Heterologous mucosal boosting with an omicron vaccine elicits a strong local mucosal and systemic antibody response, and lung T cell response. (A) Monovalent ChAdOx1 adenovirus vaccines encoding omicron BA.1 spike (“Ad-o”) or original WT spike (“Ad-WT”) sequences. (B) Vaccination schedule for the comparison of heterologous IM or IN boosting of omicron vaccine in WT vaccine–primed mice. 5 wk after boost, sera, NALT, BALF, lungs, and spleens were collected for analyses. (C) 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 % ACE-2 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 sample titration curves. Group median responses following one Ad-o^IN prime were represented on graphs as a dashed blue line, with complete data in Fig. S1. (D) Responses to earlier SARS-CoV-2 “ancestral” variants alpha (α), beta (β), gamma (γ), delta (δ), and WT in serum and NALT fluid. The dashed black line on plots represents the group median response. (E) Levels of non–cross-reactive, o-RBD–specific IgG in sera following IM and IN boosting, as measured through WT spike preabsorption (depletion) assay. The median o-RBD IgG levels in samples that were preincubated with a range of WT spike concentrations is shown at the top of the panel. The AUC values are shown on the violin plot positioned below. (F) Total number (log₁₀) of class-switched lung B cells (IgD⁻IgM⁻CD19⁺CD45^IV−) as measured by flow cytometry. (G) Frequencies of o-RBD probe–specific lung PCs (CD19⁻CD138⁺IgD⁻IgM⁻o-RBD⁺) and o-RBD probe–specific lung-resident B cells (CD45^IV−CD19⁺IgD⁻IgM⁻o-RBD⁺). (H) Total number (log₁₀) of PD-L2-, CD73-, and CD80-expressing lung B cells (CD19⁺CD45^IV−). (I) Frequency of lung cells and splenocytes that released detectable IFNγ following stimulation with omicron S1 peptides (antigen-specific), measured by IFNγ ELISpot assay. (J) Total number (log₁₀) of lung-resident memory CD8⁺ (CD45^IV−CD69⁺CD103⁺CD62L⁻CD44⁺) and CD4⁺ T cells (CD45^IV−CD69⁺CD62L⁻CD44⁺). (K) Total number (log₁₀) of lung CD8⁺ and CD4⁺ T_EM (CD62L⁻CD44⁺CD127⁺). Statistically significant differences between groups in all figures were determined through parametric t tests or nonparametric Mann–Whitney tests (*P < 0.05, **P < 0.01, ***P < 0.001); when data did not follow a normal distribution, a + was added to the left corner of the graph. On violin plots, the dashed black line represents the group median and dots represent individual mice. The results in this figure are representative of two independent vaccination experiment repeats (repeat displayed in figure: n = 6 per group, and independent repeat: n = 5 per group). AUC, area under the curve.