Fig. 1. SpFN + ALFQ induces higher frequencies of T follicular helper (Tfh) cells, polyfunctional spike-specific CD4⁺ T cells, and germinal center (GC) B cells in the draining lymph nodes (dLN).

a Timeline of vaccination and sample collection. C57BL/6 mice were vaccinated with SARS-CoV-2 antigen (SpFN) formulated with a saponin containing liposomal adjuvant formulation, ALFQ (SpFN+ALFQ) or adsorbed to aluminum hydroxide, AH (SpFN+AH) on days 0 and 21 (weeks 0 and 3) as indicated (blue dot). Blood and tissues (red dots) were collected at varying timepoints for immunological analysis. dLNs were collected from naive and vaccinated mice on days 5, 7, and 28 (week 4). Single cell suspensions were made and were stimulated with peptide pools covering the full length of the spike protein. The frequencies of spike-specific Tfh and GC B cells were determined at each time point by flow cytometry. Open and closed (black and salmon color) circles represent dLN cells from naive, SpFN + AH, and SpFN + ALFQ mice, respectively. b Frequencies of Tfh (PD-1⁺CXCR5⁺CD4⁺ T cells), c (PD-1⁺CXCR5⁺ICOS⁺CD44⁺ CD4⁺ T cells). d Percentage of IFN-γ expressing spike-specific CD4⁺ T cells. e Pie chart showing the percentage of polyfunctional spike-specific CD4⁺ T cells from SpFN + ALFQ vaccinated mice expressing IFN-γ and/or TNF-α. The slices of the pie and each slice represent the frequencies of cytokine expression in CD4⁺ T cells. The numbers below each pie chart represent the percentage of spike-specific CD4⁺ T cells expressing IFN-γ and/or TNF-α at the indicated time points. f Percentage of germinal center B cells (GL7⁺ CD45R⁺). g Percentage of plasmablasts (CD45R⁺CD138⁺) (n = 5 mice/group/time point in vaccination groups; n = 3 mice in control group); SpFN + AH dLNs were pooled into two samples for days 5 and 7 and into three samples for week 4. The flow panels and the gating strategy for the respective subsets are shown in Supplementary Tables 1 and 2, and Supplementary Fig. 1, respectively. For Immunohistochemistry (IHC) analysis (h) vaccine dLNs were obtained on week 4 from each of the vaccination groups. Sections were stained with anti-PNA, anti-Ki67, anti-PD-1, and anti-CD3. Follicular structures that were CD3⁺PD-1⁺ T follicular helper (Tfh) and PNA⁺ and Ki67⁺ were considered as germinal centers. Images were analyzed with Histowiz platform. The areas positive for the PNA, Ki67, and PD-1 were marked for ease of counting and measurement of the area. Arrows depict the GCs in the lymph node and an enlarged area of that region is shown. i Each lymph node was analyzed for the number of GCs (PNA⁺) and the area of each GC in a given lymph node and divided by the number of lymph nodes or GCs in each group. The data are represented as bar graphs (Mean ± SEM) and each dot represents the average number of GCs per dLN or the area of individual GC per dLN. The statistical differences between the two vaccinated groups were evaluated by Mann–Whitney U-test with P ≤ 0.05 considered as significant. The scale bars represent 200 and 500 μM.