Cotreatment of primary human monocytes with the NLRP3 inhibitor MCC950, the gasdermin D inhibitor necrosulfonamide or the pan-caspase inhibitor zVAD-FMK abolished the release of RNA-LPX-induced IL-1β (Fig. The production of IL-1β prompted us to ask if RNA-LPX activated the inflammasome pathway. Following RNA-LPX challenge on human peripheral blood mononuclear cells (PBMCs), a broad range of cytokines was detected in both total PBMCs and CD14 + monocytes a notable reduction in cytokine secretion was seen in CD14-depleted PBMCs, indicating that the RNA-LPX-induced cytokine response was dependent on monocytes (Fig.

To identify the factors that contribute to sensitivity to innate immune stimuli, we first studied the unmodified RNA-LPX cancer vaccine, which encodes inherent TLR7/8 agonist activity 1, 12.

Instead, the lipid components used to formulate these vaccines substituted for unmodified RNA in eliciting the IL-1 response. Surprisingly, the reactogenicity of RNA vaccines was not necessarily due to the TLR7/8 agonism, as IL-1 release was observed using vaccines containing N1-methyl-pseudouridine-modified RNA (modRNA). Unlike humans, mice preferentially upregulated anti-inflammatory IL-1ra relative to IL-1, protecting them from uncontrolled systemic inflammation. We uncovered the key role of IL-1 in triggering the release of other pro-inflammatory cytokines associated with cytokine release syndrome (CRS), with humans being markedly more sensitive than mice. We investigated the ability of lipid-formulated RNA vaccines to trigger innate immunity. The mechanisms underlying these dramatic differences have remained largely unknown. Similar observations have been made with other pro-inflammatory stimuli, creating a notable discrepancy in the dose needed to induce biological and toxicological responses in different species 1, 13, 14. Given the obvious size differences, this means that RNA-LPX doses that trigger potent systemic inflammatory responses in humans are more than 1,000-fold lower than in inbred laboratory mice 2. Even at doses of RNA (50 µg) that are well tolerated in mice, patients exhibit transient mild-to-moderate flu-like symptoms that constrain dose exploration to a narrow range and possibly limit optimal T cell responses 2, 4, 5. administration of a liposomal vaccine containing unmodified RNA (RNA-LPX) 1, 12. In contrast to humans, C57BL/6 and Balb/c mice are remarkably tolerant to RNA vaccines and only display limited systemic cytokine release following i.v. These adverse events have been observed irrespective of administration route (intravenous (i.v.) or intramuscular delivery), formulation (liposomes or lipid nanoparticles (LNPs)) or RNA modifications (unmodified uridine or N1-methyl-pseudouridine, which reduces activation of Toll-like receptors 7 and 8 (refs. As a result, RNA vaccines induce systemic elevation of pro-inflammatory cytokines and dose-dependent, transient systemic reactions such as fever and chills 1, 2, 3, 4, 5, 6, 8, 9. RNA-based vaccines activate a range of pattern recognition receptors (PRRs) due to their resemblance to infectious pathogens, thus mobilizing both adaptive and innate anti-viral mechanisms 7. Capable of eliciting exceptionally strong T cell responses 1, 2, 3, RNA has emerged as an attractive vaccine platform not only for cancer therapy, but also for prophylaxis against infectious diseases such as COVID-19 (refs. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.įor adjuvanted vaccines, induction of an innate immune response is essential to generate a protective, long-lasting adaptive immune response. Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses.

IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1β, which is dependent on both the RNA and lipid formulation. Here, we show that the ‘interleukin 1 (IL-1)–interleukin 1 receptor antagonist (IL-1ra)’ axis regulates vaccine-mediated systemic inflammation in a host-specific manner. The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies.

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