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Infection and Immunity, October 2005, p. 6372-6382, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6372-6382.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Live Nonpathogenic Parasitic Vector as a Candidate Vaccine against Visceral Leishmaniasis

Marie Breton, Michel J Tremblay, Marc Ouellette, and Barbara Papadopoulou*

Infectious Diseases Research Center, CHUL Research Center of Laval University, and Department of Medical Biology, Faculty of Medicine, Laval University, Quebec G1V 4G2, Canada

Received 4 April 2005/ Returned for modification 7 May 2005/ Accepted 21 June 2005


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ABSTRACT
 
To date, there are no proven vaccines against any form of leishmaniasis. The development of live attenuated vectors shows promise in the field of Leishmania vaccination because these organisms mimic more effectively the course of real infections and can elicit potent activation of the immune system. In the present study, we investigated the potential of a parasitic protozoan that is nonpathogenic to humans, Leishmania tarentolae, as a live candidate vaccine that efficiently targets dendritic cells and lymphoid organs, thus enhancing antigen presentation and consequently influencing the magnitude and quality of T-cell immune responses. We demonstrated that L. tarentolae activates the dendritic cell maturation process and induces T-cell proliferation and the production of gamma interferon, thus skewing CD4+ T cells toward a Th1 cell phenotype. More importantly, we found that a single intraperitoneal injection of L. tarentolae could elicit a protective immune response against infectious challenge with Leishmania donovani in susceptible BALB/c mice. These results suggest that the use of L. tarentolae as a live vaccine vector may represent a promising approach for improving the effectiveness and safety of candidate live vaccines against Leishmania infections and possibly other intracellular pathogens for which T-cell mediated responses are critical for the development of protective immunity.


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INTRODUCTION
 
Leishmania species are kinetoplastid protozoa and obligatory intracellular parasites that are transmitted to humans by the bite of an infected phlebotomine sand fly. In the mammalian host, Leishmania multiplies within mononuclear phagocytic cells, which results in clinical manifestations of a wide spectrum of diseases, depending upon the parasite species and host immune responses. These diseases range from self-limiting cutaneous leishmaniasis to visceral leishmaniasis, also known as Kala-azar, which is a fatal infection if it is left untreated (50). Leishmaniasis is an endemic disease that affects over 12 million people in 88 countries; 1.5 to 2 million new cases occur annually, and 350 million people are at risk of infection (4, 30). Visceral leishmaniasis caused by Leishmania donovani has resulted in more than 100,000 deaths in the recent epidemics in Sudan and India. Moreover, Leishmania infantum, which is endemic around the Mediterranean basin, has emerged as an opportunistic pathogen in human immunodeficiency virus type 1-infected patients (18). So far, there is no effective vaccination against human leishmaniasis, and control of this disease relies primarily on chemotherapy. The number of drugs available is limited, and each drug has various shortcomings. Pentavalent antimony has long been the pillar of anti-Leishmania chemotherapy, but resistance to this class of drug in northeast India has reached epidemic proportions (67).

Several vaccination strategies against experimental leishmaniasis have been attempted, mainly against the cutaneous form. Comparatively few vaccination strategies have been used against visceral leishmaniasis (reviewed in references 16, 27, 53, and 65). The only successful immunization strategy in humans has been leishmanization, which is based on the development of durable immunity after recovery from infection at a chosen site, usually the arm, with viable nonattenuated parasites (25, 32, 47). The use of this technique has been restricted or abandoned entirely, however, due to safety concerns. Using the mouse model, researchers have tested numerous approaches to develop safe, nonlive vaccines against Leishmania using recombinant Leishmania antigens, including GP63 (41, 74), LACK (26), PSA-2 (28), TSA/LmSTI1 (59), PFR2 (55), A2 (22), and HASPB1 (64), DNA-based vaccines (13, 26, 38, 39, 43, 52, 66), or a cDNA expression library (37). Other strategies have involved the use of live bacterial or viral recombinant vectors (1, 24, 60, 75) and/or live Leishmania administered at a low dose (11), drug-treated attenuated Leishmania (16), recombinant Leishmania expressing cytokines (20) or suicide markers (17, 46), live parasites with CpG oligodeoxynucleotide motifs (40), and genetically attenuated vaccines (3, 48, 70, 71). While each of these studies has indicated that there is some level of protection, complete, long-lived protection has not been clearly demonstrated. In clinical trials in humans, whole killed vaccines with BCG as an adjuvant failed to confer protection against cutaneous leishmaniasis (42, 58) or visceral leishmaniasis (33).

