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    Schematic representation of various cDNAs and corresponding deduced recombinant proteins evaluated in this study. Maps were derived from sequences produced after direct sequencing of the Lci2A and Lci2B, Lci3A and Lci4A cDNAs and/or from the coding genomic sequences of the Lci1A and Lci5A, available at the GeneDb (www.genedb.org). A, Lci1A cDNA, including the 5′ untranslated region (UTR) segment, which is included in the recombinant protein rLci1A-NH6. B, Lci2A cDNA, highlighting the 11 39-amino acid repetitive segments and the segment common to Lci2B cDNA and which is present in recombinant protein rLci2B-NH6. C, Lci3A cDNA highlighting the 21 repetitive 14-amino acid motif segments and recombinant protein rLci3A-R3-NH6, which contain the last three repetitive segments. D, Lci4A cDNA sequence emphasizing the last two repeats and part of the third repeat and recombinant protein rLci4A-NH6. E, Lci5A-coding sequence and the recombinant protein rLci5A-I –NH6. Arrows and open boxes, which have not been drawn to represent protein segments in scale, represent repetitive amino acid motifs and non-repetitive polypeptide segments. Hatched represent His-tag provided by the vector. ORF = open reading frame.

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    Schematic representation of the full-length sequences or sequence fragments from selected kinesins from the Leishmania donovani complex. Sequences from LdK39 (kinesin of L. donovani18), the protein encoded by LinJ14_V3.1180 (kinesin of L. infantum, GeneDB), rLcKin and rk39 (L. chagasi kinesin fragments described by Evans and others7), and the rLci2A and rLci2B kinesin fragments reported here are represented. Open boxes represent overlapping regions, and closed boxes indicate sequences in protein encoded by LinJ14_V3.1180 that are absent in LdK39. The numbers indicate the position of the amino acids at the start and end of the polypeptides.

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    Repeats found in the recombinant proteins encoded by the Lci2 and Lci3 gene fragments. A, The 39-amino acid repetitive motifs of rLci2A and rLci2B, the consensus sequences of rLci2B, and the consensus sequences rK39 are presented showing the variability of the amino acid sequences. B, The different 14-amino acid repeats found in rLci3A (the number of copies of each repeat present in the sequence is indicated in parenthesis), the consensus sequences of the rLci3A repeats, and the three repeats present in the rLci3A-R3 recombinant protein are shown.

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    Recombinant proteins evaluated in this study. Affinity-purified, His-tagged, recombinant fragments (rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, rLci4A-NH6, and rLci5A-I-NH6) were analyzed by using run in denaturing 15% SDS-PAGE stained with Coomassie Blue. Arrow indicates non-degraded rLci5A-I-NH6 protein.

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    Reactivity of a panel of canine serum with Leishmania recombinant antigens assessed by an enzyme-linked immunosorbent assay. Serum samples from 46 dogs with visceral leishmaniasis (L. chagasi-infected), 31 dogs with other infections (4 with babesiosis, 20 with erhlichiosis, and 7 with demodicosis) and 15 healthy control animals were assayed with the various recombinant antigens produced in Escherichia coli (rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, rLci4A-NH6, and rLci5A-I-NH6) or with total L. chagasi lysate as antigen (LAg). Each symbol corresponds to the result obtained with an individual serum. Solid horizontal lines indicate mean optical densities. Dashed horizontal lines indicate cutoff values, which were calculated as described in the Materials and Methods.

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    Reactivity of a panel of human sera with Leishmania recombinant antigens assessed by an enzyme-linked immunosorbent assay. Serum samples 36 patients with visceral leishmaniasis (VL), 26 patients with cutaneous leishmaniasis (CL), 40 patients with Chagas' disease (Chagas) and 50 healthy controls were assayed with the same set of antigens evaluated in Figure 5. Each symbol corresponds to the result obtained with an individual serum. Solid horizontal lines indicate mean optical densities. Dashed horizontal lines indicate cutoff values, which were calculated as described in the Materials and Methods.

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Characterization of Novel Leishmania infantum Recombinant Proteins Encoded by Genes from Five Families with Distinct Capacities for Serodiagnosis of Canine and Human Visceral Leishmaniasis

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  • Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil; Instituto Nacional de Doenças Tropicais, Salvador, Bahia, Brazil; Centro de Pesquisas Aggeu Magalhães, Recife, Pernambuco, Brazil

To expand the available panel of recombinant proteins that can be useful for identifying Leishmania-infected dogs and for diagnosing human visceral leishmaniasis (VL), we selected recombinant antigens from L. infantum, cDNA, and genomic libraries by using pools of serum samples from infected dogs and humans. The selected DNA fragments encoded homologs of a cytoplasmic heat-shock protein 70, a kinesin, a polyubiquitin, and two novel hypothetical proteins. Histidine-tagged recombinant proteins were produced after subcloning these DNA fragments and evaluated by using an enzyme-linked immunosorbent assays with panels of canine and human serum samples. The enzyme-linked immunosorbent assays with different recombinant proteins had different sensitivities (67.4–93.0% and 36.4–97.2%) and specificities (76.1–100% and 90.4–97.3%) when tested with serum samples from Leishmania-infected dogs and human patients with VL. Overall, no single recombinant antigen was sufficient to serodiagnosis all canine or human VL cases.

