Innate Immune Sensing of Modified Vaccinia Virus Ankara (MVA) Is Mediated by TLR2-TLR6, MDA-5 and the NALP3 Inflammasome

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Abstract
Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNβ-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNβ and IFNβ-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1β. Transcription of the Il1b gene was markedly impaired in TLR2−/− and MyD88−/− BMDM, whereas mature and secreted IL-1β was massively reduced in NALP3−/− BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNβ and IL-1β by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity. Modified vaccinia virus Ankara (MVA) is a highly attenuated, replication-deficient, poxvirus currently developed as a vaccine vector against a broad spectrum of infectious diseases including HIV, tuberculosis and malaria. It is well known that robust activation of innate immunity is essential to achieve an efficient vaccine response, and that poxviruses have developed numerous strategies to block the innate immune response. Yet, the precise mechanisms underlying innate immune sensing of MVA are poorly characterized. Toll-like receptors (TLR), RIG-I-like receptors (RLR) and NOD-like receptors (NLR) are families of membrane-bound and cytosolic sensors that detect the presence of microbial products and initiate host innate and adaptive immune responses. Here, we report the first comprehensive study of MVA sensing by innate immune cells, demonstrating that TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways play specific and coordinated roles in regulating cytokine, chemokine and interferon response to MVA poxvirus infection. Delineation of the pathways involved in the sensing of MVA by the host could help designing modified vectors with increased immunogenicity, which would be of particular importance since MVA is considered as a leading vaccine for HIV/AIDS vaccine following the recent failure of an adenovirus-mediated HIV vaccine trial.