Resistance to Leishmania major is linked to the H2 region on chromosome 17 and to chromosome 9.

In Leishmaniasis, as in many infectious diseases, clinical manifestations are determined by the interaction between the genetics of the host and of the parasite. Here we describe studies mapping two loci controlling resistance to murine cutaneous leishmaniasis. Mice infected with L. major show marked genetic differences in disease manifestations: BALB/c mice are susceptible, exhibiting enlarging lesions that progress to systemic disease and death, whereas C57BL/6 are resistant, developing small, self-healing lesions. F2 animals from a C57BL/6 X BALB/c cross showed a continuous distribution of lesion score. Quantitative trait loci (QTL) have been mapped after a non-parametric QTL analysis on a genome-wide scan on 199 animals. QTLs identified were confirmed in a second cross of 271 animals. Linkage was confirmed to a chromosome 9 locus (D9Mit67-D9Mit71) and to a region including the H2 locus on chromosome 17. These have been named Imr2 and Imr1, respectively.

Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania donovani(1).

To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L. donovani amastigotes. Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family was identified and characterised. The GRP78 gene was localised to chromosome 15 in L. donovani, L. major, and L. mexicana by pulse-field gel electrophoresis. The Leishmania GRP78 protein contain a carboxy-terminal endoplasmic reticulum retention signal sequence (MDDL) as does the Trypanosoma cruzi GRP78. Immunofluorescence using antibodies to the recombinant DNA-derived GRP78 protein showed staining localised to reticular material throughout the cytoplasm and in the perinuclear region of promastigotes, suggesting that the protein is localised in the endoplasmic reticulum. The protective efficacy of GRP78 was assessed in mice vaccine experiments. A GRP78 DNA vaccine primed for an immune response that protected C57Bl/6 and C3H/He mice against infection with L. major. Similarly vaccination with a recombinant form of GRP78 purified from Escherichia coli and administered with Freund's as adjuvant induced protective immunity in C57Bl/6 mice.

Treatment of murine macrophages with interferon-gamma inhibits their ability to bind leishmania promastigotes.

The binding of leishmania promastigotes to macrophages pretreated with interferon-gamma (IFN-gamma) was compared to binding to untreated (resident) cells. IFN-gamma-treated macrophages bound fewer leishmania promastigotes than did untreated cells. The decreased binding was apparent over a wide dose range of parasite inocula when the assays were performed in the absence of exogenous complement. This decrease was specific to leishmania, since treated and untreated macrophages bound comparable amounts of immunoglobulin G- and complement-coated sheep red blood cells. Decreased parasite binding occurred early in the macrophage activation pathway. Pretreatment of macrophages with IFN-gamma for as little as 6 h, a time insufficient to induce other macrophage activation parameters, significantly reduced their ability to bind leishmania promastigotes. To determine the mechanism of this decreased phagocytosis by activated cells, macrophages were pretreated with specific inhibitors before the addition of leishmania. The binding of promastigotes to untreated (resident) macrophages was inhibited by approximately 50% by reagents that blocked either of two macrophage receptors, complement receptor type 3 (Mac-1) or a leishmania species-specific lectin-like receptor. Binding to IFN-gamma-treated macrophage populations, in contrast, was substantially inhibited only by antibody to Mac-1. Saccharides that were 50% inhibitory in the resident cell population, decreased binding by less than 10% in activated cells. The lack of saccharide inhibition by IFN-gamma-treated cells was also reflected in an inability of activated macrophages to bind to beads coated with purified leishmania lipophosphoglycan (LPG). These LPG-coated beads bound well to resident macrophages but poorly to activated cells. Thus, leishmania bind to macrophages by two distinct mechanisms, one that utilizes Mac-1 and a second mechanism that does not depend on complement and is saccharide inhibitable. These two binding mechanisms are distinct and differentially regulated in resident and activated cells.

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Leishmania major proteophosphoglycan is expressed by amastigotes and has an immunomodulatory effect on macrophage function.

