Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.     

The combination of DNA vectors expressing IL-12 + IL-18 elicits high protective immune response against cutaneous leishmaniasis after priming with DNA-p36/LACK and the cytokines,followed by a booster with a vaccinia virus recombinant expressing p36/LACK

Protocols of immunization based on the DNA prime/vaccinia virus (VV) boost regime with recombinants expressing relevant antigens have been shown to elicit protection against a variety of pathogens in animal model systems, and various phase I clinical trials have been initiated with this vaccination approach. We have previously shown that mice immunized with a DNA vector expressing p36/LACK of Leishmania infantum followed by a booster with VVp36/LACK induced significant protection against Leishmania major infection. To further improve this protocol of immunization, here we investigated whether the cytokines interleukin-12 (IL-12) and IL-18 could enhance protection against L. major infection in BALB/c mice. We found that priming with DNA vectors expressing p36/LACK and either IL-12 or IL-18, followed by a booster with a VV recombinant expressing the same L. infantum LACK antigen, elicit a higher cellular immune response than by using the same protocol in the absence of the cytokines. The cytokine IL-12 triggered a higher number of IFN-gamma-secreting cells specific for p36 protein than IL-18. When immunized animals were challenged with promastigotes, the highest protection against L. major infection was observed in animals primed with DNAp36 + DNA IL-12 + DNA IL-18 and boosted with VVp36. This protection correlated with a Th1 type of immune response. Our findings revealed that in prime/booster protocols, co-expressing IL-12 and IL-18 during priming is an efficient approach to protect against leishmaniasis. This combined prime/booster immunization regime could have wide use in fighting against parasitic and other infectious diseases.     

Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.     

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.     

IL-10 from regulatory T cells determines vaccine efficacy in murine Leishmania major infection

Leishmaniasis affects 12 million people, but there are no vaccines. Immunological correlates of vaccine efficacy are unclear. Polarized Th1 vs Th2 responses in Leishmania major-infected mice suggested that a shift in balance from IL-4 to IFN-gamma was the key to vaccine success. Recently, a role for IL-10 and regulatory T cells in parasite persistence was demonstrated, prompting re-evaluation of vaccine-induced immunity. We compared DNA/modified vaccinia virus Ankara heterologous prime-boost with Leishmania homolog of the receptor for activated C kinase (LACK) or tryparedoxin peroxidase (TRYP). Both induced low IL-4 and high IFN-gamma prechallenge. Strikingly, high prechallenge CD4 T cell-derived IL-10 predicted vaccine failure using LACK, whereas low IL-10 predicted protection with TRYP. The ratio of IFN-gamma:IL-10 was thus a clear prechallenge indicator of vaccine success. Challenge infection caused further polarization to high IL-10/low IFN-gamma with LACK and low IL-10/high IFN-gamma with TRYP. Ex vivo quantitative RT-PCR and in vitro depletion and suppression experiments demonstrated that Ag-driven CD4+ CD25+ T regulatory 1-like cells were the primary source of IL-10 in LACK-vaccinated mice. Anti-IL-10R treatment in vivo demonstrated that IL-10 was functional in determining vaccine failure, rendering LACK protective in the presence of high IFN-gamma/low IL-5 responses.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.     

Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8+ T cells

Protective immunity against Leishmania major generated by DNA encoding the LACK (Leishmania homologue of receptor for activated C kinase) Ag has been shown to be more durable than vaccination with LACK protein plus IL-12. One mechanism to account for this may be the selective ability of DNA vaccination to induce CD8+ IFN-gamma-producing T cells. In this regard, we previously reported that depletion of CD8+ T cells in LACK DNA-vaccinated mice abrogated protection when infectious challenge was done 2 wk postvaccination. In this study, we extend these findings to study the mechanism by which CD8+ T cells induced by LACK DNA vaccination mediate both short- and long-term protective immunity against L. major. Mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of vaccination or infection were unable to control infection when challenge was done 2 or 12 wk postvaccination. Remarkably, it was noted that depletion of CD8+ T cells in LACK DNA-vaccinated mice was associated with a striking decrease in the frequency of LACK-specific CD4+ IFN-gamma-producing T cells both before and after infection. Moreover, data are presented to suggest a mechanism by which CD8+ T cells exert this regulatory role. Taken together, these data provide additional insight into how Th1 cells are generated and sustained in vivo and suggest a potentially novel immunoregulatory role for CD8+ T cells following DNA vaccination.     

The potency and durability of DNA- and protein-based vaccines against Leishmania major evaluated using low-dose, intradermal challenge

DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8(+) and CD4(+) T cells to the site of intradermal challenge and with IFN-gamma production by CD8(+) T cells in lymph nodes draining the challenge site. These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs.     

