IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.02032-05v1
74/12/6940    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martins, D. R. A.
Right arrow Articles by Wilson, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martins, D. R. A.
Right arrow Articles by Wilson, M. E.
Infection and Immunity, December 2006, p. 6940-6948, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.02032-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Leishmania chagasi T-Cell Antigens Identified through a Double Library Screen{triangledown}

Daniella R. A. Martins,1 Selma M. B. Jeronimo,1 John E. Donelson,2 and Mary E. Wilson3*

Department of Biochemistry and Health Graduate Program, Federal University of Rio Grande do Norte, Natal, RN, Brazil,1 Department of Biochemistry, University of Iowa,2 Departments of Internal Medicine, Microbiology, and Epidemiology, University of Iowa and Veterans' Administration Medical Center, Iowa City, Iowa 522423

Received 17 December 2005/ Returned for modification 21 January 2006/ Accepted 15 September 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1{gamma}, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leishmaniasis is a group of vector-borne diseases caused by obligate intracellular protozoa belonging to the genus Leishmania. Clinical forms of leishmaniasis include cutaneous, mucosal, and visceral leishmaniasis (30, 42). These diseases are endemic in parts of Africa, India, the Middle East, southern Europe, and Central and South America. Visceral leishmaniasis, caused by the protozoa L. donovani, L. chagasi, and L. infantum, can be fatal if left untreated. The focus of this study is the New World parasite L. chagasi, a species that may be one and the same as L. infantum from the Old World (35, 39).

When visceral leishmaniasis was first reported in the northeastern region of Brazil in 1936, most cases occurred sporadically in poor, rural farming areas (21). Due to population shifts as Brazil has become more urbanized, visceral leishmaniasis has emerged as an important health problem in the outskirts of large Brazilian cities, where thousands of people are now at risk of infection and disease (17, 31). The spectrum of manifestations due to L. chagasi infection includes at one end self-resolving infection detected by a positive delayed-type hypersensitivity (DTH) skin test response to leishmania antigen (Montenegro test) and/or antigen-specific peripheral T-cell responses in the absence of symptomatic disease. At the other end of the spectrum is symptomatic visceral leishmaniasis, a severe and potentially fatal illness in which there are high titers of antileishmanial antibodies but DTH responses are absent (45). Antibody levels decrease and specific DTH responses develop after successful treatment of the disease (5).

Antileishmanial drug treatment is problematic in that leishmanicidal drugs are toxic and costly and drug resistance is emerging, particularly in India (19, 52). Standard diagnostic procedures identifying the parasite are invasive with potential complications, and the specificity of serologic diagnosis for acute disease varies in endemic populations (5, 19). Individuals develop immunity against reinfection after clinical recovery from leishmaniasis (30, 42), suggesting that in principle vaccination is feasible. Indeed, for years individuals were "immunized" against Old World cutaneous leishmaniasis by purposeful infection with one species of Leishmania to protect against infection with another (42, 46). Despite the obvious need and considerable effort, however, there is no effective and safe vaccine approved for human use against any form of leishmaniasis. Similarly, no effective vaccine has been developed for dogs, which serve as a major reservoir for leishmaniasis in peridomestic settings of Latin America.

The Leishmania spp. protozoa are complex eukaryotic organisms that pass through both an insect (sand fly) and a mammalian host in two distinct morphological forms (promastigote and amastigote, respectively). Promastigotes modify their antigenic properties in culture, a fact that may hamper efforts to generate attenuated or gene knockout mutants as live Leishmania vaccine strains (3, 36, 51, 56). Furthermore, host immune responses occur to antigens expressed by the intracellular amastigote stage, which is difficult to raise in bulk culture (32, 57). We therefore focused on identifying antigens expressed in amastigotes that elicit immunological responses in infected hosts.

It seems likely that both a subunit vaccine and an optimized diagnostic test would benefit from inclusion of a broad range of parasite epitopes. There are many reports of antigens expressed by the promastigote (insect) stage of Leishmania that are recognized by the mammalian immune system (34). Some studies focus on antigens that are highly expressed in the intracellular amastigote, the stage present throughout mammalian infection (23, 25, 44, 49, 59). This report documents our systematic screen of an L. chagasi amastigote cDNA library to identify T-cell and T-cell-dependent B-cell antigens expressed in the mammalian parasite form.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Parasites. A Brazilian strain of Leishmania chagasi (MHOM/BR/00/1669) was maintained by serial intracardiac inoculation of amastigotes in male Syrian hamsters (58). Promastigotes were passed weekly in hemoflagellate modified minimal essential medium and used within 3 weeks of harvest from a hamster (4). A clonal line of L. chagasi that converts from promastigote to amastigote, called LcJ, was raised by alternately cycling in hemoflagellate modified minimal essential medium at 26°C and amastigote-specific medium (described for L. donovani) at 37°C (27). LcJ was passed between the promastigote and amastigote stages every 3 weeks.

Preparation and serum screening of the amastigote cDNA library. Total RNA was extracted from a pool of 1.2 x 109 amastigotes derived from 11 infected hamster spleens and used to generate a cDNA library in lambda ZAP II using a cDNA library construction kit as described by the manufacturer (Stratagene, La Jolla, CA). The library was initially immunoscreened with pooled sera from 11 Brazilian patients with visceral leishmaniasis by using standard methods (16). Patients with visceral leishmaniasis were defined by typical clinical presentation, parasite identification on bone marrow biopsy, and response to treatment (31). A total of 100,000 phage clones were screened with the serum pool followed by a secondary peroxidase-conjugated anti-human immunoglobulin G (IgG) and peroxidase substrate (4-Cl-naphthol, imidazole, and H2O2). Recombinant phages were purified in a third immunoscreen.

Phage clones encoding HSP70 or HSP90 were eliminated, since these seemed unlikely to be chosen for a vaccine. To identify these clones, the selected antigenic phages were plated in a grid and screened with [32P]DNA probes containing the sequences encoding L. chagasi HSP90 and HSP70. Probes were the HSP90-3 sequence comprising the 3' 1,359 bp of the coding region and the entire 858-bp 3'-untranslated region reported in our prior publications (20, 55) and a full-length HSP70 cDNA of 1,858 bp cloned from an L. chagasi stationary-phase promastigote cDNA library.