Live recombinant vectors are central in the development of new vaccine strategies. However, utilization of bacterial or viral recombinant vectors as candidate vaccines in humans is hampered due to problems of either preexisting immunity or inefficient antigen delivery and safety issues. The use of live attenuated Leishmania preparations as candidate vaccines is very promising because they most closely mimic the natural course of infection and may therefore elicit similar immune responses. However, the organisms may revert to virulence, and targeted deletions of essential or virulence genes result either in complete parasite destruction (70) or in mutants that induce only a delay in lesion development (61, 70). Thus, there is an urgent need for the development of new safe live vaccine vectors that are capable of enhancing antigen presentation and eliciting potent immune responses without the risk of development of disease in humans. In the present study, we used a lizard parasitic protozoan that is not pathogenic to humans, Leishmania tarentolae, as a candidate vaccine against visceral leishmaniasis. We demonstrated that this parasitic vector targets antigen-presenting cells, including dendritic cells (DCs), activates the DC maturation process, and induces T-cell proliferation and the production of gamma interferon (IFN-{gamma}), skewing CD4+ T cells toward a Th1 cell phenotype. More importantly, we showed that a single immunization of susceptible BALB/c mice with L. tarentolae could elicit a protective immune response against infectious L. donovani challenge.


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MATERIALS AND METHODS
 
Parasite growth and transfections. The L. tarentolae Tar II (= ATCC 30267), L. donovani LV9, and Leishmania major LV39 strains were grown in SDM-79 medium (12) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Wisent, St. Bruno, Quebec, Canada) and 5 µg/ml of hemin. The virulent L. donovani strain used for the challenge studies was a stabilate (passage 2) isolated from mice. L. tarentolae parasites were transfected with expression vectors containing either the luciferase gene (LUC) or the GFP gene (9, 54) by electroporation, as previously described (49). Approximately 10 to 15 µg of DNA was used for transfections, and cells were selected with G-418 (40 µg/ml).

In vitro Leishmania infection. The murine J774 and human THP1 cell lines and intraperitoneal (i.p.) macrophages were used to assess the internalization of L. tarentolae, as described previously (57). Intraperitoneal macrophages, obtained from thioglycolate-infected BALB/c mice by peritoneal washing, were incubated in slide chambers (Lab-Tek) or 24-well plates for 4 h with parasites at a parasite/macrophage ratio of 10:1. Washing was then performed to remove free parasites, and the plates were incubated at 37°C for several periods of time. Infection with fluorescent L. tarentolae expressing the green fluorescent protein (GFP) was monitored by fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis (9). To observe infection by bright-field microscopy, Diff-Quick coloration (Baxter Healthcare) was performed with Leishmania-infected macrophages.

In vivo Leishmania infection, immunization, and challenge studies. Stationary-phase L. tarentolae promastigotes (5 x 107 cells) were injected i.p., under the skin, or in the footpads of 8- to 10-week-old female SCID mice (Charles River, St. Constant, Canada) to evaluate whether L. tarentolae could induce any type of pathology in immunocompromised mice. The infection was monitored for 4 weeks; then the mice were sacrificed, and the spleens, draining lymph nodes, livers, and tissues from the site of injection were analyzed for the presence of L. tarentolae by fluorescent microscopy and by culturing the different tissues. For the immunization studies, 8- to 10-week-old female BALB/c mice (Charles River, St. Constant, Canada) (five mice per time) were injected i.p. with 5 x 106 stationary-phase L. tarentolae promastigotes. Six weeks later, the mice infected with L. tarentolae and also naïve mice were challenged with 5 x 107 virulent stationary-phase L. donovani promastigotes expressing the LUC gene (48) in the lateral tail vein. The parasite burden in the spleen and the liver was quantitatively determined 4 weeks postchallenge by measuring the luciferase activity (54) and also by microscopic evaluation of Giemsa-stained tissues. As demonstrated in previous studies (19, 54), the rate of L. donovani infection in BALB/c mice is maximal at ~4 to 5 weeks.

