Fig. 5: Pre-treating mice with a 4-fold higher saRNA^Fluc-LNP dose than the subsequent saRNA^HA-LNP vaccine dose affects both anti-HA T cell and antibody responses.

a Replicase pre-exposure and saRNA^HA vaccination schedule showing injection, blood and spleen sampling times. Anti-replicase immunity was induced in BALB/cJRj mice by two intramuscular injections of 4 µg saRNA^Fluc-LNPs (Fluc-HA group, green solid circles) given 3 weeks apart. The reference group, having no pre-existing replicase immunity, received two injections of PBS (PBS-HA group, blue solid upward triangles) 3 weeks apart. Two weeks after the last saRNA^Fluc-LNP injection the mice were vaccinated with 1 µg saRNA^HA-LNPs using a prime-boost schedule with a 3-week interval. Control group only received PBS (black open circles). Blood samples (n = 4) were collected 3 weeks after the saRNA^HA-LNP prime and 2 weeks after the saRNA^HA-LNP boost. Spleens (n = 3) were collected 7 days after the saRNA^HA-LNP boost. b Anti-HA IgG antibody titers (n = 4), c anti-HA IgG1/IgG2a ratio (n = 4) and d hemagglutination inhibition (HI) titers in serum samples (n = 4) collected 3 weeks after the prime and 2 weeks after the saRNA^HA-LNP boost. The HI detection limit was 8 and is indicated as a dashed line, and titers below this threshold are considered as undetectable titers and shown as 1. e The percentage of HA specific IFN-γ⁺ CD4⁺ (left) and IFN-γ⁺ CD8⁺ T cells (right) in splenocytes isolated 1 week after the boost (n = 3). Data are presented as geometric mean with geometric SD (b, d) or mean ± standard deviation (c, e). Statistical analysis was performed using two-way ANOVA (c, d) or one-way ANOVA (b, e) with Tukey’s multiple comparisons test. All samples are biological replicates. Exact P values are indicated above the comparison bars in the figure. All tests were two-sided. The schematic elements in (a) were created with BioRender.com. Source data are provided as a Source Data file.