Screen for T-cell antigens. Fusion proteins were prepared for T-cell assays using a method modified from that described by Mustafa et al. (40). Briefly, phage clones were plated into individual wells of a 96-well plate in SM medium and transferred to a second plate containing Escherichia coli XL1 Blue (Stratagene) using a multiwell pipettor. Bacteria were transformed at 37°C and transferred to a third plate containing 0.7% LB agar with 10 mM isopropyl-ß-D-thiogalactoside (IPTG) and top agar. Top agar containing transformed bacteria was plated in 1.5% LB agar of a fourth plate. After incubation at 42°C for 4 hours, plates were incubated overnight at 37°C to allow expression of recombinant antigens. Wells were subsequently overlaid with RPMI 1640 medium containing 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 10 µg of polymyxin B/ml. Recombinant fusion proteins were allowed to diffuse into the medium over 1 hour. Protein-containing supernatants were incorporated into replicate splenocyte proliferation assays at a 1:100 dilution. Negative control wells contained agar with no bacteria or top agar with nontransformed E. coli.

C3H.HeJ mice were infected with 107 L. chagasi promastigotes intravenously through a tail vein. Two weeks later spleens were removed from five infected mice, pooled, and made into single-cell suspensions by passage through a wire screen. B cells were removed by panning on anti-immunoglobulin, and T cells were partially purified over a nylon wool column (16, 59). This yielded a population with 60 ± 1% CD4+, 32% ± 2% CD8+, and 2.3 ± 0.7% B220+ cells (means ± standard deviations) according to fluorescence-activated cell sorter analysis. Splenocytes from uninfected syngeneic mice were treated with mitomycin C and used as a source of antigen-presenting cells.

A total of 2 x 105 column-purified T cells and 2 x 104 mitomycin C-treated splenocytes were incubated in wells at 37°C, 5% CO2. Four replicate plates contained supernatants containing recombinant proteins or the following negative controls: no supernatant, supernatant from wells with no bacteria, or supernatant from wells with negative recombinant phage clones that did not encode a protein recognized on the immunoscreen. Positive control wells contained 1 x 106 promastigotes/ml as a source of live parasite antigen. After 68 to 92 h, wells were pulsed with 0.5 µCi of [3H]thymidine/well and incubated for an additional 4 h. Stimulation indices were calculated as the ratio of [3H]thymidine incorporated in wells with recombinant proteins compared to wells containing supernatant overlaid on agarose without bacteria. The 72-hour supernatants were collected for gamma interferon (IFN-{gamma}) assay.

Murine IFN-{gamma} ELISAs. Murine IFN-{gamma} was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) as we have previously reported (59). Briefly, plates were coated with monoclonal antibody (MAb) HB170 (ATCC) and incubated with 50 µl supernatant. IFN-{gamma} was detected with polyclonal rabbit anti-IFN-{gamma} followed by biotinylated goat anti-rabbit IgG (Accurate Chemical Co., Westbury, NY), streptavidin-horseradish peroxidase (HRP), and 2,2'-azinobis(ethylbenzthiazolinesulfonic acid) substrate (ABTS; Zymed, San Francisco, CA).

Preparation of recombinant proteins. Genes encoding the above antigens, as well as the gene encoding promastigote protein KMP11 (12), were subcloned into the pQE30 expression vector (QIAGEN, Valencia, CA) in frame with an N-terminal His6 tag under control of the lacO operator. Protein expression was verified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Coomassie blue staining. Lysates with predominant bands of the correct size were assayed for reactivity with pooled antileishmania serum on immunoblots. Recombinant proteins were purified from the lysate on nickel-nitrilotriacetic acid resin and eluted with imidazole. All procedures were performed as suggested by the manufacturer. Eluted proteins were analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue stain.

Human cellular immune responses to recombinant proteins. Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll-Hypaque gradient from nonexposed control individuals (n = 3) or from individuals with immunity to L. chagasi as documented by a positive DTH skin test reaction either because of prior successful treatment of active disease (n = 3) or prior subclinical infection and residence in an household where is was endemic (n = 3). PBMCs were resuspended in RPMI 1640 culture medium (Gibco BRL, Grand Island, NY) supplemented with 10% human AB serum, 100 µg of streptomycin/ml, and 100 IU of penicillin/ml. Five hundred µl of PBMCs (2 x 106 cells/ml) was plated in 24-well plates and stimulated with L. chagasi lysate (5 µg/ml), recombinant KMP11 as a positive control (5 µg/ml), recombinant 503 (5 µg/ml), or recombinant 419 (5 µg/ml). PBMCs were incubated for 72 h at 37°C, 5% CO2, after which supernatants were collected and stored at –70°C. IFN-{gamma} in supernatants was measured by the ELISA sandwich method (R&D Systems, Minneapolis, MN). The results are shown in pg/ml.

Recombinant protein enzyme-linked immunosorbent assays. Microassay plates (Costar, Cambridge, MA) were coated overnight at 4°C with 500 ng/well recombinant L. chagasi amastigote antigens from clones 314, 503, and 419 in coating buffer, blocked with 1% Tween 20-phosphate-buffered saline, and washed in 0.1% Tween 20. Sera from 34 individuals with parasitologically confirmed visceral leishmaniasis and a positive response to recombinant K39 serology were incubated in wells at a 1:500 dilution for 1 h at room temperature. Specific antibody binding was detected with 1:4,000 anti-human IgG-horseradish peroxidase (Promega, Madison, WI) followed by substrate (ABTS, pH 4.2; Sigma, St. Louis, MO), detected based on the absorbance at 405 nm. The cutoff for a positive reaction was set at the mean + 3 standard deviations of the absorbance control sera (n = 3). Each serum sample was assayed in duplicate.

DNA sequencing and alignment. Non-HSP cDNA clones selected with the above double screen were excised into pBluescript SK(+/–) as described by the manufacturer. Inserts were sequenced using a series of primers designed from the vector and preliminary sequence data, using automated fluorescent DNA sequencing at the University of Iowa DNA Core Facility (33). Replicate sequences were aligned with homologues identified from the Sanger Centre website and the National Center for Biotechnology Information (NCBI) website (GenBank). Alignments were done using the Vector NTI Advance sequence analysis program (Invitrogen, Bethesda, MD).

