Ad-WT ^IM +Ad-o ^IN vs. Ad-o ^IN . (A) Vaccination schedule for the comparison of prime-boost Ad-WT^IM+Ad-ο^IN regimen and prime-only Ad-ο^IN regimen. 5 wk after boost, sera, NALT, and BAL fluid were collected. (B) Omicron-specific antibody responses in the serum, NALT, and BAL fluid. Levels of total omicron spike–specific IgG and IgA were measured by standardized ELISA and presented as log₁₀ ELISA units. ACE-2–competing omicron S1-specific antibodies (o-ACE2^comp-Abs) were measured by Luminex assay and presented as % competition, which was calculated by using the reduction in measured binding compared with a negative internal control. Pseudoneutralization of omicron spike–expressing lentivirus (o-NAbs) was presented as log₁₀ IC₅₀, which was calculated from titrated sample curves. (C) Responses to earlier ancestral SARS-CoV-2 ancestral variants alpha (α), beta (β), gamma (γ), delta (δ), and WT in serum. The dashed black line on plots represents the group median response. (D) IFNγ release by splenocytes stimulated with omicron S1 peptides, measured by IFNγ ELISpot assay. For all data, *P < 0.05, **P < 0.01, ***P < 0.001. To compare between groups, parametric t tests were performed. On violin plots, the dashed black line represents the group median response and dots represent individual mice. This experiment was completed once (n = 6 per group).