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis

An optimal vaccine against leishmaniasis should elicit parasite specific CD4+ and cytotoxic CD8+ T cells. In this investigation, we described a prime/boost immunization approach based on DNA and on poxvirus vectors (Western Reserve, WR, and the highly attenuated modified vaccinia virus Ankara, MVA), both expressing the LACK antigen of Leishmania infantum, that triggers different levels of specific CD8+ T cell responses and protection (reduction in lesion size and parasitemia) against L. major infection in mice. A prime/boost vaccination with DNA-LACK/MVA-LACK elicits higher CD8+ T cell responses than a similar protocol with the replication competent VV-LACK. Both CD4+ and CD8+ T cells were induced by DNA-LACK/MVA-LACK immunization. The levels of IFN-gamma and TNF-alpha secreting CD8+ T cells were higher in splenocytes from DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals. Moreover, protection against L. major was significantly higher in DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals when boosted with the same virus dose, and correlated with high levels of IFN-gamma and TNF-alpha secreting CD8+ T cells. In DNA-LACK/MVA-LACK vaccinated animals, the extent of lesion size reduction ranged from 65 to 92% and this protection was maintained for at least 17 weeks after challenge with the parasite. These findings demonstrate that in heterologous prime/boost immunization approaches, the protocol DNA-LACK/MVA-LACK is superior to DNA-LACK/VV-LACK in triggering specific CD8+ T cell immune responses and in conferring protection against cutaneous leishmaniasis. Thus, MVA-LACK is a safe and efficient vector for vaccination against leishmaniasis.     

Induction of protective CD4+ T cell-mediated immunity by a Leishmania peptide delivered in recombinant influenza viruses

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4(+) Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK(158-173) CD4(+) peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK(158-173)-specific CD4(+) T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK(158-173) triggers LACK(158-173)-specific Th1-biased CD4(+) T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2-4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4(+) T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK(158-173) led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity.     

A comprehensive analysis of LACK (Leishmania homologue of receptors for activated C kinase) in the context of Visceral Leishmaniasis

The Leishmania homologue of activated C kinase (LACK) a known T cell epitope from soluble Leishmania antigens (SLA) that confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains. LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of visceral Leishmaniasis vaccine target.     

Interactions between Leishmania parasites and host cells.

Several species of protozoa belonging to the genus Leishmania are pathogenic for humans, causing visceral and cutaneous diseases. They are transmitted by phlebotomine sandflies as flagellated promastigotes to mammals hosts, where they live as aflagellated amastigotes mainly within macrophages. Studies performed on mice infected with Leishmania major demonstrated that host defence against this infection depends on the interleukin-12-driven expansion of the T helper 1 cell subset, with production of cytokines such as interferon-gamma, which activate macrophages for parasite killing through the release of nitric oxide. The parasitocidal role of this radical is now emerging also in the human and canine model. Healing or progression of the infection is related to the genetic and immune status of the host, and to the virulence of different species and strains of Leishmania. The parasite survival ultimately depends on the ability to evade the host immune response by several mechanisms. Among them, inhibition of the signal transduction pathway of the host cells is particularly important. In fact, promastigotes inhibit protein kinase C activation, cause Ca++ influx into the host cell and decrease the levels of myristoylated alanine-rich C kinase substrate-related proteins, which are substrates for PKC. In addition, Leishmania infection blocks IFN-gamma-induced tyrosine kinase phosphorylation, with consequent impairment of signalling for IL-12 and nitric oxide production. Finally, Leishmania activates protein phosphotyrosine phosphatases, which down-regulate mitogen-activated protein kinase signalling and c-fos and nitric oxide synthase expression. New pharmacological applications, including protein tyrosine phosphatase and protein farnesyltransferase inhibitors, are being evaluated against leishmaniosis in vitro and in vivo in the murine model.     

Leishmania mexicana: LACK (Leishmania homolog of receptors for activated C-kinase) is a plasminogen binding protein

Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this parasite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen binds recombinant LACK with a K_d value of 1.6 ± 0.4 μM, and binding is lysine-dependent since it is inhibited by the lysine analog ε-aminocaproic acid. Inhibition studies with specific peptides and plasminogen binding activity of a mutated recombinant LACK have highlighted the internal motif ₂₆₀VYDLESKAV₂₆₈, similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite.     

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling

Background

Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines.

Methodology/Principal findings

We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T_H1 CD4⁺ and CD8⁺ T cells and a systemic LACK-specific T_H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T_H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T_H1 response.

Conclusions/Significance

This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.     

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasi infection. These immunogenic preparations were composed of Leishmania amazonensis or Leishmania braziliensis antigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasi by intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensis antigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasi antigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.     

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.     

Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection

In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection.     

Leishmania donovani: identification of novel vaccine candidates using human reactive sera and cell lines

Leishmaniasis is a complex of diseases caused by protozoan parasites belonging to the genus Leishmania. The development of specific resistance against re-infection after cure suggests that a vaccine approach is feasible. Various studies in humans and experimental animals strongly suggest that Th1 type of cell-mediated immune response is important for protection against the disease. A defined antigen that could elicit a specific T-cell-mediated immune response in the host would be an ideal candidate for the vaccine against this parasite. In order to select a candidate antigen, we established a screening system to identify the recombinant clone, expressing antigen having T-cell epitopes from a cDNA library. We screened the library using an established Leishmania specific cell line (LSCL) from a naive healthy human subject. The cell line with predominantly CD4+ cells behaved in a Leishmania specific manner. Fifty-two immuno-reactive clones were screened against the LSCL in vitro and we identified three cDNA clones expressing recombinant antigens that could induce proliferation of these cells to produce INFgamma. The protective efficacy of one of these recombinant proteins was investigated in a hamster model of experimental visceral leishmaniasis and showed protection against a virulent challenge. The identified antigens might be potential candidates for vaccine against Leishmania.