Immunoblot assays. LcJ promastigotes in logarithmic or stationary growth phase or LcJ axenic amastigotes were collected by centrifugation and resuspended in 200 µl of Hanks' balanced salts solution. Protein concentrations were determined by Pierce BCA protein assay, and proteins were separated on 7.5% polyacrylamide reducing gels. Proteins were transferred to a polyvinylidene difluoride membrane, stained with Ponceau S to verify equal loading, and blocked with 5% dry milk and 0.05% Tween 20 in phosphate-buffered saline. Blots were incubated with pooled serum from patients with visceral leishmaniasis (1:2,500 dilution) followed by 1:20,000 peroxidase-conjugated anti-human IgG. After washing, membranes were developed by chemiluminescence (ECL; Amersham Pharmacia Biotech, Buckinghamshire, England).

Immunoblots of purified recombinant proteins (50 µg/lane) were separated on 12% SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated with serum from individual visceral leishmaniasis patients (1:100), followed by 1:1,000 peroxidase-conjugated anti-human IgG. Blots were developed with DAB-NiCl solution [Ni(II) chloride hexahydrate-3,3'-diaminobenzidine tetrahydrochloride; Sigma].

Northern blot assays. Total mRNA was extracted from LcJ promastigotes in logarithmic or stationary growth phase or from axenic amastigotes (15). RNA was separated on formaldehyde-1.2% agarose gels and transferred by capillary action to nylon membranes. Blots were hybridized with gel-purified inserts corresponding to screen-selected cDNA clones and labeled with [{alpha}-32P]dCTP by random priming (2). Hybridization with the L. chagasi {alpha}-tubulin coding sequence was used as a loading control.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary (serum) screen of the L. chagasi amastigote cDNA library. Because it is impractical to screen 100,000 clones with T cells, we adopted a primary screen to identify phage clones that produced detectable recombinant L. chagasi proteins. The amastigote cDNA library was first screened with a pool of sera from Brazilian patients with documented visceral leishmaniasis. Patients with visceral leishmaniasis are known to produce antibodies against hundreds of parasite proteins due to the polyclonal B-cell response that develops during infection (5, 22). As expected, the pool of sera used for our library screen recognized multiple proteins in Western blot assays of lysates of both L. chagasi amastigotes and promastigotes (Fig. 1).


Figure 1
View larger version (93K):
[in this window]
[in a new window]
  		FIG. 1. Sera used for immunoscreening the amastigote cDNA library recognize multiple proteins. Replicate cultures of LcJ promastigotes or amastigotes were subjected to immunoblotting using the same pooled sera from visceral leishmaniasis patients that were used for the library immunoscreen. Proteins were separated on 7.5% SDS-polyacrylamide gels and analyzed by immunoblotting with pooled sera. Molecular weight markers are shown at the left. The sera pool was used to immunoscreen the amastigote cDNA library to detect a maximal number of clones producing recombinant proteins.

 
The primary screen yielded 242 protein-producing clones, which were purified in secondary and tertiary screens. We expected that heat shock proteins would not serve as good vaccine candidates due to their potential cross-reactivity with host proteins. Thus, clones encoding heat shock proteins were eliminated by cross-hybridization with coding sequences for L. chagasi HSP70, HSP83, and HSP90 (Table 1). Of interest, genes for these heat shock proteins were highly overrepresented among the immunoscreen-positive clones compared to their overall frequency in the amastigote cDNA library. A total of 118 of the 242 clones (49%) hybridized to one of the three L. chagasi HSP sequences. In contrast, only 38 of 5,000 (0.76%) in the cDNA library contained coding sequences for these three heat shock proteins. HSP-encoding clones were removed from the group of clones that underwent the secondary screen.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Frequency of heat shock proteins in L. chagasi amastigote cDNA clones selected by immunoscreening with polyclonal human serum

 
Secondary (T-cell) screen and T-cell antigens. The remaining 124 phages were subjected to a second screening step using immune mouse T cells. We chose to screen with T cells from C3H.HeJ mice, which are nonresponsive to lipopolysaccharide (LPS) due to a mutation in TLR4 (37), because we expected that LPS would contaminate the crude recombinant protein preparations. C3H.HeJ mice are also genetically resistant to infection with the visceralizing Leishmania spp., increasing the chance of identifying protective antigens. Four weeks after infection with L. chagasi, the time of peak parasite loads just prior to self-cure (57), T cells were purified from infected mouse spleens and stimulated with recombinant proteins in nine replicate 96-well plates. Proteins were assessed for their ability to stimulate immune T-cell proliferation. Eleven clones yielded proliferation indices more than twofold over no-insert phage controls in four replicate assays. An additional nine clones that yielded a stimulation index greater than 2.0 in only one or two of four replicate assays were not further pursued at this point.

The ends of the cDNA inserts from the 11 clones were sequenced. Three encoded heat shock proteins (two HSP70 and one HSP83) were not recognized in the above hybridization screen. One was found to encode ribosomal protein P0 and was eliminated from further analysis because it might cross-react with the mammalian protein. A final clone was eliminated due its small insert size.

Table 2 shows the results of proliferation indices and IFN-{gamma} levels released into supernatants upon stimulation with the remaining six clones studied and for a negative clone that did not generate a recombinant protein recognized by antiserum in the first screen. The positive control for proliferation was live leishmania promastigote antigen.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Proliferation indices of recombinant proteinsa

 
The remaining six clones were subjected to full sequence analysis (Table 3). The open reading frames (ORFs) were identified and compared with sequences derived from the NCBI database and from the Sanger website for the Leishmania major and L. infantum GeneDB databases (http://www.genedb.org/genedb/leish/index.jsp). The ORFs in these six cDNA clones, called by their clone numbers 314, 319, 419, 425, 503, and 648, ranged from 768 to 2,355 bp. The predicted masses of the encoded proteins were determined using the bioinformatics.org website. As summarized in Table 3, these six ORFs encode the following proteins: a transitional endoplasmic reticulum ATPase, glutamine synthetase, a hypothetical protein of unknown function, K39 (myosin-like) protein of L. chagasi, elongation factor 1{gamma} (EF-1{gamma}), and Leishmania repetitive protein A2.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Leishmania chagasi antigens identified with the double screena

 
Figure 2A shows the alignment between the 648 (A2) cDNA with its homologue L. infantum A2 and an L. infantum GeneDB (Sanger) sequence. According to Matlashewski, different members of the L. infantum A2 gene family contain between 40 and >90 repeats of a 10-amino-acid motif (14, 60, 61). Clone 648 shared 97% identity with residues 44 and 297 of the translated GeneDB sequence. The translated 648 and Sanger (GeneDB) sequences are homologous but not identical to the first A2 gene reported in the NCBI database (GenBank) (54). Differences include a different N terminus of the NCBI A2 polypeptide. Clone 648 and the Sanger sequence encode a different first amino acid residue (S) in the repeat sequence compared to the NCBI A2 gene (A), resulting in sequences of S/AVGPLSVGPQ, respectively.