Introduction

Visceral leishmaniasis (VL) is a neglected disease with an annual worldwide incidence of 500,000 human cases and which, in epidemiologic terms, can be classified into anthroponotic and zoonotic types.1 The predominant causal agent of the anthroponotic type of VL is Leishmania donovani, and the major species that cause zoonotic VL are L. chagasi in the Western Hemisphere and L. infantum in the other areas of the world.2,3 These two species that cause zoonotic VL are believed to be indistinguishable from each other.4,5 A subclinical form of the infection develops in most persons exposed to L. infantum and L. donovani,69 and the proportion of dogs that remain asymptomatic after being exposed to the parasite is not known.

The gold standard method for the diagnosis of human VL is the search for Leishmania amastigotes in smears of needle aspirates from splenic or bone marrow tissues. However, the sample collection procedure used in the method is invasive and may pose risks to patients, and the method sensitivity varies considerably.1012 Detection of canine infection and/or disease essentially can be carried out with the methods mentioned above.1316 However, because most persons with the disease produces antibodies against Leishmania, diagnoses of clinically suspected human cases are often confirmed or the infection in dogs is indicated by serologic immunoassays.1723 These assays are carried out mainly with antigens from cultured Leishmania promastigote forms, which may also react with antibodies associated with other infectious diseases such as Chagas' disease and malaria, and thus produce false-positive results.24

In the past two decades, there has been a considerable effort to produce defined antigens, especially recombinant antigens, to be used in the serodiagnosis of human and canine VL.25 Many recombinant antigens have been selected and tested for the serodiagnosis of VL (including rGP63, rHSP70, rHSP90, rK39, HASBP1, PSA, Lepp12, paple22, LiPs, and histones).25 Among these antigens, a fragment of a kinesin protein, known as K39,26 has enabled development of assays that have shown good performance in the serodiagnosis of human VL in most disease-endemic areas.27 However, it is unlikely that one recombinant antigen is recognized by antibodies from all infected or sick persons.

The repertoire of the antibody specificities against L. infantum in dogs or humans may vary with distinct conditions.2831 Moreover, even in persons with apparently the same clinical status, the fine specificity of the individual immune response is unlikely to be identical. For instance, in a panel of nine serum samples from patients with LV, no single antigen in an L. infantum extract was clearly recognized by antibodies from all serum samples when tested by using Western blotting.32 In accordance with this finding, none of the commercially available L. infantum recombinant antigen-based immunoassays has a sensitivity of 100% in different disease-endemic regions.3336 For these reasons, it is important to expand the existing L. infantum recombinant antigen panel37 and evaluate new recombinant Leishmania proteins to exploit the full potential of recombinant antigens in the serodiagnosis of VL.

With the purpose of enlarging the current panel of recombinant antigens that may be useful for the serodiagnosis, Teixiera and others screened L. infantum cDNA and genomic libraries with pools of serum from infected dogs or human patients with VL.32 In the present study, four previously obtained32 and one new recombinant antigen, encoded by five genes/genes families, were characterized and their reactivity was tested against serum from different canine and human populations.

Materials And Methods

Parasites and recombinant antigens.

Leishmania infantum promastigotes and amastigotes were generated from the MHOM/BR2000/Merivaldo2 strain and maintained as described.38 Total parasite lysate was obtained by sonication of log-phase parasites and its protein content was quantified by using the Bradford method before use in enzyme-linked immunosorbent assays (ELISAs). The lambda bacteriophage clones described in this study were isolated by two consecutive screenings of cDNA or genomic L. infantum libraries with a pool of serum from four dogs and a pool of serum from three human patients with confirmed infection by L. infantum as reported.32 The four dogs were clinically healthy mongrel animals from a visceral leishmaniasis–endemic area (Jequié, Bahia, Brazil), which in addition to antibodies against L. infantum, had delayed skin hypersensitivity reactions to Montenegro's antigen. The three humans lived in Terezina, Piauí, Brazil, and had antibodies that recognized antigens in an L. infantum lysate but did not recognize previously obtained recombinant antigens.32 Thirty-two antibody-reactive isolated recombinant clones were studied, 30 from the first screening with canine serum and 2 from the subsequent screening with human serum.

Sequencing and characterization of selected inserts.

From the selected lambda bacteriophage clones, corresponding plasmid vectors (pBK-CMV) were excised according to the manufacturer's instructions (Stratagene, La Jolla, CA). Partial (at the 5′ and 3′ ends: Lci1A and Lci5A, respectively) or full nucleotide sequences (Lci2B, Lci3A, and Lci4A) of plasmid inserts from each clone were determined and compared by using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgias) nucleotide or deduced amino acid sequences with those deposited in Leishmania and Trypanosoma genomic databases (GeneDB, http://www.genedb.org and Genebank, http://www.ncbi.nlm.nih.gov/).

Subcloning of selected inserts for production of recombinant antigens.

