Fig. 3. SpFN + ALFQ vaccine enhanced the frequency of T follicular helper (Tfh) cells, spike-specific IL-21-secreting Tfh cells and S-2P protein-specific germinal center (GC) B cells in the spleen following second vaccination (week 4).

C57BL/6 mice were vaccinated with SpFN+ALFQ or SpFN+AH on days 0 and 21 (week 3). Spleens from vaccinated (n = 6 in each group) and naive (n = 2) mice were collected on week 4 and flow cytometry analysis was performed. The frequency of a Tfh (CXCR5⁺PD-1^hi) cells in all three groups, b IL-21⁺CD40L⁺ spike-specific Tfh cells, c GC (Fas⁺GL-7⁺) B cells and d S-2P protein-specific GC B cells positive for the dual-fluorophore (S-2P-AF647 and S-2P-FITC) are shown. The data are represented as bar graphs (Mean ± SEM) and each dot represents the data from an individual spleen. The right panels show the representative contour flow plots for each subset. The flow panel and the gating strategy for the respective subsets are shown in Supplementary Tables 3 and 4 and Supplementary Figs. 2 and 3, respectively. The statistical differences between the two vaccinated groups were evaluated by Mann–Whitney U-test with P ≤ 0.05 considered as significant.