Figure 2
View larger version (36K):
[in this window]
[in a new window]
  		FIG. 2. Amino acid alignments between predicted polypeptides and L. infantum homologues. Alignments were done using the Vector NTI Advance sequence analysis program. (A) L. chagasi 648 cDNA clone aligned with two different L. infantum A2 genes, one found on the Sanger website (GeneDB) and one found on the NCBI website (GenBank). (B) L. chagasi 425 cDNA clone and L. infantum kinesin K39 amino acid alignment, showing identity. Dashes, identical residues; dots, missing residues; *, stop codon.

 
Figure 2B shows the alignment between clone 425 and L. infantum kinesin K39. The cDNA clone did not extend to the 5' end of the coding region. Eight residues near the N terminus differed between 425 and K39, and 425 had a seven-residue insert. Otherwise, the sequences were identical.

Northern blot assays. Because the original cDNA library was constructed from hamster-derived amastigotes, there was the potential that the selected clones were of hamster origin. As such, expression of these antigens by axenically grown L. chagasi parasite stages was verified on Northern blot assays of promastigotes (log and stationary phase) and axenic amastigotes. Total RNAs from wild-type L. chagasi promastigotes, LcJ promastigotes in the logarithmic and stationary phase of growth, or LcJ amastigotes were hybridized with cDNA inserts corresponding to each of the recombinant clones (Fig. 3). The {alpha}-tubulin coding region of L. chagasi served as a loading control. The slower migration of the mRNAs compared to the observed sizes of open reading frames likely corresponds to the lengths of the 5' and 3' untranslated regions on the mRNAs.


Figure 3
View larger version (62K):
[in this window]
[in a new window]
  		FIG. 3. Northern blot assays were performed with total RNA extracted from wild-type L. chagasi and LcJ promastigotes in logarithmic (L) or stationary (S) phases of growth or LcJ amastigotes (Am). RNAs were hybridized with [32P]DNA inserts from the indicated cDNA clones. A final panel was hybridized to the L. chagasi {alpha}-tubulin coding sequence to control for loading between lanes.

 
The mRNAs corresponding to clones 319 (glutamine synthetase) and 425 (K39) were expressed at similar levels in logarithmic- and stationary-phase wild-type promastigotes. The mRNAs for 314 (endoplasmic reticulum [ER] ATPase), 419 (conserved hypothetical protein), and 503 (EF-1{gamma}) displayed higher levels of expression during the logarithmic phase of growth, and 419 mRNA was low in amastigotes. As expected, the mRNA derived from clone 648, which corresponds to one of the L. chagasi A2 genes, was much more highly expressed in the amastigote than the promastigote stage. Nonetheless, all antigens are expressed in amastigotes and, as such, constitute potential targets for the immune response during mammalian infection.

Human immune responses to recombinant antigens. The predicted sizes of peptides were compared to proteins observed on immunoblots of bacterial lysates containing recombinant phage and induced with IPTG. Recombinant proteins in these lysates were recognized by a pool of sera from visceral leishmaniasis patients (Fig. 4A). The sizes of the fragments recognized by human immune sera were compatible with estimated sizes predicted from the cDNA sequences. These were visualized among background E. coli bands also recognized by the human sera. Unique bands were not visible in lanes containing transformants harboring clones 319 and 648, possibly due to low-level expression. The lack of full correspondence between predicted and observed sizes of recombinant proteins in Table 3 could be due to folding or to modifications of proteins expressed in bacteria.


Figure 4
View larger version (46K):
[in this window]
[in a new window]
  		FIG. 4. Recognition of recombinant proteins by human sera. (A) An immunoblot assay was performed with bacteria transformed with plasmids carrying L. chagasi antigens to determine the sizes of recombinant proteins selected with the double screening method. Lane a, lysate of nontransformed bacteria; lanes b and g, lysates of bacteria transformed with plasmids containing cDNAs for 648 (b, protein not visualized), 319 (c, protein not visualized), 425 (d), 503 (e), 314 (f), and 419 (g). Proteins were separated on an 8.5% SDS-polyacrylamide gel and immunoblotted with pooled sera from patients with visceral leishmaniasis. Arrows point to unique bands corresponding to recombinant proteins visualized in lanes d to g. (B) Purified recombinant proteins were generated from clones 314, 419, and 503. Recombinant proteins or total L. chagasi promastigote lysate (L.ch) was incubated with sera from individual patients with visceral leishmaniasis. A representative immunoblot shows recognition of recombinant proteins.

 
Recombinant proteins were purified and tested for reactivity with individual sera. Not all antigens expressed well in E. coli, possibly due to toxicity of the protein products for bacteria. As such, responses to 503, 314, and 419 were the only ones tested. Sera from several visceral leishmaniasis patients known to be acutely infected recognized the recombinant proteins on immunoblots (see an example in Fig. 4B). Immunoblots using sera from a nonexposed North American did not recognize recombinant 503, 419, or 314 antigens (data not shown).

To test a larger number of sera, we designed ELISAs for the three recombinant antigens (Fig. 5). All sera had already tested positive for recombinant K39 antigen, the antigen used in commercial dipstick tests (19). We previously showed that this antigen has a sensitivity of 94.7% and specificity of 98.3% in a Brazilian population (5). Negative reactions were defined using sera from individuals never exposed in the region endemic for L. chagasi.


Figure 5
View larger version (9K):
[in this window]
[in a new window]
  		FIG. 5. Recombinant protein ELISA. Sera from patients with acute visceral leishmaniasis were chosen due to their positive responses to recombinant K39. Optical density (OD) values are plotted for three recombinant proteins selected in this study and compared to responses to recombinant K39. Reactions to antigen 503 are shown on the same scale and in an expanded scale compared to other antigens. The negative cutoff value was chosen from a study of unexposed individuals and calculated as the mean + 3 standard deviations.

