Fig 1: Overview of the primary experimental design and the B and T cell responses induced by WT-LNP-mRNA vaccination against SARS-CoV-2 WT, B.1.351, and B.1.617 spikes in mice(A) Schematic of the designs of three variant-specific LNP-mRNA vaccine candidates. Functional elements are shown in the spike mRNA and translated protein of SARS-CoV-2 WT, B.1.351, and B.1.617 spikes, including protein domains, HexaPro, and variant-specific mutations.(B) 3D structure highlighting certain variant-specific mutations in B.1.351 and B.1.617 spikes. Distribution of mutations of B.1.351 and B.1.617 are shown in the structure of SARS-CoV-2 (PDB: 6VSB). Mutations of B.1.351 and B.1.617 are shown as spheres, except for those in the unstructured loop regions. Certain mutations are not visible in the structure, as they fall into floppy regions of spike.(C) Graphical representation of B.1.351-LNP-mRNA complex and B.1.617-LNP-mRNA complex formation. The spike mRNAs of B.1.351 and B.1.617 are encapsulated by LNP via NanoAssemblr Ignite. The size and encapsulation rate of the mRNA-LNP complex were measured by dynamic light scatter (DLS) and Ribogreen assay, respectively.(D) After electroporated into 293FT cells, in vitro expression of B.1.351-spike or B.1.617-spike mRNA were detected by flow cytometry using the human ACE2-Fc fusion protein and PE-anti-Fc antibody.(E and F) DLS (E) and TEM (F) of size and monodispersity characterization of LNP-mRNAs.(G) Schematic of overall design of primary experiments. Six- to 8-week-old C57BL/6Ncr mice (B.1.351-LNP-mRNA (top) and B.1.617-LNP-mRNA, n = 6 mice per group; WT-LNP-mRNA, n = 4 mice; PBS, n = 9) received 1 or 10 μg of WT-LNP mRNA, B.1.351-LNP-mRNA, or B.1.617-LNP-mRNA via the intramuscular route on day 0 (Prime) and day 21 (Boost). Blood was collected twice, 2 weeks post-prime and -boost. The binding and pseudovirus-neutralizing antibody responses induced by LNP-mRNA were evaluated by ELISA and neutralization assay. Mice were euthanized at day 40. The spleen, lymph node, and blood samples were collected to analyze immune responses by flow cytometry, bulk BCR, and TCR profiling and single-cell profiling.(H and I) Serum ELISA titers of WT-LNP mRNA-vaccinated animals (n = 4). Serum antibody titer as area under curve (AUC) of log10-transformed curve (1og10 AUC) to spike RBDs (H) and ECDs (I) of SARS-CoV-2 WT, B.1.351, and B.1.617. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance.(J) Serum neutralization titers of WT-LNP mRNA-vaccinated animals (n = 4). Cross neutralization of SARS-CoV-2 WT, B.1.351, or B.1.617 pseudovirus infection of ACE2-overexpressed 293T cells. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance.(K and L) T cell response of WT-LNP mRNA-vaccinated animals (n = 4). CD8+ (K) and CD4+ (L) T cell responses were measured by intracellular cytokine staining 6 h after addition of BFA. The unpaired parametric t test was used to evaluate the statistical significance. Note that in this figure each dot represents data from one mouse. Data are shown as mean ± SEM plus individual data points in dot plots. Statistical significance labels: n.s., not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Source data and additional statistics for experiments are provided in a supplemental excel file. See also Figures S1 and S2.