Live attenuated CHIKV 181/25 vaccine can be delivered in vitro and in vivo by using DNA vaccination. The iDNA approach appears to represent a promising vaccination strategy for CHIK and other alphaviral diseases.
DNA vaccines combine remarkable genetic and chemical stability with proven safety and efficacy in animal models, while remaining less immunogenic in humans. In contrast, live-attenuated vaccines have the advantage of inducing rapid, robust, long-term immunity after a single-dose vaccination. Here we describe novel iDNA vaccine technology that is based on an infectious DNA platform and combines advantages of DNA and live attenuated vaccines. We applied this technology for vaccination against infection with Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The iDNA vaccine is based on transcription of the full-length genomic RNA of the TC-83 live-attenuated virus from plasmid DNA in vivo. The in vivo-generated viral RNA initiates limited replication of the vaccine virus, which in turn leads to efficient immunization. This technology allows the plasmid DNA to launch a live-attenuated vaccine in vitro or in vivo. Less than 10 ng of pTC83 iDNA encoding the full-length genomic RNA of the TC-83 vaccine strain initiated replication of the vaccine virus in vitro. In order to evaluate this approach in vivo, BALB/c mice were vaccinated with a single dose of pTC83 iDNA. After vaccination, all mice seroconverted with no adverse reactions. Four weeks after immunization, animals were challenged with the lethal epidemic strain of VEEV. All iDNA-vaccinated mice were protected from fatal disease, while all unvaccinated controls succumbed to infection and died. To our knowledge, this is the first example of launching a clinical live-attenuated vaccine from recombinant plasmid DNA in vivo.
In eukaryotes, the nuclear export of mRNA is mediated by nuclear export factor 1 (NXF1) receptors. Metazoans encode additional NXF1-related proteins of unknown function, which share homology and domain organization with NXF1. Some mammalian NXF1-related genes are expressed preferentially in the brain and are thought to participate in neuronal mRNA metabolism. To address the roles of NXF1-related factors, we studied the two mouse NXF1 homologues, mNXF2 and mNXF7. In neuronal cells, mNXF2, but not mNXF7, exhibited mRNA export activity similar to that of Tipassociated protein/NXF1. Surprisingly, mNXF7 incorporated into mobile particles in the neurites that contained poly(A) and ribosomal RNA and colocalized with Staufen1-containing transport granules, indicating a role in neuronal mRNA trafficking. Yeast two-hybrid interaction, coimmunoprecipitation, and in vitro binding studies showed that NXF proteins bound to brainspecific microtubule-associated proteins (MAP) such as MAP1B and the WD repeat protein Unrip. Both in vitro and in vivo, MAP1B also bound to NXF export cofactor U2AF as well as to Staufen1 and Unrip. These findings revealed a network of interactions likely coupling the export and cytoplasmic trafficking of mRNA. We propose a model in which MAP1B tethers the NXF-associated mRNA to microtubules and facilitates their translocation along dendrites while Unrip provides a scaffold for the assembly of these transport intermediates.
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