To produce N-terminally histidine (His)–tagged recombinant proteins (NH6), whole or partial selected inserts were isolated from original pBK-CMV–derived plasmids and subcloned into prokaryotic expression vectors of the pRSET series (Invitrogen, Carlsbad, CA). The pRSET vector introduces at the N-terminus of recombinant proteins an approximately 30–amino acid peptide that includes a six histidine tag (His-tag). Inserts corresponding to only a fragment of the original insert in pBK-CMV were named with an additional letter and/or number (e.g., Lci3A-R3, Lci5A-I). Subcloning strategies used for each insert were defined by the available restriction enzyme sites and are summarized in Table 1. The only exception was Lci5A, which had to be amplified by using a polymerase chain reaction and primers flanked by Kpn I/EcoR I (forward primer 5′-CGAGGTACCGGCGCAGCGTGAGGAGCAGGC-3′; reverse primer 5′-CGAGAATTCCACCGGTGGCTCCTCCTGCTG-3′; restriction sites are underlined), before cloning into the pGEM-T Easy plasmid (Promega, Madison, WI) and sequencing and subcloning into the pRSET expression vector.

Table 1

Characterization of antigens used in immunoassays*

Group of clones/insertsProtein encoded by homolog genes of insertsGene DB accession nos. of homolog genesSubcloned DNA fragment of selected insertsFusion polypeptides used in ELISA
Lci1Heat shock protein HSP70LinJ28_V3.2950; LinJ28_V3.2960; LinJ28_V3.3000; LinJ28_V3.3060∼3,000 bp, Bam HI/Xho IrLci1A-NH6
Lci2N-kinesinLinJ14_V3.11801,285 bp, BamH I/Kpn IrLci2B-NH6
Lci3Hypothetical protein with repetitive motifsSequence similar to: LinJ34_V3.0700; LinJ34_V3.0710∼1,500 bp, Sac I/Kpn IrLci3A-R3-NH6
Lci4Poly-ubiquitinLinJ36_V3.36902,259 bp, BamH I/Kpn IrLci4A-NH6
Lci5Hypothetical protein with repetitive motifsLinJ33_V3.3230. (contains other coding DNA sequences out of frame)915 bp, Kpn I/EcoR IrLci5A-I-NH6

ELISA = enzyme-linked immunosorbent assay; bp = basepairs.

Production and purification of recombinant proteins.

To produce His-tag recombinant proteins, pRSET-derived plasmids were used to transform BL21(DE3)pLysS Escherichia coli (Invitrogen). Transformed bacteria were grown in Luria-Bertani medium and induced for protein expression with isopropyl β-d-thiogalactoside. Induced cells were centrifuged, resuspended in phosphate-buffered saline (PBS), and lysed by sonication. Protein purification was performed as described by using Ni-NTA agarose (QIAGEN, Hilden, Germany).39 Protein products were analyzed by sodium docecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a denaturing 15% polyacrylamide gel and staining of proteins with R-250 Coomassie blue. For estimation of recombinant protein concentrations, densities of bands in Coomassie blue–stained gels were compared with those of known concentrations of bovine serum albumin.

Serum samples.

Canine serum samples were obtained from two groups of dogs. The first group contained 46 dogs naturally infected with Leishmania, which was detected by splenic aspirate and culture in the disease-endemic area of Jequié (Bahia, Brazil). These dogs were polysymptomatic (21 dogs), oligosymptomatic (21 dogs), or asymptomatic (4 dogs) on the basis of defined clinical criteria.40 The second group contained 31 dogs from Leishmania-free areas in Recife, Pernambuco, Brazil, being 7, 4 and 20 dogs with demodicosis, babesiosis, and ehrlichiosis, respectively. All diseases were parasitologically confirmed.

Because the 31 animals from the Leishmania-free areas were not subjected to parasitologic examination by means of splenic aspiration, the possibility that they may have had a subclinical Leishmania infection, although unlikely, cannot be ruled out. In addition, serum from 15 healthy mongrel dogs from another non-endemic area (Salvador, Brazil) and which had negative results in splenic tissue cultures for Leishmania amastigotes, were used. The dogs were handled in accordance with the Oswaldo Cruz Foundation guidelines for experimentation on animals.

Human serum samples were obtained from three groups. The first group contained 39 clinically and parasitologically diagnosed VL patients (36 from Feira de Santana, Brazil and three from Teresina, Brazil, whose serum samples were kindly provided by Dr. Aldina Barral, Fundação Oswaldo Cruz, Salvador, Brazil). The second group contained 26 parasitologically confirmed patients with cutaneous leishmaniasis (CL). The third group contained 40 serologically confirmed chronic Chagas' disease patients. Serum samples were also obtained from 50 healthy persons of various ages from non-endemic areas in Brazil. For ethical reasons, patients with Chagas' disease, patients with CL, and healthy controls were not subjected to bone marrow or splenic aspirations for investigation of Leishmania infection. The study was approved by the appropriate Ethics Committee, and informed consent was obtained from all adults or legal guardians of children before blood was collected.

ELISA.

