Fig. 5: High-dose (HD) aerosol ChAd vaccine boosts mucosal antibody responses and induces trained innate immunity in alveolar macrophages in the airways of mRNA-vaccinated individuals with prior SARS-CoV-2 infection.

a, b Antigen-specific airway antibody responses to HD ChAd vaccine. Concentrated BALF collected at baseline (0 wk) and at 4 wk post-vaccination were subject to ELISAs using ancestral spike and RBD to generate reciprocal dilution curves. Area under curve (AUC) values for spike- and RBD-specific IgG compared between baseline and 4 wk. c, d AUC values for IgA specific to both spike and RBD are compared in concentrated BALF between baseline (0 wk) and 4 wk post-vaccination using ELISA. e Antibody-mediated neutralization assessed by a Surrogate Virus Neutralization Test (sVNT) against the ancestral RBD (left) and Omicron XBB.1.5 RBD (right). f Principal component analysis (PCA) of gene expression in alveolar macrophages (AM) from three individuals before (0 wk) and 4 wk after HD ChAd vaccination cultured with (WCL) or without (US) stimulation. Samples from the same individual are connected by colored lines. PCA visualization performed using rgl package in R. g Significantly enriched top 10 canonical pathways associated with uniquely upregulated and downregulated DEGs in WCL-stimulated 4 wk aerosol AM (Group 4/Group 2) when compared with WCL-stimulated baseline 0 wk AM (Group 3/Group 1). Horizontal bars: different pathways based on Z-scores. Red color: upregulated pathways; blue color indicates downregulated ones. Differential expression analysis performed using limma package and from each comparison significant genes (adjusted p value < 0.05) showing absolute regulation of at least 1.5-fold were selected. Obtained gene lists analyzed using STRING database to obtain lists of significantly over-represented pathways. h Heatmap shows differential-expressed genes associated with 3 selected pathways among top 10 pathways significantly enriched in WCL-stimulated 4 wk aerosol AM (Group 4/Group 2) over WCL-stimulated baseline 0 wk AM (Group 3/Group 1). i Heatmap shows 10 highly upregulated and downregulated genes unique to 4 wk WCL-stimulated inhaled aerosol vaccinated AM (Group 4)/Group 2 compared to WCL-stimulated baseline 0 wk AM (Group 3/Group 1). Each data-point in panels represents one participant. The statistical differences between groups were determined on Log-transformed data using a paired one-tailed t-test (a–e). Each data point represents a single participant in (c–e). Data are presented as mean values +/− SEM by the shaded area (error bands) in reciprocal dilution graphs. Source data are provided as a Source Data file.