Hepatic granuloma area in mice immunized with rSm29, previous infected and treated.

<p>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).</p

Experimental design.

<p>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.</p

Sm29, but Not Sm22.6 Retains its Ability to Induce a Protective Immune Response in Mice Previously Exposed to a <i>Schistosoma mansoni</i> Infection

<div><p>Background</p><p>A vaccine against schistosomiasis would have a great impact in disease elimination. Sm29 and Sm22.6 are two parasite tegument proteins which represent promising antigens to compose a vaccine. These antigens have been associated with resistance to infection and reinfection in individuals living in endemic area for the disease and induced partial protection when evaluated in immunization trials using naïve mice.</p><p>Methodology/principals findings</p><p>In this study we evaluated rSm29 and rSm22.6 ability to induce protection in Balb/c mice that had been previously infected with <i>S</i>. <i>mansoni</i> and further treated with Praziquantel. Our results demonstrate that three doses of the vaccine containing rSm29 were necessary to elicit significant protection (26%–48%). Immunization of mice with rSm29 induced a significant production of IL-2, IFN-γ, IL-17, IL-4; significant production of specific antibodies; increased percentage of CD4+ central memory cells in comparison with infected and treated saline group and increased percentage of CD4+ effector memory cells in comparison with naïve Balb/c mice immunized with rSm29. On the other hand, although immunization with Sm22.6 induced a robust immune response, it failed to induce protection.</p><p>Conclusion/significance</p><p>Our results demonstrate that rSm29 retains its ability to induce protection in previously infected animals, reinforcing its potential as a vaccine candidate.</p></div

Protection level induced by immunization with rSm22.6 plus Freund’s adjuvant in Balb/c mice previously infected/treated.

<p>^aWorm burden recovered from mice immunization with one, two or three doses. For each vaccinated group: n = 10 mice.</p><p>Comparison between total worm burden recovered^b or numbers of eggs per gram of intestine^c from IT/Sm22.6 group and IT/Saline control group, which received the same number of immunization doses and were challenged with 50 <i>S</i>. <i>mansoni</i> LE strain cercariae. NS = not significant. ND = not determined.</p><p>Protection level induced by immunization with rSm22.6 plus Freund’s adjuvant in Balb/c mice previously infected/treated.</p

Kinetics of specific anti-rSm22.6 or anti-rSm29 antibodies production induced by immunization.

<p>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.</p

Protection level induced by immunization with rSm29 or rSm22.6 plus Freund’s adjuvant in Balb/c naïve mice.

<p>^aWorm burden recovered from mice infected with 50 cercariae.</p><p>^bWorm burden recovered from mice infected with 100 cercariae. For each vaccinated group: n = 10 mice.</p><p>^cComparison between total worm burden recovered from rSm29 or rSm22.6 group and Saline control group.</p><p>Protection level induced by immunization with rSm29 or rSm22.6 plus Freund’s adjuvant in Balb/c naïve mice.</p

Protection level induced by immunization with rSm29 plus Freund’s adjuvant in Balb/c mice previously infected/treated animals.

<p>^aWorm burden recovered from mice immunization with one, two or three doses. For each vaccinated group: n = 10 mice.</p><p>Comparison between total worm burden recovered^b or numbers of eggs per gram of intestine^c from IT/rSm29 group and IT/Saline control group, which received the same number of immunization doses and were challenged with 100 <i>S</i>. <i>mansoni</i> LE strain cercariae. NS = not significant. ND = not determined.</p><p>*Statistically significant (p<0.05)</p><p>Protection level induced by immunization with rSm29 plus Freund’s adjuvant in Balb/c mice previously infected/treated animals.</p

Antibody titer in mice immunized with rSm22.6 and rSm29 after the third immunization.

<p>Antibody titer in mice immunized with rSm22.6 and rSm29 after the third immunization.</p

Frequency of memory cells in mice immunized with rSm29 or rSm22.6, or inoculated with saline.

<p>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+CD44^high or CD8CD44^high cells were selected and, within that population, the percentage of CD127⁺CD62^low cells representing CD4⁺ or CD8⁺ T effector memory cells, CD127⁺CD62^high 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.</p

Cytokine profile induced by immunized Balb/c mice, previously infected/treated, with the recombinant form of Sm22.6 and Sm29.

<p>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.</p