Protein samples were diluted in coating buffer (15 mM Na2HCO3, 28 mM NaHCO3, pH 9.6), placed into wells of 96-well microtiter plates (1 μg of parasite lysate or 0.5 μg of each recombinant antigen per well), incubated overnight at 4°C, and blocked with 0.15 M PBS, pH 7.2, containing 0.05% Tween 20 and 10% dry non-fat milk. Wells were incubated with the selected serum samples at a dilution of 1:200 (canine serum) or 1:600 (human serum). These dilutions had been shown to constitute the best compromise in terms of producing fewer false-negative and false-positive results. Wells were then washed with 0.15 M PBS, pH 7.2, containing 0.05% Tween 20. Peroxidase-conjugated goat anti-dog IgG (diluted 1:1,200) or anti-human IgG (diluted 1:15,000) (Sigma-Aldrich, St. Louis, MO) depending on the serum sample tested was added and plates were incubated for one hour at room temperature. Enzymatic activity was detected by using 0.01% hydrogen peroxide and 0.01% o-phenylenodiamine (Sigma-Aldrich) in 0.1 M phosphate-citrate buffer, pH 5.0 and read in a spectrophotometer with a 490 nm filter. The cutoff values for the ELISAs were defined as means of results obtained with serum samples from healthy donors plus 3 SD. Intensities of reactivities obtained in the ELISAs were arbitrarily classified as weak (OD values ≤ 0.299), moderate (OD = 0.300–0.899), or strong (OD values > 0.899). Means of the OD values and individual values for each serum sample are shown in the figures.

Results

Selection and identification of L. infantum antigens.

After their excision from plasmids, sequencing, and identification, inserts from 29 clones isolated during the first screening with canine serum (one clone was lost during the insert isolation process) were shown to correspond to segments of four distinct genes or gene families of L. infantum. These gene families were named Lci1 (24 clones), Lci2 (2 clones), Lci3 (2 clones), and Lci4 (1 clone). Two additional clones isolated in a second screening with a pool of three serum samples from VL patients, which in preliminary tests had not reacted with recombinant polypeptides selected in first screening,32 yielded identical inserts that corresponded to a fifth gene, which was named Lci5. All five genes were selected for further studies of the potential of the corresponding recombinant proteins for diagnosis of canine and human VL.

Characterization of selected L. infantum antigens.

Inserts of the Lci1 group encode members of the 70-kD heat shock protein (HSP70) family. The prototype clone, Lci1A, which was selected for further studies, included the full length open-reading frame encoding one of the members of the 70-kD cytosolic heat shock protein (HSP70) and additional 5′ untranslated region (UTR) and 3′UTR DNA segments (Table 1 and Figure 1A), which are known to be encoded by multiple genes within the L. infantum genome.41

Figure 1.
Figure 1.

Schematic representation of various cDNAs and corresponding deduced recombinant proteins evaluated in this study. Maps were derived from sequences produced after direct sequencing of the Lci2A and Lci2B, Lci3A and Lci4A cDNAs and/or from the coding genomic sequences of the Lci1A and Lci5A, available at the GeneDb (www.genedb.org). A, Lci1A cDNA, including the 5′ untranslated region (UTR) segment, which is included in the recombinant protein rLci1A-NH6. B, Lci2A cDNA, highlighting the 11 39-amino acid repetitive segments and the segment common to Lci2B cDNA and which is present in recombinant protein rLci2B-NH6. C, Lci3A cDNA highlighting the 21 repetitive 14-amino acid motif segments and recombinant protein rLci3A-R3-NH6, which contain the last three repetitive segments. D, Lci4A cDNA sequence emphasizing the last two repeats and part of the third repeat and recombinant protein rLci4A-NH6. E, Lci5A-coding sequence and the recombinant protein rLci5A-I –NH6. Arrows and open boxes, which have not been drawn to represent protein segments in scale, represent repetitive amino acid motifs and non-repetitive polypeptide segments. Hatched represent His-tag provided by the vector. ORF = open reading frame.

Citation: The American Society of Tropical Medicine and Hygiene 85, 6; 10.4269/ajtmh.2011.11-0102

The two Lci2 inserts, Lci2A and Lci2B, encode segments of a L. infantum kinesin that is homologous to the C-terminus of the LinJ14_V3.1180 gene product (Table 1). Comparison between the LinJ14_V3.1180–encoded protein, which is 3,279 amino acids, and the Lci2A–encoded polypeptide (524 amino acids; Lci2B completely overlapped the 3′ end of Lci2A), showed near complete homology at the C-terminus (Figure 2), except for four amino acid differences (2,782 S → A, 2,983 E → D, 2,984 L → V, and 2,991 A → T). The Lci2-encoded polypeptides possess 39-amino acid multiple tandem repetitive motifs (11 complete repeats for rLci2A and 5 for rLci2B), in addition to a 76-amino acid non-repetitive C-terminus (Figure 1B). Most of the Lci2 repeats were similar, but not fully identical, to those found in rK39 and rLcKin26 (Figure 3A) proteins and had greater sequence variability (Figure 3A).

Figure 2.
Figure 2.

