ERP149716 PRJEB64537 E-MTAB-13194 Reducing cell intrinsic immune responses to mRNA vaccine constructs improves adaptive immune response in mice The response to mRNA vaccines needs to be sufficient for immune cell activation and recruitment but moderate enough to ensure efficacious antigen expression. The choice of the cap structure and use of N1-methylpseudouridine (m1?) instead of uridine, which have been shown to reduce RNA sensing by the cellular innate immune system, has led to improved efficacy of mRNA vaccine platforms. Understanding how RNA modifications influence the cell intrinsic immune response may help in the development of more effective mRNA vaccines. In the current study, we compared mRNA vaccines in mice against influenza virus using three different mRNA formats: uridine-containing mRNA (D1-uRNA), m1?- modified mRNA (D1-modRNA), and m1?-modified mRNA with a cap1 structure (cC1-modRNA). D1-uRNA vaccine induced a significantly different gene expression profile to the modified mRNA vaccines, with an upregulation of Stat1 and RnaseL, and increased systemic inflammation. This correlated with a significantly reduced antigen specific antibody responses and reduced protection against influenza virus infection compared to D1-modRNA and cC1-modRNA. Incorporation of m1? alone without cap1 improved antibodies, but both modifications were required for the optimum response. Therefore, the incorporation of m1? and cap1 alters protective immunity from mRNA vaccines by altering the innate immune response to the vaccine material The response to mRNA vaccines needs to be sufficient for immune cell activation and recruitment but moderate enough to ensure efficacious antigen expression. The choice of the cap structure and use of N1-methylpseudouridine (m1?) instead of uridine, which have been shown to reduce RNA sensing by the cellular innate immune system, has led to improved efficacy of mRNA vaccine platforms. Understanding how RNA modifications influence the cell intrinsic immune response may help in the development of more effective mRNA vaccines. In the current study, we compared mRNA vaccines in mice against influenza virus using three different mRNA formats: uridine-containing mRNA (D1-uRNA), m1?- modified mRNA (D1-modRNA), and m1?-modified mRNA with a cap1 structure (cC1-modRNA). D1-uRNA vaccine induced a significantly different gene expression profile to the modified mRNA vaccines, with an upregulation of Stat1 and RnaseL, and increased systemic inflammation. This correlated with a significantly reduced antigen specific antibody responses and reduced protection against influenza virus infection compared to D1-modRNA and cC1-modRNA. Incorporation of m1? alone without cap1 improved antibodies, but both modifications were required for the optimum response. Therefore, the incorporation of m1? and cap1 alters protective immunity from mRNA vaccines by altering the innate immune response to the vaccine material E-MTAB-13194 in ArrayExpress https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-13194 ENA-FIRST-PUBLIC 2023-10-03 ENA-LAST-UPDATE 2023-10-03