Two-dimensional profiles of the total extracts from <i>Leishmania infantum</i> promastigote and amastigote-like stages.

<p>2DE gels were obtained after separation of promastigote (in A) and amastigote-like (in B) protein extracts (150 µg, each one) by 2DE (first dimension: IEF pH range 4–7, second dimension: 12% SDS-PAGE), and staining with colloidal Coomassie Brilliant Blue G-250. 2DE gels were derived from three independent protein preparations. One representative preparation of each parasite stage was used in this study.</p

Proteins of <i>Leishmania infantum</i> amastigotes-like identified by an immunoproteomic approach.

<p>a) Sera samples of dogs with VL. b) Name of the identified protein and species: Lmj, <i>L. major</i>; Lbr, <i>L. braziliensis</i>; Li, <i>L. infantum</i>; Ld, <i>L. donovani</i>. c) Accession numbers according to NCBI. d) Experimental expected and predicted molecular weights (<i>M</i>r, in KDa). e) Experimental expected and predicted isoeletric points (p<i>I</i>).</p

Comparison of spots identified in protein extracts from promastigote and amastigote-like stages of <i>Leishmania infantum</i>.

<p>Diagrams show the percentage of protein spots identified in either individual or combined parasite stages. In A, the percentage of total proteins identified by asymptomatic (19/18%), symptomatic (64/62%), and a combination of both sera classes (21/20%). In B, the percentage of proteins from the promastigote stage identified by asymptomatic (10/20%), symptomatic (25/49%), and a combination of both sera classes (16/31%). In C, the percentage of proteins from amastigote-like stage identified by asymptomatic (9/17%), symptomatic (39/74%), and a combination of both sera classes (5/9%).</p

Immunoproteomic analyses of the protein extract from the <i>Leishmania infantum</i> amastigote-like stage.

<p>2DE gels obtained after the separation of total protein extracts (150 µg) of amastigote-like stages by 2DE (first dimension: IEF pH range 4–7, second dimension: 12% SDS-PAGE), and staining with colloidal Coomassie Brilliant Blue G-250 (A, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g001" target="_blank">Figure 1</a>). Immunoblots of reactive spots were identified after incubation of the membrane with pools of sera of asymptomatic (B) or symptomatic (C) VL dogs. Bound antibodies were detected with goat anti-dog IgG antibodies at a 1∶5.000 dilution. The x-axis represents the tentative isoeletric point (<i>pI</i>), while the y-axis represents the approximate molecular weight (kDa) as determined by a commercial 2DE gel marker (BenchMark Protein Ladder). Protein spots were numbered, and their identities are listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g006" target="_blank">Figure 6</a>. Immunoblots are a reliable representation of three independent experiments.</p

Proteins of <i>Leishmania infantum</i> promastigotes identified by an immunoproteomic approach.

<p>a) Sera samples of dogs with VL. b) Name of the identified protein and species: Lmj, <i>L. major</i>; Lbr, <i>L. braziliensis</i>; Li, <i>L. infantum</i>; Ld, <i>L. donovani</i>. c) Accession numbers according to NCBI. d) Experimental expected and predicted molecular weights (<i>M</i>r, in KDa). e) Experimental expected and predicted isoeletric points (p<i>I</i>).</p

Immunoproteomic analyses of the protein extract from the <i>Leishmania infantum</i> promastigote stage.

<p>2DE gels obtained after separation of total protein extract (150 µg) of promastigote stage by 2DE (first dimension: IEF pH range 4–7, second dimension: 12% SDS-PAGE), and staining with colloidal Coomassie Brilliant Blue G-250 (A, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g001" target="_blank">Figure 1</a>). Immunoblots of reactive spots were identified after incubation of the membrane with pools of sera of asymptomatic (B) or symptomatic (C) VL dogs. Bound antibodies were detected with goat anti-dog IgG antibodies at a 1∶5.000 dilution. The x-axis represents the tentative isoeletric point (<i>pI</i>), while the y-axis represents the approximate molecular weight (kDa) as determined by a commercial 2DE gel marker (BenchMark Protein Ladder). Protein spots were numbered, and their identities are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001430#pntd-0001430-g005" target="_blank">Figure 5</a>. Immunoblots are a reliable representation of three independent experiments.</p

Antibody titers and cytokines production against SLA after infection in control and vaccinated mice groups.

<p>The humoral and cellular response against soluble <i>Leishmania</i> antigens (SLA) was determined at week seven after <i>L</i>. <i>major</i> infection in mice (n = 8 per group) previously vaccinated with the LiPABP1 + LiPABP2 + LiPABP3 combined genetic vaccine (pcDNA3-PABPs) or immunized with the pcDNA3 empty vector (pcDNA) or that received the vaccine diluent (Saline) prior to infection. Anti—SLA IgG (A) or IgG1 and IgG2a (B) titers were individually determined by assaying sera from 1/50 to 1/820,000 and using a horseradish peroxidase-conjugated anti-mouse IgG, IgG1 or IgG2a secondary antibody at 1/2,000 dilution. Results are shown as whisker (min to max) plots. For cytokine determinations spleen cells were cultured for 72 h at 37°C, 5% CO₂ in the absence or in the presence or soluble <i>Leishmania</i> antigen (SLA; 12 μg/ml). Levels of IFN-γ, IL-10 and IL-4 were assessed by ELISA in culture supernatants (C). Each bar represents the mean ± standard deviation (SD). The IFN-γ/IL-10 and IFN-γ/IL-4 ratios were determined and represented as the mean ± standard deviation (SD) (D). In (A) ⁺⁺ (<i>P</i> < 0.01) and ** (<i>P</i> < 0.01) statistical decrease in anti-SLA response in the LiPABPs vaccinated group with respect saline or pcDNA3 control groups, respectively. In (B) ** (<i>P</i> < 0.01) and *** (<i>P</i> < 0.001) statistical differences between IgG1 and IgG2a anti-SLA titers within each group (Mann-Whitney test). In (C and D) ⁺⁺⁺ <i>P</i> < 0.001 and *** <i>P</i> < 0.001 significant differences between LiPABP vaccinated and saline and pcDNA3 control groups, respectively (Student T-test). Results are representative of two independent experiments.</p