Schematic representation of the full-length sequences or sequence fragments from selected kinesins from the Leishmania donovani complex. Sequences from LdK39 (kinesin of L. donovani18), the protein encoded by LinJ14_V3.1180 (kinesin of L. infantum, GeneDB), rLcKin and rk39 (L. chagasi kinesin fragments described by Evans and others7), and the rLci2A and rLci2B kinesin fragments reported here are represented. Open boxes represent overlapping regions, and closed boxes indicate sequences in protein encoded by LinJ14_V3.1180 that are absent in LdK39. The numbers indicate the position of the amino acids at the start and end of the polypeptides.

Citation: The American Society of Tropical Medicine and Hygiene 85, 6; 10.4269/ajtmh.2011.11-0102

Figure 3.
Figure 3.

Repeats found in the recombinant proteins encoded by the Lci2 and Lci3 gene fragments. A, The 39-amino acid repetitive motifs of rLci2A and rLci2B, the consensus sequences of rLci2B, and the consensus sequences rK39 are presented showing the variability of the amino acid sequences. B, The different 14-amino acid repeats found in rLci3A (the number of copies of each repeat present in the sequence is indicated in parenthesis), the consensus sequences of the rLci3A repeats, and the three repeats present in the rLci3A-R3 recombinant protein are shown.

Citation: The American Society of Tropical Medicine and Hygiene 85, 6; 10.4269/ajtmh.2011.11-0102

Inserts of the Lci3 group encode for multiple copies of 14-amino acid tandem repeats (22 copies for Lci3A and 15 copies for Lci3B, which is the smallest of the two inserts and completely overlaps the 3′ end of Lci3A) and a non-repetitive 235-amino acid C-terminal end (Figures 1C and 3C). There is no annotated gene sequence in the L. infantum genome database that fully matches the Lci3 or cDNA fragments (Table 1). However, a L. major hypothetical protein (3,167 amino acids long with a predicted molecular mass of 358 kD; LmjF34.0690) was found. It displayed 87% identity and 92% similarity with the non-repetitive Lci3 C-terminus.

The insert of the fourth clone group, Lci4A, corresponds to the 3′ end of the L. infantum homolog of the LinJ36_V3.3690 polyubiquitin gene (Table 1). In addition to LinJ36_V3.3690, the L. infantum genome includes three genes encoding one (monoubiquitin, LinJ31_V3.1930 and LinJ31_V3.2070) or several (polyubiquitin, LinJ09_V3.0950) of the 76 amino-acid ubiquitin repetitive motifs. The Lci4A insert encompasses slightly more than the last two 76 amino-acid repeats at the C-terminal end of the gene and has a long 3′UTR (Figure 1D).

The two Lci5 inserts encode an internal segment (ranging from residues 486 to 1,160) of a large protein (3,296 amino acids) encoded by the L. infantum LinJ33_V3.3230 gene (Table 1). This hypothetical protein consists of a 98-residue N-terminus and multiple sets of repeats, some clearly related among themselves but others being dissimilar. A similar organization with multiple copies of distinct repeats, mostly conserved, was also observed in an L. major ortholog (LmjF33.3070), although a non-repetitive C-terminus was observed in this protein. The N-terminal half of the L. infantum protein composed of seven copies of the first repeat (148 amino acids) plus five copies of the second repeat (72 amino acids) is shown in Figure 1E.

Recombinant protein expression.

To produce recombinant polypeptides for evaluation in immunologic assays, we expressed and affinity-purified recombinant His-tagged proteins, which represented each of the five groups of cloned DNA sequences, in E. coli. Results of representative analysis by SDS-PAGE with all five expressed recombinant proteins are shown in Figure 4 and are also schematically shown in Figure 1. The bands observed by SDS-PAGE were shown to be recombinant proteins rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, rLci4A-NH6, rLci5A-I-NH6, or their degradation products by Western blotting with antibodies against histidine (data not shown).

Figure 4.
Figure 4.

Recombinant proteins evaluated in this study. Affinity-purified, His-tagged, recombinant fragments (rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, rLci4A-NH6, and rLci5A-I-NH6) were analyzed by using run in denaturing 15% SDS-PAGE stained with Coomassie Blue. Arrow indicates non-degraded rLci5A-I-NH6 protein.

Citation: The American Society of Tropical Medicine and Hygiene 85, 6; 10.4269/ajtmh.2011.11-0102

The rLci1A-NH6 protein consists of an N-terminus encoded by the vector, which is common to all five recombinant proteins, plus a 52-amino acid segment encoded by the gene 5′UTR (Figure 1A) and the entire protein open reading frame. The rLci2B-NH6 polypeptide contains 293 amino acids, five complete 39-amino acid repeats, and the protein C-terminus (Figures 1B and 3A). The rLci3A-R3-NH6 polypeptide includes the last three 14-amino acid repeats and the entire C-terminus from rLci3A (Figures 1C and 3B). The polypeptide encoded by the Lci4A fragment consists of slightly more than two 76-amino acid repeats (rLc4A-NH6, Figure 1D). The Lci5A recombinant fragment generated a polypeptide with two copies of the 148-amino acid repeat (rLci5A-I-NH6, Figure 1E).

Recognition of recombinant proteins by canine serum samples.

