Respiratory mucosal immunity is still elevated at 3 months following IN boosting but wanes with time. (A) Vaccination schedule comparison of immune responses at 5 wk or 15 wk after an Ad-ο^IN boost, with an IM prime-only group as a comparison. (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 % ACE-2 competition, which was calculated by using the reduction in measured binding compared with a negative internal control. ELISA titer limit of detection (“Blank”) is noted for reference. (C) Total number (log₁₀) of CD8⁺ and CD4⁺ T cells in the lung (left) and proportion (group median) of T_RM⁺ (CD8⁺: CD69⁺CD103⁺CD62L⁻CD44⁺ and CD4⁺: CD69⁺CD62L⁻CD44⁺) and T_EM⁺ (CD62L⁻CD44⁺CD127⁺ for both CD4⁺ and CD8⁺) cells (right). (D) Total number (log₁₀) of lung-resident memory CD8⁺ and CD4⁺ T cells (CD8⁺: CD69⁺CD103⁺CD62L⁻CD44⁺ and CD4⁺: CD69⁺CD62L⁻CD44⁺) and effector memory CD8⁺ and CD4⁺ T cells (CD62L⁻CD44⁺CD127⁺). (E) Total number (log₁₀) of lymphocytes in BAL fluid (top) and proportion (group median) of CD8⁺ and CD4⁺ T cells, CD19⁺ B cells, and CD138⁺CD19⁻ PCs (bottom). (F) Total number (log₁₀) of CD8⁺ and CD4⁺ T cells in the BAL fluid (left) and proportion (group median) of T_RM⁺ and T_EM⁺ cells (defined as in C) (right). (G) Total number (log₁₀) of BAL-resident memory CD8⁺ and CD4⁺ T cells and effector memory CD8⁺ and CD4⁺ T cells (defined as in C). (H) Proportion of BAL effector memory CD8⁺ and CD4⁺ T cells expressing CXCR3. For all data, *P < 0.05, **P < 0.01, ***P < 0.001, ****P ≤ 0.0001. Parametric t tests or one-way ANOVA tests with multiple comparison testing were performed. On violin plots, the dashed black line represents the group median response and dots represent individual mice. The data shown in this figure are pooled from two independent experiments where test groups “5 WK” and “15 WK” were included (n = 4–5 per group). The “control” test group (n = 5) was uniquely included in one of the independent experiments.