TLR7 Agonists Display Potent Antiviral Effects against Norovirus Infection via Innate Stimulation

Norovirus infections are a significant health and economic burden globally, accounting for hundreds of millions of cases of acute gastroenteritis every year. In the absence of an approved norovirus vaccine, there is an urgent need to develop antivirals to treat chronic infections and provide prophylactic therapy to limit viral spread during epidemics and pandemics. Toll-like receptor (TLR) agonists have been explored widely for their antiviral potential, and several are progressing through clinical trials for the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and as adjuvants for norovirus viruslike particle (VLP) vaccines. However, norovirus therapies in development are largely direct-acting antivirals (DAAs) with fewer compounds that target the host. Our aim was to assess the antiviral potential of TLR7 agonist immunomodulators on norovirus infection using the murine norovirus (MNV) and human Norwalk replicon models. TLR7 agonists R-848, Gardiquimod, GS-9620, R-837, and loxoribine were screened using a plaque reduction assay, and each displayed inhibition of MNV replication (50% effective concentrations [EC₅₀s], 23.5 nM, 134.4 nM, 0.59 μM, 1.5 μM, and 79.4 μM, respectively). RNA sequencing of TLR7-stimulated cells revealed a predominant upregulation of innate immune response genes and interferon (IFN)-stimulated genes (ISGs) that are known to drive an antiviral state. Furthermore, the combination of R-848 and the nucleoside analogue (NA) 2'C-methylcytidine elicited a synergistic antiviral effect against MNV, demonstrating that combinational therapy of host modulators and DAAs might be used to reduce drug cytotoxicity. In summary, we have identified that TLR7 agonists display potent inhibition of norovirus replication and are a therapeutic option to combat norovirus infections.