To assess the potential of recombinant antigens for serodiagnosis of canine infection by Leishmania, we evaluated various his-tagged polypeptides by using ELISAs with serum from dogs naturally infected with L. infantum (n = 46), healthy controls (n = 15), and dogs with other infectious diseases (n = 31). Serum samples were assayed against total Leishmania antigens. As expected, mean reactivities of serum samples from L. infantum-infected dogs and healthy controls were strong and weak, respectively. The reactivity of serum samples from dogs with other infectious diseases was similar to that of the healthy control group, although 5 of 31 dogs in this group showed reactions above the cutoff value (Figure 5).

Figure 5.
Figure 5.

Reactivity of a panel of canine serum with Leishmania recombinant antigens assessed by an enzyme-linked immunosorbent assay. Serum samples from 46 dogs with visceral leishmaniasis (L. chagasi-infected), 31 dogs with other infections (4 with babesiosis, 20 with erhlichiosis, and 7 with demodicosis) and 15 healthy control animals were assayed with the various recombinant antigens produced in Escherichia coli (rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, rLci4A-NH6, and rLci5A-I-NH6) or with total L. chagasi lysate as antigen (LAg). Each symbol corresponds to the result obtained with an individual serum. Solid horizontal lines indicate mean optical densities. Dashed horizontal lines indicate cutoff values, which were calculated as described in the Materials and Methods.

Citation: The American Society of Tropical Medicine and Hygiene 85, 6; 10.4269/ajtmh.2011.11-0102

Mean reactivities of serum samples from dogs infected with L. infantum were evaluated with recombinant antigens and showed strong reactivity with rLci1A-NH6 (n = 43: 27 strong, 9 moderate, and 7 weak), rLci2B-NH6 (n = 44: 29 strong, 10 moderate, and 5 weak), and rLci4A-NH6 (n = 37: 33 strong and 4 moderate) antigens and moderate reactivity with rLci3A-R3-NH6 (n = 37: 8 strong, 14 moderate, and 13 poor) and rLci5A-I-NH6 (n = 46: 12 strong, 14 moderate, and 23 poor) antigens (Figure 5). No significant differences between reactivities of serum samples from four asymptomatic Leishmania-infected dogs and 42 symptomatic dogs were observed. Mean reactivity of serum samples from healthy control dogs was mostly weak for the different antigens (rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, and rLci5A-I-NH6), with the exception of rLci4A-NH6, which showed a result classified as moderate (n = 15: 8 moderate and 7 poor). Mean binding activities were weak for all recombinant antigens with serum samples from dogs with other infectious diseases. Sensitivities and specificities of Leishmania lysate antigens and rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, rLci4A-NH6, and rLci5A-I-NH6 (used to detect natural infections in dogs) are shown in Table 2. Interestingly, recombinant antigens that showed the best performance with dog serum samples were rLci3A-R3-NH6 and rLci4A-NH6, which showed sensitivities and specificities > 90% (Table 2).

Table 2

Serodiagnostic performance of recombinant antigens with canine serum samples*

Fusion polypeptides used in ELISASerum from naturally Leishmania-infected dogsSerum from healthy controls or dogs with other infectious diseases
No. samples testedNo. positive samplesSensitivity (%)No. samples testedNo. positive samplesSpecificity (%)
LAg434195.344489.1
rLci1A-NH6434093.0461176.1
rLci2B-NH6433683.746784.8
rLci3A-R3-NH6373491.946295.7
rLci4A-NH6373491.9460100
rLci5A-I-NH6463167.439294.9

ELISA = enzyme-linked immunosorbent assay.

Recognition of recombinant proteins by human serum samples.

We evaluated human serum samples with different antigens to investigate the potential of these antigens for diagnosis of VL. Mean reactivities of serum samples from patients with VL and from healthy controls to total Leishmania antigens were strong and weak, respectively, as expected (Figure 6). Mean reactivities of serum samples from patients with CL and Chagas' disease were classified as moderate. The mean reactivities of serum samples from patients with VL were moderate with rLci1A-NH6 (n = 36: 9 strong, 18 moderate, and 9 poor), rLci2B-NH6 (n = 36: 17 strong, 17 moderate, and 2 poor), and rLci3A-R3-NH6 (n = 21: 7 strong, 3 moderate, and 11) and strong with rLci4A-NH6 (n = 22: 15 strong, moderate 5, and 2 poor) and rLci5A-I-NH6 (n = 22: 16 strong, 3 moderate, and 3). In contrast, mean binding of antibodies from serum samples of healthy controls, patients with CL, and patients with Chagas' disease was weak for recombinant antigens tested, with the exception of rLci4A-NH6 and rLci5A-I-NH6, which showed results defined as moderate with serum samples from patients with Chagas' disease (Figure 6). Sensitivities and specificities of Leishmania lysate antigens, rLci1A-NH6, rLci2B-NH6, rLci3A-R3-NH6, rLci4A-NH6, and rLci5A-I-NH6 in detecting antibodies in serum samples (diluted 1:600) from patients with VL are shown in Table 3. Recombinant antigen rLci2B-NH6 had the best overall performance (sensitivity and specificity > 90%), and all recombinant antigens tested had a high specificity > 90%.

