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. Overall design: Single Cell RNA-Seq of cells from lymph nodes of mice left naïve, or treated with DNA vaccine, mRNA vaccine, or adjuvanted protein vaccine基因载体疫苗在过去数年间重要性与日俱增。然而，针对用于递送DNA尤其是mRNA的脂质纳米颗粒（lipid nanoparticle, LNP）制剂之间的差异，目前尚未得到系统研究。相较于mRNA包封制剂，对LNP包封DNA的表征研究可进一步完善现有的基因递送策略。 本研究针对四组分可电离脂质纳米颗粒33（ionizable LNP33）包封的质粒DNA制剂（DNA-LNPs）展开研究，结果表明该制剂在低剂量下即可诱导强效天然免疫与适应性免疫，其免疫效力与mRNA-LNPs及佐剂蛋白疫苗相当。以携带流感病毒血凝素的编码载体（HA）为模型，我们证实此类DNA-LNPs可通过cGAS-STING-TBK1信号通路驱动强效炎症反应，且该过程不依赖Toll样受体9（TLR9）。 经HA DNA-LNPs预免疫后，迁移性树突状细胞（migratory DC, mDC）亚群被显著激活，同时迁移性树突状细胞与中性粒细胞的比例显著上调。转录组学分析显示，经DNA-LNPs预免疫后，多种先天免疫细胞群体中促迁移因子的表达与激活均显著上调。相较于其他疫苗平台，HA DNA-LNPs可特异性诱导更优异的HA特异性CD8+ T细胞应答。 此外，HA DNA-LNPs可诱导强烈的生发中心应答，尽管其应答频率低于mRNA-LNPs与佐剂蛋白疫苗，但所诱导的血清抗体功能与二者相当。将该研究发现拓展至另一病原体抗原后，我们发现编码新冠病毒（SARS-CoV-2）刺突蛋白的DNA-LNPs可产生与mRNA-LNPs相当的保护效力。 综上，本研究阐明了DNA-LNPs免疫后的激活机制并表征了其免疫表型，为疫苗开发提供了新的研究方向。 整体实验设计：对未免疫的空白对照小鼠，以及经DNA疫苗、mRNA疫苗或佐剂蛋白疫苗免疫的小鼠的淋巴结细胞进行单细胞RNA测序（Single Cell RNA-Seq）