Investigations on the molecular mode of action of the novel immunostimulator ZelNate: Activation of the cGAS-STING pathway in mammalian cells

Bovine respiratory disease (BRD) is usually prevented or treated with vaccines and/or antibiotics. The use of antibiotics is, however, of concern due to the potential promotion of microbial resistance and the occurrence of residues. Recently an alternative aid in the treatment of BRD, the cationic lipid/bacterial plasmid DNA liposome-based immunomodulator ZelNate, has entered the veterinary market. In the present study, we provide data on the molecular mode of action of ZelNate. Despite the presence of numerous non-methylated CpG motifs in its plasmid DNA, ZelNate proved to be inactive on human and mouse toll-like receptor 9 (TLR9) in cell culture, in both recombinant and natural cellular receptor settings. However, in the human monocyte cell line THP1 and in the mouse melanoma cell line B16, ZelNate activates strongly the stimulator of interferon genes (STING) pathway, which is known to lead predominantly to interferon response factor 3 (IRF3) activation. Further analysis in THP1 cells suggests that the ZelNate plasmid DNA activates STING via interaction with cyclic guanylate adenylate synthase (cGAS), but not via interferon induced gene 16 (IFI16). Our in vitro observations suggest that ZelNate may act predominantly via the cGAS/STING/IRF3 pathway.