CNF1 activity confers curative immunoadjuvant properties.

<p>BALB/c mice were first infected with 3x10⁶ of stationary phase parasites. Fourteen days post-infection, mice were immunized via the nasal route with promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), PL plus either WT CNF1 (WT CNF1 + PL) or catalytically inactive CNF1 (mCNF1 + PL). The controls represent infected but non-immunized animals. Twenty-one and twenty-eight days post infection, the mice were immunized again. At day 42, the mice were sacrificed, and parasite numbers were determined by quantitative PCR using mouse spleen DNA extracts. The bars represent the mean cytokine production ± SEM. *: p<0.05, **: p<0,01, ***: p<0,001. n = 5.</p

Immunoadjuvant Properties of the Rho Activating Factor CNF1 in Prophylactic and Curative Vaccination against <i>Leishmania infantum</i>

<div><p>There is a need to develop new effective immunoadjuvants for prophylactic or therapeutic vaccines against intracellular pathogens. The activation of Rho GTPases by bacterial cytotoxic necrotizing factor 1 (CNF1) elicits humoral protective responses against protein antigens. Here, we set out to investigate whether CNF1 activity initiates humoral immunity against co-administered parasite antigens and anti-microbial immune signaling. We report that co-administration of wild-type (WT) CNF1 with <i>Leishmania</i> (<i>L</i>.) promastigote antigens at the nasal mucosa triggered prophylactic and curative vaccine responses against this parasite. Vaccination of the mucosa with promastigote lysate antigens combined with WT CNF1 conferred protection against high inoculum <i>L</i>. <i>infantum</i> infection, which reached 82% in the spleen. Immune parameter analysis by antigen recall indicated robust T-helper (Th)1 polarization of immune memory cells, with high IL-2 and IFN-γ production combined with decreased IL-4 production. Additionally, we explored the curative effect of WT CNF1 on previously infected animals. We observed that PL combined with WT CNF1, but not the inactive C866S mutant CNF1 (mCNF1), induced a 58% decrease in the parasite burden in the spleen.</p></div

<i>In vitro</i> antigen recall experiments.

<p>Spleen homogenates from mice immunized via the nasal route with promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1) and infected with 10⁸ stationary phase <i>L</i>. <i>infantum</i> metacyclic parasites were challenged with 50 μg/ml PL for 48 hours. The supernatants were collected and assayed for IL-2 (A), IFN-γ (B) and IL-4 (C) by ELISA. The bars represent the mean cytokine production ± SEM. *: p<0.05, **: p<0,01, ***: p<0,001. n = 7.</p

Antibody responses to <i>L</i>. <i>infantum</i> antigens post-vaccination.

<p>The anti-PL IgG antibody responses were measured post-vaccination by ELISA (one day before infection). Mice were immunized intranasally with 3x15 μg promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). The controls represent infected but non-immunized animals. Serum samples were tested at a 1/100 dilution and evaluated using HRP-labeled anti-mouse IgG. The interquartile ranges as well as the 10–90% percentiles are presented for each group. ***: p<0.001. The results are representative of 2 independent experiments. n = 7.</p

<i>In vitro</i> antigen recall for curative experiments.

<p>BALB/c mice were first infected with 3x10⁶ stationary phase parasites. Fourteen days post-infection, the mice were immunized via the nasal route with promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), or promastigote lysate plus either WT CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). Twenty-one and 28 days post infection, the mice were immunized again. The controls represent infected but non-immunized animals. At day 42, the mice were sacrificed, and spleen homogenates were challenged with 50 μg/ml PL for 48 hours. Supernatants were collected and assayed for IL-2 (A), IFN-γ (note, on graph it is INF) (B) and IL-4 (C) by ELISA. The bars represent the mean cytokine production ± SEM. *: p<0.05, *: p<0.05, **: p<0,01, ***: p<0,001. n = 5.</p

Antibody responses to <i>L</i>. <i>infantum</i> antigens post-infection.

<p>Anti-PL IgG antibody responses measured by ELISA post-infection in vaccinated mice. Mice were immunized intranasally with 3x15 μg promastigote lysate plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). Serum samples were collected one month after infection and tested at a 1/100 dilution and were evaluated using HRP-labeled anti mouse IgG. The interquartile ranges as well as the 10–90% percentiles are presented for each group. ***: p<0.001. The results are representative of 2 independent experiments. n = 7.</p

Monitoring of <i>L. infantum</i> infection using BLI and application for leishmanicidal drug screening.

