Fig. 2

RNH1-S1139 elicits long-term and cross-reactive antibodies and is safe in animal models. A Immunization strategy and bleeding schedule of mice. S1139-specific IgG titer measured by ELISA. B Titers of IgG1 and IgG2a antibody isotypes in immune serum measured by ELISA. C Binding antibody titers of RBD or RNH1-S1139 immunization group against S trimers from MERS-CoV, SARS-CoV and SARS-CoV-2 and its variants. Values were detected by ELISA and normalized to the SARS-CoV-2 S trimer group. D ELISA assay showing changes of binding antibody titers elicited by RNH1-S1139 against S1139 epitopes with single point mutation in S1139 or epitopes from different coronavirus strains. Each line represents a biological replicate. E Left, the crystal structure of mAb B6 Fab in complex with SARS-CoV-2 stem helix peptide (part of S1139). Middle, zoomed-in view showing how epitope forms salt bridge with Fab heavy and light chain (PDB 7M53). Structural model was prepared by PyMol. Right, superposition of S1139 epitope and D1153Y substitution. F Tissues of mice heart, liver, spleen, lung, kidney, brain, and muscle collected from PB, low-dose RNH1-S1139 (5 μg/Ms) and high-dose RNH1-S1139 (50 μg/Ms) groups 3 and 60 days post initial immunization were stained with H&E or Sirius red. N = 6 mice per group; data are presented as mean values with SD in A and B. p values were determined by One-way ANOVA followed by Dunnett’s multiple comparison test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001