Plasmid DNA or mRNA formulated in lipid nanoparticles drive unique innate immune activation to promote adaptive immunity

Gene-vectored vaccines grew in importance over the past several years. However, understanding the differences between of lipid nanoparticle (LNP) formulations for delivering DNA and mRNA in particular has not been studied. Characterization of LNP-formulated DNA compared with mRNA could build upon current genetic delivery approaches. Here, we study a four-component ionizable LNP33 plasmid DNA formulation (DNA-LNPs) which we demonstrate induces potent innate and adaptive immunity at low doses with similar potency to mRNA-LNPs and adjuvanted protein. Using an influenza virus hemagglutinin-encoding construct (HA), we show that these DNA-LNPs drive potent inflammation dependent on the cGAS-STING-TBK1 pathway but independent of TLR9. Priming with HA DNA-LNP demonstrated robust activation in migratory DC (mDC) subpopulations and significant upregulation of mDCs and neutrophils. Transcriptomics elucidated activation and upregulation of pro39 migration factors among multiple innate immune populations after priming with DNA-LNP. HA DNALNP uniquely induced superior HA-specific CD8+ 40 T cell responses relative to other platforms. HA DNA41 LNP additionally induced robust germinal center responses attenuated in frequency to mRNA-LNPs and adjuvanted protein, but with equivalent functional serum antibodies. Extending these findings to an additional pathogen antigen, SARS-CoV-2 spike-encoding DNA-LNP elicited protective efficacy comparable to spike mRNA-LNPs. Thus, this study identifies priming mechanisms and characterizes immune phenotypes after DNA-LNP immunization, suggesting additional avenues for vaccine development.