ROC curve of antibody levels predict the positivity thresholds against rHSP70 and rH2A.

<p>ROC curves were constructed using data obtained with sera from TL (n = 102) (A), ML (n = 53) (B) and CL (n = 49) as patients and sera from individuals with Chagas' disease (n = 30), SLE (n = 10), leprosy (n = 30) and tuberculosis (n = 22) as negative control. The solid lines represent the area under the curve and the dotted line represents the identity line curves. The tables show the detailed information obtained from each ROC curve (cut-off values chosen, area under the curve, the p values, and the sensitivity and specificity with a confidence interval of 95% and the likelihood ratio).</p

Anti-<i>Leishmania</i> IgG antibodies levels TL patients.

<p>Sera from mucosal <i>Leishmania</i>sis (ML) (n = 53) and cutaneous <i>Leishmania</i>sis (CL) (n = 49) patients, and sera from healthy individuals from endemic (n = 39) and from non-endemic (n = 49) areas were tested against SLA (A), rHSP70 (B), rH2A (C), rH2B (D), rH3 (E) rH4 (F) and rKMP11 antigens (G). The cut-off was calculated by comparison of the reactivity values from patient's serum samples and from normal volunteers from endemic area and non-endemic areas. The cut-off value for negative and positive samples is indicated in dotted line. The solid lines represent the median values. Each point represents the mean of the duplicate OD values for the same serum with a standard deviation lower than 20%. Significance was compared using Kruskal-Wallis test with Dunn's post test for multiple comparisons. * p<0.05, ** p<0.001 **, p<0.0001.</p

Epidemiological characteristics of individuals involved in the study.

<p>CL = Cutaneous Leishmaniasis; ML = Mucosal Leishmaniasis; Non-endemic ctls = Non-endemic controls; LES = Lupus Erythematosus Systemic.</p

Specificity of ELISA employing parasite antigens.

<p>Sera from patients with Chagas' disease (n = 30), SLE (n = 10), leprosy (n = 30) and tuberculosis (n = 22) were tested against SLA (A), rHSP70 (B), rH2A (C), rH2B (D), rH3 (E) rH4 (F) and rKMP11 antigens (G). The cut-off was established from the TL ROC curve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066110#pone-0066110-g002" target="_blank">Fig. 2</a>) (dotted line). The solid lines represent median values. Each point represents the mean of duplicate values with a standard deviation lower than 20%.</p

ROC curve of antibody levels predict the positivity thresholds against parasite antigens.

<p>The ROC curves were constructed using data obtained in ELISA performed with antigens and sera from TL (n = 102) (A), ML (n = 53) (B) or CL (n = 49) (C) patients and control subjects from an endemic (n = 39) and from a non-endemic (n = 49) area. The solid lines represent the area under the curve and the dotted line represents the identity line curves. The tables show the detailed information obtained for each ROC curve (cut-off values chosen, area under the curve, the values of p, and the sensitivity and specificity with a confidence interval of 95% and the likelihood ratio).</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

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

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

Genes modulated in High- and Low- IFN-γ producers discriminate subclinical <i>L</i>. <i>braziliensis</i> infection from active CL.

<p>(A) PBMCs from CL patients (<i>n</i> = 5) and SC individuals (<i>n</i> = 8) were stimulated with <i>L</i>. <i>braziliensis</i> for 72h. Relative expression of <i>IFI27</i>, <i>IFIT1</i>, <i>TLR2</i>, <i>IRF1</i>, <i>JAK2</i> and <i>IL6</i> was evaluated by qRT-PCR. Gene expression is represented as fold change of stimulated over unstimulated cultures, normalized to a housekeeping gene and each symbol represents one individual. (B) A heat map was designed to depict the pattern of gene expression [shown in (A)] of SC individuals) vs. active CL and two-way hierarchical cluster analysis (Ward’s method) of differentially expressed genes was performed. Expression scale for each gene represents the log2-fold change from the mean. (C) Principal component analysis of the differentially expressed genes [depicted in (A)] showing PC1 (x axis), PC2 (y axis) and PC3 (z axis).</p