Proteophosphoglycan (PPG) is a newly described mucin-like glycoprotein found on the surface of Leishmania major promastigotes and secreted in the culture supernatant. We show here that antigenically similar PPGs are present in several Leishmania species. PPG could also be detected on the surface of amastigotes and in small, parasite-free vesicles in infected macrophages. Because of the similarity of its carbohydrate chains to lipophosphoglycan, a parasite receptor for host macrophages, PPG was tested for binding to macrophages. PPG bound to macrophages and was internalized in a time-dependent manner. PPG inhibited the production of tumor necrosis factor-alpha and synergized with interferon-gamma to stimulate the production of nitric oxide by macrophages. PPG may contribute to the binding of Leishmania to host cells and may play a role in modulating the biology of the infected macrophage at the early stage of infection.

Mice unresponsive to GM-CSF are unexpectedly resistant to cutaneous Leishmania major infection.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to play a protective role in leishmanial infection. Mice with a null mutation in the gene for the beta common (beta c) chain of the receptors for GM-CSF, interleukin(IL)-3 and IL-5 (beta c-null mice) display normal steady state hemopoiesis and develop lung disease similar to the human condition, alveolar proteinosis, due to a lack of signaling by GM-CSF. We therefore expected to observe a heightened sensitivity to Leishmania major in the beta c-null mice. Surprisingly, the beta c-null mice were more resistant to cutaneous infection than wild-type (wt) mice. Upon intradermal injection of L. major promastigotes, fewer beta c-null mice developed cutaneous lesions than wt mice and these lesions were smaller and healed more rapidly than in wt mice. This resistance to disease was associated with a reduced percentage of in vitro infected beta c-null macrophages. Macrophages from beta c-null mice displayed a more activated phenotype and produced increased amounts of nitric oxide following infection with L. major, both in vivo and in vitro. Paradoxically, however, the parasite burden in the draining lymph nodes was similar in both beta c-null and wt mice, suggesting that at least a subpopulation of cells was susceptible to the parasite. The mechanism preventing normal lesion development remains to be elucidated.

Cell biology of Leishmania.

Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly, where they grow as motile flagellated promastigotes in the gut, and the mammalian macrophage, where they survive and grow intracellularly as non-flagellated amastigotes in the phagolysosome. Leishmaniasis is the outcome of an evolutionary 'arms race' between the host's immune system and the parasite's evasion mechanisms, which ensure survival and transmission in the population. The diverse spectrum of patterns and severity of disease reflect the varying contributions of parasite virulence factors and host responses, some of which act in a host protective manner while others exacerbate disease. This chapter describes the interaction of the Leishmania with their hosts, with emphasis on the molecules and mechanisms evolved by the parasites to avoid, subvert or exploit the environments in the sandfly and the macrophage, and to move from one to the other.

Nitrocellulose-based assays for the detection of glycolipids and other antigens: mechanism of binding to nitrocellulose.

A variety of simple and rapid assays for the detection of glycolipids by direct binding to nitrocellulose or binding to antibody-coated nitrocellulose, and probing with monoclonal antibodies are described. These include dot-blotting, charge shift electrophoresis and electroblotting. It is shown that the direct binding of the Leishmania major glycolipid to nitrocellulose is dependent on its lipid moiety, indicating that the mechanism of binding is probably via hydrophobic interactions. However, the L. major glycolipid from which the lipid moiety has been removed can still be detected by blotting onto nitrocellulose precoated with a monoclonal antibody directed to a carbohydrate epitope. The general approach of blotting onto antibody-coated nitrocellulose thus extends the usefulness of these techniques to cases in which the antigen to be detected does not bind directly to nitrocellulose.

Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major.

This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major. The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L. major cloned line V121. The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced. At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT. The LmYPT gene was present as a single copy gene in both the L. major and L. donovani genomes. Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L. major isolate L119. The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite. Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein. Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.

Isolation and characterization of infective and non-infective clones of Leishmania tropica.

