N1-methylpseudouridine enhances immunogenicity of RNA/lipidnanoparticle vaccines targeting SARS-CoV-2 spike and the model antigen ovalbumin

The recent success of RNA/lipid nanoparticle (LNP)-based vaccine formulations in the clinic against SARS-CoV-2 and RSV has highlighted the potential of this platform.  While RNA-LNP vaccines share the fundamental principle of using lipid nanoparticles to deliver RNA molecules into host cells to induce antigen expression and antigen-specific immune responses, several factors including RNA type (i.e., messenger, self-amplifying), RNA chemistry and/or nanoparticle formulation can influence the vaccine’s stability, tolerability and overall activity.  Although these differences may influence clinical performance, the lack of head-to-head comparative studies limits the ability to identify which factors have contributed most to the success of the clinically approved vaccines. Herein, we evaluated the influence of N1-methylpseudouridine incorporation into the RNA molecule on the overall immunogenicity of RNA vaccines encoding either the pathogen derived antigen SARS-CoV-2 spike or the model antigen ovalbumin.  Across repeated in vivo mouse studies, we demonstrate that the replacement of canonical uridine with N1-methylpseudouridine in the RNA molecules leads to more immunogenic vaccine formulations, corresponding to higher antigen-specific antibody titers as well as neutralizing activity.  Both types of RNA vaccines induced antigen-specific T cells, with the chemically modified RNA eliciting significantly higher levels of spike-specific CD4+ T cells.  As such, the incorporation of N1-methylpseudouridine can enhance vaccine immunogenicity, providing insight into the activity of previously evaluated formulations and supporting its continued use in vaccines under development.