Worms recovered and the percentage of worm reduction after PZQ pressure.

<p> = 20 mice were used for each group.^, p<0.05 (Non-selected vs. Selected). N</p><p> The initial control group is the same for the Non-selected and Selected populations.</p><p>% Percentage of reduction of Selected (S) and Non-selected (N) worms obtained after treatment with PZQ at 2×100 mg/kg and 3×300 mg/kg. For each round, eggs produced by the worms that had survived the drug treatment were used to infected snails, and the cercariae obtained were used to infect a new set of mice. Mean numbers of worms recovered from treated and untreated mice after the first, sixth and seventh rounds of PZQ treatment. </p><p>≤0.05 indicates a significant difference between Selected and Non-selected parasites in the percent worm reduction upon PZQ treatment (Mann Whitney).^, P</p><p> SD: standard deviation.</p

Primers used for microsatellite genotyping, and genomic localization.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002596#pntd.0002596-Zerlotini1" target="_blank">[54]</a>. The genomic localization of microsatellites was identified using Blast Search in SchistoDB </p

Expected and observed heterozygosity in the Selected and Non-selected populations.

<p>≤0.05. Guo and Thompson test p</p><p> Microsatellite markers were tested for expected (He) and observed (Ho) heterozyzosity in Non-selected (N) and Selected (S) populations.</p

Allele and Genotype numbers in Non-selected (N) and Selected (S) populations.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002596#s2" target="_blank">Materials and Methods</a> by using specific primers for each microsatellite locus. PCR reactions were performed in S and N worms obtained as described in the legend of <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002596#pntd-0002596-t002" target="_blank">Table 2</a>. Number of adult worms tested, in the Non-selected (N) and Selected (S) populations after 7 passages. The number of alleles and genotypes was determined by PCR. Scoring of the polymorphic microsatellite was conducted as described in </p

Ratio of male/female adult worms recovered from the Non-selected and Selected populations after 11 passages.

<p>×300 mg/kg of PZQ. Ratio between male or female parasites recovered after treatment of the Non-selected and Selected populations and control populations. Average number of male and female worms recovered after treatment in the Non-selected and Selected populations. SD: standard deviation. Treatment was carried out with 3</p

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

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

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 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

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