Infection of BALB/c mice.

<p>Mice (n = 8) were infected subcutaneously with 1×10⁶ stationary promastigotes of <i>Leishmania amazonensis</i>. Lesion development in the infected footpads was monitored weekly, up to 8 weeks after infection. Mean ± standard deviation (SD) are shown in (A). Parasite load in the infected footpads, spleen, and liver was analyzed in all animals (B). Other mice (n = 8, per group) were subcutaneously infected with 1×10⁶ stationary promastigotes of <i>L. amazonensis</i> obtained from R0 or R30 passages, and the lesion development was monitored up to 8 weeks after infection. Mean ± SD of the groups are shown (C). The parasite load in the infected footpads, spleen, and liver was also evaluated in these groups (D). The experiments were repeated three times, and presented similar results. *Significant difference between the R0 and R30 groups (<i>P</i><0.05).</p

Two-dimensional profiles of cultures from <i>Leishmania amazonensis</i>.

<p>The 2-DE gels were obtained after the separation of stationary promastigotes extracts (R0, R10, R20, and R30 passages; 650 µg of each extract) by 2-DE (first dimension: IEF pH range 4–7; second dimension: 12% SDS-PAGE) and staining with colloidal Coomassie Brilliant Blue G-250. The gel fragments in the lower portion of the figures represent evaluated amplifications (see within the dotted lines). 2-DE gels of each passage were derived from four independent protein preparations of each passage. One representative preparation of each sample is showed in this study.</p

Immunoblotting validation of some proteins in <i>Leishmania amazonensis</i>.

<p>Representative immunoblotting of some proteins that presented a significant decrease or increase in their expression content between R0 and R30 passages, using promastigote and amastigotes-like forms of <i>L. amazonensis</i>, are shown here. For each protein [α-tubulin, in A; paraflagellar rod protein 1D, in B; glucose-regulated protein 78 (GRP78) in C, and heat shock protein 83 (HSP83), in D], this image presents one example of correspondent 2-DE spot of promastigote form obtained from R0 or R30 passages. The antibodies used to validate each spot are described in the material and methods section. Asterisks represent the comparison between the expression of the protein in the R0 condition in relation to the R30 sample in each parasite stage, applying the Student's t-test (<i>P</i><0.05), and the numbers represent the relative variation of each protein in comparison to R0 of each parasite stage. All experiments were performed in triplicate.</p

Identification of proteins that presented a significant increase in their expression content.

a<p>⁾ Spots match ID number obtained from ImageMaster Platinum;</p>b<p>⁾ Name of the identified protein;</p>c<p>⁾ Uniprot identification code;</p>d<p>⁾ Experimentally predicted and expected isoelectric point (<i>pI</i>);</p>e<p>⁾ Experimentally predicted and expected molecular weight (<i>Mr</i>, in kDa);</p>f<p>⁾ Number of identified peptides by MS;</p>g<p>⁾ Percentage of the protein sequence covered by identified peptides;</p>h<p>⁾ Normalized data from R0 represented by mean values of each condition divided by R30 value;</p>i<p>⁾ Fold represents the maximum spot intensity mean value of the conditions divided by the smallest value;</p>j<p>) One-way ANOVA (<i>P</i><0.01) obtained from spot analysis;</p>k<p>⁾ Biological functions according to NCBI, UniProt, and Gene Ontology databases;</p>l<p>⁾ Biological activity and/or immunological application described in other studies: <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Joshi1" target="_blank">[53]</a> Joshi et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Tielens1" target="_blank">[54]</a> Tielens et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Soto1" target="_blank">[55]</a> Soto et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Lackovic1" target="_blank">[56]</a> Lackovic et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Scher1" target="_blank">[57]</a> Scher et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-SnchezCaete1" target="_blank">[58]</a> Sánchez-Cañete et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Peris1" target="_blank">[59]</a> Peris et al., 1994; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Steiner2" target="_blank">[60]</a> Steiner et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Jaramillo1" target="_blank">[61]</a> Jaramillo et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-DuclertSavatier1" target="_blank">[62]</a> Duclert-Savatier et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Celeste1" target="_blank">[63]</a> Celeste et al., 2004; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Liu1" target="_blank">[64]</a> Liu et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Leblanc1" target="_blank">[65]</a> Leblanc et al., 1998; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Priest1" target="_blank">[66]</a> Priest et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-BakkerGrunwald1" target="_blank">[67]</a> Bakker-Grunwald, 1992; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Banerjee1" target="_blank">[68]</a> Banerjee et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Casanova1" target="_blank">[69]</a> Casanova et al., 2008; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Jensen1" target="_blank">[70]</a> Jensen et al., 2001. The proteins were identified through the data included in the NCBI database (dated June 2012) for <i>Leishmania spp.</i></p

Identification of proteins that presented a significant decrease in their expression content.