Culture and differentiation of dendritic cells. Immature monocyte-derived dendritic cells (iMDDCs) were obtained by cytokine differentiation of CD14-positive cells as previously described (23). Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from human blood by density gradient centrifugation on Ficoll-Hypaque. The layer of lymphocytes/monocytes was collected and washed to perform AutoMACS (Miltenyi Biotec Inc., Auburn, CA) CD14-positive separation as recommended by the manufacturer. The CD14-positive cells were cultured at 37°C, in the presence of 5% CO2 for 7 days at a concentration of 1 x 106 cells/ml in RPMI 1640 containing 10% FBS (HyClone, Logan, Utah) with interleukin-4 (IL-4) (200 U/ml) and granulocyte-macrophage colony-stimulating factor (1,000 U/ml) to allow differentiation in the iMDDCs. Monocyte-derived macrophages were obtained by addition of macrophage colony-stimulating factor to the culture of CD14-positive cells.

FACS analyses and antibodies. Flow cytometry analyses were performed with macrophages or DCs to monitor L. tarentolae infection based on GFP fluorescence from L. tarentolae-GFP recombinant parasites. Flow cytometric analysis was performed with stimulated monocyte-derived DCs an L. tarentolae incubated with 500 µl of phosphate-buffered saline (PBS) containing a saturating amount of each mouse anti-human monoclonal antibody (anti-HLA-DR, anti-CD40, anti-80, anti-CD83, and anti-CD86) or rat anti-mouse conjugated antibody (phycoerythrin-anti-CD4, Cy5-anti-CD8, phycoerythrin-anti-CD25, and fluorescein isothiocyanate-anti-CD69) at 4°C for 30 min. Then the cells were washed with PBS and labeled with 100 µl of a saturating amount of R-phycoerythrin-conjugated goat anti-mouse antibody at 4°C for 30 min for cells incubated with nonconjugated first antibody. Finally, the cells were washed, fixed with 2% paraformaldehyde, acquired on a cytofluorometer (EPICS Elite ESP; Coulter Electronics), and analyzed with WinMDI.

Cell proliferation assays. Spleens obtained from individual mice on day 0 (just prior to infection) or at weeks 1, 2, 4, and 8 postinfection were homogenized, and red blood cells were removed with an isotonic ammonium chloride solution. Splenocytes were washed and resuspended in 200 µl of RPMI medium (RPMI 1640 supplemented with 10% FBS [HyClone], 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM ß-mercaptoethanol) to a concentration of 2.5 x 105 cells/ml in 96-well flat-bottom microplates (Costar). The cells were then stimulated with 4 x 105 Leishmania cells, and proliferation was detected on day 4 by [3H]thymidine incorporation. The cells were pulsed with 1 µCi [3H]thymidine (Amersham) per well for 24 h on day 3 and were harvested on day 4 with a Harvester 96 (Tomtec). The counts were evaluated with a 1205 BetaPlate liquid scintillation counter (Amersham). The stimulation index was calculated for each sample by comparing the mean counts per minute for the stimulated and unstimulated cells.

Cytokine production. Culture supernatants from stimulated splenocytes cultured under the same conditions that were used for T-cell proliferation were collected in triplicate and assayed for IFN-{gamma} and IL-4 by a sandwich enzyme-linked immunosorbent assay (ELISA), using the appropriate combination of antibodies from R&D Systems (Minneapolis, Minn.), as previously described (48).

Air pouch system. Air pouches were created by injecting 2.5 ml of sterile air on days 0 and 3 into the dorsum of 8-week-old CD-1 mice (Charles River, St. Constant, Canada), as previously described (21, 35). Briefly, 5 x 107 stationary-phase L. tarentolae or L. major parasites in 1 ml of endotoxin-free PBS (Sigma) were injected into the pouches on day 6. The negative control was inoculated with 1 ml of endotoxin-free PBS, and the positive control was inoculated with 100 ng of lipopolysaccharide (LPS) in 1 ml of endotoxin-free PBS (five mice per group). Six hours following injection, the mice were sacrificed, the pouch contents were recovered, and the cells were counted with a hemacytometer. The differential cell counts were determined by microscopy. A total of 1 x 105 cells were centrifuged at 500 rpm onto slides using a Cytospin 3 (Shandon, ThermoCorporation). Cytospin preparations were stained with Diff-Quick (Baxter Healthcare) to allow quantification of the leukocyte subpopulations.

