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 1␤ 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.The World Health Organization reports that ϳ170 million people worldwide are infected with hepatitis C virus (HCV) (29). The disease affects primarily the liver, with 10 to 20% of chronic infections leading to cirrhosis and 1 to 5% leading to liver cancer. The recommended standard therapy for HCV is a once-weekly injection of pegylated interferon alpha 2a (IFN␣2a) plus daily oral ribavirin. The sustained viral response is approximately 50% (23), and the therapy is associated with significant side effects including flu-like symptoms, depression, and injection site reactions (9). In addition, some patients develop neutralizing antibodies to recombinant IFN, which may contribute to treatment failures (2, 13). There is therefore a need to discover new drugs with improved efficacy and reduced side effects.Toll-like receptors (TLRs) are a family of receptors that play a key role in innate immunity. On binding microbial ligands, they induce a signaling cascade resulting in the induction of type I IFNs and other cytokines, which drive an inflammatory response and activate the adaptive immune system (for a review, see reference 27). Whereas current IFN therapy involves the administration of a single IFN-␣ subtype (2a or 2b), TLR activation induces a range of different IFN subtypes. For example, the TLR7 agonist imiquimod has been shown to induce IFN-␣1, -␣2...

Pharmacokinetic and Pharmacodynamic Properties of GS-9620, a Novel Toll-Like Receptor 7 Agonist, Demonstrate Interferon-Stimulated Gene Induction without Detectable Serum Interferon at Low Oral Doses

“…cytokines and chemokines that are consistent with the biology of TLR7. At concentrations of GS-9620 higher than 10 mM, additional cytokines such as TNF-a were induced in PBMCs, which is consistent with stimulation of TLR8 (Gorden et al, 2005;Thomas et al, 2007). Similarly, oral administration of GS-9620 to cynomolgus monkeys in vivo preferentially induced IFN-a and a pattern of antiviral serum cytokines and chemokines at low oral doses, whereas TNF-a was only observed following relatively high oral doses.…”

“…This is consistent with GS-9620 selectivity for TLR7 versus TLR8 at lower concentrations and combined TLR7 and TLR8 stimulation at higher concentrations of GS-9620. TLR7 agonism preferentially mediates secretion of IFN-a in PBMC cultures, whereas TLR8 stimulation in cDC and monocyte PBMC subsets is viewed as the dominant trigger for secretion of inflammatory cytokines, e.g., TNF-a (Gorden et al, 2005;Thomas et al, 2007). Although GS-9620 induced IFN-a at a mean (6 S.D.)…”

“…Unlike human and cynomolgus monkey dendritic cell subsets in which TLR7 is exclusively expressed by pDCs and TLR8 expression is predominantly found in cDCs, the pattern of murine TLR7 expression is notably different, as expression of murine TLR7 is found in both pDCs and cDCs (Edwards et al, 2003;Ketloy et al, 2008). As a consequence, the response profiles observed with a TLR7 agonist in mice or mouse splenocytes are more reminiscent of profiles observed with dual TLR7/TLR8 agonists in monkeys and humans (Ito et al, 2002;Gorden et al, 2005;Thomas et al, 2007).…”

Impaired Toll-Like Receptor 3-Mediated Immune Responses from Macrophages of Patients Chronically Infected with Hepatitis C Virus

“…In murine models, TLR3 mediates robust cytokine responses to the related flavivirus West Nile virus (49) and to influenza A virus (although such responses are detrimental in that context [50]) for cross-priming of viral antigens to cytotoxic T cells (51). TLR3 in human hepatocytes has been shown to trigger IFN responses upon infection with HCV (52), and TLR3 agonists have been shown to stimulate antiviral activity in human PBMCs (53). Our studies suggest that TLR3 may provide effective anti-HCV responses and this pathway may provide an effective boost to viral clearance.…”

Endogenous IFNγ in chronic HCV genotype 4 patients treated with PEG-IFNα and ribavirin

“…Prepared PBMCs were resuspended into RPMI 1640 media as previously described [22].Stimulation of PBMCs or lymphocyte subsets was performed using freshly isolated PBMCs which were seeded at 2 × 10 6 /mL (2 mL) into 96-well microtiter plates, and phytohemagglutinin (PHA) (Sigma Aldrich, Saint Louis, USA) at a concentration of 10 µg/mL was added to each well. For stimulation, the final ratio of PBMCs and PHA was 10:1.…”