a<p>⁾ Spots match ID number obtained from ImageMaster Platinum;</p>b<p>⁾ Name of the identified protein;</p>c<p>⁾ Uniprot identification code;</p>d<p>⁾ Experimentally predicted and expected isoelectric point (<i>pI</i>);</p>e<p>⁾ Experimentally predicted and expected molecular weight (<i>Mr</i>, in kDa);</p>f<p>⁾ Number of identified peptides by MS;</p>g<p>⁾ Percentage of the protein sequence covered by identified peptides;</p>h<p>⁾ Normalized data from R0 represented by mean values of each condition divided by R30 value;</p>i<p>⁾ Fold represents the maximum spot intensity mean value of the conditions divided by the smallest value;</p>j<p>⁾ One-way ANOVA (<i>P</i><0.01) obtained from spot analysis;</p>k<p>⁾ Biological functions according to NCBI, UniProt, and Gene Ontology databases;</p>l<p>⁾ Biological activity and/or immunological application described in other studies: <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Tull1" target="_blank">[22]</a> Tull et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Oliveira1" target="_blank">[23]</a> Oliveira et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Daifalla1" target="_blank">[24]</a> Daifalla et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-IyerJ1" target="_blank">[25]</a> Iyer et al., 2008; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-HungerGlaser1" target="_blank">[26]</a> Hunger-Glaser et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Werbovetz1" target="_blank">[27]</a> Werbovetz et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Feng1" target="_blank">[28]</a> Feng et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Niemirowicz1" target="_blank">[29]</a> Niemirowicz et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-HungerGlaser2" target="_blank">[30]</a> Hunger-Glaser et al., 1997; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Alcolea1" target="_blank">[31]</a> Alcolea et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Bhaskar1" target="_blank">[32]</a> Bhaskar et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Khanra1" target="_blank">[33]</a> Khanra et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Berberich1" target="_blank">[34]</a> Berberich et al., 2003; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Moore1" target="_blank">[35]</a> Moore et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Swenerton1" target="_blank">[36]</a> Swenerton et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Hummadi1" target="_blank">[37]</a> Hummadi et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Martins1" target="_blank">[38]</a> Martins et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Burns1" target="_blank">[39]</a> Burns et al., 1993; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Kushawaha1" target="_blank">[40]</a> Kushawaha et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Misra1" target="_blank">[41]</a> Misra et al., 2005; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Bringaud1" target="_blank">[42]</a> Bringaud et al., 1995; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Eggleson1" target="_blank">[43]</a> Eggleson et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Mureev1" target="_blank">[44]</a> Mureev et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Steiner1" target="_blank">[45]</a> Steiner et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-MartnezRodrguez1" target="_blank">[46]</a> Martínez-Rodríguez et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Drummelsmith2" target="_blank">[47]</a> Drummelsmith et al., 2004; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Achour1" target="_blank">[48]</a> Achour et al., 2002; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Buda1" target="_blank">[49]</a> Buda et al., 2013; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Alcolea2" target="_blank">[50]</a> Alcolea et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Martn1" target="_blank">[51]</a> Martín et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Silva1" target="_blank">[52]</a> Silva et al., 2012. The proteins were identified through the data included in the NCBI database (dated June 2012) for <i>Leishmania spp</i>.</p

Cellular and humoral response induced in BALB/c mice by immunization with rLiHyp1 plus saponin.

<p>Single cells suspensions were obtained from the spleens of mice, four weeks after vaccination. Cells were non-stimulated (medium; background control) or stimulated with rLiHyp1 (20 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 levels were measured in culture supernatants by capture ELISA (<b>A</b>). Each bar represents the mean ± standard deviation (SD) of data from four individual mice per group. Statistically significant differences in the IFN-γ, IL-12 and GM-CSF levels between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>B</b>); and between IL-12/IL-10 and IL-12/IL-4 levels (<b>C</b>) are also showed. Statistically significant differences in the ratios between the rLiHyp1 plus saponin group and control groups were observed (*** <i>P</i><0.0001). The ratio between rLiHyp1-specific IgG1 and IgG2a antibodies was obtained for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and control groups was also observed (* <i>P</i><0.005) (<b>D</b>).</p

Analysis of the cellular and humoral response and of the involvement of IL-12, CD4 and CD8 T cells in the IFN-γ production after <i>L. infantum</i> challenge.

<p>Single cells suspensions were obtained from the spleens of mice, 10 weeks after infection. Cells were non-stimulated (medium; background control) or stimulated with <i>L. infantum</i> SLA (25 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. Levels of IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 were measured in culture supernatants by capture ELISA. Mean ± standard deviation (SD) of the cytokines levels determined in four individual mice per group is shown (<b>A</b>). Statistically significant differences between the rLiHyp1 plus saponin group and the control mice (saline and saponin groups) were observed (*** <i>P<0.0001</i>). The analysis of the involvement of IL-12 and CD4 and CD8 T cells in the IFN-γ production is showed (<b>B</b>). Levels of IFN-γ in the supernatants of spleen cells cultures stimulated with SLA, as explained above, in the absence (positive control) or in the presence of anti-IL-12, anti-CD4, or anti-CD8 monoclonal antibodies were measured. Statistically significant differences between non-treated control cells and cultures incubated with anti-CD4 and anti-IL-12 monoclonal antibodies were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>C</b>), and between IL-12/IL-10 and IL-12/IL-4 levels (<b>D</b>), are also showed. Statistically significant differences between the rLiHyp1 plus saponin group and the control groups were observed (***<i>P</i><0.0001). The ratio between SLA-specific IgG1 and IgG2a antibodies levels were calculated for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and the control groups was also observed (* <i>P</i><0.005) (<b>E</b>).</p

Protection of BALB/c mice vaccinated with rLiHyp1 plus saponin against <i>L. infantum</i>.

<p>Mice inoculated with saline, saponin, or rLiHyp1 plus saponin were subcutaneously infected with virulent 1×10⁷ stationary-phase promastigotes of <i>L. infantum</i>. The number of parasites in the liver (<b>A</b>), spleen (<b>B</b>), bone marrow (<b>C</b>), and paws' draining lymph nodes (<b>D</b>) was measured, 10 weeks after challenge by a limiting-dilution technique. Mean ± standard deviation (SD) of four mice in each group is shown. Statistically significant differences in the parasite load in all evaluated organs between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) are showed (in numbers). Data shown in this study are representative of two independent experiments, which presented similar results.</p