Synthetic peptides as a novel approach for detecting antibodies against sand fly saliva.

BACKGROUND: Hosts repeatedly bitten by sand flies develop antibodies against sand fly saliva and screening of these immunoglobulins can be employed to estimate the risk of Leishmania transmission, to indicate the feeding preferences of sand flies, or to evaluate the effectiveness of vector control campaigns. Previously, antibodies to sand fly saliva were detected using whole salivary gland homogenate (SGH) or recombinant proteins, both of which also have their disadvantages. This is the first study on sand flies where short peptides designed based on salivary antigens were successfully utilized for antibody screening. METHODOLOGY/PRINCIPAL FINDINGS: Specific IgG was studied in hosts naturally exposed to Phlebotomus orientalis, the main vector of Leishmania donovani in East Africa. Four peptides were designed by the commercial program EpiQuest-B, based on the sequences of the two most promising salivary antigens, yellow-related protein and ParSP25-like protein. Short amino acid peptides were synthesised and modified for ELISA experiments. Specific anti-P. orientalis IgG was detected in sera of dogs, goats, and sheep from Ethiopia. The peptide OR24 P2 was shown to be suitable for antibody screening; it correlated positively with SGH and its specificity and sensitivity were comparable or even better than that of previously published recombinant proteins. CONCLUSIONS/SIGNIFICANCE: OR24 P2, the peptide based on salivary antigen of P. orientalis, was shown to be a valuable tool for antibody screening of domestic animals naturally exposed to P. orientalis. We suggest the application of this promising methodology using species-specific short peptides to other sand fly-host combinations

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

BACKGROUND:Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS:Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. CONCLUSIONS/SIGNIFICANCE:Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species

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

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

<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

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

Evaluation of recombinant proteins by correlation analysis.

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