Fig. 4: High-dose (HD) aerosol ChAd vaccine induces greater magnitude and broader antigen-specific T cell responses over the LD in the airways of mRNA-vaccinated individuals with prior SARS-CoV-2 infection.

a, b Total cell counts and differential cell type frequencies in BALF from low dose (LD) and high dose (HD) ChAd vaccine cohorts. c, d Antigen-specific CD8 and CD4 responses assessed after stimulation with S1, NC, or POL peptide pools. Scatter plots compare total numbers of cytokine + (calculated using Boolean gating, which sums non-overlapping single and multiple cytokine producers) CD8 (c) and CD4 (d) T cells. e Pie charts depict mean frequencies of S1- and NC-specific CD8 and CD4 T cells for 3+, 2+, and 1+ cytokines, out of total CD8 and CD4 T cells, identified by the Boolean operator on single IFN-γ + /TNF + /IL-2 + expressing cells at 4 wk post-vaccination in the HD cohort. f Dot plots show CD8 + CD69 + T cells gated for CD103 and IFN-γ after mega pool stimulation. Scatter plot compares frequencies of tissue-resident memory CD8 T cells (CD69 + CD103 + IFN-γ +) among total IFN-γ + CD8 + T cells across cohorts. g t-SNE plots show airway VLA-4 + (CD29 + CD49d +) CD8 T cell distribution at baseline (0 wk) and 4 wk post-vaccination in the HD cohort. Dimensionality reduction performed in FlowJo on live BAL cells stimulated with the mega pool. Colours indicate T cell subset: Green: (CD8 +), orange (CD8 + VLA-4 + IFN-γ −), red: (CD8 + VLA-4 + IFN-γ +). Scatter plot compares frequencies of both antigens-specific and non-specific CD8 + VLA-4 + T cells. h Cross-reactivity of HD ChAd-induced S1-specific CD8 T cells with Omicron XBB.1.5 assessed by peptide stimulation. Dot plots and pie charts show multifunctional profiles; scatter plot compares ancestral vs XBB.1.5-specific responses. Each data point in all panels represents one participant. Three participants for LD and six for HD. Lung-homing molecules were assessed in five participants in the HD cohort, due to cell limitations. XBB.1.5 cross-reactivity was evaluated in three participants in HD cohort. All scatter plots show the median (horizontal line). The statistical differences between groups were determined using ordinary two-way ANOVA with Šidák multiple comparisons test for a and on Log-transformed data for (c, d, e, and h). Two-tailed Mann–Whitney test was used to analyze statistical difference between vaccine doses for (f) and timepoints for (g). Significant differences (p < 0.05) are shown. Source data are provided as a Source Data file.