mRNA-LNP COVID-19 vaccine lipids induce low level complement activation and production of proinflammatory cytokines: Mechanisms, effects of complement inhibitors, and relevance to adverse reactions

Messenger RNA-containing lipid nanoparticles (mRNA-LNPs) enabled widespread COVID-19 vaccination with a small fraction of vaccine recipients displaying acute or sub-acute inflammatory symptoms. The molecular mechanism of these adverse events (AEs) remains undetermined. Here we report that the mRNA-LNP vaccine, Comirnaty, triggers low-level complement (C) activation and production of inflammatory cytokines, which may be key underlying processes of inflammatory AEs. In serum, Comirnaty and the control PEGylated liposome (Doxebo) caused different rises of C split products, C5a, sC5b-9, Bb and C4d, indicating stimulation of the classical pathway of C activation mainly by the liposomes, while a stronger stimulation of the alternative pathway was equal with the vaccine and the liposomes. Spikevax had similar C activation as Comirnaty, but viral or synthetic mRNAs had no such effect. In autologous serum-supplemented peripheral blood mononuclear cell (PBMC) cultures, Comirnaty caused increases in the levels of sC5b-9 and proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1β < TNF-α < IL-6 < IL-8, whereas heat-inactivation of serum prevented the rises of IL-1α, IL-1β, and TNF-α. Clinical C inhibitors, Soliris and Berinert, suppressed vaccine-induced C activation in serum but did not affect cytokine production when applied individually. These findings suggest that the PEGylated lipid coating of mRNA-LNP nanoparticles can trigger C activation mainly via the alternative pathway, which may be causally related to the induction of some, but not all inflammatory cytokines. While innate immune stimulation is essential for the vaccine's efficacy, concurrent production of C- and PBMC-derived inflammatory mediators may contribute to some of the AEs. Pharmacological attenuation of harmful cytokine production using C inhibitors likely requires blocking the C cascade at multiple points. ### Competing Interest Statement The authors affiliated with SeroScience LLC are involved in the contract research service activity of the company providing studies that were applied here. H.F. is an advisor for the companies Behring, Takeda, Pharming, Kalvista, Ono, Pharvaris, Astria, Intellia, and Biocryst.