Analysis of the humoral response induced against the LiPABPs after challenge.

<p>IgG1 and IgG2a antibody titers specific for a mix of the LiPABPs (A; Mix), LiPABP1 (B), LiPABP2 (C) and LiPABP3 (D) were individually determined by ELISA in mice (n = 8) immunized with a mixture of pcDNA3 plasmids encoding the LiPABP1, LiPABP2 and LiPABP3 proteins (pcDNA-LiPABPs), or controls groups inoculated with saline (Saline) or immunized with pcDNA3 vector (pcDNA) and challenged with <i>L</i>. <i>major</i> one month after last immunization. Prior to titer determination, sera were assayed at 1/200 dilution to monitor the reactivity against the LiPABP proteins. For sera showing moderate reactivity titers were determined from 1/50 to 1/12,8000. For sera showing high reactivity, titers were determined from 1/500 to 1/1,024,000. Horseradish peroxidase-conjugated anti-mouse IgG1 or IgG2a were used as the secondary antibodies at 1/2,000. Results are shown as whisker (min to max) plots. * (<i>P</i> < 0.05) and ** (<i>P</i> < 0.01) statistical differences between IgG1 and IgG2a reactivity values evaluated by the Mann-Whitney test. Results of each panel are representative of two independent experiments.</p

Course of <i>L</i>. <i>major</i> infection in BALB/c mice vaccinated with the LiPABP DNA combined vaccine.

<p>Mice (n = 8 per group) vaccinated with the LiPABP1 + LiPABP2 + LiPABP3 combined genetic vaccine (pcDNA3-PABPs) or inoculated with the pcDNA3 empty vector (pcDNA) or receiving the vaccine diluent (Saline) were challenged in the footpad with 5 × 10⁴ <i>L</i>. <i>major</i> stationary promastigotes one month after the last immunization. Footpad swelling is represented as the mean ± SD of the difference of thickness between the infected and the uninfected contra-lateral footpads (A). The numbers of viable parasites in the popliteal lymph nodes draining the infected legs (DLN) or in spleens were individually determined by limiting dilution at week seven post-challenge. Mean ± SD of the parasite burdens in the complete organ is shown (B). ⁺ <i>P</i> < 0.05, ⁺⁺ <i>P</i> < 0.01 and ⁺⁺⁺ <i>P</i> < 0.001 significant differences between LiPABP vaccinated and saline control mice; * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 significant differences between LiPABP vaccinated mice and pcDNA3 immunized control mice (Student T-test). Results in each panel are representative of two independent experiments.</p

The immunization of the LiPABP genetic combined vaccine induces a predominant Th1-biased response against the LiPABP family in BALB/c mice.

<p>Mice (n = 16 per group) were immunized three times with phosphate saline buffer (saline), with the pcDNA3 plasmid or with the LiPABP1 + LiPABP2 + LiPABP3 combined genetic vaccine. The presence of anti—LiPABP1, anti-LiPABP2, anti-LiPABP3 and anti-LiPABPs IgG1 and IgG2a immunoglobulins were individually determined four weeks after the last doses. Sera were assayed from 1/100 to 1/820,000 and horseradish peroxidase-conjugated anti-mouse IgG1 or IgG2a were used as the secondary antibodies at 1/2,000 (A). Results are shown as whisker (min to max) plots. * (<i>P</i> < 0.05) and ** (<i>P</i> < 0.01) statistical differences between IgG1 and IgG2a reactivity values evaluated by the Mann-Whitney test. At the same time <b>s</b>pleen cell cultures were established from eight mice. Splenocytes (5 x 10⁶ cells/ml) were cultured for 72 h at 37°C, 5% CO₂ in the presence of parasite LiPABP1, LiPABP2 and LiPABP3 as independent stimuli (12 μg/ml) or a mixture of the three proteins (12 μg/ml total protein, 4 μg/ml each one). As background control, parallel cultures were maintained without stimulation (Medium). The levels of IFN-γ, IL-10 and IL-4 were determined by capture ELISA in culture supernatants (B). Each bar represents the mean ± standard deviation (SD) of data taken from eight individual mice. ⁺ <i>P</i> < 0.05, ⁺⁺ <i>P</i> < 0.01 and ⁺⁺⁺ <i>P</i> < 0.001 significant differences between LiPABP vaccinated and saline control mice; ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 significant differences between LiPABP vaccinated mice and pcDNA3 immunized control mice (Student T-test). Results are representative of two independent experiments.</p