Cloning by limit dilution of an isolate of Leishmania tropica (LRC-L137) that is infective for mice resulted in 7 stable clones, only one of which was infective in BALB/c mice. Three of the non-infective clones that were examined for survival in BALB/c macrophages in vitro seemed to be killed more readily, suggesting failure to establish in macrophages as the basis for non-infectivity in vivo. Promastigotes from three non-infective clones and one infective clone were biosynthetically labelled or surface radioiodinated, and the detergent lysates were analyzed by 2-dimensional gel electrophoresis. The pattern of the radiolabelled cytoplasmic and membrane proteins of promastigotes from all L. tropica clones was similar, with minor differences. All clones as well as the uncloned population bound to the same extent to a series of lectins with galactose and N-acetylgalactosamine as specificities. They also bound in a solid-phase radioimmunoassay to 9 monoclonal antibodies raised against the uncloned L. tropica (LRC-L137). The genetic characterization of four L. tropica clones was attempted by analysis of their isolated kinetoplast DNA. The clones from two schizodemes since they possess kinetoplast DNAs which exhibit similar restriction endonuclease fingerprints and show extensive DNA sequence homology, suggesting that the four clones are closely related and that the non-infective variants may be derived from the infective presumptive parental clone L137-7-121. Further characterization of the clones of L. tropica should allow a better understanding of the genetic basis of parasite virulence in cutaneous leishmaniasis.

Epitope mapping of monoclonal antibodies directed against lipophosphoglycan of Leishmania major promastigotes.

Monoclonal antibodies (MAbs) were generated against Leishmania major promastigote lipophosphoglycan (LPG) to use as tools in defining functional epitopes of this major cell surface glycoconjugate. Epitope mapping of four MAbs, designated 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2, revealed that the phosphorylated oligosaccharide repeat unit PO4-6[Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, P3, is a highly immunogenic epitope which has previously been demonstrated, by chemical analyses, to be a repeat unit specific to L. major. Two antibodies, 4A2-A2 and 5E10-F2, also recognised the repeat unit PO4-6[Ara(beta 1-2)Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, 4Pa, with less affinity than P3, while 2G11-A3 recognised P4a with greater affinity than for P3. The L. major metacyclic-specific antibody 3F12 only recognised repeat units terminating with arabinose residues. In particular, 3F12 recognised P4a, which is upregulated in metacyclic LPG compared to the procyclic form of the molecule. The oligosaccharides P3, P4a and P5a are specific to L. major LPG. The epitopes of 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2 were found on the cell surface and in the flagellar pocket of both procyclic and metacyclic V121 promastigotes, but were only detected at very low levels on amastigotes. The repeat unit P3 is able to inhibit attachment of procyclic promastigotes to the midgut of the sandfly vector, but neither Fab fragments of the four antibodies nor purified P3 could inhibit attachment of metacyclic promastigotes to the macrophage cell line J774. It was also shown that human sera from patients with cutaneous leishmaniasis recognised purified P3. The data suggests that while P3 is an immunogen in the natural course of infection of the human host, P3 plays no role in attachment and internalisation of promastigotes into the macrophages of the mammalian host.

Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein.

Antibodies against Leishmania major wheat germ agglutinin-binding glycoproteins were used to select from a genomic lambda gt11 expression library a clone coding for a L. major glycoprotein. The partial DNA sequence indicated the presence of a mosaic of repetitive sequences. Southern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to chromosome band 18. Northern blot analysis of L. major mRNA detected a major transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies affinity-purified on the fusion protein identified a complex of two water-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polypeptides in other Leishmania species, in Crithidia lucilliae and very weakly in Leptomonas. The apparent molecular weight of these polypeptides, while conserved within each species, varied between species. A peptide map of the two polypeptides from L. major generated an identical pattern suggesting a close relatedness at the protein level. This protein complex was not hydrolysed by N-glycanase and was not affected by tunicamycin, but treatment with anhydrous hydrogen fluoride suggested that it is O-glycosylated. The glycan moiety appears to be N-acetylglucosamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was capable of adding [3H]galactose to it. This was susceptible to beta elimination and beta-galactosidase treatment. Taken together, the data indicates that gp96/92 belongs to the newly described class of cytoplasmic and nuclear glycoproteins containing O-linked N-acetylglucosamine.

