AIDS Res Hum Retroviruses. 2013 Jun;29(6):907-18.

Gardiquimod: a Toll-like receptor-7 agonist that inhibits HIV type 1 infection of human macrophages and activated T cells.


Buitendijk M, Eszterhas SK, Howell AL.

V.A. Medical Center, White River Junction, VT 05009, USA.



Immune response modifiers are being studied as therapeutic agents for viral infections and cancer. These molecules include agonists for the Toll-like receptors (TLR), a family of innate immune receptors. TLR7 and 8, located in cellular endosomes, bind single-stranded RNA characteristic of viral genomes, and trigger intracellular signaling pathways that induce inflammatory cytokines and antiviral innate immune factors. We studied the anti-HIV-1 effects of gardiquimod, a specific TLR7 agonist when used at concentrations below 10 μM, in macrophages and activated peripheral blood mononuclear cells (PBMCs). Gardiquimod, added prior to or within 2 days after infection with X4, R5, or dual-tropic (R5/X4) strains of HIV-1, significantly reduced infection in these cells. Cocultures of activated PBMCs added to gardiquimod-treated and HIV-1-exposed macrophages demonstrated minimal HIV-1 replication for up to 10 days, suggesting that gardiquimod inhibited activated PBMCs viral amplification from HIV-1-exposed macrophages. Gardiquimod treatment of both activated PBMCs and macrophages induced interferon-alpha (IFN-α) transcription within hours of addition, and sustained IFN-α protein secretion for several days. Treatment of cells with a peptide inhibitor to the MyD88 adaptor protein blocked the induction of IFN-α by gardiquimod, and partially reversed the anti-HIV effects in activated PBMCs. Blocking the IFN-α receptor with a neutralizing antibody also reduced the anti-HIV effect of gardiquimod. Gardiquimod inhibited HIV-1 reverse transcriptase, an early step in the life cycle of HIV-1. These findings suggest that gardiquimod, functioning as both an immune system modifier and a reverse transcriptase inhibitor, could be developed as a novel therapeutic agent to block systemic and mucosal transmission of HIV-1.

PMID: 23316755



This manuscript reported on the potential for small molecules that function as immune system modifiers to suppress HIV-1 infection and replication in susceptible cell populations. Immune cells possess a number of innate immune receptors that when activated by ligand, induce rapid responses to block pathogen infection. Innate immune responses are triggered by the binding of a microbial pathogen to one or more of a family of receptors termed “pattern recognition receptors” (PRR) that includes the toll like receptor (TLR) family. The advantages of innate immune responses are two-fold: On the one hand, they trigger intracellular signaling pathways that induce cytokine production that can suppress or inhibit a pathogen infection. In addition, many of the cytokines that are induced by PRR activation serve to enhance antigen presentation by innate immune cells to T and B cells. Gardiquimod is not only a TLR7 ligand, but also functions as a reverse transcriptase inhibitor to block HIV-1 infection in macrophages. We showed that gardiquimod added to macrophages either prior to or up to two days after HIV-1 exposure was highly effective at suppressing infection of these cells.

We extended these studies to other TLR located in the endosomes of immune cells, including TLR3, 7, 8 and 9 (Figure 1, below). In phytohemagglutinin (PHA)- activated peripheral blood mononuclear cells (PBMC), agonists to these TLR all induced expression of the type I interferons and interferon stimulated genes, although to variable levels depending on the agonist used. In addition, these agonists inhibited HIV-1 infection and replication in these cells. Interestingly, an agonist to TLR9 also induced type II interferon as well as the anti-HIV molecules Apobec3G and SAMHDI. The addition of an inhibitor to intracellular activation pathways showed that the anti-HIV activity was not entirely mediated by TLR activation, but likely by the activation of additional anti-viral sensors in the cytoplasm of HIV targets cells. Our findings suggest that innate immune receptors and their activation products could be harnessed as an anti-HIV therapy, especially at sites of initial infection.
Slide1Figure 1. TLR agonists inhibit HIV-1 replication in PBMC.  PHA-activated PBMC were treated once with agonists to TLR 3 (Poly (I:C)), TLR7 (CL264), TLR8 (ssPolyU RNA) or TLR9 (ODN2395 DNA) either 24 hours before, immediately after, or 24, 48 or 72 hours post-HIV infection.  p24 levels in culture supernatant were measured by ELISA on day 7 post-infection. All experiments were performed in triplicate using cells from three different donors. ** = p < 0.005.


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