Heterologous IM boosting of omicron vaccine results in a poor omicron-specific antibody response in alpha spike vaccine–primed mice. (A) Monovalent ChAdOx1 adenovirus vaccines encoding omicron BA.1 spike (Ad-ο) or alpha spike (“Ad-α”) sequences. (B) Vaccination schedule for the comparison of heterologous IM prime-boost regimens (Ad-α^IM+Ad-ο^IM and Ad-ο^IM+Ad-α^IM) and homologous IM prime-boost regimens (Ad-α^IM+Ad-α^IM and Ad-ο^IM+Ad-ο^IM); homologous and heterologous experiments were performed separately following identical schedules. 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. (C) 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. 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^IM+GFP^IM). (D) Levels of total α spike–specific IgG and α-ACE2^comp-Abs were measured and presented as in C. For IgG and ACE2^comp-Abs data in C and D, Ad-α^IM+Ad-ο^IM was compared against all other groups statistically. 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). For all data, **P < 0.01, ***P < 0.001, and ****P ≤ 0.0001. 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).