome of treatment in chronic HCV infection, there continues to be an unmet medical need for improved therapies in difficult-to-treat patients as well as liver graft infection. Viral entry is a promising target for antiviral therapy.

Design: Aiming to explore the role of entry inhibitors for future clinical development, we investigated the antiviral efficacy and toxicity of entry inhibitors in combination with DAAs or other host-targeting agents (HTAs). Screening a large series of combinations of entry inhibitors with DAAs or other HTAs, we uncovered novel combinations of antivirals for prevention and treatment of HCV infection.

Results: Combinations of DAAs or HTAs and entry inhibitors including CD81-, scavenger receptor class B type I (SR-BI)- or claudin-1 (CLDN1)-specific antibodies or small-molecule inhibitors erlotinib and dasatinib were characterised by a marked and synergistic inhibition of HCV infection over a broad range of concentrations with undetectable toxicity in experimental designs for prevention and treatment both in cell culture models and in human liver-chimeric uPA/SCID mice.

Conclusions: Our results provide a rationale for the development of antiviral strategies combining entry inhibitors with DAAs or HTAs by taking advantage of synergy. The uncovered combinations provide perspectives for efficient strategies to prevent liver graft infection and novel interferon-free regimens.

Keywords: HCV; HEPATITIS C; LIVER.

Figure 1

Combination of direct-acting antivirals and entry inhibitors results in a synergistic activity in preventing HCV infection. Huh7.5.1 cells were preincubated for 1 h with serial concentrations of (A) telaprevir, boceprevir, simeprevir or danoprevir, (B) daclatasvir or (C) mericitabine or sofosbuvir and 0.01 µg/mL of receptor-specific (anti-CD81, anti-SR-BI or anti-CLDN1) mAbs or 0.1 µM protein kinase inhibitors (erlotinib or dasatinib) before incubation with HCVcc Luc-Jc1 in the presence of both compounds. HCVcc infection was analysed by luciferase activity as described. The CIs at IC₅₀, IC₇₅ and IC₉₀ are indicated in online supplementary table S1. Means±95% credible intervals from three independent experiments performed in triplicate are shown.

Figure 2

Antiviral synergy of entry and protease inhibitors. Combination (A) telaprevir and erlotinib, (B) boceprevir or (C) simeprevir and anti-CLDN1 mAb was performed as described in figure 1. (D–F) Synergy was confirmed using the Prichard and Shipman method.

Figure 3

Synergy of entry inhibitors and daclatasvir. Combination of daclatasvir and (A) anti-SR-BI mAb, (B) anti-CLDN1 mAb or (C) erlotinib was performed as described in figure 1. (D–F) Synergy was confirmed using the Prichard and Shipman method.

Figure 4

Entry inhibitors enhance the antiviral activity of sofosbuvir in a synergistic manner. Combination of sofosbuvir and (A) anti-SR-BI mAb, (B) anti-CLDN1 mAb, (D) dasatinib or (E) erlotinib was performed as described in figure 1. (C, F) Synergy was confirmed using the Prichard and Shipman method.

Figure 5

Synergy of host-targeting agents on HCV infection. (A) Combination of receptor-specific (anti-CD81, anti-SR-BI or anti-CLDN1) mAbs and protein kinase inhibitors (erlotinib or dasatinib) was performed as described in figure 1 and the CIs at IC₅₀, IC₇₅ and IC₉₀ are indicated in online supplementary table S2. Means±95% credible intervals from three independent experiments performed in triplicate are shown. (B) Combination of anti-CLDN1 mAb and dasatinib (left) or erlotinib (right). (C) Synergy was confirmed using the Prichard and Shipman method. (D) Combination of alisporivir and entry inhibitors was performed as described in figure 1 and the CIs at IC₅₀, IC₇₅ and IC₉₀ are indicated in online supplementary table S2. Means±95% credible intervals from three independent experiments performed in triplicate are shown. (E) Combination of alisporivir with erlotinib (left) or anti-CLDN1 mAb (right). (F) Synergy was confirmed using the Prichard and Shipman method.

Figure 6

Combination of direct-acting antivirals (DAAs), host-targeting agents and entry inhibitors results in synergy in treatment of established HCV infection. Huh7.5.1 cells were infected for 5 days with HCVcc prior to treatment with DAAs or alisporivir and entry inhibitors as described in ‘Materials and methods’. Synergy between (A) telaprevir and anti-CLDN1 mAb (left) or erlotinib (right), (B) daclatasvir and anti-SR-BI mAb (left) or anti-CLDN1 mAb (right), (C) sofosbuvir and anti-CLDN1 mAb (left) or erlotinib (right), (D) alisporivir and anti-SR-BI (left) or anti-CLDN1 mAb (right) was assessed using the Prichard and Shipman method.

Figure 7

Synergistic effect of combining direct-acting antivirals and entry inhibitors in treatment of HCV infection using persistently infected DMSO-differentiated hepatoma cells. HCV-infected cell culture was established by transfecting RNA encoding wild-type HCV Luc-Jc1 into Huh7.5.1 cells. The cells were then differentiated in the presence of 1% DMSO for 5 days before treatment with the combinations of (A) telaprevir+anti-CLDN1 mAb (left) or erlotinib (right), (B) daclatasvir+anti-SR-BI mAb (left) or anti-CLDN1 mAb (right), (C) sofosbuvir+anti-CLDN1 mAb (left) or erlotinib (right), (D) simeprevir+erlotinib (left) or anti-CLDN1 mAb (right). HCVcc infection was assessed by luciferase activity 5 days after treatment. Synergy was assessed using the Prichard and Shipman method.

Figure 8

Entry inhibitors limit viral rebound from direct-acting antiviral therapy. Persistently HCV (Jc1) infected and DMSO-differentiated cells described in ‘Materials and methods’ were first treated with 500 nM simeprevir for 8 days followed by incubation with either 1% DMSO control medium (mock), 10 µg/mL anti-CD81 mAb or 10 µM erlotinib sequentially for another 12 days. Cells maintained in 1% DMSO throughout the experiment were used as control (CTRL). Viral load was assessed by HCV-specific RT-PCR every 4 days. The limit of quantification, indicated by a dashed line, was 10³ copies/mL. Means±SD from a representative experiment performed in triplicate are shown. An asterisk indicates one out of three samples was HCV RNA negative; two asterisks indicate two samples were HCV RNA negative.

Figure 9

Effect of combination of an entry inhibitor and direct-acting antiviral on HCV infection in vivo. Human liver-chimeric uPA/SCID mice engrafted with primary human hepatocyte were inoculated with HCVcc (Jc1) 8 weeks before the experiment as described in ‘Materials and methods’. Nine mice were grouped randomly into control group (two mice), telaprevir monotherapy group (two mice), scavenger receptor B type I (SR-BI)-specific mAb (NK8-H5-E3) monotherapy group (two mice) and combination therapy group (three mice). Mice received telaprevir monotherapy (300 mg/kg PO BID), anti-SR-BI mAb monotherapy (500 µg IP twice a week), combination treatment of telaprevir and anti-SR-BI mAb or control treatments as described in ‘Materials and methods’. Indicated by an asterisk, two mice from the group treated with combination of telaprevir and anti-SR-BI mAb had to be sacrificed before the end of the experiment due to bleeding and injury. (A) HCV RNA from mouse sera was measured by RT-PCR every 5–10 days. Viral load of each individual mouse is shown. (B) Human albumin (HA) concentration in the blood of human liver-chimeric uPA/SCID mice. HA was measured during the treatment as described in ‘Materials and methods’.