Nucleoside-modified mRNAs were encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA (pH 4

Nucleoside-modified mRNAs were encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA (pH 4.0) was rapidly mixed with an aqueous solution of lipids (ionizable lipid/distearoylphosphatidylcholine (DSPC)/cholesterol/PEGylated lipid) dissolved in ethanol. for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD)15, formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies. Introduction The emergence of SARS-CoV-2 in late 201916 and the subsequent COVID-19 pandemic highlighted the need for ultrapotent and rapidly scalable vaccine platforms11,17,18. Lipid nanoparticle (LNP)-encapsulated, nucleoside-modified mRNA vaccines encoding prefusion-stabilized, membrane-anchored Spike (S-2P) were found to be safe, effective, and manufacturable at scale, leading to emergency use authorization less than a year after the sequence of the viral genome was available3,5,19C22. These first-generation mRNA vaccines saved many lives and lessened the global health and economic burden of SARS-CoV-223. Although mRNA-LNPs were not the only vaccine modality utilized in response to the COVID-19 pandemic24, the subsequent introduction of several booster vaccines to keep up with emerging immune-evasive viral PX20606 trans-isomer variants25 has further emphasized the sequence-invariant manufacturing advantages of mRNA vaccines26,27. We previously described an with a peptide pool containing overlapping peptides from the Wuhan-Hu-1 SARS-CoV-2 Spike protein. Following stimulation, intracellular staining was performed to detect the production of individual FGFR3 cytokines (Extended Data Fig. 6). For a baseline, we also isolated and stimulated lung and spleen lymphocytes from naive (i.e., unimmunized) mice. As expected, we observed minimal to undetectable antigen-specific CD4 or CD8 T cells in either the lungs or spleens of these mice (Fig. 3b, ?,cc). Open in a separate window Fig. 3. mRNA vaccines induce robust antigen-specific CD8 T cell responses in C57BL/6 mice.a, Study design and groups (ICS, intracellular cytokine staining); n=10 mice/group received either AddaVax-adjuvanted protein (equimolar amounts of RBD: 0.9 g of RBD per dose for Rpk9-based constructs, 5 g of Spike per dose for S-2P-foldon) or nucleoside-modified, LNP-encapsulated mRNA (1 g per dose). b, Percentage of CD4 T cells producing IFN, IL-2, and TNF in response to a Wuhan-Hu-1 SARS-CoV-2 S peptide pool of overlapping 15-mers. PX20606 trans-isomer c, Percentage of CD8 T cells producing IFN, IL-2, and TNF in response to a Wuhan-Hu-1 SARS-CoV-2 S peptide pool of overlapping 15-mers. b-c, Each symbol represents an individual animal. Error bars represent mean SEM. The dotted vertical line separates the protein and mRNA immunized groups. Statistical analyses have been omitted for clarity but can be found in Supplementary Information. With regard to antigen-specific CD4 T cell responses, mRNA-delivered membrane-anchored S-2P induced the highest levels of IFN production in the lungs (~0.8% IFN+ CD4 T cells), although higher levels of IL-2 and TNF production were observed with the protein-delivered vaccines (~1.2C1.7% IL-2+ CD4 T cells and ~1.8C2.6% TNF+ CD4 T cells) (Fig. 3b). In the spleen, mRNA-delivered membrane-anchored S-2P induced the highest levels of IFN, IL-2, and TNF production among all groups (~0.5% IFN+, ~0.4% IL-2+, and ~0.6% TNF+ CD4 T cells). In both the lungs and spleen, we also observed that protein-delivered S-2P-foldon induced slightly higher levels of IFN, IL-2, and TNF production compared to the other adjuvanted protein vaccines, although the differences were not statistically significant. We attribute these observations in part to the fact that the Spike-based immunogens comprise all of the possible T cell epitopes PX20606 trans-isomer in the overlapping peptide library used for stimulation, whereas the RBD-based immunogens comprise only a fraction of the epitopes53,54. With regard to antigen-specific CD8 T cell responses, all three mRNA vaccines induced comparable levels of IFN, IL-2, and TNF production in the lungs (~24C28% IFN+, ~2C4% IL-2+, and ~18C24% TNF+ CD8 T cells), as well as similar PX20606 trans-isomer IL-2 production in the spleen (~1.5C1.6% IL-2+ CD8 T cells) (Fig. 3c). However, PX20606 trans-isomer mRNA-delivered membrane-anchored S-2P induced higher levels.