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

<div><p>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.</p></div

TLR9 and MyD88 contribute to the induction of type I IFN in cDCs by MVA.

<p>GM-CSF-BMDCs were generated from MyD88^−/− (A), TLR9^−/− (B), TLR7^−/− (C) mice, and their age-matched WT controls. Cells (1×10⁶) were either stimulated with CpG or infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 6). The combined results of three independently performed experiments are shown. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; comparisons were made between WT cells and various knockout cells as indicated.</p

MVA induces type I IFN production in conventional dendritic cells (cDCs).

<p>(A) Murine pDCs and cDCs were purified from Flt3L-BMDCs using FACS. 2×10⁵ pDCs (CD11c⁺B220⁺PDCA-1⁺) and 1×10⁶ cDCs (CD11c⁺B220⁻PDCA-1⁻) were either stimulated with CpG (at a final concentration of 10 µg/ml) or infected with either WT VAC or MVA at a MOI of 10. Supernatants were collected at 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 6). A representative experiment is shown, repeated at least twice. (B) GM-CSF-BMDCs (1×10⁶) were infected with WT VAC or MVA at a MOI of 10. Supernatants were collected at 1, 4, 8, 14, and 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated once. (C) GM-CSF-BMDCs (1×10⁶) were infected with MVA at a MOI of 0.25, 0.5, 1, 5, or 10. Supernatants were collected at 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated once. (D) GM-CSF-BMDCs (1×10⁶) were infected with MVA or WT VAC at a MOI of 10. Cells were collected at 6 h post infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. ***, <i>p</i><0.001; comparisons were made between MVA and WT VAC infected cells. (E) GM-CSF-BMDCs (1×10⁶) were infected with MVA or WT VAC at a MOI of 10. Cells were collected at 1, 2, 4, and 8 h post infection. Western blot analysis was performed using anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3, and anti-IRF3. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.</p

Transcription factors IRF3/IRF7 and the type I IFN positive feedback loop mediated by IFNAR1 are required for the induction of type I IFN in murine cDCs by MVA.

<p>GM-CSF-BMDCs were generated from IRF3^−/− (A), IRF7^−/− (B), IFNAR1^−/− (C) mice, and their age-matched WT controls. Cells (1×10⁶) were stimulated with CpG or infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; comparisons were made between WT cells and various knockout cells as indicated.</p

Endosomal and lysosomal acidification and lysosomal enzyme activities are required for type I IFN production by BMDCs in response to MVA infection.

<p>(A) GM-CSF-BMDCs (1×10⁶) were infected with MVA at a MOI of 10 for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of chloroquine (50 µM), bafilomycin A1 (100 nM), or CA-074-Me (10 µM). Supernatants were collected 22 h later. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM (n = 6). The combined results of three independently performed experiments are shown. **, <i>p</i><0.01; ***, <i>p</i><0.001; compared with no drug treatment. (B) GM-CSF-BMDCs were generated from Cathepsin B KO (CathB^−/−) and age-matched WT controls (CathB^+/+). Cells (1×10⁶) were infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. **, <i>p</i><0.01; comparisons were made between CathB^+/+ and CathB^−/− cells as indicated.</p

STING is required for the induction of type I IFN and IRF3 phosphorylation by MVA in BMDCs.

<p>GM-CSF-BMDCs were generated from WT mice (Sting^+/+) and the N-ethyl-N-nitrosourea (ENU)-induced <i>Goldenticket</i> (<i>Gt</i>) mutant mice (Sting^Gt/Gt) harboring a single nucleotide variant of Sting resulting in a functionally null allele. (A) Cells (1×10⁶) were either stimulated with LPS or infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). Results shown are representative of three experiments. ***, <i>p</i><0.001; comparisons were made between Sting^+/+ and Sting^Gt/Gt as indicated. (B) Cells (1×10⁶) were infected with MVA at a MOI of 10. Cells were collected at 6 h post infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. **, <i>p</i><0.01; ***, <i>p</i><0.001; comparisons were made between Sting^+/+ and Sting^Gt/Gt as indicated. (C) Western blot analysis of BMDCs from Sting^+/+ and Sting^Gt/Gt infected with MVA at a MOI of 10, or mock infected. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3, and anti-IRF3. GAPDH was used as a loading control. “hpi”, hours post infection. “M”, mock infection control. (D) Sting^Gt/Gt, IRF3^−/− and age-matched WT C57B/6 control mice were infected with MVA (2×10⁷ pfu) via intravenous inoculation. Serum levels of IFN-α and IFN-β were determined by ELISA. Data are means ± SD. Results shown are representative of two independent experiments.</p

cGAS is the critical cytosolic DNA sensor for MVA infection of cDCs.