The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle.

The promastigote surface antigen 2 (PSA-2) complex comprises a family of antigenically similar polypeptides of M(r) 96,000, 80,000 and 50,000, anchored to the membrane with glycosylphosphatidylinositol. Although PSA-2 was initially detected only in promastigotes, Northern blot analysis indicated that mRNA transcripts are also present in amastigotes. Unlike the situation in promastigotes, where at least four major transcripts (2.6-5.3 kb) were detected, only one major (2.6 kb) and two minor transcripts were present in amastigotes. A cDNA clone encoding a member of the PSA-2 family expressed in amastigotes was isolated using DNA probes. The predicted protein sequence of M(r) 40,000 is distinct from promastigote sequences, but shows significant similarity to previously described members of the family from L major and L amazonensis. Antibodies to the carboxyl terminal sequence conserved in all L major PSA-2 studied to date, as well as antibodies affinity purified on the amastigote cDNA-derived polypeptide recognized a major M(r) 50,000 amastigote polypeptide. Immuno-electron microscopy localized both promastigote and amastigote PSA-2 to the cell surface. The expression of PSA-2 polypeptides during the transformation of amastigotes into promastigotes was ordered in a time-dependent manner, with the promastigote M(r) 80000 polypeptide appearing first, followed by the M(r) 96000 polypeptide. In contrast to the glycosylphosphatidylinositol anchor of promastigote PSA-2, which could be hydrolysed by phosphatidylinositol-specific phospholipase C, the amastigote form was resistant to this enzyme.

An antigenically distinct lipophosphoglycan on amastigotes of Leishmania major.

We show that lipophosphoglycan (LPG) on the surface of amastigotes of Leishmania major is antigenically and biochemically distinct from promastigote LPG. A rabbit antiserum raised against the amastigote integral membrane fraction detected LPG spanning the region of Mr 55,000-100,000 on Western blots of the amastigote integral membrane fraction, but did not recognize the promastigote integral membrane fraction. WIC 79.3, a monoclonal antibody which recognizes L. major metacyclic promastigote LPG, did not recognize the amastigote integral membrane fraction on Western blots. The antigen recognized by this rabbit antiserum was shown to be LPG by its migration pattern on SDS-PAGE, the presence of terminal galactose residues, recognition by a monoclonal antibody to LPG, WIC 108.3, the biosynthetic incorporation of label from [3H]glucose and [32P]phosphate, a hydrophobic chromatography elution profile similar to promastigote LPG, and the presence of a lipid anchor sensitive to phosphatidylinositol-specific phospholipase C. The temporal regulation of LPG expression during parasite differentiation was studied in vitro. During amastigote-to-promastigote transformation, the amastigote-specific form of LPG disappeared after subculture at 48 h. The WIC 79.3 epitope was not detected by Western blotting on transforming parasites until 48 h in culture. During promastigote-to-amastigote transformation, the amastigote-specific form of LPG was detected 12 h after infection. WIC 79.3 epitopes gradually diminished over 48 h. The results demonstrate the developmentally regulated expression of an antigenically distinct LPG on amastigotes of L. major.

Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species.

