Matching the prime-boost vaccination route dampens omicron-specific antibody responses induced by a heterologous omicron booster vaccine. (A) Vaccination schedule for the comparison of heterologous IM prime-boost regimens (Ad-WT^IM+Ad-ο^IM and Ad-ο^IM+Ad-WT^IM) and homologous IM prime-boost regimens (Ad-WT^IM+Ad-WT^IM and Ad-ο^IM+Ad-ο^IM); heterologous and homologous experiments were performed separately following identical vaccination schedules. (B–D) Heterologous vaccination experiment was repeated (n = 5–6 per group), with data representative of one repeat experiment (n = 6 per group) present in figures (B–D). Sera were collected from mice in the homologous prime-boost regimens 4 wk after prime to measure prime-only responses, and 3 wk after boost for prime-boost regimens. (B) Levels of total omicron spike–specific IgG were measured by standardized ELISA and presented as log₁₀ ELISA units (EU). ACE-2–competing omicron S1-specific antibodies (o-ACE2^comp-Abs) were measured by Luminex assay and presented as % ACE-2 competition. Pseudoneutralization of omicron spike–expressing lentivirus (o-NAbs) was presented as log₁₀ IC₅₀. Median responses of negative control sera from mice vaccinated twice with an irrelevant vaccine (ChAdOx1-GFP) were included as a green dashed line on graphs (GFP+GFP). (C) Levels of total WT spike–specific IgG, WT-ACE2^comp-Abs, and WT-NAbs were measured, presented as in B. (D) Levels of non–cross-reactive, o-RBD–specific IgG in sera following Ad-WT^IM+Ad-ο^IM and Ad-ο^IM+Ad-WT^IM, as measured through WT spike preabsorption (depletion) assay. The median o-RBD IgG levels in samples that were preincubated with a range of full-length WT spike concentrations are shown on the left. The AUC values are shown on the right. (E) Vaccination schedule for the comparison of heterologous IN prime-boost where the order of immunization of Ad-ο^IN and Ad-WT^IN was reversed. Data are pooled from two independent experiments (n = 5–6 per group per experiment). (F) Levels of total omicron spike–specific IgG, omicron spike–specific IgA, o-ACE2^comp-Abs, and o-NAbs were measured, presented as in B. For IgG, ACE2^comp-Abs and NAbs data in B Ad-WT^IM+Ad-ο^IM were compared against all other groups and (C) Ad-ο^IM+Ad-WT^IM was the comparator. Parametric one-way ANOVA tests were completed when data were normally distributed; otherwise, a nonparametric Kruskal–Wallis test was performed (indicated with a + at the top left of the graph). A parametric t test was used to compare groups in D and F, or a nonparametric Mann–Whitney U test was used (indicated with a + at the top left of the graph). For all data, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P ≤ 0.0001. On violin plots, the dashed black line represents the group median and dots represent individual mice. AUC, area under the curve.