Defining Composition–Cytokine Relationships Enables the Design of Lipid Nanoparticles with Programmed Immunogenicity

Lipid nanoparticles (LNPs) are central to next-generation vaccines, yet candidate selection remains largely empirical, limiting early identification of formulations associated with rare adverse events such as myocarditis. A better understanding of LNP composition-immunogenicity relationships is therefore critical for rational vaccine design. Here, we profiled a panel of clinically relevant LNP formulations across complementary in vitro and in vivo models to define mechanisms underlying innate immune activation and adaptive responses. We identified three distinct cytokine programs: (i) a monocyte chemoattractant protein-1 (MCP-1)-dominated inflammatory response associated with cytotoxic stress; (ii) inflammasome-dependent interleukin-1 beta (IL-1β) secretion requiring pro-inflammatory priming; and (iii) type I and II interferon-dependent responses in which LNPs synergize with interferon gamma (IFNγ) to amplify interferon gamma-induced protein 10 (IP-10) production. Using a design of experiments (DoE) framework with formulation feature analysis, we found that polyethylene glycol-conjugated (PEGylated) lipid content and ionizable lipid identity are key modulators of the IFNγ/IP-10 axis, previously implicated in vaccine-associated myocarditis. In vivo validation showed that innate cytokine responses are strongly influenced by lipid composition, whereas adaptive humoral and cellular responses correlate with transgene expression rather than cytokine magnitude. Collectively, these findings define relationships among LNP composition, cytokine induction, and vaccine efficacy, and provide a framework for rational design and screening of LNP-based vaccines that maximize immunogenicity while minimizing reactogenicity.