Figure 6.
Figure 6.

Reactivity of a panel of human sera with Leishmania recombinant antigens assessed by an enzyme-linked immunosorbent assay. Serum samples 36 patients with visceral leishmaniasis (VL), 26 patients with cutaneous leishmaniasis (CL), 40 patients with Chagas' disease (Chagas) and 50 healthy controls were assayed with the same set of antigens evaluated in Figure 5. Each symbol corresponds to the result obtained with an individual serum. Solid horizontal lines indicate mean optical densities. Dashed horizontal lines indicate cutoff values, which were calculated as described in the Materials and Methods.

Citation: The American Society of Tropical Medicine and Hygiene 85, 6; 10.4269/ajtmh.2011.11-0102

Table 3

Serodiagnostic performance of recombinant antigens with human serum samples*

Fusion polypeptides used in ELISASerum from patients with VLSerum from healthy controls or patients with CL or Chagas' disease
No. samples testedNo. positive samplesSensitivity (%)No. samples testedNo. positive samplesSpecificity (%)
LAg363597.21132181.4
rLci1A-NH6362672.2113793.8
rLci2B-NH6363597.2113397.3
rLci3A-R3-NH622836.4115496.5
rLci4A-NH6221881.8115793.9
rLci5A-I-NH6221986.41151190.4

ELISA = enzyme-linked immunosorbent assay; VL = visceral leishmaniasis; CL = cutaneous leishmaniasis.

Discussion

In this study, lambda bacteriophage clones encoding five gene or gene family members (Lci1, Lci2, Lci3, Lci4, and Lci5) and selected from an L. infantum cDNA or genomic library were characterized and evaluated for their reactivity against a panel of canine or human serum samples. Four of these clones (Lci2, Lci3, Lci4, and Lci5) encode proteins that have tandem amino acid repeats, an observation reported by other investigators using a different approach.37,42 The only exception we identified was Lci1, which encodes a protein homolog to members of the cytoplasmic HSP70 family43 and has also been identified in L. donovani and L. infantum using similar approaches.4447 Cytoplasmic HSP70s are highly conserved proteins in eukaryotes,43 and canine and human HSP70s have each approximately 70% identity with L. infantum HSP70.47,48 Recombinant protein rLi1A-NH6 reacted strongly with antibodies from most dogs naturally infected with Leishmania.

Although this protein also cross-reacted with antibodies from a certain proportion of dog with other infectious diseases, the OD values observed were low. In contrast, it reacted only moderately with antibodies from most patients with VL, which resulted in an ELISA with much less sensitivity than the ELISA for L. infantum-infected dog serum samples. Low cross-reactivity was observed with some serum samples from patients with CL and patients with Chagas' disease.

The Lci2 inserts encode C-terminal fragments of the ≈358-kD protein that belongs to the kinesin superfamily of motor proteins,49 of which the complete coding sequence has been recently determined from a L. donovani genomic cosmid library clone.50 The N-terminal segments of this protein correspond to the recombinant proteins K39 and Lckin, which were isolated from a L. infantum genomic library.26 These N terminally derived kinesin fragments have been used to develop immunodiagnostic assays that showed good performance (high sensitivity and specificity).9,27,51,52 The performance of the C-terminal region of the kinesin (Lci2) as ELISA antigen in the serodiagnosis of human VL, as shown in the present study, also showed good performance. Serum samples from 3 of 26 patients with CL showed a positive result in the Lci2-based ELISA. However, this result is consistent with results obtained with rK39, which reacted with 1 of 13 serum samples from patients with CL.34 The Lci2-based ELISA performance with canine serum samples was inferior (in sensitivity and specificity) compared with that observed with human serum samples and also with other recombinant antigens evaluated in this study.

The Lci3 inserts are encoded by a gene within a dicistronic operon that is conserved in L. major, L. infantum, and L. braziliensis; the second cistron is also homologous to that of Lci3. On the basis of the non-repetitive C-terminus, several related proteins were found and showed various degrees of homology with various trypanosomatid species. A possible ortholog from Trypanosoma cruzi (TCR3) is also composed of 14-amino acid repeats and a non-repetitive C-terminus homologous to the one found in Lci3.53 Ortholog TCR3 is located in the paraflagellar region, between the body of the parasite and the flagellum.54 Likewise, the Lci3 ortholog in T. brucei has been classified as a flagellar attachment zone protein (Tb927.4.3740). The ELISA with rLci3A-R3-NH6 antigen displayed high sensitivity and specificity for dogs naturally infected with Leishmania. However, among all ELISAs evaluated, it had the poorest capacity for the diagnosis of patients with VL.

The ubiquitin gene product is one of the most conserved proteins among eukaryotes,55 and the L. infantum sequence shares 97% identity with canine and human ubiquitin.56 If one considers only one repeat, L. infantum polyubiquitin has only two and four amino acid differences (at positions 14, 52, 70, and 77) relative to canine and human ubiquitin, respectively. Nevertheless, it has been shown that patients with chronic Chagas' disease produce antibodies against T. cruzi ubiquitin,57 the monomer of which has only three amino acid differences relative to human ubiquitin. The ELISA developed with the ubiquitin-derived rLci4A-NH6 antigen showed the best performance in serodiagnosis of VL in dogs. However, it also showed moderate cross-reactivity with serum samples from a few patients with CL or Chagas' disease or healthy control persons.

