Temporal release of a three-component protein subunit vaccine from polymer multilayers.

Sustained antigen and adjuvant availability have been shown to improve antiviral immune responses following vaccination. Transcutaneous delivery of vaccines using microneedles has also shown promise and may be particularly relevant for mosquito-borne viruses. We aim to combine these traits to create a three-component Protein Subunit vaccine on Microneedle Arrays (PSMNs) for transcutaneous delivery using layer-by-layer (LbL) assembly. Polymer multilayer thin films were generated to co-deliver a model combination of three chemically distinct vaccine components, a dengue virus Envelope protein Domain III (EDIII) subunit antigen and two adjuvants, a double-stranded RNA (Poly (inosinic:cytidylic acid) (PolyI:C)) and an amphiphilic hexapeptide, Pam3CSK4. Following application of PSMNs to the skin, implanted thin films facilitated sustained and temporal release of individual vaccine components from polymer multilayers. By modulating LbL composition and architecture, component release profiles in the skin could be independently tuned to allow release of adjuvants and antigen from days up to two weeks. Uptake of antigen and adjuvant from implanted vaccine films by antigen-presenting cells was demonstrated using in vivo mouse and ex vivo human skin models. Overall, we believe that such modular vaccine strategies offer design principles for enhancing the immunogenicity of protein subunit vaccines.

Erratum: Author Correction: Mimicking immune signatures of flavivirus infection with targeted adjuvants improves dengue subunit vaccine immunogenicity.

[This corrects the article DOI: 10.1038/s41541-019-0119-3.].

Mimicking immune signatures of flavivirus infection with targeted adjuvants improves dengue subunit vaccine immunogenicity.

Neutralizing antibodies (nAbs) are a critical component for protection against dengue virus (DENV) infection, but little is known about the immune mechanisms governing their induction and whether such mechanisms can be harnessed for vaccine development. In this study, we profiled the early immune responses to flaviviruses in human peripheral blood mononuclear cells and screened a panel of toll-like receptor (TLR) agonists that stimulate the same immune signatures. Monocyte/macrophage-driven inflammatory responses and interferon responses were characteristics of flavivirus infection and associated with induction of nAbs in humans immunized with the yellow fever vaccine YF-17D. The signatures were best reproduced by the combination of TLR agonists Pam3CSK4 and PolyI:C (PP). Immunization of both mice and macaques with a poorly immunogenic recombinant DENV-2 envelope domain III (EDIII) induced more consistent nAb and CD4+ T-cell responses with PP compared to alum plus monophosphoryl lipid A. Induction of nAbs by PP required interferon-mediated signals in macrophages in mice. However, EDIII + PP vaccination only provided partial protection against viral challenge. These results provide insights into mechanisms underlying nAb induction and a basis for further improving antigen/adjuvant combinations for dengue vaccine development.

Microneedle-based intradermal delivery of stabilized dengue virus.

Current live-attenuated dengue vaccines require strict cold chain storage. Methods to preserve dengue virus (DENV) viability, which enable vaccines to be transported and administered at ambient temperatures, will be decisive towards the implementation of affordable global vaccination schemes with broad immunization coverage in resource-limited areas. We have developed a microneedle (MN)-based vaccine platform for the stabilization and intradermal delivery of live DENV from minimally invasive skin patches. Dengue virus-stabilized microneedle arrays (VSMN) were fabricated using saccharide-based formulation of virus and could be stored dry at ambient temperature up to 3 weeks with maintained virus viability. Following intradermal vaccination, VSMN-delivered DENV was shown to elicit strong neutralizing antibody responses and protection from viral challenge, comparable to that of the conventional liquid vaccine administered subcutaneously. This work supports the potential for MN-based dengue vaccine technology and the progression towards cold chain-independence. Dengue virus can be stabilized using saccharide-based formulations and coated on microneedle array vaccine patches for storage in dry state with preserved viability at ambient temperature (VSMN; virus-stabilized microneedle arrays).

Flaviviral RNA Structures and Their Role in Replication and Immunity.

More than simple vectors of genetic information, flaviviral RNAs have emerged as critical regulators of the virus life cycle. Viral RNAs regulate interactions with viral and cellular proteins in both, mosquito and mammalian hosts to ultimately influence processes as diverse as RNA replication, translation, packaging or pathogenicity. In this chapter, we will review the current knowledge of the role of sequence and structures in the flaviviral RNA in viral propagation and interaction with the host cell. We will also cover the increasing body of evidence linking viral non-coding RNAs with pathogenicity, host immunity and epidemic potential.

Correction: G3BP1, G3BP2 and CAPRIN1 Are Required for Translation of Interferon Stimulated mRNAs and Are Targeted by a Dengue Virus Non-coding RNA.

[This corrects the article DOI: 10.1371/journal.ppat.1004242.].

Flaviviral RNAs: weapons and targets in the war between virus and host.

Flaviviruses are a genus of (+)ssRNA (positive ssRNA) enveloped viruses that replicate in the cytoplasm of cells of diverse species from arthropods to mammals. Many are important human pathogens such as DENV-1-4 (dengue virus types 1-4), WNV (West Nile virus), YFV (yellow fever virus), JEV (Japanese encephalitis virus) and TBEV (tick-borne encephalitis). Given their RNA genomes it is not surprising that flaviviral life cycles revolve around critical RNA transactions. It is these we highlight in the present article. First, we summarize the mechanisms governing flaviviral replication and the central role of conserved RNA elements and viral protein-RNA interactions in RNA synthesis, translation and packaging. Secondly, we focus on how host RNA-binding proteins both benefit and inhibit flaviviral replication at different stages of their life cycle in mammalian hosts. Thirdly, we cover recent studies on viral non-coding RNAs produced in flavivirus-infected cells and how these RNAs affect various aspects of cellular RNA metabolism. Together, the article puts into perspective the central role of flaviviral RNAs in modulating both viral and cellular functions.

G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3' UTR structures.

Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines.  The DENV-2 positive-strand RNA genome contains 5' and 3' untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins.  In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5' and 3' UTRs as "bait", combined with quantitative mass spectrometry.  We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs.  Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex.  Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3' UTR, possibly by formation of a pseudoknot structure.  Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3' UTR.  These results suggest that the DENV-2 3' UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3' UTR is required for DENV replication.