Weiyi Wang

Modified Vaccinia Virus Ankara Triggers Type I IFN Production in Murine Conventional Dendritic Cells via a cGAS/STING-Mediated Cytosolic DNA-Sensing Pathway

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.

Binding-Pocket and Lid-Region Substitutions Render Human STING Sensitive to the Species-Specific Drug DMXAA.

SUMMARY The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) showed therapeutic promise against solid tumors in mouse models but subsequently failed in human clinical trials. DMXAA was later discovered to activate mouse, but not human, STING, an adaptor protein in the cyclic dinucleotide cGAMP-mediated signaling pathway, inducing type I interferon expression. To facilitate the development of compounds that target human STING, we combined structural, biophysical, and cellular assays to study mouse and human chimeric proteins and their interaction with DMXAA. We identified a single substitution (G230I) that enables a DMXAA-induced conformational transition of hSTING from an inactive “open” to an active “closed” state. We also identified a substitution within the binding pocket (Q266I) that cooperates with G230I and the previously identified S162A binding-pocket point substitution, rendering hSTING highly sensitive to DMXAA. These findings should facilitate the reciprocal engineering of DMXAA analogs that bind and stimulate wild-type hSTING and their exploitation for vaccine-adjuvant and anti-cancer drug development.

Innate Immune Response of Human Plasmacytoid Dendritic Cells to Poxvirus Infection Is Subverted by Vaccinia E3 via Its Z-DNA/RNA Binding Domain

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.

Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells.

Inactivated vaccinia virus induces antitumor immunity that can overcome resistance to checkpoint inhibitors. A viral STING operation for tumors The immune response to viruses can be harnessed to attack tumors. Here, Dai et al. demonstrate that inactivated modified vaccinia virus Ankara (iMVA) can induce antitumor responses in two different models of cancer in mice. This effect relied on both Batf3-dependent dendritic cells and the cGAS-STING cytosolic DNA-sensing pathway. Moreover, intratumor injection of iMVA overcame tumor resistance to checkpoint inhibitors. Viral-induced innate immune responses may therefore tip the scale to successful cancer immunotherapy. Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immunosuppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as immunotherapeutic agents. We show that infection of conventional dendritic cells (DCs) with heat- or ultraviolet-inactivated MVA leads to higher levels of interferon induction than MVA alone through the cGAS (cyclic guanosine monophosphate–adenosine monophosphate synthase)–STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA) was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than in wild-type mice, indicating that both cytosolic DNA sensing and Batf3-dependent CD103+/CD8α+ DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral tumor implantation models as well as in a unilateral large established tumor model. Our results suggest that inactivated vaccinia virus could be used as an immunotherapeutic agent for human cancers.

Abstract B031: Heat-inactivated modified vaccinia virus ankara induces type I IFN and antitumor immunity via the cytosolic DNA-sensing pathway

Type I interferon (IFN), well known for its antiviral activity, is a critical component of cancer immune surveillance through its direct actions on cancer cells as well as on tumor microenvironment. Advanced melanoma remains a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immune suppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses are cytosolic DNA viruses that have been investigated as oncolytic and immunotherapeutic agents. We recently reported that the highly attenuated modified vaccinia virus Ankara (MVA), a safe vaccine for smallpox, triggers type I IFN production in conventional dendritic cells (cDCs) via the cytosolic DNA sensor cGAS and its adaptor STING, and that it requires transcription factors IRF3 and IRF7. Here we show that infection of cDCs with heat-inactivated MVA leads to higher levels of IFN induction than MVA. This induction is also mediated by the cytosolic DNA-sensing pathway cGAS/STING/IRF3/IRF7. In addition, we found that intratumoral injection of Heat-MVA caused tumor eradication in a murine B16 melanoma model as well as the generation of adaptive anti-tumor immunity. Furthermore, Heat-MVA-induced anti-tumor therapy is less effective in STING, IRF7, or Batf3-deficient mice than in wild-type mice, indicating that both the innate immune-sensing pathway and CD8α+DCs are essential for Heat-MVA-based immunotherapy. Lastly, the combination of intratumoral delivery of Heat-MVA with systemic delivery of anti-CTLA-4, PD-1 or PD-L1 antibodies achieved enhanced efficacy in tumor eradication and survival than Heat-MVA alone. Our results have strong implications for the development of poxvirus-based cancer immunotherapeutics as well novel strategies to overcome resistance to immune checkpoint blockade therapy. Citation Format: Peihong Dai, Weiyi Wang, Cristian Serna-Tamayo, Dimitriy Zamarin, Stewart Shuman, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Heat-inactivated modified vaccinia virus ankara induces type I IFN and antitumor immunity via the cytosolic DNA-sensing pathway. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B031.

Abstract A007: Intratumoral delivery of inactivated vaccinia virus is more efficacious than live oncolytic vaccinia virus in murine bilateral tumor implantation models

Poxviruses are large cytoplasmic DNA viruses that have potentials as vaccine vectors and oncolytic agents. Although almost all of the oncolytic vaccinia viruses that are being investigated in clinical trials are replication competent, it is still unclear how these viruses exert antitumor immunologic effects, and whether viral replication is even necessary to achieve systemic antitumor efficacy. In this study, we compared therapeutic efficacy of the intratumoral delivery of the live, attenuated, replication competent oncolytic vaccinia virus expressing mGM-CSF (VC-TK−-mGM-CSF)vs. its heat-inactivated counterpart (iVC-TK−-mGM-CSF) in two bilateral tumor implantation models- B16-F10 murine melanoma and MC38 colon adenocarcinoma. We found that intratumoral injection of inactivated virus exerted stronger systemic antitumor effects than the live virus. FACS analyses of tumor infiltrating immune cells revealed that intratumoral injection of iVC-TK−-mGM-CSF lead to increased percentages and numbers of activating effector CD8+ and CD4+ T cells and reduced percentages and numbers of CD4+Foxp3+ regulatory T cells in the non-injected tumors compared with intratumoral delivery of the live virus. Infection of bone marrow-derived dendritic cells (DCs) with iVC-TK−-mGM-CSF induced type I IFN and proinflammatory cytokine and chemokine production and DC maturation, whereas the live virus did not. Taken together, our results demonstrate that intratumoral delivery of inactivated vaccina virus is safer and more efficacious than live oncolytic vaccinia virus expressing mGM-CSF in two murine bilateral tumor implantation models. Our findings have important implications for the design of poxviral-based immunotherapeutics for patients with metastatic cancers. Note: This abstract was not presented at the conference. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Intratumoral delivery of inactivated vaccinia virus is more efficacious than live oncolytic vaccinia virus in murine bilateral tumor implantation models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A007.