The hypothetical protein encoded by the Lci5 gene has not been described in the literature, apart from the automatic annotation performed with sequenced trypanosomatid genomes. This conserved protein showed ≈30% identity in the N-terminal half with L. major and T. brucei orthologs (whose sequences are known). The rLci5A-I-NH6 protein showed strong reactivity in an ELISA with serum samples from patients with VL, although it also showed cross-reactivity with 10 of 40 serum samples from patients with Chagas' disease and with 1 of 49 serum samples from healthy controls. In contrast, its ELISA performance was much lower with canine serum samples (lower sensitivity and weaker reactivities).

Sensitivities and specificities of rLci1A-NH6, rLci2B-NH6, rLci3A-NH6, rLci4A-NH6, and rLci5A-I-NH6 ranged from 36.4% to 97.2% and 90.4% to 97.3% in detecting human VL and from 67.4% to 93.0% and 76.1% to 100% in detecting canine infection or disease, respectively. The recombinant antigen that showed the best performance for serodiagnosis of human disease by ELISA was rLci2B-NH6 (sensitivity = 97.2% and specificity = 97.3%) The recombinant antigens that showed the best performance for serodiagnosis of canine infection or disease were rLci3A-NH6 and the rLci4A-NH6 (sensitivities = 91.9% and specificities = 95.7% and 100.0%, respectively). Specificities and sensitivities of the ELISAs with these recombinant antigens were comparable with those of other ELISAs with recombinant antigens previously evaluated for serodiagnosis of canine and human VL.52,5861

Three of the recombinant antigens in the present study were derived from fragments of genes that encode relatively large native Leishmania proteins (Lci2, Lci3, and Lci5). To the best of our knowledge, these large proteins have not been identified as antigens by Western blotting performed with parasite lysates and patient serum samples. This finding is in contrast to the fact that rK39, rLci2, rLci3, and rLci5 are clearly recognized by antibodies in patient serum samples and could be explained by denaturation of native proteins, possible cleavage during preparation of parasite antigens, or relatively low efficiency of transfer of high molecular mass proteins from polyacrylamide gels to nitrocellulose membranes.

As detailed above, the recombinant proteins evaluated in this work displayed different capacities to react with antibodies from dogs naturally infected with L. infantum and from humans with VL. The present work and data reported by other investigators62 suggest that repertoires of humoral immune responses in dogs and humans infected with L. infantum are different, and that this difference should be taken into account when developing serodiagnostic methods. Because no antigen, even if it has a high sensitivity and specificity in ELISAs for detection of antibodies against L. infantum, was able to detect all dogs or humans that acquired the infection and produced specific antibodies, immunodiagnostic assays should be developed on a platform appropriate to show reactivity to several recombinant antigens simultaneously (http://www.chembio.com/newtechnologies.html). A single platform could be potentially useful with human and canine serum samples. Alternatively, species-specific platforms could be developed that differ for those antigens that induce stronger and more specific humoral immune responses in different host species.

ACKNOWLEDGMENTS:

We thank Professor Yung-Fu Chang (Cornell University, Ithaca, NY) for helping with the Leishmania genomic library construction and Marco Silvany and Rafael Dhalia for providing reagents.

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Author Notes

*Address correspondence to Geraldo G. S. Oliveira, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, No. 121, Candeal, CEP 40.296-710, Salvador, Bahia, Brazil. E-mail: ggileno@bahia.fiocruz.br

Financial support: This study was supported by the Programa Rede Nordeste de Biotecnologia (RENORBIO); BNB-FINEP-MCT, Brazil; PDTIS (RID17), FIOCRUZ, Brazil; and Instituto Nacional de Ciencia e Tecnologia de Doenças Tropicais, CNPq (INCT-DT grant 573839/2008-5).

Authors' addresses: Geraldo G. S. Oliveira, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, No. 121, Candeal, CEP 40.296-710, Salvador, Bahia, Brazil, and Instituto Nacional de Ciencia e Tecnologia de Doenças Tropicais, Salvador, Bahia, Brazil, E-mail: ggileno@bahia.fiocruz.br. Márcia C. A. Teixeira, Andrea M. Pereira, Cristiane G. M. Pinheiro, Lenita R. Santos, Washington L. C. dos-Santos, Edson D. Moreira Jr, and Lain C. Pontes de Carvalho, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Av. Waldemar Falcão, No. 121, Brotas, CEP 40.296-710, Salvador, Bahia, Brazil. Franklin B. Magalhães, Marília B. Nascimento, Cheila N. G. Bedor, Alessandra L. Albuquerque, Yara M. Gomes, Maria E. F. Brito, and Osvaldo P. Melo Neto, Centro de Pesquisas Aggeu Magalhães, Fiocruz, Av. Moraes Rego s/n, Campus UFPE, CEP 50670-420, Recife, Pernambuco, Brazil.

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