Fig. 2. Intranasal immunization with Nsp1-K164A/H165A induces mucosal and systemic humoral immunity and cellular immunity.

Male (4-month-old, n = 8) Syrian hamsters were intranasally vaccinated with 10⁴ PFU Nsp1-K164A/H165A. Animals were bled at 14 and 28 DPI to collect sera and n = 4 animals were euthanized at each time point to collect broncho-alveolar lavage fluid (BALF). Anti-RBD IgG titers against WA1/2020 (blue) or BA.1 (red) in serum (a) and BALF (b) were measured by ELISA. Anti-RBD IgA (***p = 0.0002) was likewise quantified in serum (c) and BALF (***p = 0.0003) (d). e Serum nAb titers at 28 DPI were measured against WA1/2020 (WA1), Delta (B.1.617.2), as well as Omicron subvariants (BA.1, BA.2.12.1, BA.4, BA.5) using a 50% focus reduction neutralization (FRNT50) assay. f Splenocytes were isolated from naive and vaccinated hamsters at 14 DPI and pulsed with 10 μM WA1/2020 Spike (S) and Nucleocapsid (N) antigen pools for 48 h. IFNγ-secreting splenocytes were enumerated by ELISPOT. Bar graphs represent mean values with standard deviation for samples collected at two time points from the same animals in a single experiment with dots representing individual animals. *p = 0.0246, **p = 0.0026. ELISPOT data were compared using two-way ANOVA with Sidak’s multiple comparison test. Unless otherwise indicated, statistical differences were calculated using ordinary one-way analysis of variance (ANOVA) in GraphPad Prism 9.4.0 with Tukey’s multiple comparisons tests. For statistical significance, *p < 0.05, **p < 0.01, and ****p < 0.0001. DPI days post-infection.