IFN- γ expression by T cells and NK cells following infected sand fly bites.

<p>Ear cells were recovered from naïve and exposed mice at 6 h, 24 h, 48 h and one week after transmission and cultured for 16 h. Brefeldin, PMA and ionomycin were added during the last four hours. The percent and absolute number of CD4+ T cells (<b>A</b>) or NK cells (<b>B</b>) expressing IFN-γ. Percentages shown are representative of three independent experiments; absolute numbers shown represent the mean ± SEM from three independent experiments. Five mice were used per group per experiment. *<i>p</i><0.05.</p

Characterization of the early leukocyte infiltrate following vector-transmission of <i>L. major</i>.

<p>Ear cells were recovered from naïve and exposed mice at 6 h, 24 h, 48 h and one week after transmission. At the indicated times after challenge, cells were stained <i>ex vivo</i> and analyzed by FACS for surface expression of TCR-β, CD4, NK1.1, F4/80, CD11b, and Ly-6G to identify specific leukocyte populations. (<b>A</b>) Absolute number of leukocytes per ear; (<b>B</b>) The gating strategy used to identify cells (NK1.1^posTCR-β^neg); CD4⁺ T cells (TCR-β^posCD4^pos), neutrophils (F4/80^neg CD11b^highLy6G^high), Gr1⁺ inflammatory monocytes (F4/80^posCD11b^highLy6G^high) and macrophages (F4/80^highLy6G^negCD11b^high); (<b>C</b>) Absolute number of neutrophils, NK cells, Gr1⁺ monocytes, macrophages and CD4⁺ T cells per ear. The numbers shown represent mean ± SEM from 3 independent experiments. Five mice were used per group per experiment. *<i>p</i><0.05.</p

Mice exposed to uninfected <i>P. duboscqi</i> bites are protected against vector-transmitted <i>L. major</i>.

<p>(<b>A</b>) Representative parasite load and percent metacyclics in the gut of infected <i>P. duboscqi</i> sand flies the day of transmission. The mean ± SEM are shown. (<b>B–C</b>) Mice exposed to uninfected bites in the right ear (▪) or are naïve () were challenged with 10 <i>L. major</i>-infected <i>P. duboscqi</i> sand flies in the left ear two weeks after the last exposure. (<b>B</b>) Weekly measurement of ear lesions after transmission. The mean ± SEM for 10 mice per group are shown. (<b>C</b>) The number of parasites per ear for five mice at four weeks post-transmission determined by limiting dilution assay. The bar represents the mean parasite burden per ear. Data are representative of three independent experiments. *<i>p</i><0.02; **<i>p</i> = 0.008.</p

Immunity to <i>Lutzomyia whitmani</i> Saliva Protects against Experimental <i>Leishmania braziliensis</i> Infection

<div><p>Background</p><p>Previous works showed that immunization with saliva from <i>Lutzomyia intermedia</i>, a vector of <i>Leishmania braziliensis</i>, does not protect against experimental infection. However, <i>L</i>. <i>braziliensis</i> is also transmitted by <i>Lutzomyia whitmani</i>, a sand fly species closely related to <i>Lu</i>. <i>intermedia</i>. Herein we describe the immune response following immunization with <i>Lu</i>. <i>whitmani</i> saliva and the outcome of this response after <i>L</i>. <i>braziliensis</i> infection.</p><p>Methods and findings</p><p>BALB/c mice immunized with <i>Lu</i>. <i>whitmani</i> saliva developed robust humoral and cellular immune responses, the latter characterized by an intense cellular infiltrate and production of IFN-γ and IL-10, by both CD4⁺ and CD8⁺ cells. Mice immunized as above and challenged with <i>L</i>. <i>braziliensis</i> plus <i>Lu</i>. <i>whitmani</i> saliva displayed significantly smaller lesions and parasite load at the challenge site. This protection was associated with a higher (p<0.05) IFN-γ production in response to SLA stimulation. Long-term persisting immunity was also detected in mice immunized with <i>Lu</i>. <i>whitmani</i> saliva. Furthermore, individuals residing in an endemic area for cutaneous leishmaniasis (CL) presented antibody responses to <i>Lu</i>. <i>whitmani</i> saliva. However CL patients, with active lesions, displayed a lower humoral response to <i>Lu</i>. <i>whitmani</i> saliva compared to individuals with subclinical <i>Leishmania</i> infection.</p><p>Conclusion</p><p>Pre-exposure to <i>Lu</i>. <i>whitmani</i> saliva induces protection against <i>L</i>. <i>braziliensis</i> in a murine model. We also show that <i>Lu</i>. <i>whitmani</i> salivary proteins are immunogenic in naturally exposed individuals. Our results reinforce the importance of investigating the immunomodulatory effect of saliva from different species of closely related sand flies.</p></div

