Fig 1.
The assessment of the protection (A) and the immune response (B) triggered by rSm22.6 or rSm29 immunization in previously infected and treated Balb/c mice were performed as indicated in the figure.
Table 1.
Protection level induced by immunization with rSm22.6 plus Freund’s adjuvant in Balb/c mice previously infected/treated.
Table 2.
Protection level induced by immunization with rSm29 plus Freund’s adjuvant in Balb/c mice previously infected/treated animals.
Table 3.
Protection level induced by immunization with rSm29 or rSm22.6 plus Freund’s adjuvant in Balb/c naïve mice.
Fig 2.
Hepatic granuloma area in mice immunized with rSm29, previous infected and treated.
Representative picture of granuloma reaction (A) and the measurement of granuloma area of each group (B). To determine granuloma area, approximately 100 granulomas from IT/rSm29 group and from its control group (IT/Saline), with a single well-defined egg (exudative stage), were measured. Total area of the granulomas was expressed in square micrometers (μm2). Scale bar = 100 μm (x100).
Fig 3.
Kinetics of specific anti-rSm22.6 or anti-rSm29 antibodies production induced by immunization.
Sera from 10 animals/group were collected forty-five days after infection, fifteen days after treatment and two weeks after each immunization dose. The levels of specific IgG, IgG1, IgG2a, IgE against rSm22.6 or against rSm29 were determined by ELISA. Sera dilution was: 1:1.000 (IgG and IgG1—rSm29 tests), 1:100 (IgG2a—rSm29 test), 1:600 (IgG—rSm22.6 test), 1: 1.000 (IgG1—rSm22.6 test), 1:400 (IgG2a—rSm22.6 test) or 1:40 (IgE—rSm22.6 and rSm29 test). Bars represent the mean absorbance values measured at 450 nm ± SD (Standard Deviation). Arrows indicate the timing of infection, treatment and immunization. Statistically significant differences between rSm22.6 or rSm29 group and saline control group is denoted in the graphic by one asterisk (p<0.05), two asterisks (p<0.01) or three asterisks (p<0.001). Statistically significant difference between the immunization doses is pointed in the graphic. &: difference compared to infected mice; #: difference compared to treated mice.
Table 4.
Antibody titer in mice immunized with rSm22.6 and rSm29 after the third immunization.
Fig 4.
Recognition of native Sm22.6 and Sm29 in schistosomula surface by sera from immunized mice.
Twenty newly-transformed schistosomula (A) were incubated with sera from mice inoculated with saline + CFA/IFA, immunized with rSm29 + CFA/IFA or immunized with rSm22.6 + CFA/IFA. Antibody reactivity to Sm29 and Sm22.6 surface proteins were detected by a secondary anti-mouse IgG-FITC-conjugated antibody. As an experimental control, parasites were incubated with anti-mouse IgG-FITC-conjugated antibody. The fluorescence in the schistosomula tegument, after incubation, was observed by fluorescence microscopy. Scale bar = 25 μm (x400). A significant fluorescence measurement was detected (B) in the schistosomulum incubated with sera from rSm22.6 and rSm29 immunized mice compared to parasite incubated with sera from saline inoculated animals. Fluorescence intensity in the schistosomula tegument was measure using ImageJ software. The graphic represent box plot with whiskers from minimum to maximum values of relative fluorescence (Arbitrary unit). Significant differences were denoted in the graphic.
Fig 5.
Cytokine profile induced by immunized Balb/c mice, previously infected/treated, with the recombinant form of Sm22.6 and Sm29.
One week after the last immunization dose, spleen cells were obtained and cultured for cytokine measurement. Spleens from naïve mice immunized with rSm22.6 or rSm29 (white bars) or from non-immunized infected and treated mice (cross-hatched bars) were also obtained as a control for the experiment. IL-2 (A), TNF-α (B), IFN-ɣ (C), IL-4 (D), IL-6 (E), IL-17 (F) and IL-10 (G) production in response to rSm22.6 (25μg/mL), rSm29 (25μg/mL) or medium alone (control) was assessed. Cytokine levels were measured by the CBA Th1/Th2/Th17 Kit. Bars represent the median with interquartile range of the difference on cytokine production in response to rSm22.6 or rSm29 and the basal cytokine production (observed in nonstimulated splenocytes); gray bars represent the cytokine production in the saline group in response to rSm22.6 or rSm29 in vitro stimulation; black bars represent the cytokine production in rSm29 or rSm22.6 immunized group in response to rSm22.6 or rSm29 in vitro stimulation. Statistically significant difference between saline and rSm22.6 or Sm29 group is pointed in the graphic. Statistical analyses were performed using Mann-Whitney test.
Fig 6.
Activation status of spleen cells from mice immunized with rSm29 or rSm22.6, or inoculated with saline.
One week after the last immunization, spleen cells from IT/Saline, IT/rSm22.6, IT/rSm29, rSm22.6 and rSm29 groups were labeled to evaluate the frequency of activated lymphocytes, and were acquired in a flow cytometer. Data analysis was carried out as follows (A): within singlet cells/lymphocyte region, cells expressing CD4, CD8 or CD19 molecules were selected and the expression of CD25 and CD69 activation molecules were evaluated in TCD4+ or TCD8+ cells. B-cells activation status was evaluated in CD19+ cells expressing the activation marker CD86. (B): Bars represent the median with interquartile range of the percentage of cells expressing the activation molecules. Statistically significant difference is pointed in the graphic. Statistical analyses were performed by the Mann-Whitney test.
Fig 7.
Frequency of memory cells in mice immunized with rSm29 or rSm22.6, or inoculated with saline.
One week after the last immunization, spleen cells from IT/Saline, IT/rSm22.6, IT/rSm29, rSm22.6 and rSm29 groups were labeled to assess the frequency of memory lymphocytes and were acquired in flow cytometer. Data analysis was carried out as follows (A): within singlet cells/lymphocyte population, CD4+CD44high or CD8CD44high cells were selected and, within that population, the percentage of CD127+CD62low cells representing CD4+ or CD8+ T effector memory cells, CD127+CD62high population representing the CD4+ or CD8+ T central memory cells were determined. Furthermore, within the lymphocytes population, CD19+ cells were selected and the percentage of CD19+CD27+ cells was determined. (B): The results are expressed in bars as median with interquartile range of the percentage of memory cells. Statistically significant difference is pointed in the graphic. Statistical analyses were performed by the Mann-Whitney test.