Antibodies raised against a Leishmania major recombinant promastigote surface antigen 2 (PSA-2) fragment recognized three major polypeptides of approximate M(r) 96,000, 80,000 and 50,000 in promastigotes of three Israeli isolates of L. major including the cloned line LRC-L137-V121, but detected a different array of polypeptides in other L. major isolates. The pattern was different both in number of polypeptides detected and their molecular weight. The antibodies to L. major PSA-2 also recognized polypeptides in L. tropica, L. donovani and very weakly in L. mexicana promastigotes and in Crithidia lucilliae. The number and size of the polypeptides was different in each species. In addition to the membrane-bound PSA-2 polypeptides we identified water-soluble forms of PSA-2 released in promastigote culture supernatants. Peptide maps of the various L. major PSA-2 membrane polypeptides showed they were different from each other. N-terminal amino acid sequence of the three polypeptides expressed by L. major showed they are similar but distinct, consistent with being members of a polymorphic family. Because of the extensive sequence similarity between the PSA-2 genes it has been difficult to assign protein products to individual genes. As a first step towards solving this problem, we have transfected into L. mexicana a genomic clone of a L. major PSA-2 gene and shown that it produces a M(r) 35,000 polypeptide recognized by monoclonal and polyclonal antibodies to L. major PSA-2.

Lipophosphoglycan expression and virulence in ricin-resistant variants of Leishmania major.

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats jointed by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the Mr 63,000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies. All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.

Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.

Cloning, expression and antigenicity of the L. donovani reductase.

The protozoan parasite Leishmania undergoes a morphological and biochemical transformation from the promastigote to the amastigote form during its life cycle, which is reflected in the expression of stage-specific proteins. One of these proteins shows homology to a superfamily of reductase proteins. We have cloned the reductase gene from L donovani and have shown that it differs in only one nucleotide from the L. major homologue, resulting in one amino acid change. A cytosine (C) to guanine (G) transposition in the coding sequence leads to a nonconserved substitution of asparagine (N) for lysine (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant reductase protein. Interestingly, 6 of 10 PBMC isolated from Danish controls proliferated in the presence of the reductase protein. Intracellular IFNgamma was found in a significant percentage of cells in all the tested PBMC cultures from Danes, whereas IL4 was only found in a small proportion of cells, or not at all. The results indicate the presence of cross-reacting CD45R0 memory T-cells in individuals not exposed to Leishmania. Several previous studies have shown that T-cells from nonexposed individuals often respond to crude Leishmania antigen preparations. The present study suggests that this reactivity is partly caused by T-cells recognising L. donovani reductase.

Cellular and humoral immune responses in a population from the Baringo District, Kenya to Leishmania promastigote lipophosphoglycan.

In a cross-sectional house-to-house study in a leishmaniasis-endemic area in Kenya, the cellular and humoral immune response to Leishmania lipophosphoglycan (LPG) was determined. Clinical data, peripheral blood mononuclear cells, and plasma were obtained from 50 individuals over the age of eight years. Lymphoproliferation and interferon-gamma (IFN-gamma) production by these cells were examined. It was shown that cells from all six individuals in the population with a history of kala-azar responded to LPG in the lymphocyte proliferation assay, and four of these six responded in the IFN-gamma assay. In contrast, cells from 12 of 44 individuals from the study area with no history of kala-azar and none of the five Danish control samples responded to LPG. Antibodies against LPG were detected by enzyme-linked immunosorbent assay in 45 of 50 plasma samples. Our findings clearly show that mononuclear cells from kala-azar patients cured of infection were able to respond to the LPG preparation. The finding of a specific cellular immune response to LPG in 12 of 44 individuals with no history of kala-azar is consistent with previous epidemiologic studies, in which it has been shown that a proportion of L. donovani infections run a subclinical course. The high frequency of individuals with antibodies against LPG might indicate that a majority of the population had been exposed to the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)

The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major.

The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes. Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-gamma and tumour necrosis factor-beta, and little interleukin-4, thereby showing a Th1 cytokine pattern. Parallel cultures showed clear Th1 and Th2 response patterns to purified protein derivative of tuberculin or tetanus toxoid, respectively. Flow cytometric analysis revealed that PSA-2 induced blastogenesis in the CD3 positive population and that these cells were the major source of interferon-gamma. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen-specific Th1-like cells, PSA-2 might be considered a vaccine candidate for human leishmaniasis.