Statistical analyses. Statistical analyses were performed with a paired Student t test. A P value of <0.05 was considered significant.


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RESULTS
 
L. tarentolae efficiently targets antigen-presenting cells. As a lizard parasite, L. tarentolae has never been associated with any pathology in humans, and no previous studies have investigated the capacity of this parasitic protozoan to infect antigen-presenting cells and lymphoid tissues and/or its potential to stimulate T-cell immune responses in a mouse model. We first tested whether this parasitic vector could infect murine and human macrophages, as well as iMDDCs. L. tarentolae infection was monitored both by microscopic examination and by FACS analysis using recombinant parasites expressing the gene encoding the GFP. Our results indicated that L. tarentolae could be phagocytosed efficiently by both mouse and human macrophages and differentiated into intracellular amastigote-like forms inside these cells (Fig. 1A, B, and E). Infected macrophages indeed contained a large number of amastigotes (average, 8 to 10 parasites/cell) (Fig. 1A, D, and E and data not shown). More than 31% of J774 murine macrophages were infected by L. tarentolae at 24 h postinfection (Fig. 1C). Intraperitoneal injection of L. tarentolae into BALB/c mice resulted in rapid uptake of the free GFP-expressing parasites. Approximately 5 h following infection, free parasites were no longer observed in the peritoneum, but many parasites were present inside the macrophages (Fig. 1D and data not shown). Nearly 34% of the human THP1 macrophage cells were found to be infected with stationary-phase GFP-expressing L. tarentolae 24 h following infection in vitro (Fig. 1F). Microscopic examination revealed a similar percentage of infected macrophages (Fig. 1G). Five days following the initial infection of THP1 cells with L. tarentolae, the rate of infection decreased by approximately 40% (Fig. 1G), but many parasites were still detected inside these cells. We also tested whether L. tarentolae could infect monocyte-derived macrophages obtained from CD14 purification of human PBMCs. As shown by FACS analysis, nearly 43% of these cells were infected by L. tarentolae at 24 h postinfection (Fig. 1H).



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  		FIG. 1. L. tarentolae uptake by murine and human macrophages. Murine monocytic cells, murine monocyte-derived macrophages, and the human monocytic cell line TPH1 were used to evaluate the ability of L. tarentolae to infect cells in vitro. The ability of L. tarentolae to enter intraperitoneal macrophages was evaluated in vivo. (A) Giemsa staining of J774 infected macrophages with L. tarentolae. (B) J774 macrophages stained with Evans blue and viewed by confocal microscopy. (C) FACS evaluation of the percentage of GFP-expressing L. tarentolae-infected macrophages 24 h following infection. (D) Giemsa staining of L. tarentolae-infected intraperitoneal macrophages. (E) Giemsa staining of THP1 macrophages infected with L. tarentolae 24 h following infection. (F) FACS evaluation of the percentage of GFP-expressing L. tarentolae-infected THP1 macrophages 24 h postinfection. (G) Percentage of THP1 macrophages infected by L. tarentolae as monitored by microscopic evaluation of Giemsa-stained tissues at different times following infection. On average, 200 macrophages per time were counted. (H) FACS evaluation of the percentage of monocyte-derived macrophages infected with GFP-expressing L. tarentolae, as determined by cytokine differentiation of CD14 purification of human PBMCs 24 h postinfection.

L. tarentolae infection elicits DC maturation. Leishmania is primarily found in macrophages, although it has been reported that several Leishmania species can be phagocytosed by DCs and that parasites can survive and even multiply within these cells (reviewed in references 10 and 56). To determine whether L. tarentolae could target immature monocyte-derived DCs, iMDDCs obtained by cytokine differentiation of CD14-purified monocytes from human PBMCs were infected with GFP-expressing L. tarentolae. The phenotype of the iMDDCs was monitored by analyzing the expression profile of multiple surface markers, including CD14, DC-SIGN, CD40, B7.1, B7.2, CD83, CD1a, HLA-DR, ICAM-3, and LFA-1 (data not shown). L. tarentolae parasites were indeed capable of infecting iMDDCs (Fig. 2A), and as monitored by FACS analysis, nearly 30% of these cells remained infected 48 h following infection (Fig. 2B). On average, 6 to 12 parasites were found in each infected iMDDC (Fig. 2A and data not shown).



