Figure 1.
Immunolocalization of Sm29 antigen on male and female adult worm and lung-stage schistosomula of S. mansoni.
Polyclonal anti-Sm29 antibodies, serum from mice that received Freund´s adjuvant as negative control, and Cy5-conjugated anti-mice IgG were used. Actin was visualized by falloidin-Alexa fluor 488. The parasites were fixed in Omnifix II and used to whole-mount or section immunolocalization. (A and B) Whole-mount immunolocalization of Sm29 antigen on the surface (outer tegument) of male adult worm and (C) lung-stage schistosomula of S. mansoni. (D) Immunolocalization of Sm29 on the surface (outer tegument) of male adult worm, and (E) on the surface (outer tegument) and in some internal tissues on the female adult worm using deparaffinized sections of the parasites. Localization of Sm29 is identified by the orange color and actin filaments by the green color.
Figure 2.
Identification of Sm29 on skin-stage schistosomula tegument by Western blot.
Two micrograms of purified skin-stage schistosomula tegument was applied onto 12% SDS-PAGE and transferred to a nitrocellulose membrane by western blot. After that, the membrane was probed with serum from rSm29 vaccinated mice or naïve animals diluted 1∶500 in TBST. Arrow indicates the native Sm29. The molecular mass markers, from top to bottom, are: 97, 66, 45, 30, 20.1, and 14.4 kDa.
Figure 3.
Kinetics of specific anti-Sm29 IgG induced in mice immunized with rSm29.
Sera of ten immunized mice per group were collected at days 15, 30, 45, and 90 after the first immunization and assayed by ELISA. Arrows indicate the timing of vaccination. Results are presented as the mean absorbance measured at 492nm for each group. Results represent the mean of two independent experiments. Statistically significant differences of vaccinated mice compared to PBS+adjuvant control group is denoted by one asterisk for (p<0.05).
Table 1.
IgG1 and IgG2a immune profile induced by vaccination with recombinant Sm29.
Figure 4.
Sm29 induces IL-12 and TNF-α production by macrophages from TLR4 knockout mice.
Levels of IL-12 (p40) (A) or TNF-α (B) were measured in the supernatants of inflammatory macrophages from TLR4 KO or wild-type mice stimulated for 24 hrs with rSm29 (25 μg/ml), E. coli LPS (1 μg/ml) or medium alone. Significant differences from stimulated TLR4 KO or C57BL/6 macrophages in relation to non-stimulated cells are denoted by an asterisk (*) for p<0.05.
Table 2.
Parasitologic data, cytokine profile, intestinal egg counts and liver granuloma analysis of C57BL/6 mice vaccinated with rSm29 and challenged with 100 Schistosoma mansoni cercariae.
Figure 5.
Transcript level changes in selected genes of S. mansoni worms recovered from mice vaccinated with rSm29.
The figure shows real-time RT-PCR validation experiment for six genes down-regulated in S. mansoni worms recovered from rSm29+adjuvant immunized mice (black bars) compared to worms from PBS+adjuvant injected control mice (white bars). The selected genes were: CD36-CD36-like class B scavenger receptor [S. mansoni]; Sm23-Sm23 kDa integral membrane protein [S. mansoni]; Sm14-Fatty acid-binding protein Sm14 [S. mansoni]; Superox-Superoxide dismutase [S. mansoni]; TGF-β RIP-TGF-β receptor interacting protein 1 [C. sinensis]; B-cell RAP-B-cell receptor-associated protein-like protein [S. mansoni]. Statistically significant differences of vaccinated mice compared to PBS+adjuvant control group is denoted by one asterisk (p<0.03).
Table 3.
Putative identity, categories of biological function and fold change of a subset of down-regulated genes identified in worms recovered from animals vaccinated with rSm29 associated to adjuvant.