Extending the prime-boost interval inhibits the efficacy of intramuscular Ad-omicron boost. (A) Vaccination schedule to vary the interval between prime and boost. Mice were given Ad-WT^IM+Ad-ο^IM with either 4 or 17 wk between the prime and boost. Serum was collected 3–5 wk after the boosting immunization. Data are pooled from two independent experiments (n = 4–5 per group per experiment). (B) WT and omicron-specific IgG antibody responses in the serum measured by standardized ELISA and presented as log₁₀ ELISA units. (C) Omicron-specific antibody responses in the serum. Levels of total omicron spike–specific IgG were measured by standardized ELISA and presented as log₁₀ ELISA units. (D) Omicron-specific antibody responses in the serum before and after Ad-ο^IM boosting immunization. Preboost is defined as 1 day before boost vaccination. After boost is defined as 3–5 wk postboost vaccination. Data in B were analyzed by paired one-way ANOVA, in C were analyzed by t test, and in D by mixed-effects ANOVA. *P < 0.05, **P < 0.01, ****P < 0.01. On violin plots, the dashed black line represents the group median response and dots represent individual mice.