 
Sera from patients with acute visceral leishmaniasis and positive rK39 serology were tested against three of the recombinant proteins, excluding antigen 425, which is the K39 homologue. For the three antigens tested, 21, 21, or 23 of the 34 sera yielded positive reactions to the three recombinant antigens, respectively. Among the 34 sera, 33 were positive for at least one antigen and 26 were positive for two or three of the recombinant antigens tested.

Cellular immune assays were performed with two recombinant antigens (Table 4). Two of six individuals tested responded to parasite lysate with IFN-{gamma} (mean, 64.2 ± 10.4 ng/ml), two of six secreted IFN-{gamma} when stimulated with a control antigen, KMP11 (mean, 8.4 ± 0.7 pg/ml) (12), three of six responded to antigen 419 with IFN-'{gamma} (mean, 10.9 ± 6.0 pg/ml), and one of six responded to antigen 503 by release of IFN-{gamma} (16.8 pg/ml). Overall, the selected antigens yielded serologic and/or cytokine responses in all humans naturally infected with L. chagasi that were tested.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Human immune responses to recombinant proteinsa

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The estimated worldwide incidence of leishmaniasis is 2 million new cases each year, and an estimated 1/10 of the world's population is at risk for infection (World Health Organization Leishmaniasis Control home page [http://who.int/ctd/html/leis.html]). Unfortunately, many individuals living in regions where it is endemic still do not have adequate access to treatment of these diseases. When available, therapy can cause toxic side effects, and drug resistance is emerging (19, 47). Efforts at controlling leishmaniasis, therefore, have been aimed both at discovering less toxic and more affordable drugs and at developing a preventive vaccine (42).

The diagnosis of visceral leishmaniasis is hampered by the lack of a prompt diagnostic method. The "gold standard" diagnostic procedure is still the parasitological confirmation in specimens collected either from spleen or bone marrow aspirates. Serologic diagnosis with rK39 antigen strips varies from 67 to 100% sensitivity and 93 to 100% specificity. In Brazil and in our hands, the rK39 ELISA has proven a very efficient means of diagnosis, whereas in India there have been cases of weak positive responses after cure or false-positive responses in healthy persons with the rK39 dipstick test (5, 19).

Although there are no currently approved human or canine vaccines against leishmaniasis, cure of disease results in long-term protective immunity to reinfection in normal hosts (42). There is considerable interest in development of an antileishmanial vaccine that will mimic this natural protective immunity. Human trials with parasite antigen preparations with or without BCG adjuvant have been disappointing (1). Several alternate vaccination strategies, many based on strategies to deliver a subunit vaccine, are under consideration. Because the Leishmania spp. are complex protozoa able to modify their antigenic characteristics in vitro and in vivo (18), it seems likely that a cocktail of antigens must be included if a subunit vaccine is to be effective.

Antigens that might be appropriate for a subunit vaccine or for diagnostic purposes have been identified through a variety of approaches, including differential expression in parasite stages or recognition by antibodies and T cells (reviewed in reference 34). We reasoned that the field would benefit from a systematic screen for T-cell antigens and T-dependent B-cell antigens expressed in the intracellular amastigote stage of the parasite, the stage present in the mammalian host. Therefore, the aim of the present study was to conduct such a screen of an L. chagasi amastigote cDNA library.

We patterned a two-step library screening procedure after the method of Mustafa et al. (40). Individuals with acute visceral leishmaniasis develop a polyclonal B-cell response generating antibodies to a large number of parasite antigens (22). Therefore, our L. chagasi amastigote cDNA library was first immunoscreened with pooled serum from Brazilians with visceral leishmaniasis to select as many clones producing recombinant parasite proteins as possible. Once accomplished, we eliminated heat shock proteins by cross-hybridization, since we reasoned that the ubiquitous nature of heat shock proteins would limit their potential utility in vaccines. Among the 242 clones identified in the first screen, 49% encoded members of the heat shock protein HSP70, HSP86, and HSP90 families. This represented a marked enrichment over the 0.76% frequency of these sequences in the general library.

The remaining non-heat shock proteins were subjected to a secondary screen for their ability to efficiently induce a T-cell response. The "probe" consisted of purified T lymphocytes from a mouse strain that is resistant to L. chagasi infection and which is genetically nonresponsive to LPS due to a mutation in the TLR4 gene (53). In initial assays, 20 clones (16% of the total) were identified for their ability to cause proliferation. After eliminating clones for which the proliferation response was not replicated, residual heat shock proteins missed on the hybridization screen, and a ribosomal structural protein, six unique clones encoding proteins that repeatedly stimulated lymphocyte responses in all assays were chosen for further study.

The choice of murine T cells for the secondary screen warrants some discussion. There are clearly differences between human and murine lymphocyte responses. We chose to screen for murine responses because of the availability of LPS-hyporesponsive mouse strains and the confounding influence that LPS contaminating the recombinant proteins would undoubtedly cause when incubated with human T lymphocytes. The fact that we selected homologues of two antigens of interest in the literature, i.e., a homologue of K39 which induces B-cell responses during acute visceral leishmaniasis, and A2, which causes protective immunity in mice, served as a positive validation that the screen was successful in picking potentially useful antigens. The selection of an A2 gene that diverges from that used in prior immunization studies through this unbiased screen (Fig. 3) indicates its potential validity as a vaccine candidate.

The literature contains reports of many antigens that induce type 1 immune responses and/or protect against parasites causing cutaneous leishmaniasis (e.g., L. major and L. amazonensis). Immunization of genetically susceptible BALB/c mice with L. major LACK (Leishmania homologue of mammalian RACKs receptors for activated C kinase) was effective in protecting BALB/c mice against progressive L. major infection (28). LeIF, a Leishmania species gene with homology to eukaryotic ribosomal protein eIF4A, is a potent stimulus of IFN-{gamma} production by T lymphocytes from lymph nodes draining L. major in BALB/c mice (48). A cocktail vaccine containing LeIF, LmSTI1, and TSA induced protection in both murine and nonhuman primate models of cutaneous leishmaniasis (9). Other proteins that induce potentially protective T-cell responses in models of cutaneous leishmaniasis have included GP63 (54), WD protein (8), GP46 (PSA) (13), P4 (7), P8 (49), and PSA-2 (29), among others (26, 34).

