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.

 

ABSTRACT:

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

 

SUPPLEMENT:

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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