Fig. 4.

PUUC NPs delivered intramuscularly with spike protein enhance humoral responses. Female BALB/c mice were immunized I.M. into both tibialis anterior muscles at day 0 (1st dose) with soluble spike protein at doses of 80 ng, 200 ng, 1000 ng with or without adjuvant-NPs (4 mg) loaded with PUUC (+P, 20 ng PUUC dose). Peripheral blood was sampled on day 26. On day 28, mice received a 2nd dose of protein subunit vaccines. Mice received the same formulations, except for two groups that received 80 ng spike protein as a 1st dose received 1000 ng spike protein as a 2nd antigen dose (80/1000 and 80/1000 +P). Mice were euthanized on day 36 for to collect blood and popliteal LNs. A) Anti-spike IgG in post-1st dose sera at various dilutions measured by absorbance at 450 nm during ELISA assays and B) comparison of area under the curve (AUC). C–D) Anti-spike IgG in post-2nd dose sera measured by absorbance at 450 nm and comparison of AUC. E) ACE-2 signal measured by absorbance at 450 nm in spike protein neutralization assay with post-2nd dose sera. Absorbance was normalized to a blank well in each row of a 384 well plate to correct for plate effects. Lower absorbance values indicate higher spike-neutralizing antibody levels in sera. Percentages of cells expressing F) Bcl6⁺ out of B220⁺ cells, G) GL7⁺ out of B220⁺ cells and H) CXCR5⁺ out of B220⁻ cells from combined popliteal lymph nodes. B,D) Normality was assessed with the Kolmogorov-Smirnov test. Statistical significance was determined with the Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons. E–H) Statistical significance calculated with One-Way ANOVA and Tukey post-hoc test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 for all graphs.