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. 2022 Aug 26;17(9):2559–2571. doi: 10.1021/acschembio.2c00497

Figure 4.

Figure 4

Individually encapsulated cGAMP and admixed cGAMP and CL075-PSs enhanced rHA-specific neonatal humoral and cell-mediated immune responses. (A) Infant C57BL/6 mice were immunized i.m. on DOL (day of life) 7 and 14. All groups received 1 μg of each of the Flublok quadrivalent antigens (rHA), except the PBS group. rHA was given alone or in combination with cGAMP (1 μg) and CL075 (164 μM) delivered in admixture or single-loaded or dual-loaded PEG-b-PPS nanocarriers. (B) Antibody titers for rHA-specific IgG (B), IgG1(C), and IgG2c (D) were determined by ELISA in serum samples collected at DOL 21. (E–G) Murine CD4+ T cell responses after rHA stimulation. Splenocytes from rHA and adjuvanted vaccinates were isolated, stimulated with 10 μg/mL of Flublok along with CD28 (1 μg/mL) and CD49d (1 μg/mL) for 12 h followed by 6 h of BFA stimulation to block the extracellular cytokine secretion. After stimulation, cells were harvested, stained (intracellular cytokine staining), and analyzed by flow cytometry. Plots were gated on CD44+ CD4+ lymphocytes and analyzed for all combinations of simultaneous IFNγ, TNF, and IL-2 productivity. Statistical comparison was performed either using one-way ANOVA or nonparametric Kruskal-Wallis test corrected for multiple comparisons; ns denoted non-significant, *p < 0.033, **p < 0.002, ***p < 0.001 (n = 5–12 per group). The study was inclusive of two independent repeats.

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