During the design and synthesis of a series of 8-hydroxy-2-(2-methoxyethoxy)-adenine derivatives bearing various substituted −RCOOH groups at the 9-position, we identified a TLR7-inert ligand, which does not activate TLR7 signaling pathway. Of interest, the coupling of weakly immunogenic antigens via the −RCOOH group was able to significantly enhance the immunogenicity of the antigens. Herein, an inert ligand, 9-(3-carboxypropyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine (5, GD2), was synthesized and conjugated to 5 different weakly immunogenic antigens (BSA, OVA, MSA, MG7, and thymosin). Compared with the GD2 and the potent agonist UC-1 V150, all conjugates demonstrated potent immunogenicity in vitro and in vivo. All conjugates induced prolonged increases, while UC-1 V150 showed a rapid decline in the levels of proinflammatory cytokines following initial increases. These data indicate that the immunostimulatory activity of TLR7-inert ligands could be amplified and prolonged by conjugation to antigens, thus broadening the potential therapeutic application of these agents.Keywords: adenine derivative; immunostimulatory activity; Toll-like receptor agonist

•TLR7 ligand (SZU101) prevents PRRSV replication in PAMs.•SZU101 treatment activates NF-κB and induces antiviral cytokines and genes expression.•Blocking the activation of NF-κB reverses the suppression effect of SZU101 on PRRSV replication.•Inhibiting the proper subcellular localization of TLR7 using specific inhibitor reverses the anti-PRRSV effect of SZU101.•Co-administration of SZU101 and PRRSV enhances the humoral and cellular immune responses against PRRSV antigens in mice.Porcine reproductive and respiratory syndrome virus (PRRSV), a common viral pathogen, causes huge annual economic losses to the swine industry worldwide. After triggering by specific ligands, the Toll-like receptor 7 (TLR7), a type of pattern-recognition receptor (PRR), induces antiviral cytokines production. Previously, we synthesized an adenine analog, designated SZU101, a TLR7-specific ligand. In this study, we assessed the inhibitory effect of SZU101 on PRRSV infection in vitro. SZU101 significantly suppressed PRRSV infection in primary porcine alveolar macrophages (PAMs) in a dose-dependent manner. Moreover, SZU101-induced inhibition involved NF-κB pathway activation in PAMs to initiate expression of TLR7-mediated cytokines and induce expression of downstream signaling IFN-stimulated genes (ISGs). Chloroquine, a TLR7 inhibitor, and BAY 11-7082, an NF-κB inhibitor, reversed both the SZU101-induced antiviral effect and induction of cytokine genes and ISGs expression. Therefore, SZU101 antiviral effects depend at least in part on TLR7-NF-κB signaling pathway. Additionally, administration of SZU101 enhanced the humoral and cell-mediated immune responses against PRRSV antigens in mice. Given these results, SZU101 holds promise as an antiviral agent and a vaccine adjuvant to prevent PRRSV infection in pigs.