The pulmonary immune system consists of a network of tissue-resident cells as well as immune cells that are recruited to the lungs during infection and/or inflammation. How these immune components function during an acute poxvirus infection is not well understood. Intranasal infection of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we report that vaccinia C7 is a crucial virulence factor that blocks activation of the transcription factor IRF3. We provide evidence that type II alveolar epithelial cells (AECIIs) respond to pulmonary infection of vaccinia virus by inducing IFN-β and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic acid-sensing pathways and the type I IFN positive feedback loop. This leads to the recruitment and activation of CCR2+ inflammatory monocytes in the infected lungs and subsequent differentiation into Lyve1− interstitial macrophages (Lyve1− IMs), which efficiently engulf viral particles and block viral replication. Our results provide insights into how innate immune sensing of viral infection by lung AECIIs influences the activation and differentiation of CCR2+ inflammatory monocytes to defend against pulmonary poxvirus infection.

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.

Effective depletion of immune suppressive regulatory T cells (Tregs) in the tumor microenvironment without triggering systemic autoimmunity is an important strategy for cancer immunotherapy. Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus with a long history of human use. Here, we report rational engineering of an immune-activating recombinant MVA (rMVA, MVA∆E5R-Flt3L-OX40L) with deletion of the vaccinia E5R gene (encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase, cGAS) and expression of two membrane-anchored transgenes, Flt3L and OX40L. Intratumoral (IT) delivery of rMVA (MVA∆E5R-Flt3L-OX40L) generates potent antitumor immunity, dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and type I IFN signaling. Remarkably, IT rMVA (MVA∆E5R-Flt3L-OX40L) depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Single-cell RNA-seq analyses of tumors treated with rMVA showed the depletion of OX40hiCCR8hi Tregs and expansion of IFN-responsive Tregs. Taken together, our study provides a proof-of-concept for depleting and reprogramming intratumoral Tregs via an immune-activating rMVA.

The pulmonary immune system consists of a network of tissue-resident cells as well as immune cells that are recruited to the lungs during infection and/or inflammation. How these immune components communicate during an acute poxvirus infection is not well understood. Intranasal infection of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we provide evidence that type II alveolar epithelial cells (AECIIs) function as the sentinels of pulmonary infection of vaccinia virus by inducing IFN-β and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic acid-sensing pathways and the type I IFN positive feedback loop. This leads to the recruitment and activation of CCR2+ inflammatory monocytes in the infected lungs and their differentiation into Lyve1- interstitial macrophages (Lyve1- IMs), which efficiently engulf viral particles and block viral replication. Our results provide novel insights into how innate immune-sensing of viral infection by lung AECIIs influences the activation and differentiation of CCR2+ inflammatory monocytes to defend pulmonary poxvirus infection.