Figure 2.

Molecular mechanisms of DNA vaccines. Transfected double stranded B DNA (dsDNA) is sensed by cytoplasmic DNA receptors such as interferon-inducible protein 16 (IFI16), DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41) and the cGAMP synthase (cGAS), each of which can activate the STING▸TBK1▸IRF3 pathway to induce type 1 interferon production.¹⁴³ An additional cytoplasmic innate immune pathway activated nonspecifically by transfected dsDNA is the cytoplasmic AIM2 inflammasome.¹⁵⁷ Other dsDNA receptors and innate immune activation pathways exist,¹⁴³ including a recently identified STING/IRF7 signaling pathway required for DNA vaccine immunogenicity.¹⁵⁸ By contrast, the endosomal innate immune receptor TLR9 recognizes specific unmethylated CpG DNA motifs in DNA vaccines. To improve innate immune activation, addition of optimized immunostimulatory CpG motifs in the vector backbone may be used to increase TLR9 activation. Immunostimulatory RNA expressed from the vector may be used to activate alternative RNA sensing innate immune receptors such as RIG-I using an additional RNA Polymerase III RNA expression cassette¹¹⁷ (plasmid backbone adjuvant) or incorporation of RNA recognizing TLR agonist motifs such as CpG RNA into the 3′ UTR.¹⁵² Due to limited transgene expression after DNA vaccination in large animals, vector modifications (e.g., <500 bp bacterial region Nanoplasmid™ vectors; intronic bacterial region MIP vectors) and deliveries (e.g., Electroporation) that improve transgene expression also improve adaptive immunity.^62,125,159 Adapted under a Creative Commons Attribution license from Williams, 2013.¹⁶⁰