Specificity of recombinant proteins.

<p>Results from ELISA are presented as mean optical densities ± standard deviation of IgG antibody reaction with <i>P</i>. <i>orientalis</i> salivary gland homogenate (SGH) and three recombinant proteins (rPorSP24, rPorSP67, and rPorSP76) in mice experimentally bitten by <i>Ph</i>. <i>orientalis</i>, <i>Ph</i>. <i>papatasi</i>, or <i>Se</i>. <i>schwetzi</i>, and non-exposed control mice. Four mice per sand fly colony and four non-exposed controls were used. Asterisks (*) indicate significant differences (p<0.05, calculated with non-parametric Wilcoxon Rank-Sum Test) of IgG levels between mice bitten by <i>Ph</i>. <i>orientalis</i> and mice bitten by other sand fly species or non-bitten controls.</p

Identification of <i>Phlebotomus orientalis</i> salivary antigens in dogs.

<p><i>Ph</i>. <i>orientalis</i> salivary proteins were separated under non-reducing conditions by SDS-PAGE on a 12% gel and incubated with two different pools of sera from naturally-exposed Ethiopian dogs (Et), and one pooled sera from non-exposed control dogs (neg). Each pool was a mixture of 5 individual samples. Five antigenic proteins (PorSP65, PorSP24, PorSP15, PorSP76, and PorSP67) were identified by successive proteome analysis and mass spectrometry. Molecular weights of standard (STD) are indicated. UIB means unidentified bands.</p

Validation of recombinant proteins.

<p>Selected recombinant proteins were validated in ELISA tests with sera of indicated domestic animals naturally-exposed to <i>Phlebotomus orientalis</i>. The analysis was based on comparison with anti-<i>Ph</i>. <i>orientalis</i> SGH IgG as a standard. The table provides cut-off values, mean values of optical density ± standard deviation of IgG levels in animals exposed to <i>Ph</i>. <i>orientalis</i>, correlation coefficients between IgG levels against SGH and a recombinant protein (ρ), positive predictive values (PPV), negative predictive values (NPV), sensitivity, and specificity. Asterisks (*) indicate significant correlations: *p<0.05, **p<0.01, ***p<0.001. Combinations with the correlation coefficient lower than 0.7 in evaluation experiments (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004553#pntd.0004553.t002" target="_blank">Table 2</a>) were excluded from the validation experiments. For each combination, the lowest cut-off value, the highest correlation coefficient, and the highest PPV, NPV, sensitivity, and specificity values are shaded grey. N.A. means not applicable.</p

<i>Phlebotomus orientalis</i> salivary proteins expressed in <i>Escherichia coli</i>.

<p><i>Phlebotomus orientalis</i> salivary proteins expressed in <i>Escherichia coli</i>.</p

Evaluation of recombinant proteins by correlation analysis.

<p>Evaluation of recombinant proteins by correlation analysis.</p

Correlation analyzes between IgG antibodies against SGH and recombinants rPorSP65 and rPorSP24 in ELISA.

<p>For each animal species from validation experiments (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004553#pntd.0004553.g003" target="_blank">Fig 3</a>), the protein with the highest positive correlation was displayed: A: rPorSP65 (ParSP25-like protein) tested with canine sera (n = 50), B: rPorSP24 (yellow-related protein) tested with goat sera (n = 248), C: rPorSP24 (yellow-related protein) tested with sheep sera (n = 209). Sera from naturally-exposed Ethiopian animals together with sera from non-exposed controls were included in this analysis. Correlation coefficients and p-values from Spearman-Rank analysis are indicated.</p

Visualization of the tunnel in 3Q6K_Llon1.

<p>The structure was visualized in PyMOL using the MOLE script for calculating the tunnel. The 3Q6K_Llon1 protein is drawn in green, the tunnel through this protein structure is in black mesh and the red-stick molecule represents serotonin.</p

Sand fly species with published salivary glands–cDNA libraries.

<p>Sand fly species with published salivary glands–cDNA libraries.</p

Identified sand fly salivary YRPs.

<p>Identified sand fly salivary YRPs.</p

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

<div><p>Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against <i>Leishmania</i> parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from <i>Lutzomyia longipalpis</i>. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-<i>Leishmania</i> vaccines or as an antigen to measure host exposure to sand flies.</p></div