Fig. 4: PSs stimulate robust cellular and humoral immunity as a two-dose subunit vaccine, with elevated CD8⁺ T cell response and enhanced type-1 immunity.

a, Vaccination timeline with prime-boost schedule in C57BL/6 mice (n = 6). Four hocks were injected, and dLNs and the spleen were collected post-killing. All treatments were 10 µg OVA adjuvanted with 20 µg CpG ODN 1826, except saline and empty PSs (T33M10-empty). Encapsulated formulations consisted of 100 µg polymer and are benchmarked to the unencapsulated, adjuvanted OVA control. b, IFNγ-secreting CD8⁺ T cells were measured in dLNs and spleen after 6 h SIINFEKL restimulation using intracellular staining, with percentages reported by subtracting unstimulated from stimulated to remove non-specific responses. c, Double-positive TNF⁺IFNγ⁺-secreting CD8⁺ T cells measured in dLNs and spleen after 6 h SIINFEKL restimulation. d, A representative gating strategy for 6 h restimulation for intracellular cytokines. Samples are first gated on single, live CD8⁺ T cells. e, The gating strategy for SIINFEKL/MHC-I tetramer staining on antigen-experienced CD8⁺CD44⁺ T cells to measure antigen specificity. f, Quantification of antigen-specific CD8⁺ T cells. g,h, Splenocytes stimulated for 3 days using the full OVA protein, where IFNγ (g) and TNF (h) levels were measured in the supernatants to indicate a type-1 immune response. i, Total OVA-specific IgG levels measured by ELISA and reported as logAUC from serum samples taken 2 weeks post prime vaccination (day 14) and 1 week post boost (day 27). Data are biological replicates plotted as box plots with max/min, median and quartiles, and compared using ordinary one-way ANOVA with Tukey’s post-test for multiple comparisons (b and c) and a Kruskal–Wallis test with Dunn’s post-test for multiple comparisons (f). Welch’s one-way ANOVA with Dunnett post-test was used for g and h and two-way ANOVA with Sidak’s post-test was used for i. Panel a created with BioRender.com.