Fig 2: Challenging ChAdOx1-NP + M1-RSVF-vaccinated mice with H1N1.a Vaccination and challenge schematic for the assessment of the protective capacity of ChAdOx1-NP + M1-RSVF against H1N1 infection and subsequent disease in mice. Mice were prime-boost-vaccinated, then challenged with H1N1, and culled 5 days later with tissues and fluids harvested. Blood sampling was performed 4 weeks post-priming and 3 weeks post-boosting. b Weight change in mice over time post-challenge, as measured by % of pre-challenge weight. The significant difference between IM-IN and IM-IM mouse groups is represented with ## (##=p < 0.01). c Viral load in lungs 5 days post-challenge (number of M gene copies/μg lung RNA (log10)). d H1N1 NP-specific IgG and IgA levels in serum, NWs, BALF and LHS post-challenge, as measured ELISA (log10 EU). Median control values are displayed as dashed lines on graphs. e Levels of antigen-specific CD8+ TRM cells, and relative levels of antigen-specific CD8+ TEM and TRM, in BAL and lungs post-challenge. TRM and TEM cells were defined as CD3+CD8+CD44+CD62L-CD103+CD69+, and CD3+CD8+CD44+CD62L-, respectively, and positive for influenza pentamer H-2Kd TYQRTALV. In boxplots, a “+” symbol represents the group mean. Two mice in group IM-IM did not have detectable BAL TRM, and one mouse in group IM-IM did not have detectable Lung TRM. Group differences for all data were analysed using non-parametric Kruskal-Wallis tests (*=p < 0.05, **=p < 0.01, ***=p < 0.001). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Fig 3: Cellular immunogenicity of ChAdOx1-NP + M1-RSVF.a Cytokine responses in CD4+ and CD8+ splenocytes that were separately stimulated with influenza A (H1N1) NP + M1- and RSVF-spanning peptides; % cytokine+ T cells were determined through intracellular staining. Basal frequencies of cytokine+ T cells (unstimulated sample) were subtracted from stimulated sample frequencies. Median responses in each group are displayed as the top lines of bars on graphs on the left-hand side of the figure, with symbols representing individual mice, grey-shaded bars prime-boost regimens and white bars prime-only regimens. b Cytokine responses in CD4+ and CD8+ lung cells harvested from mice, separately stimulated with influenza A (H1N1) NP + M1- and RSVF-spanning peptides. c Vaccination schematic for the continued assessment of the cellular immunogenicity of ChAdOx1-NP + M1-RSVF. d IFNγ responses in splenocytes stimulated with NP + M1- and RSVF-spanning peptides (IFNγ spot-forming cells (SFCs)/million splenocytes), as measured by IFNγ enzyme-linked immunosorbent spot (ELISpot) assay. e Total counts of CD8+ TRM cells (left), as well as relative counts of CD8+ TEM and TRM cells (right) in lungs post-vaccination (all non-antigen-specific). TRM cells were defined as CD8+CD44+CD62L-CD103+CD69+, and negative for intravenous (IV) circulatory CD3+ T cell stain. TEM cells were defined as CD8+CD44+CD62L-CD127+, and negative for IV circulatory CD3+ T cell stain. Group differences of data in (a), (b), (d) and (e) were analysed using non-parametric Kruskal-Wallis tests (*=p < 0.05, **=p < 0.01, ***=p < 0.001). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Fig 4: Challenging ChAdOx1-NP + M1-RSVF-vaccinated mice with H3N2.a Vaccination and challenge schematic for the assessment of the protective capacity of ChAdOx1-NP + M1-RSVF against X31 (H3N2) infection and subsequent disease in mice. Mice were prime-boost-vaccinated, then challenged with X31, and culled 6 days later with tissues and fluids harvested. Blood sampling was performed 4 weeks post-prime and 3 weeks post-boost. b Weight change in mice over time post-challenge, measured as % of pre-challenge weight. Significant differences at timepoints between IM-IN and control mouse groups are represented with *, between IM-IN and IM-IM as # and between IN-IN and unvaccinated as @ (*, @ or # =p < 0.05, **=p < 0.01). c Viral load in lungs 6 days post-challenge (M gene copies/μg lung RNA (log10)). d H3N2 NP-specific IgG and IgA levels in serum, NWs, BALF and LHS post-challenge, as measured by ELISA (log10 EU). Median negative control values are displayed as dashed lines. e Levels of antigen-specific CD8+ TRM cells, and relative levels of antigen-specific CD8+ TEM and TRM, in BAL and lungs post-challenge. TRM and TEM cells were defined as CD3+CD8+CD44+CD62L-CD103+CD69+, and CD3+CD8+CD44+CD62L-, respectively, and positive for influenza pentamer H-2Kd TYQRTALV. In boxplots, a “+” symbol represents the group mean. One mouse in group IM-IN did not have detectable Lung TRM. Group differences for all data were analysed using non-parametric Kruskal-Wallis tests (*=p < 0.05, **=p < 0.01). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.