with an individual dose of 30?g of mRNA-LNP vaccines. (ACC) Spleen and YH249 lungs were harvested and stimulated with SARS-CoV-2?S protein peptide pools 10?days after immunization. demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T?cell responses, as well as long-lived plasma and memory B?cell responses. Additionally, we detect strong and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of contamination Characterization of SARS-CoV-2 Nucleoside-Modified mRNA Constructs We designed and produced mRNAs encoding three potential SARS-CoV-2 vaccine antigens: full-length S protein (wild-type [WT]), full-length S protein with a deleted furin cleavage site (furin), and a short construct encoding the soluble RBD of S?protein. The furin mutant was included as a potential way to stabilize the full-length S and to maintain the covalent association of the S1 and S2 subunits (Kirchdoerfer et?al., 2016), while the RBD was investigated as it is a critical target of neutralizing antibodies against SARS-CoV-2. Protein expression from mRNAs was confirmed by cell transfection studies. RBD protein secretion was exhibited by ELISA using supernatant from RBD mRNA-transfected 293F cells YH249 (Physique?1 A). YH249 Because the full-length WT and furin S proteins contain the transmembrane domain name, they were expressed on the surface of transfected 293F cells. Thus, we used circulation cytometry to assess binding of full-length WT and furin S proteins by an anti-RBD monoclonal antibody, D001, and a human ACE2-Fc (hACE2-Fc) fusion protein. Interestingly, we found that the full-length furin S protein showed higher binding capacity to D001 and hACE2-Fc compared to its WT counterpart, indicating that it may be a better vaccine antigen, due either to higher expression or favorable antigenicity (Physique?1B). Therefore, we selected the full-length furin construct to evaluate in immunization studies along with RBD. Open in a separate window Physique?1 Characterization of SARS-CoV-2 Nucleoside-Modified mRNA Constructs (A) Supernatant from 293F cells transfected with RBD-encoding mRNA or mock was tested for binding reactivity to D001 and hACE2-Fc by ELISA. Data shown are area under curve of the log-transformed concentrations (log AUC). Symbols represent independent experiments. (B) 293F cells were transfected with mRNA encoding SARS-CoV-2 full-length WT and furin S protein. Binding reactivity of full-length WT and furin S proteins to D001, hACE2-Fc, and unfavorable control CH65 (an anti-influenza YH249 neutralizing antibody) was measured by circulation cytometry. Binding capacity was expressed in mean fluorescence intensity (MFI). Each dot represents an independent experiment. p?value indicates a paired t test; ?p?< 0.05. Data symbolize imply plus SEM. SARS-CoV-2 mRNA Vaccines Induce Strong T Cell Responses in the Spleen and Lungs BALB/c mice were injected with a single i.m. dose of 30?g of mRNA-LNPs encoding full-length furin, RBD, or firefly luciferase (Luc, negative control) mRNA-LNPs, and S protein-specific CD4+ and CD8+ T?cell responses were evaluated after 10?days by intracellular cytokine staining (Figures 2 , S1, and S2). Both spike mRNA constructs elicited antigen-specific, polyfunctional CD8+ (Physique?2A) and CD4+ (Physique?2B) T?cells expressing type 1 (Th1) immune response cytokines (interferon [IFN]-, tumor necrosis factor [TNF], and interleukin [IL]-2) as?well as CD8+ T?cells with cytotoxic markers (granzyme B+?CD107a+) (Physique?2C) in both the spleen and lungs. These responses were particularly strong in the lungs, especially for CD8+ T?cells. We also noted that the vast majority of the CD8+ T?cell response in BALB/c mice was directed at epitopes in the N-terminal half of the S protein, while CD4+ T?cells recognized epitopes in both halves of the protein (Figures S2A and S2B). Because S protein-specific PR52B lung-infiltrating T?cell responses may contribute to SARS-CoV-2 YH249 vaccine protection as seen with SARS-CoV-1 (Zhao et?al., 2016), we next examined whether vaccine-induced lung T?cells were truly infiltrating into the lung parenchyma. We performed intravenous (i.v.) labeling with a CD45-specific antibody in order to differentiate between vascular (i.v. label-positive).