<p>GM-CSF-BMDCs were generated from cGAS^−/− mice and its age-matched WT controls. (A) Cells (1×10⁶) were infected with MVA at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice (***, <i>p</i><0.001). (B) Cells (1×10⁶) were infected with MVA at a MOI of 10. Cells were collected at 6 h post infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice (***, <i>p</i><0.001). (C) Western blot analysis of cGAS^+/+ and cGAS^−/− cDCs infected with MVA at a MOI of 10, or mock infected. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3, and anti-IRF3. GAPDH was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.</p

Vaccinia N1 virulence factor plays an inhibitory role in the type I IFN gene induction.

<p>GM-CSF-BMDCs (1×10⁶) were infected with MVA or MVA-N1L at a MOI of 10. (A) Cells were collected at 6 h post-infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM. A representative experiment is shown, repeated twice. (B) Western blot analysis of GM-CSF-BMDCs infected with MVA or MVA-N1L at a MOI of 10. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3 and anti-IRF3. GAPDH was used as a loading control. “hpi”, hours post infection. “M”, mock infection control. (C) Cells (1×10⁶) were infected with MVA or MVA-N1L at a MOI of 10. Supernatants were collected 22 h later. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice (**, <i>p</i><0.01).</p

MVAΔE3L induces high levels of type I IFN gene expression in BMDCs than MVA does.

<p>(A) GM-CSF-BMDCs were generated from 6–8 week-old female WT C57B/6 mice. Cells (1×10⁶) were infected with MVA or MVAΔE3L at a MOI of 10. Cells were collected at 6 h post infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. ***, <i>p</i><0.001; comparisons were made between MVA and MVAΔE3L infected cells. (B) GM-CSF-BMDCs were generated from IRF3^−/− mice and age-matched WT C57B/6 mice. Cells were infected with MVA or MVAΔE3L at a MOI of 10, or mock infected. Cells were collected at 6 h post infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. **, <i>p</i><0.01; ***, <i>p</i><0.001; comparisons were made between IRF3^+/+ and IRF3^−/− cells. (C) Western blot analysis of GM-CSF-BMDCs infected with MVA or with MVAΔE3L at a MOI of 10, or mock infected. Whole-cell lysates were prepared. Equal amount of proteins were subjected to SDS-PAGE and immunoblotting with anti-phosphoserine-396 of IRF3. β-actin was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.</p

Radiation followed by lymphocyte infusion leads to improved persistence of responses to tumor vaccination and to cure of some mice with established tumors.

<p>A) Mice were treated with radiation and lymphocyte infusion, and were then immunized with VP22-Opt-TRP1 DNA vaccine starting 1 day afterwards for 3 immunizations. Either 5, 12, or 19 days after the last immunization, splenocytes were restimulated with TRP1_455-463 peptide and IFNγ production from CD8⁺ T cells was quantified by flow cytometry. n=3/group, results shown from one of two experiments with similar results. B) Mice were treated as in A with hTRP2 or VP22-Opt-TRP1 DNA vaccine starting 1 day after radiation and lymphocyte infusion. Either 5, 12, or 19 days after the last immunization, mice were challenged intradermally with B16 melanoma. Mice were then monitored for development of palpable tumors. n=10-15/group, results shown from one of two experiments with similar results. C) Mice were inoculated intradermally with B16 melanoma. Three days later, some mice received radiation and lymphocyte infusion, followed 1 day later by immunizations with VP22-Opt-TRP1 DNA vaccine every 5 days. Mice were harvested on day 21 after irradiation and splenic and tumor-infiltrating lymphocytes were evaluated by flow cytometry. Additional mice were treated similarly for a total of 8 immunizations, n=10/group, and followed for overall survival, with results shown from one of three experiments with similar results. D) Tg(Grm1)EPv-transgenic mice began treatment at 8-12 weeks of age with 5 weekly vaccinations of VP22-Opt-TRP1 DNA or an empty control plasmid; some mice were pre-treated with irradiation and lymphocyte infusion one day prior. Mice were evaluated weekly for development of tail and ear melanomas. n=4-5/group, with combined results from 2 experiments.</p