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  		FIG. 2. L. tarentolae uptake by iMDDCs. Target cells were obtained by cytokine differentiation (IL-4, granulocyte-macrophage colony-stimulating factor) of CD14 purified from human PBMCs by MACS magnetic bead isolation. The ability of L. tarentolae to enter these cells was evaluated by adding GFP-expressing L. tarentolae to iMDDCs at a parasite/macrophage ratio of 10:1. The infection was monitored 12 h after infection by FACS. (A) Confocal microscopy of infected iMDDCs. (B) Flow cytometry profile showing the percentage of infected iMDDCs. (C) Expression of activation markers 48 h following in vitro stimulation of iMDDCs with L. tarentolae (Ltar) at a macrophage/parasite ratio of 10:1 or with 100 ng of LPS. The expression of the different surface markers was evaluated by FACS analysis by incubating the cells with the following monoclonal antibodies: anti-HLADR, anti-CD40, anti-CD80 (B7.1), anti-CD86 (B7.2), and anti-CD83.

We also tested whether L. tarentolae infection could promote DC maturation by monitoring the expression of activation markers on the surface of the iMDDCs. Of the different markers examined, MHC-II, CD40, and CD83 were found to be up-regulated 48 h following L. tarentolae infection (Fig. 2C). The MHC-II up-regulation implied that there was an increase in antigen presentation on the cell surface, and the CD40 up-regulation implied that there was activation of the DCs. An increase in the expression of CD83 indicated that there was maturation of DCs. Also, the morphology of the iMDDCs changed to the mature forms following infection with L. tarentolae (data not shown). Moreover, L. tarentolae-infected DCs expressed higher levels of the costimulatory molecules CD80 (B7.1) and CD86 (B7.2) (Fig. 2C). Overall, these studies demonstrated that L. tarentolae is able to infect all types of murine or human phagocytic cells and, most interestingly, DCs.

L. tarentolae infection induces an early inflammatory response in CD-1 mice. Early inflammatory responses are often required to induce host innate responses (reviewed in reference 5). To assess whether L. tarentolae could induce a proinflammatory response at the site of inoculation, we used the air pouch mouse model system. The results of the experiment with L. tarentolae were compared to the results for L. major infections that are known to recruit a high number of leukocytes at the site of inoculation and to induce accumulation of proinflammatory cytokines (35). Six hours postinoculation in the air pouch, which is known to be the maximal peak of recruitment, a major accumulation of cells was observed. Indeed, a 14-fold increase in the recruitment of leukocytes was obtained with L. tarentolae compared with the PBS control. This increase in the overall number of leukocytes was similar to the increase obtained with the pathogenic L. major strain (Fig. 3A and B). More than 75% of the recruited cells were neutrophils, 13% were eosinophils, and 11% were monocytes/macrophages. Lymphocytes were almost absent because they are part of the adaptive immunity and they usually appear later at the lowest levels. Injection of L. tarentolae into lymph nodes that drained the footpad in mice resulted in local inflammation that was resolved after a few days (data not shown). The presence of transient inflammation is important for recruitment of the immune cells and establishment of an immune response.



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  		FIG. 3. Leukocyte accumulation in the air pouch mouse system in response to Leishmania inoculation. Air pouches were raised on the dorsum of 6- to 8-week-old female CD-1 mice. One milliliter of endotoxin-free PBS with or without LPS or Leishmania (5 x 107 cells) was injected into the pouches, and the exudates were collected 6 h after inoculation. The nonpathogenic species L. tarentolae (Ltar) and the pathogenic species L. major (Lm) were used in these studies. (A) Number of leukocytes, as determined microscopically with a hemacytometer. (B) Sum of the recruited neutrophils, monocytes/macrophages, eosinophils, and lymphocytes, as determined on slides by microscopic analysis using Cytospin stained with a Diff-Quick solution. The proportion of each cell type was determined by examining 300 cells. The data represent the means ± standard errors of two independent experiments (five mice each). Asterisks and daggers indicate statistically significant differences between experimental mice and the PBS control mice (asterisks, P < 0.01; daggers, P < 0.05).

