Figure 1.
Secretory expression of recombinant Sj23LHD-GST by S. typhimurium.
Diagram of plasmid constructs used in the study (A). nirB, pagC and pMohly represent nirB-sopE-Sj23LHD-GST, pagC-sopE-Sj23LHD-GST and pMohly1-Sj23LHD-GST constructs, respectively. Plasmids expressing the recombinant protein of Sj23LHD-GST fused to type III secretion signal of Salmonella outer protein E (SopE) (sopE-Sj23LHD-GST) or type I secretion signal of α-hemolysin A (HlyA) (hlyA-Sj23LHD-GST) were transformed into Salmonella. Whole bacteria lysates and cultured supernatants of these strains were examined for the presence of the chimeric protein sopE–Sj23LHD-GST or hlyA-Sj23LHD-GST by Western blotting as described in Methods (B). Mouse macrophage cell line RAW 264.7 was infected with Salmonella harboring plasmids expressing EGFP as a marker for the expressed recombinant proteins. The presence and location of EGFP in the cell were examined (C).
Figure 2.
Humoral response elicited by vaccination with recombinant Salmonella strains.
BALB/c mice were orally immunized with nirB strain, pagC strain, pMohly strain, vector strain or PBS (control) 3 times at 2 weeks interval. Two weeks after the last immunization, serum samples were collected and assayed by ELLISA. The recombinant Sj23LHDGST protein was used as the coating antigen. (A) The total antigen-specific IgG production in serial dilutions (1∶50, 1∶100, 1∶200, and 1∶400). Each bar and symbol represents the mean ± SD of sera from 8 mice. (B) The ratio of IgG2a to IgG1 in mouse serum (diluted 1∶50) from each group was calculated. The column represents the mean absorbance of IgG2a divided by the mean IgG1 absorbance of 8 animals. *P<0.05 and **P<0.01.
Figure 3.
Colonization of the spleens (A) and Peyer's patches (B) of BALB/c mice following oral inoculation with recombinant S. typhimurium vaccines and wild type S. typhimurium carrying no plasmid (Sal) (white bar).
The organs were collected from 3 animals 3, 10 or 20 d after inoculation and homogenized in PBS and plated onto agar for bacteria counting. Data are presented as mean ± SD, n = 3; N.D. represents no detection. **P<0.01 and ***P<0.001 when compared with non-transformed S. typhimurium (Sal).
Figure 4.
Expression of CD44 and activation of CD69.
BALB/c mice were orally immunized 3 times, 2 weeks interval, with nirB strain, pagC strain, pMohly strain, vector strain or PBS (control). Splenocytes collected 2 weeks after the 3rd immunization were stained with CD44-PE and assayed by flow cytometry. Histograms (A) and quantitation (D) are shown. For CD69 activation of CD4 and CD8 T cells in the presence of recombinant heterologous antigens, splenocytes collected 2 weeks after the 3rd immunization were stimulated by the sj23LHDGST protein for 16 h and then doubly stained with CD4-PEcy5 and CD69-FITC or CD8-PEcy5 and CD69-FITC. Dot plots assayed by flow cytometry (B, C) and the percentage of CD8+CD69+ double positive cells (E) and CD4+CD69+ double positive cells (F) are showed. Each column represents the mean ± SD of 3 independent experiments in duplicate. *P<0.05 and ***P<0.001.
Table 1.
S. japonicum burden in mice immunized with recombinant S. typhimurium vaccine strains.
Figure 5.
IFN-γ production by splenocytes of immunized mice.
BALB/c mice were orally immunized 3 times, 2 weeks interval, with nirB strain, pagC strain, pMohly strain, vector strain or PBS (control). The splenocytes of immunized mice were collected at 2 weeks after the 3rd immunization and stimulated with recombinant Sj23LHD protein (10 µg/ml), GST protein (10 µg/ml), conA (10 µg/ml) or media alone. The content of IFN-γ in the culture supernatant was measured by ELISA kit. Each column represents the mean±SD of 3 independent experiments. ***P<0.001 when compared with vector group.
Table 2.
The cytokines produced by splenocytes of mice vaccinated with heterologous prime–boost regime.
Figure 6.
Granulomas formation in the liver.
6 weeks after S. japonicum challenge, the livers of mice immunized with PBS (control), vector, rSj23LHD-GST, nirB strain, nirB prime-boost or vector prime-boost were collected, fixed and sectioned for hematoxylin-eosin (H&E) staining. (A) Representative granuloma was shown (magnification, ×400) and (B) granuloma area was measured, Mean ± SD, n = 12–15. *P<0.05, and ***P<0.001.
Table 3.
S. japonicum burden in mice immunized with heterologous prime-boost vaccination.