PROJECT SUMMARY The immunogenicity of nanomaterials is directly related to their performance and toxicity. However, there is still a lack of systematic studies of how nanomaterials interact with the immune system. In this work, we will focus on the immunogenicity of lipid nanoparticles (LNPs). Various studies have raised concerns about the adverse event of LNPs from PEG lipids, ionizable lipids, and even helper phospholipids, such as anti-polyethylene glycol (PEG) antibodies found in BioNTech/Pfizer and Moderna COVID vaccines. During our recent studies of LNP- based mRNA cancer vaccines, the side effects, including ulcerative dermatitis, were also found on vaccinated mice after subcutaneous administrations. Most studies of LNPs via screening or design today have mainly focused on LNP efficacy while few on LNP immunogenicity. Very limited human data on very limited formulations in the context of COVID-19 vaccines following intramuscular administration have very limited scope in immunogenicity such as accessing the overall outcome of immunogenicity only. The investigations have not extensively delved into the underlying causes of the widely reported adverse effects associated with LNP-based mRNA vaccines, the specific immunological pathways of immunogenicity, and the underlying relationship between immunogenicity and components of LNPs. Currently, the mechanism of how LNPs induce adverse events is yet to be fully understood. Here we will formulate three libraries of LNPs covering widely used lipids and vary chemical properties of each component. We will perform immunological assays on primary murine cells to evaluate cytokine secretion and profile inducible gene expression. Similar tests will be performed in human peripheral blood mononuclear cells (hPBMCs). Toll-like receptor (TLR) and NOD-like receptor (NLR)-dependent immune response will be evaluated by reporter assays and validated in deficient mouse primary cells. Furthermore, the immunogenicity of LNPs will be profiled in the setting of intramuscular injections by assessing local inflammatory responses at the injection sites, and intravenous injections by evaluating the systemic response including accelerated blood clearance effects. Through these studies, we will identify key components that are responsible for LNP immunogenicity, understand how the chemistry of each LNP component alters the level of immune activation, and discover any synergistic effects on the immunogenicity among the components. The success of this work will advance current LNP technologies and provide clinical benefits for applications from vaccines to therapeutics.

Project Narrative The objective of the proposed work is to study the immunogenicity of lipid nanoparticles (LNPs) and to map out the relationship between lipid structures and their inflammatory responses. The success of this work will provide a fundamental understanding of the immunogenicity of LNPs, identify lipid components responsible for LNP immunogenicity, and offer principles for the design of low-immunogenicity LNPs for a broad range of applications from vaccines to therapeutics.