L. tarentolae infection induces a Th1-type response in mice. Given that L. tarentolae efficiently targets antigen-presenting cells (Fig. 1 and 2) or lymph nodes (data not shown) and induces early inflammatory responses (Fig. 3), it is likely that this parasitic vector also stimulates T-cell-mediated immune responses. We therefore measured the effect of L. tarentolae infection on cellular proliferation and the production of effector cytokines by splenocytes at different times postinfection. As determined by the thymidine uptake assay, proliferation of splenic cells from L. tarentolae-infected mice at 4 weeks was 17-fold greater in mice previously infected with L. tarentolae than in uninfected mice (Fig. 4A). Proliferation of splenic cells to a similar extent was observed even at 12 weeks postinfection upon restimulation with live L. tarentolae parasites (Fig. 4A). Analysis of the supernatants of these cultures by ELISA demonstrated that splenocytes from L. tarentolae-infected mice produced significant levels of IFN-{gamma} upon in vitro restimulation with live parasites (Fig. 4B). The amounts of IFN-{gamma} were high up to 8 weeks postinfection (Fig. 4B). Thus, our data suggest that L. tarentolae can elicit T-cell proliferation and skew CD4+ T cells toward a Th1 cell phenotype via the production of IFN-{gamma}.



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  		FIG. 4. L. tarentolae induces T-cell proliferation and IFN-{gamma} production in mice. (A) Proliferation of lymphocytes from BALB/c mice previously infected with L. tarentolae. A total of 5 x 105 splenocytes were isolated from immunized BALB/c mice at 1, 2, 4, 8, and 12 weeks postinfection, grown in culture, and restimulated for 4 days with L. tarentolae live promastigotes (Ltar). Three days following stimulation, 1 µCi of [3H]thymidine was added to each culture for 24 h, and then [3H]thymidine incorporation was measured as described in Materials and Methods. (B) IFN-{gamma} cytokine production by the splenocytes at different times following vaccination of mice with 5 x 106 L. tarentolae cells. Levels of IFN-{gamma} were measured by a sandwich ELISA in culture supernatants of splenocytes 4 days following in vitro restimulation with L. tarentolae promastigotes. The data are the means ± standard errors for levels of cytokine production in three independent experiments (five mice each). Asterisks indicate that the P value is <0.01 for a comparison of stimulated and unstimulated splenocytes.

Vaccination with L. tarentolae protected BALB/c mice against an infectious L. donovani challenge. Given that L. tarentolae was capable of eliciting a Th1-type cytokine response in mice, we tested whether vaccination with this vector could protect against pathogenic Leishmania species for which Th1-type responses are critical in developing resistance to infection (2, 72). Groups of BALB/c mice were immunized i.p. with 5 x 106 L. tarentolae cells. At 6 weeks postimmunization, the mice were challenged in the tail vein with 5 x 107 virulent late-stationary-phase L. donovani promastigotes expressing the luciferase gene (LUC). One month following L. donovani challenge, mice were sacrificed, and the parasite burden in the liver and the spleen was quantitatively estimated by measuring the luciferase activity of LUC-expressing parasites (Fig. 5). Naïve control mice developed progressive disease; in contrast, mice previously immunized with L. tarentolae displayed significant cross-protection to L. donovani infectious challenge (Fig. 5). Indeed, an 80% to 85% decrease in parasite burdens was observed in the livers or spleens of mice previously immunized with a single intraperitoneal dose of L. tarentolae (Fig. 5). Microscopic analysis of liver smears revealed the same decrease in the parasite burden in immunized mice (data not shown). Thus, our results indicated that L. tarentolae-infected BALB/c mice showed significant protection against infectious challenge with L. donovani. Protection was greater when L. tarentolae parasites were delivered by the intraperitoneal route than when they were delivered by a subcutaneous route (data not shown).



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  		FIG. 5. Protective immunity in mice preimmunized with L. tarentolae following a challenge with a virulent L. donovani strain. Six weeks after immunization with L. tarentolae, 5 x 107 stationary-phase LUC-expressing L. donovani promastigotes (Ld) were injected into the tail vein of BALB/c mice. Naïve control mice were treated similarly. At 1 month postchallenge, the spleen and the liver were harvested, and the luciferase activity was measured as an indicator of the presence of parasites. The data are the means ± standard errors for five mice per group and are representative of three experiments in which similar results were obtained. Asterisks and daggers indicate statistically significant differences between experimental mice and control mice (asterisks, P < 0.01; daggers, P < 0.05).

