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      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.

          Author Summary

          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.

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          Most cited references55

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          Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.

          Interferons (IFNs) are critical for protection from viral infection, but the pathways linking virus recognition to IFN induction remain poorly understood. Plasmacytoid dendritic cells produce vast amounts of IFN-alpha in response to the wild-type influenza virus. Here, we show that this requires endosomal recognition of influenza genomic RNA and signaling by means of Toll-like receptor 7 (TLR7) and MyD88. Single-stranded RNA (ssRNA) molecules of nonviral origin also induce TLR7-dependent production of inflammatory cytokines. These results identify ssRNA as a ligand for TLR7 and suggest that cells of the innate immune system sense endosomal ssRNA to detect infection by RNA viruses.
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            RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates.

            Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5'-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.
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              Differential Roles of TLR2 and TLR4 in Recognition of Gram-Negative and Gram-Positive Bacterial Cell Wall Components

              Toll-like receptor (TLR) 2 and TLR4 are implicated in the recognition of various bacterial cell wall components, such as lipopolysaccharide (LPS). To investigate in vivo roles of TLR2, we generated TLR2-deficient mice. In contrast to LPS unresponsiveness in TLR4-deficient mice, TLR2-deficient mice responded to LPS to the same extent as wild-type mice. TLR2-deficient macrophages were hyporesponsive to several Gram-positive bacterial cell walls as well as Staphylococcus aureus peptidoglycan. TLR4-deficient macrophages lacked the response to Gram-positive lipoteichoic acids. These results demonstrate that TLR2 and TLR4 recognize different bacterial cell wall components in vivo and TLR2 plays a major role in Gram-positive bacterial recognition.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                June 2009
                June 2009
                19 June 2009
                : 5
                : 6
                : e1000480
                Affiliations
                [1 ]Infectious Diseases Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland
                [2 ]Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
                [3 ]Department of Biochemistry, University of Lausanne, Epalinges, Switzerland
                [4 ]Centro Nacional de Biotecnología, CSIC, Ciudad Universitaria Cantoblanco, Madrid, Spain
                [5 ]Laboratory of AIDS Immunopathogenesis, Immunology and Allergology Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland
                University of Florida, United States of America
                Author notes

                Conceived and designed the experiments: JD TR QGST TC. Performed the experiments: JD DLR MKR. Analyzed the data: JD TR QGST DLR TC. Contributed reagents/materials/analysis tools: JD TR QGST SA VP CEG BP JT GP ME TC. Wrote the paper: JD TR ME TC.

                Article
                08-PLPA-RA-1419R3
                10.1371/journal.ppat.1000480
                2691956
                19543380
                553fe4e2-754f-4e31-be32-34ae040b3582
                Delaloye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 12 November 2008
                : 21 May 2009
                Page count
                Pages: 15
                Categories
                Research Article
                Immunology
                Immunology/Immune Response
                Immunology/Immunity to Infections
                Immunology/Innate Immunity
                Infectious Diseases
                Infectious Diseases/HIV Infection and AIDS
                Infectious Diseases/Viral Infections
                Virology/Effects of Virus Infection on Host Gene Expression
                Virology/Host Antiviral Responses
                Virology/Immune Evasion
                Virology/Vaccines

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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