Dear Editor, The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need to develop effective and safe vaccines. Similar to SARS-CoV, SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as receptor for host cell entry. 1, 2 SARS-CoV-2 spike (S) protein consists of S1, including receptor-binding domain (RBD), and S2 subunits. 3, 4 We previously demonstrated that RBDs of SARS-CoV and MERS-CoV serve as important targets for the development of effective vaccines. 5, 6 To identify an mRNA candidate vaccine, we initially designed two mRNA constructs expressing S1 and RBD, respectively, of SARS-CoV-2 S protein (Fig. 1 a). Both culture supernatants and lysates of cells transfected with S1 or RBD mRNA reacted strongly with a SARS-CoV-2 RBD-specific antibody (Supplementary information, Fig. S1a), demonstrating expression of the target proteins.

To detect whether S1 and RBD mRNAs durably express antigens in multiple cell types, we constructed N-terminal mCherry-tagged SARS-CoV-2 S1 and RBD mRNAs, encapsulated them with lipid nanoparticles (LNPs)(Supplementary information, Fig. S1b), and tested mCherry expression. Relative to the control, both RBD-and S1-mCherry mRNAs showed robust protein expression in cells for at least 160h, with higher expression of the RBD construct (Supplementary information, Fig. S2a). In addition, these mRNAs expressed proteins efficiently in a variety of human (A549, Hep-2, HEP-G2, Caco-2, HeLa, 293T), monkey (Vero E6), and bat (Tb1-Lu) cell lines (Supplementary information, Fig. S2b). Particularly, the expression of RBD-mCherry protein was higher than that of S1-mCherry protein in all cell lines tested (Supplementary information, Fig. S2b). These data indicate long-term and broad expression of mRNA-encoding proteins, particularly RBD, in target cells. We then characterized LNP-encapsulated S1 and RBD mRNAs for stability and subcellular localization. The mCherry-tagged S1 and RBD showed strong and stronger fluorescence intensity, respectively, irrespective of incubation temperature (4 or 25 C) and culture time (0, 24, or 72h)(Supplementary information, Fig. S3a). S1-and RBD-mCherry proteins were not colocalized with nuclei but associated with lysosomes (Supplementary information, Fig. S3b). These results suggest that LNP-encapsulated SARS-CoV-2 S1 and RBD mRNAs are stable at various temperatures and may be resistant to lysosomal degradation. We next evaluated T follicular helper (Tfh), germinal center (GC) B, and plasma cell responses induced by SARS-CoV-2 S1 and RBD mRNA-LNPs in BALB/c mice. Mice were intradermally (ID) prime and boost immunized with each mRNA-LNP (30 μg/mouse) or empty LNP control, and draining lymph nodes or spleens were tested for Tfh, GC B, or plasma cells 10 days post-2nd immunization (Supplementary information, Fig. S4a). The percentages of Tfh cells (Supplementary information, Fig. S5a) and GC B cells (Supplementary information, Fig. S5b) were higher or