Antigens from the parasites causing visceral leishmaniasis have also been reported to induce partial protective immunity. Immunization of mice with killed L. donovani crude antigen fractions, purified dp72 and gp70-2 L. donovani membrane proteins (43), recombinant LCR1 (59), Ldp23 (10), A2 (25), or HASPB1 (50) offered partial protection against parasite challenge. A2, which was also identified in our screen, includes a group of proteins expressed exclusively in amastigotes with an as-yet-elusive function (24). L. infantum A2 proteins contain repeated peptide epitopes varying from 10 to 90 in different A2 genes (60). Parasites causing cutaneous leishmaniasis (L. major, L. tropica, and L. braziliensis) contain A2 homologues that lack the repeated sequences (23). Somewhat predictably, immunization with A2 protects against L. donovani but not L. major infections in mice (25), whereas LACK protects against L. major but not L. donovani infections (38). A2 and LACK may therefore represent species-specific antigens that influence the course of visceral or cutaneous leishmaniasis but might be "ignored" during the immune response to parasites causing the other clinical form.

Among the six L. chagasi amastigote antigens identified in this study, A2 was exclusively expressed in amastigotes and the mRNAs for three other proteins (EF-1{gamma} [11], ER ATPase, and a conserved hypothetical protein) were represented more highly in logarithmic- than stationary-phase promastigotes. It should be mentioned that clone 503 is not the same as the elongation factor (EF-1{alpha}) previously reported to be released from the parasitophorous vacuole into the infected macrophage cytoplasm (41). Glutamine synthetase and the homologue of K39 (6) were expressed similarly between the stages. As with other Leishmania spp. genes, the level of mRNA expression is not necessarily expected to correlate with protein abundance.

It is remarkable that the antigens recognized by T cells are not all predicted to localize on the promastigote surface or be secreted. Indeed, glutamine synthetase and EF-1{gamma} might be primarily cytoplasmic, ER ATPase might localize to organelles, and K39 might be found in the cytoplasm or cytoskeleton. It is possible that small amounts of glutamine synthetase are expressed on the parasite surface, and A2 might be secreted. Nonetheless, it is possible that intracellular parasites release more antigens into the dendritic cell or macrophage antigen presentation pathways than we have as yet appreciated.

Humans naturally exposed to L. chagasi mounted both humoral and cellular responses to some of the above antigens. The fact that no one antigen elicited responses in all exposed persons, whether symptomatic or asymptomatic, underscores the fact that a vaccine formulation, and possibly an optimized diagnostic test, may require a mixture of parasite antigens to induce or detect an immune response. Further studies are clearly needed to characterize the efficacy of individual amastigote antigens, or the combination of antigens, in inducing both humoral and cellular immune responses in infected humans from regions where it is endemic.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health AI32135 and AI059451 (J.E.D. and M.E.W.), AI045540, AI048822, AI067874, and TW01369 (M.E.W.), and P50 AI-30639 (M.E.W. and S.M.B.J.), as well as a Merit Review from the Department of Veterans' Affairs (M.E.W.). It was performed during the tenure of D.R.M. as a fellow of the NIH Fogarty Foundation, grant TW-00-007.

We thank Betty Young for technical support.

We disclose a patent application regarding vaccine development using data reported in this work.


   FOOTNOTES
 
* Corresponding author. Mailing address: Dept. of Internal Medicine, University of Iowa, SW34-GH, 200 Hawkins Dr., Iowa City, IA 52242. Phone: (319) 356-3169. Fax: (319) 384-7208. E-mail: mary-wilson{at}uiowa.edu. Back

