Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach

In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruziinfected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The study’s findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis of L. infantum infection in dogsThis work was supported by grants from the Pró-Reitoria de Pesquisa from UFMG (Edital 07/2012), Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-NANOBIOFAR, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (CBB-APQ-02364-08, CBB-APQ-00356-10, CBB-APQ-00496-11, and CBB-APQ-00819-12), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (APQ-472090/2011-9), and the Instituto Nacional de Ciência e Tecnologia em Vacinas (INCT-V). E.A.F.C. and A.P.F. are CNPq grant recipients. M.A.C.-F. is a FAPEMIG/CAPES grant recipient. This study was supported in Spain, in part, by grants from the Ministerio de Ciencia e Innovación (FIS/PI1100095)

Identification of differentially expressed proteins from Leishmania amazonensis associated with the loss of virulence of the parasites.

BACKGROUND: The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach. METHODOLOGY/PRINCIPAL FINDINGS: Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified. CONCLUSIONS/SIGNIFICANCE: The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis

Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis.

BackgroundThe present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.Methodology/principal findingsThe LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.Conclusions/significanceThe present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL

Correction: Antigenicity and Protective Efficacy of a Leishmania Amastigote-specific Protein, Member of the Super-oxygenase Family, against Visceral Leishmaniasis.

[This corrects the article on p. e2148 in vol. 7.]

Two-dimensional profiles of cultures from <i>Leishmania amazonensis</i>.

<p>The 2-DE gels were obtained after the separation of stationary promastigotes extracts (R0, R10, R20, and R30 passages; 650 µg of each extract) by 2-DE (first dimension: IEF pH range 4–7; second dimension: 12% SDS-PAGE) and staining with colloidal Coomassie Brilliant Blue G-250. The gel fragments in the lower portion of the figures represent evaluated amplifications (see within the dotted lines). 2-DE gels of each passage were derived from four independent protein preparations of each passage. One representative preparation of each sample is showed in this study.</p

Infection of BALB/c mice.

<p>Mice (n = 8) were infected subcutaneously with 1×10⁶ stationary promastigotes of <i>Leishmania amazonensis</i>. Lesion development in the infected footpads was monitored weekly, up to 8 weeks after infection. Mean ± standard deviation (SD) are shown in (A). Parasite load in the infected footpads, spleen, and liver was analyzed in all animals (B). Other mice (n = 8, per group) were subcutaneously infected with 1×10⁶ stationary promastigotes of <i>L. amazonensis</i> obtained from R0 or R30 passages, and the lesion development was monitored up to 8 weeks after infection. Mean ± SD of the groups are shown (C). The parasite load in the infected footpads, spleen, and liver was also evaluated in these groups (D). The experiments were repeated three times, and presented similar results. *Significant difference between the R0 and R30 groups (<i>P</i><0.05).</p

Immunoblotting validation of some proteins in <i>Leishmania amazonensis</i>.

<p>Representative immunoblotting of some proteins that presented a significant decrease or increase in their expression content between R0 and R30 passages, using promastigote and amastigotes-like forms of <i>L. amazonensis</i>, are shown here. For each protein [α-tubulin, in A; paraflagellar rod protein 1D, in B; glucose-regulated protein 78 (GRP78) in C, and heat shock protein 83 (HSP83), in D], this image presents one example of correspondent 2-DE spot of promastigote form obtained from R0 or R30 passages. The antibodies used to validate each spot are described in the material and methods section. Asterisks represent the comparison between the expression of the protein in the R0 condition in relation to the R30 sample in each parasite stage, applying the Student's t-test (<i>P</i><0.05), and the numbers represent the relative variation of each protein in comparison to R0 of each parasite stage. All experiments were performed in triplicate.</p

Identification of proteins that presented a significant increase in their expression content.

a<p>⁾ Spots match ID number obtained from ImageMaster Platinum;</p>b<p>⁾ Name of the identified protein;</p>c<p>⁾ Uniprot identification code;</p>d<p>⁾ Experimentally predicted and expected isoelectric point (<i>pI</i>);</p>e<p>⁾ Experimentally predicted and expected molecular weight (<i>Mr</i>, in kDa);</p>f<p>⁾ Number of identified peptides by MS;</p>g<p>⁾ Percentage of the protein sequence covered by identified peptides;</p>h<p>⁾ Normalized data from R0 represented by mean values of each condition divided by R30 value;</p>i<p>⁾ Fold represents the maximum spot intensity mean value of the conditions divided by the smallest value;</p>j<p>) One-way ANOVA (<i>P</i><0.01) obtained from spot analysis;</p>k<p>⁾ Biological functions according to NCBI, UniProt, and Gene Ontology databases;</p>l<p>⁾ Biological activity and/or immunological application described in other studies: <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Joshi1" target="_blank">[53]</a> Joshi et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Tielens1" target="_blank">[54]</a> Tielens et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Soto1" target="_blank">[55]</a> Soto et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Lackovic1" target="_blank">[56]</a> Lackovic et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Scher1" target="_blank">[57]</a> Scher et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-SnchezCaete1" target="_blank">[58]</a> Sánchez-Cañete et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Peris1" target="_blank">[59]</a> Peris et al., 1994; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Steiner2" target="_blank">[60]</a> Steiner et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Jaramillo1" target="_blank">[61]</a> Jaramillo et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-DuclertSavatier1" target="_blank">[62]</a> Duclert-Savatier et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Celeste1" target="_blank">[63]</a> Celeste et al., 2004; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Liu1" target="_blank">[64]</a> Liu et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Leblanc1" target="_blank">[65]</a> Leblanc et al., 1998; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Priest1" target="_blank">[66]</a> Priest et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-BakkerGrunwald1" target="_blank">[67]</a> Bakker-Grunwald, 1992; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Banerjee1" target="_blank">[68]</a> Banerjee et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Casanova1" target="_blank">[69]</a> Casanova et al., 2008; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Jensen1" target="_blank">[70]</a> Jensen et al., 2001. The proteins were identified through the data included in the NCBI database (dated June 2012) for <i>Leishmania spp.</i></p