<p>A. <i>Follow up of infection by BLI and drug screening in vivo</i>. Groups of 2 BALB/c mice were IV infected with 1×10⁸ or 3×10⁸ of stationary phase LUC-parasites and animals were imaged at the indicated times. BLI of mice shows the typical liver infection followed by spleen parasite colonization. By day 40, 3 mice with different levels of spleen infection were treated with miltefosine during 5 days (+) and reimaged 2 days later. Miltefosine efficacy was illustrated by the loss of luminescence signal compared to a control non treated mouse. Efficacy was confirmed by parasite counts (number of parasites/mg organ) measured by ELISA following dissection (indicated below each mouse tested). The illustration is representative of three different experiments. B. <i>In vitro drug screening</i>. PMA-differentiated THP-1 cells in 96 well microplates were incubated for 3 h with stationary phase LUC-parasites at a parasite-to-THP-1 cell ratio of 10∶1. After 48 h incubation, miltefosine at concentrations ranging from 10⁻⁴ to 10⁻⁸ M were delivered. 48 h later, the plate was washed and wells were lysed with 50 µl of reporter lysis buffer. 20 µl of supernatant was assayed for luminescence activity and showed a clear negative correlation between the drug concentration and luminescence of parasites. IC 50 was measured at 1.5 µM. Cell viability measured by trypan blue exclusion was 83% and 96% for miltefosine concentration of 100 µM and 10 µM, respectively. The dose dependency of miltefosin efficacy on parasite killing is representative of 3 different experiments.</p

Infectivity of LUC-parasites and sensitivity of bioluminescence <i>in vivo</i> and <i>ex vivo</i>.

<p>A. <i>Infectivity of Luc-parasites as compared to L. infantum WT parasites</i>. Both luciferase transgenic and wild type <i>L. infantum</i> stationary phase promastigotes establish themselves in BALB/c mice. Groups of 4 BALB/c mice were given either 0.12×10⁸ to 3×10⁸ stationary phase luciferase-transgenic or wild type <i>L. infantum</i>. One month later, mice were imaged, the bioluminescence being recorded before their sacrifice. <i>L. infantum</i> burdens were estimated by ELISA in the liver and the spleen. Burdens were calculated as parasite/mg organ x organ weight (in mg). Data representative of three experiments are presented as box whisker plots. For statistical analysis Man Whitney Wilcoxon test was performed and did not show any significant difference between parasite loads of mice inoculated with WT or luciferase parasites; ns =  not-significant. B. <i>Bioimaging of BALB/c mice one month post the IV inoculation of luciferase transgenic L. infantum</i>. Mice inoculated with LUC-parasite and with parasite loads depicted in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001323#pntd-0001323-g001" target="_blank">figure 1A</a>, were given luciferin via IP. The photon emission was recorded once the anesthetized mice were deposited in the imaging chamber of the Photon Imager. Red zones are ROIs that delineate the liver or spleen. C. <i>Sensitivity of BLI.</i> Luminescence (photon/s/cm²) was recorded in ROIs corresponding to liver and spleen of the 11 mice infected with LUC-parasites depicted in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001323#pntd-0001323-g001" target="_blank">figure 1A</a>. Luminescence recorded in ROIs was plotted versus parasite density measured by ELISA as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001323#pntd-0001323-g001" target="_blank">figure 1A</a> to generate regression curves. The threshold sensitively of BLI calculated as three times luminescence of naïve mice (indicated by the gray zone) was 20,000 to 40,000 parasites/mg for spleen and liver respectively. D. <i>Ex-vivo quantification of parasite loads by bioluminescence</i>. Luminescence of equal numbers of the LUC-parasite under exponentially growing promastigote form or liver and spleen amastigotes was analyzed using a luminometer. Equal numbers of liver and spleen amastigotes from mice infected with WT promastigotes were used as specificity control. Sensitivity of <i>ex vivo</i> analysis of parasite density by bioluminescence (1,000 to 6,000 amastigotes/mg for spleen and liver respectively) was calculated as parasite numbers corresponding to twice luminescence background values. E. <i>Accuracy of ex vivo bioluminescence analysis for estimation of parasite density</i>. Liver and spleen samples from 18 IV infected BALB/c mice were detergent extracted and assayed both by ELISA and bioluminescence. Values generated by both techniques were plotted to generate regression curves.</p

Localization of bioluminescent parasites in intra-abdominal mesenteric fat in BALB/c mice infected by IP route.

<p>4 Mice were infected with 5×10⁸ of LUC-parasites by IP route. Infection was monitored by BLI. On day 40 post infection (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001323#pntd-0001323-g003" target="_blank">Fig 3A</a>), mice showed intraperitoneal parasites localisation by BLI. One representative mouse (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001323#pntd-0001323-g003" target="_blank">Fig 3B</a>) was dissected and the adipose tissue localization detected by bioluminescence was verified <i>in situ</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001323#pntd-0001323-g003" target="_blank">Fig 3C</a>) and after removing the adipose tissue and reimaging (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001323#pntd-0001323-g003" target="_blank">Fig 3D</a>). The illustrations are representative of at least three different experiments.</p