{triangledown} Published ahead of print on 25 September 2006. Back

Editor: W. A. Petri, Jr.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1. Armijos, R. X., M. M. Weigel, M. Calvopina, A. Hidalgo, W. Cevallos, and J. Correa. 2004. Safety, immunogenicity, and efficacy of an autoclaved Leishmania amazonensis vaccine plus BCG adjuvant against New World cutaneous leishmaniasis. Vaccine 22:1320-1326.[CrossRef][Medline]
2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 2006. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
3. Beetham, J. K., K. S. Myung, J. J. McCoy, M. E. Wilson, and J. E. Donelson. 1997. Glycoprotein 46 mRNA abundance is post-transcriptionally regulated during development of Leishmania chagasi promastigotes to an infectious form. J. Biol. Chem. 28:17360-17366.
4. Berens, R. L., R. Brun, and S. M. Krassner. 1976. A simple monophasic medium for axenic culture of hemoflagellates. J. Parasitol. 62:360-365.[CrossRef][Medline]
5. Braz, R. F. S., E. T. Nascimento, D. R. A. Martins, M. E. Wilson, R. D. Pearson, S. G. Reed, and S. M. B. Jeronimo. 2002. The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection. Am. J. Trop. Med. Hyg. 67:344-348.[Abstract]
6. Burns, J. M., Jr., W. G. Shreffler, D. R. Benson, H. W. Ghalib, R. Badaro, and S. G. Reed. 1993. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. Natl. Acad. Sci. USA 90:775-779.[Abstract/Free Full Text]
7. Campbell, K., H. Diao, J. Ji, and L. Soong. 2003. DNA immunization with the gene encoding P4 nuclease of Leishmania amazonensis protects mice against cutaneous leishmaniasis. Infect. Immun. 71:6270-6278.[Abstract/Free Full Text]
8. Campbell, K., V. Popov, and L. Soong. 2004. Identification and molecular characterization of a gene encoding a protective Leishmania amazonensis Trp-Asp (WD) protein. Infect. Immun. 72:2194-2202.[Abstract/Free Full Text]
9. Campos-Neto, A., R. Perrozzi, and K. Greeson. 2001. Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease. Infect. Immun. 69:4103-4108.[Abstract/Free Full Text]
10. Campos-Neto, A., L. Soong, J. L. Cordova, D. Sant'Angelo, Y. A. W. Skeiky, N. H. Ruddle, S. G. Reed, C. Janeway, and D. McMahon-Pratt. 1995. Cloning and expression of an Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages. J. Exp. Med. 182:1423-1433.[Abstract/Free Full Text]
11. Canavate, C., M. el Bali, Y. Montoya, D. C. Barker, and J. Alvar. 2002. Isolation and characterization of Leishmania infantum cDNA encoding a protein homologous to eukaryotic elongation factor 1 gamma. Trans. R. Soc. Trop. Med. Hyg. 96:S41-S47.
12. Carvalho, L. P., M. Soto, S. M. B. Jeronimo, B. Dondji, C. Alonso, A. Ribeiro de Jesus, and E. M. Carvalho. 2003. Characterization of the immune response to Leishmania infantum recombinant antigens. Microbes Infect. 5:7-12.[CrossRef][Medline]
13. Champsi, J., and D. McMahon-Pratt. 1988. Membrane glycoprotein M-2 protects against Leishmania amazonensis infection. Infect. Immun. 52:3272-3279.
14. Charest, H., and G. Matlashewski. 1994. Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene. Mol. Cell. Biol. 14:2975-2984.[Abstract/Free Full Text]
15. Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.[Medline]
16. Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober. 1991. Current protocols in immunology. John Wiley and Sons, New York, N.Y.
17. Costa, C. H., H. F. Pereira, and M. V. Araujo. 1990. Epidemia de leishmaniose visceral no estado do Piauí, Brasil. Rev. Saude Publica 24:361-372.[Medline]
18. Coulson, R. M. R., and D. F. Smith. 1990. Isolation of genes showing increased or unique expression in the infective promastigotes of Leishmania major. Mol. Biochem. Parasitol. 40:63-76.[CrossRef][Medline]
19. Davies, C. R., P. M. Kaye, S. L. Croft, and S. Sundar. 2003. Leishmaniasis: new approaches to disease control. BMJ 326:377-382.[Free Full Text]
20. De Andrade, C. R., L. V. Kirchhoff, J. E. Donelson, and K. Otsu. 1992. Recombinant Leishmania Hsp90 and Hsp70 are recognized by sera from visceral leishmaniasis patients but not Chagas' disease patients. J. Clin. Microbiol. 30:330-335.[Abstract/Free Full Text]
21. Evans, T. G., M. J. Teixeira, I. T. McAuliffe, I. De Alencar Barros Vasconcelos, A. Wilson Vasconcelos, A. De Queiroz Sousa, J. Wellington de Oliveira Limaqq, and R. D. Pearson. 1992. Epidemiology of visceral leishmaniasis in northeast Brazil. J. Infect. Dis. 166:1124-1132.[Medline]
22. Galvao-Castro, B., J. A. S. A. Ferreira, M. C. Marzochi, S. G. Marzochi, S. G. Coutinho, and P. H. Lambert. 1984. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human American visceral leishmaniasis. Clin. Exp. Immunol. 56:58-66.[Medline]
23. Ghedin, E., W.-W. Zhang, H. Charest, S. Sundar, R. T. Kenney, and G. Matlashewski. 2005. Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis. Clin. Diagn. Lab. Immunol. 4:530-535.
24. Ghosh, A., S. Labrecque, and G. Matlashewski. 2001. Protection against Leishmania donovani infection by DNA vaccination: increased DNA vaccination efficiency through inhibiting the cellular p53 response. Vaccine 19:3169-3178.[CrossRef][Medline]
25. Ghosh, A., W. W. Zhang, and G. Matlashewski. 2001. Immunization with A2 protein results in mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infection. Vaccine 20:59-66.[CrossRef][Medline]
26. Ghosh, M., and S. Bandyopadhyay. 2003. Present status of antileishmanial vaccines. Mol. Cell Biochem. 253:199-205.[CrossRef][Medline]
27. Goyard, S., H. Segawa, J. Gordon, M. Showalter, R. Duncan, S. J. Turco, and S. M. Beverley. 2003. An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans. Mol. Biochem. Parasitol. 130:31-42.[CrossRef][Medline]
28. Gurunathan, S., D. L. Sacks, D. R. Brown, S. L. Reiner, H. Charest, N. Glaichenhaus, and R. A. Seder. 2005. Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major. J. Exp. Med. 186:1137-1147.
29. Handman, E., A. H. Noormohammadi, J. M. Curtis, T. Baldwin, and A. Sjolander. 2000. Therapy of murine cutaneous leishmaniasis by DNA vaccination. Vaccine 18:3011-3017.[CrossRef][Medline]
30. Herwaldt, B. L. 1999. Leishmaniasis. Lancet 354:1191-1199.[CrossRef][Medline]
31. Jeronimo, S. M. B., P. Duggal, R. F. S. Braz, C. Cheng, G. R. G. Monteiro, E. T. Nascimento, F. L. Bezerra, T. M. Karplus, M. F. F. M. Ximenes, C. G. C. Oliveira, V. G. Pinheiro, D. R. A. Martins, J. M. Peralta, J. M. Sousa, I. M. Medeiros, R. D. Pearson, T. L. Burns, E. W. Pugh, and M. E. Wilson. 2004. An emerging peri-urban pattern of infection with Leishmania chagasi, the protozoan causing visceral leishmaniasis in northeast Brazil. Scand. J. Infect. Dis. 36:443-449.[CrossRef][Medline]
