Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.

The interactions of viruses with the skin immune system are not well understood. The interface of poxviruses with skin is especially intriguing, given that (i) skin infection and disfigurement are prominent features of smallpox disease; (ii) successful smallpox immunization is achieved by intentional skin infection with vaccinia virus; and (iii) eczema vaccinatum is a major severe complication of vaccination in people with atopic dermatitis (AD), a condition associated with defects in skin barrier function and antiviral innate immunity (25,26,69). Concerns about smallpox as a bioterrorism threat against an unvaccinated population, the persistence of monkeypox in Africa and episodes of its extracontinental spread, and the imperative to improve smallpox vaccination strategies all focus attention on poxvirus-host dynamics, particularly with skin. A recent case of life-threatening eczema vaccinatum in a child with AD who became infected by mere household contact with a smallpox vaccinee and then passed the infection on to a third party (68) highlights just how delicate the balance is between poxvirus virulence and skin immunity.Keratinocytes comprise the predominant cell type in the epidermis. Liu et al. (43) reported that vaccinia virus had limited replicative capacity in human keratinocytes and that infection induced keratinocytes to produce the Th2 cytokines transforming growth factor ␤, interleukin-10 (IL-10), and IL-13, which suggested that vaccinia virus might downregulate skin immune responses. Human keratinocytes express Toll-like receptors (TLRs) that initiate innate immune signaling by binding to ligands referred to as pathogen-associated molecular patterns (30,40,47,49). Human keratinocytes produce a repertoire of cytokines, chemokines, and antimicrobial peptides in response to TLR stimulation (6,40,66). Such cytokines and chemokines augment innate and acquired immunity mediated by dendritic cells, neutrophils, macrophages, NK cells, and T cells, which reside in the skin or are recruited to the skin in the setting of infection or inflammation (39).The type I interferons (IFN-␣ and IFN-␤) are key mediators of antiviral innate immunity (65). Double-stranded RNA (dsRNA) introduced during virus infection is a potent inducer of the type I IFN response. dsRNA can trigger distinct signaling pathways by engaging either (i) the endosomal membrane-bound receptor TLR3 (42), (ii) the soluble cytoplasmic receptors 29,31,73), or (iii) the dsRNA-dependent protein kinase PKR (16). Signaling through TLR3 leads to activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-B via the adaptor molecule TRIF. As a consequence, IRF3 is phosphorylated and then moves to the nucleus to activate IFN-␤ expression (59). In contrast, cytoplasmic dsRNA produced during replication of RNA viruses binds to RIG-I or MDA5 and triggers activation of IRF3 and NF-B through the mitochondrial antiviral signaling protein, MAVS

No abstract

No abstract

Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.