<i>Lutzomyia whitmani</i> saliva immunization induces protection against experimental <i>Leishmania braziliensis</i> infection.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) (red circles) or were inoculated with saline (control) (black circles), in the right ear, at two week intervals. Two weeks after the last immunization mice challenged in the opposite ear with 10⁵ <i>L</i>. <i>braziliensis</i> plus SGS (equivalent to one pair of salivary glands). (<b>A</b>) Lesion development was monitored weekly. Data, shown as mean ± SEM, are from one representative experiment, performed with five mice in each group. (<b>B</b>) Parasite load was determined ten weeks after challenge via a limiting dilution assay. Data (bar at mean ± SEM) are shown individually. (<b>C</b>) Ten weeks after challenge, spleen cells were stimulated with Soluble <i>Leishmania</i> Antigen (SLA) and IFN- levels were measured in culture supernatants by ELISA, 72h later. Data, shown as mean ± SEM, are from one representative experiment, performed with five mice in each group. * p<0.05; **, p<0.01; ***, p< 0.001.</p

Human serum IgG response against <i>Lutzomyia whitmani</i> SGS.

<p>(<b>A</b>) ELISA was performed against <i>Lu</i>. <i>whitmani</i> SGS using human sera were from individuals from a Cutaneous Leishmaniasis (CL) endemic area (n = 264) or from control individuals (n = 13). (<b>B</b>) ELISA was performed against <i>Lu</i>. <i>whitmani</i> SGS with human sera from CL individuals (n = 30), sera from healthy individuals with either a negative (n = 231) or a positive (n = 33) <i>Leishmania</i> skin test (LST). The dotted line represents the cut-off value for the assay. Data are shown individually, bar at mean. p<0.01; ***, p< 0.001.</p

Persistent immunity induced by <i>Lutzomyia whitmani</i> saliva protects against experimental <i>Leishmania braziliensis</i> infection.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) (red circles) or were inoculated with saline (control) (black circles), in the right ear, at two week intervals. Twelve weeks after the last immunization mice challenged in the opposite ear with 10⁵ <i>L</i>. <i>braziliensis</i> plus SGS (equivalent to one pair of salivary glands). (<b>A</b>) Lesion development was monitored weekly. Data, shown as mean ± SEM, are from one representative experiment, performed with five mice in each group. (<b>B</b>) Parasite load was determined eight weeks after challenge via a limiting dilution assay. Data (bar at mean ± SEM) are shown individually. (<b>C</b>) IgG response to <i>Lu</i>. <i>whitmani</i> SGS determined by ELISA at twelve weeks before challenge. Data are shown individually, from one representative experiment (bar at mean ± SEM). * p<0.05; **, p<0.01; ***, p< 0.001.</p

Immune response to <i>Lutzomyia whitmani</i> salivary molecules.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) (red circles) or were inoculated with saline (control) (black circles), in the right ear, at two week intervals. (A) IgG respose to <i>Lu</i>. <i>whitmani</i> SGS determined by ELISA at different time points. Data are shown individually, from one representative experiment (bar at mean ± SEM). (B) Western blot analysis of <i>Lu</i>. <i>whitmani</i> salivary proteins using sera from immunized mice and SDS-PAGE depicting <i>Lu</i>. <i>whitmani</i> salivary proteins. (C) Two weeks after the last immunization, mice were challenged in the opposite ear with <i>Lu</i>. <i>whitmani</i> SGS and DTH response was measure 48h later. Data, shown as (median ± SD), are from two experiments performed with five mice in each group (D) Ear sections were obtained 48h later and stained with H&E. Sections were analyzed by optical microscopy under (100X). ** p<0.01.</p

Frequency and absolute number of cytokine-producing cells in mice immunized with <i>Lutzomyia whitmani</i> SGS.

<p>BALB/c mice were immunized three times with <i>Lu</i>. <i>whitmani</i> SGS (equivalent to 1 pair of salivary glands) or were inoculated with saline (control), in the right ear, at two week intervals. Forty eight hours after SGS inoculation in the left ear dermis, mice were euthanized and draining lymph node cells were stimulated with anti-CD3 and anti-CD28 for 16h. Cells were subsequently stained for determination of frequency and absolute numbers of (<b>A</b>) CD4⁺TCR⁺IFN-⁺ and CD8⁺TCR⁺IFN-⁺ and (<b>B</b>) CD4⁺TCR⁺IL-10⁺ and CD8⁺TCR⁺IL-10⁺ T cells. The numbers shown represent mean ± SEM from three independent experiments, each performed with three mice. Frequency plots are representative from one experiment.</p

Antibody responses induced by vaccination with KSAC+GLA-SE or L110f+GLA-SE.

<p>BALB/c mice were vaccinated by the subcutaneous route three times every three weeks with either 20 µg adjuvant alone (GLA-SE), 10 µg KSAC+20 µg GLA-SE, or 10 µg L110f+20 µg GLA-SE. Serum was obtained one day before each immunization and 20 days after the last immunization. (A) KSAC- and L110f-specific IgG1 and IgG2a antibody levels in mice vaccinated with the respective antigen. (B) Ratio of antigen-specific IgG2a∶IgG1 levels in mice vaccinated with KSAC+GLA-SE or L110f+GLA-SE. The OD values are presented after subtraction of the OD value of mice vaccinated with GLA-SE alone. Ten mice were used in each group. Data are representative of two independent experiments.</p