C57BL/6 mice (n = 8/group) received a 1 µg intramuscular injection on days 0 and 28 of lipid inorganic nanoparticle (LION)/replicating RNA (repRNA) encoding either the native conformation of spike derived from A.1, or B.1 viruses, or the pre-fusion-stabilized conformation of spike derived from B.1, B.1.1.7, or B.1.351 viruses, corresponding to repRNAs 600, 675, 676, 673, and 671, respectively. Mice were bled 14 days after the boost immunization and (A) A.1 spike (S)-, S1 domain-, S2 domain-, or receptor-binding domain (RBD)-binding antibody responses measured by enzyme linked immunosorbent assay (ELISA). Neutralizing antibody responses (B–F) measured by 80% plaque reduction neutralization tests (PRNT80) against A.1, B.1, B.1.1.7, or B.1.351 viruses in samples from mice vaccinated with native A.1 (B), native B.1 (C), pre-fusion B.1 (D), pre-fusion B.1.1.7 (E), or pre-fusion B.1.351 (F) spike-derived vaccines. Data in A are presented as geometric means (±geometric standard deviations) along with each individual sample and differences in S-, S1-, S2-, or RBD-binding titers between the 600 A.1 group and other groups were compared by two-way ANOVA (*p < 0.05). Data in B–F are presented as each individual sample connected by black lines with the geometric mean depicted in red and differences between geometric means were compared by Student’s t test.