Identification of proteins that presented a significant decrease in their expression content.

a<p>⁾ Spots match ID number obtained from ImageMaster Platinum;</p>b<p>⁾ Name of the identified protein;</p>c<p>⁾ Uniprot identification code;</p>d<p>⁾ Experimentally predicted and expected isoelectric point (<i>pI</i>);</p>e<p>⁾ Experimentally predicted and expected molecular weight (<i>Mr</i>, in kDa);</p>f<p>⁾ Number of identified peptides by MS;</p>g<p>⁾ Percentage of the protein sequence covered by identified peptides;</p>h<p>⁾ Normalized data from R0 represented by mean values of each condition divided by R30 value;</p>i<p>⁾ Fold represents the maximum spot intensity mean value of the conditions divided by the smallest value;</p>j<p>⁾ One-way ANOVA (<i>P</i><0.01) obtained from spot analysis;</p>k<p>⁾ Biological functions according to NCBI, UniProt, and Gene Ontology databases;</p>l<p>⁾ Biological activity and/or immunological application described in other studies: <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Tull1" target="_blank">[22]</a> Tull et al., 2010; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Oliveira1" target="_blank">[23]</a> Oliveira et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Daifalla1" target="_blank">[24]</a> Daifalla et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-IyerJ1" target="_blank">[25]</a> Iyer et al., 2008; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-HungerGlaser1" target="_blank">[26]</a> Hunger-Glaser et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Werbovetz1" target="_blank">[27]</a> Werbovetz et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Feng1" target="_blank">[28]</a> Feng et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Niemirowicz1" target="_blank">[29]</a> Niemirowicz et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-HungerGlaser2" target="_blank">[30]</a> Hunger-Glaser et al., 1997; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Alcolea1" target="_blank">[31]</a> Alcolea et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Bhaskar1" target="_blank">[32]</a> Bhaskar et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Khanra1" target="_blank">[33]</a> Khanra et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Berberich1" target="_blank">[34]</a> Berberich et al., 2003; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Moore1" target="_blank">[35]</a> Moore et al., 1996; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Swenerton1" target="_blank">[36]</a> Swenerton et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Hummadi1" target="_blank">[37]</a> Hummadi et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Martins1" target="_blank">[38]</a> Martins et al., 2006; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Burns1" target="_blank">[39]</a> Burns et al., 1993; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Kushawaha1" target="_blank">[40]</a> Kushawaha et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Misra1" target="_blank">[41]</a> Misra et al., 2005; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Bringaud1" target="_blank">[42]</a> Bringaud et al., 1995; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Eggleson1" target="_blank">[43]</a> Eggleson et al., 1999; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Mureev1" target="_blank">[44]</a> Mureev et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Steiner1" target="_blank">[45]</a> Steiner et al., 2007; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-MartnezRodrguez1" target="_blank">[46]</a> Martínez-Rodríguez et al., 2012; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Drummelsmith2" target="_blank">[47]</a> Drummelsmith et al., 2004; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Achour1" target="_blank">[48]</a> Achour et al., 2002; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Buda1" target="_blank">[49]</a> Buda et al., 2013; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Alcolea2" target="_blank">[50]</a> Alcolea et al., 2011; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Martn1" target="_blank">[51]</a> Martín et al., 2009; <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002764#pntd.0002764-Silva1" target="_blank">[52]</a> Silva et al., 2012. The proteins were identified through the data included in the NCBI database (dated June 2012) for <i>Leishmania spp</i>.</p

Cellular and humoral response induced in BALB/c mice by immunization with rLiHyp1 plus saponin.

<p>Single cells suspensions were obtained from the spleens of mice, four weeks after vaccination. Cells were non-stimulated (medium; background control) or stimulated with rLiHyp1 (20 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 levels were measured in culture supernatants by capture ELISA (<b>A</b>). Each bar represents the mean ± standard deviation (SD) of data from four individual mice per group. Statistically significant differences in the IFN-γ, IL-12 and GM-CSF levels between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>B</b>); and between IL-12/IL-10 and IL-12/IL-4 levels (<b>C</b>) are also showed. Statistically significant differences in the ratios between the rLiHyp1 plus saponin group and control groups were observed (*** <i>P</i><0.0001). The ratio between rLiHyp1-specific IgG1 and IgG2a antibodies was obtained for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and control groups was also observed (* <i>P</i><0.005) (<b>D</b>).</p