Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection

Abstract

Toll-like receptors (TLRs) are key mediators of innate immunity, and their activation by microbial components leads to the production of cytokines and interferons. Recombinant alpha interferon has been used to treat several viral diseases and is the current standard of care for hepatitis C virus (HCV) infection. Recently, agonists of TLR7 and TLR9 have been shown to have clinical efficacy in HCV patients, and this is correlated with their ability to induce endogenous type I interferon production. We have carried out a comprehensive study of agonists of TLRs 1 to 9 to determine if any additional TLRs can induce antiviral molecules from human peripheral blood mononuclear cells (PBMCs). The agonists were incubated with PBMCs, and the supernatant was then removed and added to HCV replicon cells to assess antiviral activity. Agonists of TLRs 3, 4, 7, 8, and 9 were found to be potent inducers of antiviral activity in PBMC supernatants, and the activity correlated with the induction of alpha interferon and the interferon-induced antiviral biomarker 2',5'-oligoadenylate synthase. Antiviral activity of TLR7 and TLR8 agonists was blocked by an antibody that binds to the type I interferon receptor, confirming that the antiviral activity results from type I interferon induction. TLR4 and TLR8 agonists were found to strongly induce the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha at concentrations similar to those inducing antiviral activity. This raises concerns about adverse side effects if these were to be used as antiviral agents. We therefore conclude that TLRs 3, 7, and 9 represent the most attractive targets for the development of new HCV therapies.

FIG. 1.

Antiviral activity and OAS induction from untreated PBMC supernatants. (A) PBMCs from four donors were pooled and incubated for the indicated times, and the supernatant was then added to HCV replicon cells to determine antiviral activity. HCV levels were quantified by luciferase assay. (B) The PBMC cell pellet from A was used to determine OAS gene expression levels by quantitative PCR. (C) PBMCs from a single donor and pooled from donors were incubated for 24 h, and the supernatant was then added to HCV replicon cells to determine antiviral activity. Data are means of duplicate samples ± standard deviations. RLU, relative light units.

FIG. 2.

Antiviral activity of resiquimod in PBMCs from a single donor versus PBMCs pooled from four donors. Resiquimod was incubated with PBMCs from four separate donors and a mixture of the four donors for 24 h, and supernatants were then transferred to HCV replicon cells to determine antiviral activity. Duplicate data points at each concentration are plotted. RLU, relative light units.

FIG. 3.

Determining the optimal time point for endpoint measurements. (A) Pooled PBMCs from four donors were incubated with 20 nM or 1 μM resiquimod for 1, 6, or 24 h, and supernatants were added to HCV replicon cells to determine antiviral activity. Data are a single measurement from each sample. (B) Cells from the 1 μM resiquimod treatment were used to quantify OAS RNA levels. Data are means of triplicate samples ± standard deviations. (C) Supernatants from the 1 μM resiquimod treatment were used to quantify cytokine levels. Data are means of duplicate samples ± standard deviations. RLU, relative light units.

FIG. 4.

Antiviral activity of TLR ligands. Various concentrations of the ligands were incubated with PBMCs for 24 h, and supernatants were then added to HCV replicon cells to measure antiviral activity (circles). As a control for nonspecific effects, the ligands were also added directly to HCV replicon cells (triangles). Data points are means of duplicate samples ± standard deviations. The TLR ligand is indicated at the top of each graph, with the TLR that it activates following in parentheses. RLU, relative light units.

FIG. 5.

Cytokine induction by TLR ligands. TLR ligands were incubated with PBMCs at various concentrations for 24 h, and the supernatant was then removed and used to quantify levels of cytokines. The maximum cytokine level induced over the ligand concentration range is plotted. The concentrations at which these cytokine levels were induced are shown in Table 2. Untreated PBMC supernatants contained 4 to 20 pg/ml IL-6 and no detectable IFN-α, IL-1β, or TNF-α. Data are means of duplicate samples ± standard deviations. (a) IFN-α. (b) IL-6. (c) IL-1β. (d) TNF-α.

FIG. 6.

Induction of IFN-α and OAS by TLR ligands. TLR ligands were incubated with PBMCs at various concentrations for 24 h. The supernatant was removed and used to quantify IFN-α, and the remaining cell pellet was used to quantify OAS by quantitative PCR. Induction (n-fold) was calculated relative to untreated samples. Squares, OAS; triangles, IFN-α. (A) The eight ligands that induced complete and potent antiviral activity. The dashed line indicates the antiviral EC₅₀. (B) The four ligands that induced only weak, partial antiviral activity. All data points are means of duplicate samples ± standard deviations. The TLR ligand is indicated at the top of each graph, with the TLR that it activates following in parentheses.

FIG. 7.

Proinflammatory cytokine induction relative to antiviral activity. TLR ligands were incubated with PBMCs at various concentrations for 24 h. The supernatant was removed and added to HCV replicon cells to quantify antiviral activity. The levels of TNF-α in the same supernatants were also measured. Circles, antiviral activity; triangles, TNF-α. Data points are means of duplicate samples ± standard deviations. The TLR ligand is indicated at the top of each graph, with the TLR that it activates following in parentheses. RLU, relative light units.

FIG. 8.

Effect of type I IFN receptor antibody on antiviral activity of TLR ligands. TLR ligands were incubated with PBMCs for 24 h. The concentrations used were 50 IU/ml IFN-α, 100 nM resiquimod, 3 μM 3M002, 20 μM imiquimod, and 200 nM SM360320. The supernatant was removed and added to HCV replicon cells in the presence of type I IFN receptor antibody. White bars, no antibody; gray bars, 1 μg/ml antibody; black bars, 10 μg/ml antibody. Data are means of duplicate samples ± standard deviations. RLU, relative light units.