Protection elicited by L. tarentolae is associated with IFN-{gamma} production and suppression of the IL-4 response. To determine the nature of the immune response induced after L. tarentolae-immunized mice were challenged with L. donovani, splenocytes from naïve and immunized mice 4 weeks following challenge were restimulated in vitro with L. donovani promastigotes, and the production of IFN-{gamma} and IL-4 was assessed by ELISA. As Fig. 6 shows, mice immunized with a single i.p. injection of L. tarentolae and challenged with a virulent L. donovani strain produced much larger amounts of IFN-{gamma} than the naïve control mice produced when they were restimulated in vitro with L. donovani parasites. We were unable, however, to detect IL-4 production in the group of L. tarentolae-immunized mice (Fig. 6). Thus, the protection observed in mice previously immunized with L. tarentolae should probably be attributed to more efficient activation of macrophages by IFN-{gamma}-secreting T cells in the absence of IL-4 production.



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  		FIG. 6. Cytokines produced by BALB/c mice initially infected with L. tarentolae after challenge with L. donovani. The IFN-{gamma} and IL-4 cytokine levels produced by splenocytes nearly 12 weeks following vaccination with 5 x 106 L. tarentolae and 6 weeks following challenge with 5 x 107 L. donovani were determined. Cytokines were measured by an ELISA by using culture supernatants, as described in the legend to Fig. 4, except that the experiments were conducted 6 weeks after challenge with L. donovani. The bars indicate the mean cytokine production of three independent experiments (five mice each), and the error bars indicate standard errors.


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DISCUSSION
 
In the present study, we investigated the potential of a nonpathogenic parasitic protozoan, L. tarentolae, as a live vaccine candidate vector to efficiently target DCs and lymphoid organs, thus enhancing antigen presentation and consequently influencing the magnitude and quality of T-cell immune responses. We showed that the L. tarentolae-DC interaction activates the DC maturation process and induces T-cell proliferation and the production of IFN-{gamma}, thus skewing CD4+ T cells toward a Th1 cell phenotype and acting as an immunostimulatory adjuvant. More importantly, we showed that a single intraperitoneal injection of L. tarentolae could elicit a protective immune response against infectious challenge with L. donovani in susceptible BALB/c mice.

The ability of macrophages and especially DCs to efficiently present antigens to T cells is one of the key features in vaccination for priming naïve T cells and inducing protection against intracellular pathogens for which cellular immunity is required. DCs are the most potent professional antigen-presenting cells that have important implications for antigen uptake and processing and the establishment of an effective adaptive immune response (reviewed in reference 7). Given the central role of DCs in shaping the phenotype of a pathogen-specific immune response, candidate vaccines should be aimed at efficiently targeting and stimulating DCs, which in turn should activate the different arms of the cellular immune response in order to provide broad, cross-reactive, and long-lasting immunity. Leishmania is one of the few pathogens that infect macrophages and DCs (10, 36). Interaction with DCs occurs mainly through the DC-SIGN receptor (14, 15, 77). We report here that L. tarentolae can successfully target macrophages/monocytes and DCs like its pathogenic counterparts (29, 51, 73) and that it can elicit DC maturation, as measured by increased expression of major histocompatibility complex class II products and costimulatory or adhesion molecules on the surface of DCs. The maturation process enables DCs to migrate to the T-cell areas of lymphoid organs, where they present antigens to naïve T cells and modulate their responses (7). Mice immunized with L. tarentolae displayed inflammation at the site of injection along with recruitment of neutrophils, monocytes/macrophages, and eosinophils during the first few hours, after which no dermal pathology was observed. Neutrophils instruct DC recruitment and activation, leading in turn to Th1 cell activation and ultimately immunity to microbial infection (8).

The major attribute of the L. tarentolae vector is its capacity to induce protection against a virulent L. donovani challenge in susceptible mice. Protection against L. donovani challenge in L. tarentolae-vaccinated mice is associated with a predominant Th1 response, as measured by in vitro IFN-{gamma} production following restimulation with live L. donovani parasites and the lack of IL-4 production. IFN-{gamma} seems to play a key role in resistance to L. donovani infection, as determined by both murine and human studies (31, 44, 45, 63). A suppressive Th2 cell-associated immune response with mainly IL-4 has been detected in sera from Indian patients with visceral leishmaniasis (68). Individuals who have a subclinical L. donovani infection develop antigen-specific T-cell responsiveness and IFN-{gamma} production (6). Moreover, IFN-{gamma} treatment alone or combined with chemotherapy leads to better control of the murine visceral infection in vivo (34, 45, 69) and in humans (62).

