title: Recombinant modified vaccinia virus Ankara generating excess early double-stranded RNA transiently activates protein kinase R and triggers enhanced innate immune responses.
abstract: Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-β in murine and human cells in the presence of an intact E3L gene. IFN-β induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-α, IFN-γ, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo.
  
  
  OBJECTIVE
  Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector.
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Wolferstätter
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Schweneker
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Späth
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Lukassen
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Marieken
authors.initials: M
authors.lastname: Klingenberg
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Kay
authors.initials: K
authors.lastname: Brinkmann
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Wielert
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Lauterbach
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Hubertus
authors.initials: H
authors.lastname: Hochrein
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Chaplin
authors.affiliation: University of Zurich, Zurich, Switzerland.
authors.forename: Mark
authors.initials: M
authors.lastname: Suter
authors.affiliation: Infectious Diseases Division, Bavarian Nordic GmbH, Martinsried, Germany juergen.hausmann@bavarian-nordic.com.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hausmann
mesh_terms: D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D012330:RNA, Double-Stranded / Q000276:immunology* / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics / Q000276:immunology*; D019892:eIF-2 Kinase / Q000276:immunology* / Q000378:metabolism
chemical_list: D016207:Cytokines; D012330:RNA, Double-Stranded; D019892:eIF-2 Kinase