Abstract
Lipid nanoparticles (LNPs) represent the leading delivery platform for mRNA vaccines with advantageous biocompatibility, scalability, adjuvant activity and often an acceptable safety profile. Here we investigate the physicochemical characteristics and adjuvanticity of four-component LNPs. Previous vaccine studies have demonstrated that altering the ionizable lipid influences the adjuvanticity of an LNP; however, the impact of the polyethylene glycol lipid and phospholipid has received less attention. Our mRNA–LNP vaccine formulations utilized different phospholipids and varying ratios of polyethylene glycol lipid, whereas the ionizable lipid and cholesterol remained approximately constant. We demonstrate that such modifications impact the magnitude and quality of the vaccine-elicited immune responses. We also dissect the underlying mechanisms and show that the biodistribution and cellular uptake of LNPs correlate with the magnitude and quality of the immune responses. These findings support the rational design of novel LNPs to tailor immune responses (cellular or humoral focused) based on the vaccine application.
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Data availability
Source data are provided with this paper. The primary datasets for Supplementary Figs. 1–18, along with detailed results of the statistical analyses, are provided in Supplementary Data 1.
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Acknowledgements
We thank the Cell & Developmental Biology Microscopy Core at the University of Pennsylvania. Additionally, the flow cytometry data were generated in the Penn Cytomics and Cell Sorting Shared Resource Laboratory at the University of Pennsylvania and is partially supported by a Abramson Cancer Center NCI Grant (P30 016520). The research identifier number is RRid:SCR_022376. The Pardi laboratory was supported by the National Institute of Allergy and Infectious Diseases (NIAID; R01AI146101, R01AI153064, P01AI158571 and U19AI181968). Z.L. was supported by the National Laboratory for Biotechnology (2022-2.1.1-NL-2022-00008) and the Hungarian Academy of Sciences (Lendület Program Grant (LP2017-7/2017)). K.A.L. and P.B. were supported by R01AI 152236. C.G.R. was supported by the NIH-NCI National Cancer Institute; R01CA283736 (C.G.R. and N.P.), the Parker Institute for Cancer Immunotherapy, 20221408 (C.G.R. and N.P.), and the University of California, Los Angeles, Immunology Advisory Committee (IAC) Award (C.G.R.). We also thank Y. Du, H. Sun and E. L. Prak of the Human Immunology Core (HIC) at the Perelman School of Medicine at the University of Pennsylvania for assistance with the Luminex assays, and M. Eldabbas, E. Maddox, T. Sinha and J. Shu for the preparation of deidentified primary human monocytes. The HIC is supported in part by NIH P30 AI045008 and P30 CA016520. HIC RRID: SCR_022380. We also thank M. Katona at the University of Pittsburgh for guidance in confocal imaging quantification methods; L. Palmer, H. Mahmoud, A. Martin, A. Liu and K. McClintock for their contributions and support throughout this project; and the Proteomics Research Group of the HUN-REN BRC Core Facility for performing the liquid chromatography/mass spectrometry analysis and validation of recombinant proteins.
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N.P. and J.H. conceived the study. N.P. designed the vaccine antigens. H.M. produced the mRNA vaccine antigens. P.S. encapsulated the mRNAs into LNPs and O.D. performed the immunization studies of PEG lipid titration. M.V. designed, performed and analysed the total IgG ELISA, IVIS and Luminex. K.A.L. and P.B. designed, performed and analysed the FRNT assay. B.T.G., M.V. and D.A. designed, performed and analysed the B cell studies. M.V., E.F.D., K.R., H.R.L. and C.G.R. designed, performed and analysed the T cell studies. M.V., W.Z., K.L., V.V.S., V.R.M., E.Á., G.C., A.H.B., K.N., T.M.L., T.L. and C.G.R. performed, designed and analysed the biodistribution studies. E.F.D., J.X., X.H. and M.J.M. designed, performed and analysed the in vitro LNP uptake studies. E.F.D. performed and analysed the IgG subtypes, IgA ELISA and innate cell infiltration to the injection site studies. E.F.D. and W.Z. designed, performed and analysed the endosomal escape assay. W.Z. performed and analysed the LNP protein corona assay. N.D.L., D.C., E.B. and M.L. designed, performed and analysed the in vivo LNP uptake studies. E.Á. and Z.L. produced the recombinant proteins. A.S. and T.M. helped with the mouse blood collection. M.V., K.L., E.F.D. and N.P. wrote the paper with help from co-authors.
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N.P. served on the mRNA strategic advisory board of Sanofi Pasteur in 2022 and Pfizer in 2023–2024. N.P. is also a member of the Scientific Advisory Board of AldexChem and BioNet, and has consulted for Vaccine Company Inc., Optimeos and Pasture Bio. K.L., J.H., O.D., W.Z. and P.S. are employees of Genevant Sciences Corp. and own shares or options of Genevant’s parent company. The remaining authors declare no competing interests.
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Supplementary Figs. 1–18, Tables 1–8, Methods and references.
Source data
Supplementary Data 1
Primary datasets for Supplementary Figs. 1–18, along with detailed results of statistical analyses.
Source Data Fig. 1
Primary datasets for Fig. 1, along with detailed results of statistical analyses.
Source Data Fig. 2
Primary datasets for Fig. 2, along with detailed results of statistical analyses.
Source Data Fig. 3
Primary datasets for Fig. 3, along with detailed results of statistical analyses.
Source Data Fig. 4
Primary datasets for Fig. 4, along with detailed results of statistical analyses.
Source Data Fig. 5
Primary datasets for Fig. 5, along with detailed results of statistical analyses.
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Vadovics, M., Zhao, W., Daley, E.F. et al. Tailoring the adjuvanticity of lipid nanoparticles by PEG lipid ratio and phospholipid modifications. Nat. Nanotechnol. 20, 1312–1322 (2025). https://doi.org/10.1038/s41565-025-01958-5
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DOI: https://doi.org/10.1038/s41565-025-01958-5
