After conjugation, PLGA ａnd Chitosan nanoparticles were precipitated ａnd the free amounts of rHCA59 protein were determined by a BCA protein assay kit. It was found that 89% ａnd 71% of rHCA59 were conjugated to PLGA ａnd Chitosan, respectively. In other words, a total of 1.78 mg of protein bound to PLGA, ａnd 1.42 mg of protein bound to Chitosan.A scanning electron microscopy was used to identify the size ａnd morphology of PLGA ａnd Chitosan nanoparticles. The SEM results showed that both PLGA ａnd Chitosan nanoparticles’ appearances were smooth. The size of rHCA59-PLGA NPs was from 180 nm to 402 nm (Figure 1A) ａnd the size of rHCA59-CS NPs was from 200 nm to 500 nm (Figure 1B).

Notes: (1): PBS group (blank control). (2): pET-32a protein group. (3): PBS-PLGA group. (4): PBS-CS group. (5): PBS-CFA group. (6): rHCA59 group. (7): rHCA59-PLGA group. (8): rHCA59-CS group. (9): rHCA59-CFA group. (10): rHCA59-CS-PLGA group. (A) IL-12. (B) IL-17. (C) IFN-γ. (D) IL-4. (E) TGF-β. Data are representative of triplicate independent experiments (*p < 0.05, **p < 0.01, ａnd ***p < 0.001). Abbreviations: PLGA, poly-D,L-lactide-co-glycolide; CS, Chitosan; CFA, complete Freund’s adjuvant; IL, interleukin; ELISA, enzyme linked immunosorbent assay

Notes: rHCA59 loaded nanoparticles could affect the proportions of CD4+ T cells ａnd CD8+ T cells in immunized mice. The percentages of CD4+ T cells (A1–A10) ａnd CD8+ T cells (B1–B10) in ten groups were investigated by flow cytometry on day 14 after immunization. (C ａnd D) Different treatments affected the proportions of CD4+ T cells ａnd CD8+ T cells. (A1 ａnd B1) PBS group (blank control). (A2 ａnd B2) PBS-CFA group. (A3 ａnd B3) PBS-PLGA group. (A4 ａnd B4) PBS-CS group. (A5 ａnd B5) pET-32a protein group. (A6 ａnd B6) rHCA59 group. (A7 ａnd B7) rHCA59-CFA group. (A8 ａnd B8) rHCA59-CS group. (A9 ａnd B9) rHCA59-PLGA group. (A10 ａnd B10) rHCA59-CS-PLGA group. The results shown here are from three independent experiments (*p < 0.05, **p < 0.01, ａnd ***p < 0.001). Abbreviations: CD4, cluster of differentiation 4; CD8, cluster of differentiation 8; T cells, thymus cells; PLGA, poly-D,L-lactide-co-glycolide; CS, Chitosan; CFA, complete Freund’s adjuvant.