Anti-leishmanial activity of canine monocyte-derived macrophages in non-immune and immune dogs.

<p>The ability of pre-infected canine monocyte-derived macrophages to kill <i>Leishmania</i> parasites when they were exposed to autologous peripheral lymphocytes derived from PBMC was expressed as the percentage of parasitic index inhibition after <i>in vitro</i> infection with <i>Leishmania infantum</i> promastigotes (MHOM/MA/67/ITMAP-263) and 72 h incubation with and without autologous lymphocytes. Anti-leishmanial activity of co-cultured canine macrophages was evaluated immediately before immunization and two months after the third dose. Values represent means +/- standard deviation of duplicate experiments (* <i>p</i>< 0.05, ** <i>p</i><0.01, *** <i>p</i><0.001).</p

Vaccine specific serological responses as detected by Enzyme-Like Immunosorbent Assay (ELISA).

<p>Evolution of levels of (A) anti-rPSA, (B) anti-Cter-rPSA and (C) anti-<i>Li</i>ESAp specific IgG2 antibodies was assessed in serum samples isolated from dogs of each group immediately before immunization and at different times post-immunization: first month after the second dose and two months after the third dose. Each serum sample was tested in triplicates. Cut-off value was calculated using the following formula: mean OD in sera from all dogs before immunization + 3 standard deviations. Values represent means OD +/- standard deviation of triplicate experiments (* <i>p</i>< 0.05, ** <i>p</i><0.01, *** <i>p</i><0.001).</p

Parasitological evaluation of placebo and vaccinated dogs.

<p>The presence of (A) live <i>Leishmania</i> parasites was highlighted by subculture analysis of bone marrow aspirates isolated from dogs of placebo (n = 5), rPSA/QA-21 (n = 9) and Cter-rPSA (n = 5) groups at 2, 4 and 6 months post-challenge (PC). A sample was considered as positive when <i>Leishmania</i> parasites were detected during the seeding or subculture analysis. The presence of (B) <i>Leishmania</i> DNA and (C) the parasite load in bone marrow aspirates of dogs of each group were assessed by quantitative PCR. Dogs were considered as positive when the titer was superior to 40 parasites per mL. Data are expressed as (B) the number of dogs with positive PCR at each time points post-challenge and (C) the mean number of parasites per mL of bone marrow aspirates at different times post-challenge (4 and 6 months) (* <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001).</p

Total Soluble <i>Leishmania</i> antigen (TSLA) specific IFN-γ, granzyme B, TNF-α responses.

<p>IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g002" target="_blank">Fig. 2a</a>), granzyme B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g002" target="_blank">Fig. 2b</a>), were detected and quantified from culture supernatants of PBMC exposed for 120h to local TSLA (10 µg/ml) and TSLA Ldd8 (10 µg/ml), by Cytokine Beads Array test (CBA) using Flow cytometry. Statistically significant differences between stimulated and non stimulated cultures and between groups (p≤0.03) are showed.</p

SDS-PAGE and silver staining of purified recombinant <i>La</i>-PSA38S protein expressed in <i>L. tarentolae</i>.

<p>Purified <i>La</i>-PSA38S was separated by electrophoresis in a 12% SDS-PAGE and silver stained under reducing (R) and non reducing (NR) conditions. The arrow indicates the position of <i>La</i>-PSA-38S (45 kDa) and Molecular Weight markers were marked in kDa.</p

<i>La</i>PSA-38S specific IFN-γ, granzyme B, TNF-α and IL-10 responses.

<p>IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3a</a>), granzyme B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3b</a>), TNF-α (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3c</a>) and IL-10 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3d</a>) were detected and quantified from culture supernatants of PBMC exposed for 120 h to SLA (10 µg/ml) or <i>La</i>PSA-38S (10 µg/ml) using Cytokine Beads Array test (CBA). Data were analyzed by Flow cytometry. PHA (10 µg/ml) was used for all cultures as positive control (data not shown). Statistically significant differences between stimulated and non stimulated cultures (p≤0.003) and between groups (p≤0.01) are showed.</p

Total IgG prevalence against <i>La</i>PSA-38S in different endemic areas of leishmaniasis.

<p>The cut-off value of reactivity for <i>La</i>PSA-38S antigen was calculated as the mean plus 3 SD of the OD values observed in naive controls from each country. These cut-off values allowed us to distinguish between positive and negative sera and consequently to estimate performance parameters of each ELISA test.</p

Study population.

<p>The recruitment and sampling collection (186 donors) of different groups (patients, cured, immunes without clinical symptoms and naives) were performed in endemic and non-endemic areas in each country.</p><p>*CVLd: Cured Visceral Leishmaniasis due to <i>L. donovani</i> (India), aVLd: active Visceral Leishmaniasis due to <i>L. donovani</i> (India), HLR-I: Healthy Low Responders from India, CCLb: Cured Cutaneous Leishmaniasis due to <i>L. brasiliensis</i> (Peru), aCLb: active Cutaneous Leishmaniasis due to <i>L. brasiliensis</i> (Peru), HLR-P: Healthy Low Responders from Peru, CCLm: Cured Cutaneous Leishmaniasis due to <i>L. major</i> (Tunisia), HHR-Lm: Healthy High Responders living in an endemic area for <i>L. major</i> (Tunisia), HHR-LiT: Healthy High Responders living in an endemic area for <i>L. infantum</i> (Tunisia), aVLiT: active Visceral Leishmaniasis due to <i>L. infantum</i> (Tunisia), H-T: Healthy Low Responders from Tunisia, HHR-LiF: Healthy High Responders living in an endemic area for <i>L. infantum</i> (France), HLR-F: Healthy Low Responders from France, HHR-LiS: Healthy High Responders living in an endemic area for <i>L. infantum</i> (Spain), aVLiS: active Visceral Leishmaniasis due to <i>L. infantum</i> (Spain), HLR-S: Healthy Low Responders from Spain.</p