Although L. tarentolae efficiently targets antigen-presenting cells and survives within these cells for several days, as indicated by infection assays in vitro, experimental L. tarentolae infection in BALB/c mice suggests that this parasitic species does not persist for a long time. Indeed, cultures of spleen or liver tissues isolated from infected BALB/c mice 1 month postinfection remained negative, and no fluorescent parasites (L. tarentolae expressing GFP) were detected by FACS analysis (data not shown). However, we did detect by microscopic examination parasites in infected human lymphoid tissue cultured ex vivo at least 2 weeks postinfection (data not shown). Although our data suggest that L. tarentolae parasites are not persistent in mice, we cannot rule out the possibility that parasites are present at very low levels that cannot be detected by culturing or by fluorescence-based techniques. Reverse transcription-PCR-based techniques may be more suitable for detecting low numbers of the parasite. As a lizard parasite, L. tarentolae is not evolutionarily adapted to promote infection in mice, and this could very well explain the observed lack of persistence. L. tarentolae was unable to cause any pathology even in severely compromised immunodepressed SCID mice that lacked B and T cells, and this was independent of the dose and the route of administration of the parasite (data not shown).

An ideal live vaccine has to elicit long-term effective immune responses without persisting for a long time in the vaccinated host, as safety is an important criterion for the use of live vaccines in humans. Although continuous exposure to the antigen was believed to be very important for development of a T-cell memory immune response (78), recently it has been shown that other parameters could also participate in this process. For example, the type of activated cells and the level and duration of stimulation could be important for the development of T-cell memory immunity (76). In mice vaccinated with parasites lacking dihydrofolate reductase-thymidylate synthase that are auxotrophic for thymidine and therefore cannot survive within the host, effector T-cell responses are lost if parasites are eliminated, but central memory T cells that can develop into effector T cells and provide protection are maintained (76). Thus, protection of L. tarentolae-immunized mice against a virulent L. donovani challenge in the absence of detectable persistent parasites may be explained by the development of central memory T cells that could mediate long-lasting protection. Experiments to verify this possibility need to be performed, however.

The current data suggest that protection against Leishmania infection requires effective activation of several cell populations, including macrophages, DCs, and antigen-specific CD4+ and CD8+ T cells. As shown in this study, L. tarentolae is capable of promoting DC maturation and activating T-cell lymphocytes to produce IFN-{gamma}. It is worth noting that the genomic DNA of L. tarentolae contains CpG nonmethylated motifs (data not shown) that could act as pathogen-associated molecular patterns capable of activating innate immunity. More importantly, vaccination of susceptible BALB/c mice with L. tarentolae provided significant levels of protection against virulent L. donovani challenge. The L. tarentolae-based vaccination strategy could be further improved either by generating recombinant L. tarentolae expressing selected Leishmania immunodominant epitopes or by combining the L. tarentolae recombinant parasite with a DNA vaccine as part of a prime-boost strategy to elicit more effective and long-lasting protection against reinfection with virulent L. donovani strains. Overall, L. tarentolae appears to be a promising live candidate vector for development of an effective vaccine against Leishmania infections and possibly infections by other intracellular pathogens for which T-cell-mediated immunity is critical for protection.


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ACKNOWLEDGMENTS
 
We thank Philippe Tessier and Pascal Rouleau for helping us with the air pouch model system.

This work was supported by the Canadian Vaccine Centre of Excellence (CANVAC) and by Canadian Institutes of Health Research (CIHR) GR-14500 grants to B.P., M.J.T., and M.O. M.B. is the recipient of a CIHR studentship. B.P. is a Burroughs Wellcome Fund in Molecular Parasitology and Senior FRSQ Scholar. M.J.T. holds the Senior Canada Research Chair in Human Immuno-Retrovirology. M.O. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology and holds the Senior Canada Research Chair in Antimicrobial Resistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Diseases Research Center, CHUL Research Center, CHUQ, 2705 Laurier Blvd., Quebec (QC), Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: barbara.papadopoulou{at}crchul.ulaval.ca. Back

Editor: W. A. Petri, Jr.


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Infection and Immunity, October 2005, p. 6372-6382, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6372-6382.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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