<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

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

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

<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-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

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