32. Kaye, P. M. 2002. The immunology of visceral leishmaniasis: current status, p. 137-150. In J. P. Farrell (ed.), Leishmania, vol. 4. Kluwer Academic Publishers, Boston, Mass.
33. MacBeath, J. R. E., S. S. Harvey, and N. J. Oldroyd. 2001. Automated fluorescent DNA sequencing on the ABI PRISM 377, p. 119-152. In C. A. Graham and A. J. M. Hill (ed.), DNA sequencing protocols, vol. 167. Humana Press, Inc., Totowa, N.J.[CrossRef]
34. Mauel, J. 2002. Vaccination against Leishmania infections. Curr. Drug Targets Immune Endocr. Metab. Disord. 2:201-226.
35. Mauricio, I. L., J. R. Stothard, and M. A. Miles. 2000. The strange case of Leishmania chagasi. Parasitol. Today 16:188-189.[CrossRef][Medline]
36. McKean, P. G., P. W. Denny, E. Knuepfer, J. K. Keen, and D. F. Smith. 2001. Phenotypic changes associated with deletion and overexpression of a stage-regulated gene family in Leishmania. Cell. Microbiol. 3:511-523.[CrossRef][Medline]
37. Means, T. K., D. T. Golenbock, and M. J. Fenton. 2000. The biology of Toll-like receptors. Cytokine Growth Factor Rev. 11:219-232.[CrossRef][Medline]
38. Melby, P. C., J. Yang, W. Zhao, L. E. Perez, and J. Cheng. 2001. Leishmania donovani p36(LACK) DNA vaccine is highly immunogenic but not protective against experimental visceral leishmaniasis. Infect. Immun. 69:4719-4725.[Abstract/Free Full Text]
39. Momen, H., R. S. Pcheco, E. Cupolillo, and G. Grimaldi. 1993. Molecular evidence for the importation of Old World Leishmania into the Americas. Biol. Res. 26:249-255.[Medline]
40. Mustafa, A. S., F. Oftung, A. Deggerdal, H. K. Gill, R. A. Young, and T. Godal. 1988. Gene isolation with human T lymphocyte probes. Isolation of a gene that expresses an epitope recognized by T cells specific for Mycobacterium bovis BCG and pathogenic mycobacteria. J. Immunol. 141:2729-2733.[Abstract]
41. Nandan, D., T. Yi, M. Lopez, C. Lai, and N. E. Reiner. 2002. Leishmania EF-1{alpha} activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. J. Biol. Chem. 277:50190-50197.[Abstract/Free Full Text]
42. Pearson, R. D., and A. Q. Sousa. 1996. Clinical spectrum of leishmaniasis. Clin. Infect. Dis. 22:1-13.[Medline]
43. Rachamin, N., and C. L. Jaffe. 1999. Pure protein from Leishmania donovani protects mice against both cutaneous and visceral leishmaniasis. J. Immunol. 150:2322-2331.
44. Rafati, S., A. A. Baba, M. Bakhshayesh, and M. Vafa. 2000. Vaccination of Balb/c mice with Leishmania major amastigote-specific cysteine proteinase. Clin. Exp. Immunol. 120:134-138.[CrossRef][Medline]
45. Reed, S. G., R. Badaro, H. Masur, E. M. Carvalho, R. Lorenco, A. Lisboa, R. Teixeira, W. D. Johnson, and T. C. Jones. 1986. Selection of a skin test antigen for American visceral leishmaniasis. Am. J. Trop. Med. Hyg. 35:79-85.[Abstract/Free Full Text]
46. Reed, S. G., and A. Campos-Neto. 2003. Vaccines for parasitic and bacterial diseases. Curr. Opin. Immunol. 15:456-460.[CrossRef][Medline]
47. Singh, N., R. T. Singh, and S. Sundar. 2003. Novel mechanism of drug resistance in kala-azar field isolates. J. Infect. Dis. 188:600-607.[CrossRef][Medline]
48. Skeiky, Y. A. W., M. Kennedy, D. Kaufman, M. M. Borges, J. A. Guderian, J. K. Scholler, P. J. Ovendale, K. S. Picha, P. J. Morrissey, K. H. Grabstein, A. Campos-Neto, and S. G. Reed. 1998. LeIF: a recombinant Leishmania protein that induces an IL-12-mediated Th1 cytokine profile. J. Immunol. 161:6171-6179.[Abstract/Free Full Text]
49. Soong, L., S. M. Duboise, P. Kima, and D. McMahon-Pratt. 1995. Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis. Infect. Immun. 63:3559-3566.[Abstract]
50. Stager, S., D. F. Smith, and P. M. Kaye. 2000. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J. Immunol. 165:7064-7071.[Abstract/Free Full Text]
51. Streit, J. A., T. J. Recker, F. G. Filho, S. M. Beverley, and M. E. Wilson. 2001. Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms. J. Immunol. 166:1921-1929.[Abstract/Free Full Text]
52. Sundar, S. 2001. Drug resistance in Indian visceral leishmaniasis. Trop. Med. Int. Health 6:849-854.[CrossRef][Medline]
53. Vogel, S. N., D. Johnson, P.-Y. Perera, A. Medvedev, L. Lariviere, S. T. Qureshi, and D. Malo. 1999. Cutting edge: functional characterization of the effect of the C3H/HeJ defect in mice that lack an Lpsn gene: in vivo evidence for a dominant negative mutation. J. Immunol. 162:5666-5670.[Abstract/Free Full Text]
54. Walker, P. S., T. Scharton-Kersten, E. D. Rowton, U. Hengge, A. Bouloc, M. C. Udey, and J. C. Vogel. 1998. Genetic immunization with glycoprotein 63 cDNA results in helper T cell type 1 immune response and protection in a murine model of leishmaniasis. Hum. Gene Ther. 9:1899-1907.[Medline]
55. Wilson, M. E., K. A. Andersen, and B. E. Britigan. 1994. Response of Leishmania promastigotes to oxidant stress. Infect. Immun. 62:5133-5141.[Abstract/Free Full Text]
56. Wilson, M. E., K. K. Hardin, and J. E. Donelson. 1989. Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes. J. Immunol. 143:678-684.[Abstract]
57. Wilson, M. E., S. M. B. Jeronimo, and R. D. Pearson. 2005. Immunopathogenesis of infection with the visceralizing Leishmania species. Microb. Pathog. 38:147-160.[CrossRef][Medline]
58. Wilson, M. E., and R. D. Pearson. 1986. Evidence that Leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism. J. Immunol. 136:4681-4688.[Abstract]
59. Wilson, M. E., B. M. Young, K. P. Andersen, J. V. Weinstock, A. Metwali, K. M. Ali, and J. E. Donelson. 1995. A recombinant Leishmania chagasi antigen that stimulates cellular immune responses in infected mice. Infect. Immun. 63:2062-2069.[Abstract]
60. Zhang, W. W., and G. Matlashewski. 1996. The developmental expression of Leishmania donovani A2 amastigote-specific genes is post-transcriptionally mediated and involves elements located in the 3'-untranslated region. J. Biol. Chem. 271:17081-17090.[Abstract/Free Full Text]
61. Zhang, W.-W., H. Charest, E. Ghedin, and G. Matlashewski. 1996. Identification and overexpression of the A2 amastigote-specific protein in Leishmania donovani. Mol. Biochem. Parasitol. 78:79-90.[CrossRef][Medline]

Infection and Immunity, December 2006, p. 6940-6948, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.02032-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.02032-05v1
74/12/6940    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martins, D. R. A.
Right arrow Articles by Wilson, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martins, D. R. A.
Right arrow Articles by Wilson, M. E.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals