Leish-111f, a Recombinant Polyprotein Vaccine That Protects against Visceral Leishmaniasis by Elicitation of CD4⁺ T Cells

Abstract

The Leishmania-derived recombinant polyprotein Leish-111f or its three component proteins, thiol-specific antioxidant (TSA), Leishmania major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF), have previously been demonstrated to be efficacious against cutaneous or mucosal leishmaniasis in mice, nonhuman primates, and humans. In this study we demonstrate that Leish-111f is also a vaccine antigen candidate against visceral leishmaniasis (VL) caused by Leishmania infantum. We evaluated the immune response and protection induced by Leish-111f formulated with monophosphoryl lipid A in a stable emulsion (Leish-111f+MPL-SE) and demonstrated that mice developed strong humoral and T-cell responses to the vaccine antigen. Analysis of the cellular immune responses of immunized, uninfected mice demonstrated that the vaccine induced a significant increase in CD4⁺ T cells producing gamma interferon, interleukin 2, and tumor necrosis factor cytokines, indicating a Th1-type immune response. Experimental infection of immunized mice and hamsters demonstrated that Leish-111f+MPL-SE induced significant protection against L. infantum infection, with reductions in parasite loads of 99.6%, a level of protection greater than that reported for other vaccine candidates in animal models of VL. Taken together, our results suggest that this vaccine represents a good candidate for use against several Leishmania species. The Leish-111f+MPL-SE product we report here is the first defined vaccine for leishmaniasis in human clinical trials and has completed phase 1 and 2 safety and immunogenicity testing in normal, healthy human subjects.

Leishmaniasis is a spectrum of diseases varying from a fatal visceral form to a localized self-healing cutaneous lesion. With an estimated 350 million people at risk for acquiring infection with Leishmania parasites, a worldwide prevalence estimated at approximately 12 million cases, with 1.5 million new reports of cutaneous leishmaniasis (CL), and 500,000 new reports of visceral leishmaniasis (VL) each year, the World Health Organization considers leishmaniasis to be one of the most serious, epidemic-prone parasitic infectious diseases afflicting the poor and disadvantaged.

Recent epidemics of VL, also known as kala-azar in Sudan and India, have resulted in over 100,000 deaths (10). Failure of the pentavalent antimonials, the main form of chemotherapy currently used worldwide, is attributed to the emergence of antimonial-resistant Leishmania strains, resulting in frequent relapses after treatment (22). In India, clinical resistance to antimony has long been recognized as a major problem, and 65% of VL patients fail to respond or promptly relapse (47). Alternative chemotherapeutic regimens with amphotericin B and its lipid formulation have severe limitations due to their toxic effects and prohibitively high cost of treatment(22). In vitro studies have shown that Leishmania also develops resistance against miltefosine, a recently approved oral drug effective in VL treatment, by changes in or loss of a leishmanial P-type phospholipid translocase (30). Growing limitations in the available chemotherapeutic strategies due to emerging resistant strains and lack of an effective vaccine strategy against VL deepen the crisis.

Because of the lack of effective and low-cost treatments, considerable effort has been devoted to vaccine development. Killed or live attenuated parasites, as well as a large number of leishmanial antigens from different species, have been identified and tested as vaccines. Studies of recombinant protein vaccines in mice demonstrated that antigens such as the proteins GP63, p36/LACK, A-2, PSA-2, and KMP11 induced strong immune responses but weak and short-lived protection against Leishmania infection (1, 3, 29, 35, 38, 39). Other studies have identified several antigens, including lmd29 and 584C, that reproducibly exacerbated leishmania disease (37). Few studies have been directed at the potential for a vaccine derived from one Leishmania species to provide cross-protection against another species. Initial results using sequential infections with distinct species have suggested complex cross-protection relationships. For example, immunization of mice with heat-killed L. donovani can induce protection against a subsequent infection with L. major (2). Few antigens, including LACK (11, 12, 19), dp72 (33), and P4 nuclease (5), have been tested for cross-protection in mice, with varied success.

In previous studies, we showed that Leish-111f, a polyprotein antigen composed of three candidate antigens, Leishmania elongation initiation factor (LeIF) (4, 32, 40, 41), L. major stress-inducible protein (LmSTI1) (49, 50), and thiol-specific antioxidant (TSA) (48), fused in tandem (42), when combined with monophosphoryl lipid A-stable emulsion (MPL-SE), induces a potent Th1-type immune response. This immune response protects mice against CL when challenged at 3 or 12 weeks postimmunization (7, 8). In the present study, we thus evaluated the use of animal models of VL, T- and B-cell enzyme-linked immunosorbent spots (ELISPOTs), and seven-parameter flow cytometry to investigate the mechanism of the protective immune response induced by the Leish-111f+MPL-SE vaccine against L. infantum. Indeed, it is important to develop a defined vaccine that is effective against more than one species of Leishmania because of the overlapping regions of parasite endemicity and the simplicity of administering a broadly effective vaccine.

MATERIALS AND METHODS

Animals and parasites.

Female BALB/c, C57BL/6, and C57BL/10 mice (6 to 8 weeks old) as well as LVG golden Syrian hamsters (30 to 33 days old) were purchased from Charles River Laboratories (Portage, MI). Animals were maintained under pathogen-free conditions in a level 2 physical containment facility and were used for experiments beginning at between 7 and 10 weeks of age. L. infantum was maintained by continuous passages in hamsters. For the preparation of L. infantum promastigotes, the parasites were grown at 25°C in complete HOMEM (minimal essential medium [MEM] supplemented with 0.5× MEM amino acids solution [Invitrogen], 1× MEM nonessential amino acids solution [Invitrogen], 1 mM sodium pyruvate, 8.3 mM glucose, 26 mM sodium bicarbonate, 100 pg/ml 6-biotin, 1 μg/ml para amino benzoic acid, 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid [HEPES], 50 μg/ml gentamicin, 6 μg/ml hemin, and 10% heat-inactivated fetal bovine serum). In all experiments, promastigotes were used after one to three passages in vitro.

Prophylactic immunization with Leish-111f+MPL-SE and determination of parasite numbers after challenge.

Groups of eight mice were immunized with 10 μg of the Leish-111f protein alone or with 20 μg of MPL-SE (GlaxoSmithKline Biologicals, Rixensart, Belgium) in a volume of 0.1 ml. Control groups received either adjuvant alone or saline. Subcutaneous injections were given in the right footpad and at the base of the tail at 3-week intervals over a total of 6 weeks. At 4 weeks after the last immunization, animals were challenged by intravenous injection of 5 × 10⁶ late-log-stage promastigotes of L. infantum (MHOM/BR/82/BA-2) obtained from the spleens of infected hamsters. The number of living L. infantum parasites in infected tissues was determined with the parasite-limiting dilution assay. Briefly, serial dilutions of the spleen and liver homogenates were twofold serially distributed in complete HOMEM in replicate wells with Novy-Nicolle-McNeal blood agar, and the plates were incubated at 26°C. After 10 to 12 days, the number of parasites was evaluated microscopically, and the number per organ was determined.

LVG golden Syrian hamsters between 4 to 6 weeks of age were used for experimental purposes with prior approval of the institutional animal care and use committee. For immunization, hamsters were divided into three groups; one group was immunized three times with Leish-111f+MPL-SE at 3-week intervals, and the other two groups were injected with saline or MPL-SE adjuvant alone. For evaluating the protection against challenge in hamsters, animals were challenged by intracardiac injection with 5 × 10⁶ L. infantum promastigotes. Spleen and liver sections from immunized and control animals were taken 1 month postinfection and assessed microscopically after staining with hematoxylin and eosin. Briefly, sections from each spleen (n = 5 hamsters per group) were examined by counting 25 consecutive microscopic fields per section at a magnification of ×40.

ELISA for anti-Leish-111f immunoglobulin G.

Serum samples were taken from all animals, and antigen-specific enzyme-linked immunosorbent assays (ELISAs) were performed for the identification of immunoglobulin G1 (IgG1) and IgG2a specific for recombinant (r)Leish-111f, rTSA, rLmSTI1, and rLeIF. Briefly, 96-well microtiter plates (Costar) were coated with 100 μl/well of each recombinant antigen at 2 μg/ml in phosphate-buffered saline (PBS) and incubated for 4 h at 37°C. Plates were washed and blocked overnight at 4°C with 200 μl/well 10% fetal calf serum (FCS) in PBS-Tween. Serum samples were diluted to 1:100 with PBS-Tween-10% FCS and applied to plates in twofold serial dilutions. Plates were incubated at 37°C for 4 h. Plates were washed, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates Ltd., Birmingham, AL) was added at a 1:2,000 dilution and incubated for 2 h at 37°C. Plates were detected using TMB substrate (3,3′,5,5′-tetramethylbenzidine; Kirkegaard and Perry, Gaithersburg, MD). The optical density was determined at 450 nm, using 570 nm as a reference wavelength.

ELISPOT assay.

Bone marrow (BM) IgG1- and IgG2a-secreting cells were enumerated by an antigen-specific ELISPOT assay, as described previously (13). In brief, Leish-111f-specific IgG1 and Leish-111f-specific IgG2a antibody-secreting cells (ASCs) were enumerated by an Ig ELISPOT assay. After isolation of BM cells, red blood cells were lysed via incubation with ammonium chloride Tris. Of the remaining population, 10⁶ cells/well were apportioned to Leish-111f-coated (at 10 μg/ml) Multiscreen 96-well plates (Millipore), and threefold serial dilutions were made before incubation. Plates were incubated for 4 h at 37°C. After the incubation, plates were washed in 0.1% Tween 20 and in nanopure water. ASCs were detected by HRP-conjugated anti-mouse IgG1 and HRP-conjugated anti-mouse IgG2a (Southern Biotechnology Associates, Inc.). ELISPOTs were developed by a peroxidase 3-amino-9-ethylcarbazole (Vector) chromogen substrate. ELISPOTs were enumerated via Immunospot software (Cellular Technology Ltd., Laboratories, LLC).

Culture of spleen cells.

Proliferative and cytokine responses were measured by preparing single-cell suspensions of spleens using Lympholyte-M density gradient centrifugation (CedarLane Labs, Hornby, Ontario, Canada). Splenic mononuclear cells were resuspended in complete medium (RPMI 1640 supplemented with 10% FCS, 50 μg/ml gentamicin, 2 mM l-glutamine, and 5 × 10⁻⁵ M β-mercaptoethanol) and plated at 2 × 10⁶ cells/ml in 96-well flat-bottom plates (Costar). The spleen cells were stimulated in vitro at 37°C in 5% CO₂ with rLeish-111f, rTSA, rLmSTI1, or rLeIF at 10 and 2 μg/ml or with soluble Leishmania antigen (10 μg/ml) or with medium alone. Concanavalin A at a concentration of 3 μg/ml was used in all experiments as a positive control for cell viability. The elicitation of NO, quantified by the accumulation of nitrite in the culture medium, was measured 48 h later by mixing 100 μl of supernatant with an equal volume of Griess reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% H₃PO₄]. Absorbance at 540 nm was measured after cells were incubated at room temperature for 10 min. Supernatants were also taken after 72 h of stimulation in culture and tested by ELISA for interleukin 4 (IL-4) and gamma interferon (IFN-γ) elicitation, as previously described (6).

Intracellular cytokine staining.

Splenocytes were plated in 24-well plates and stimulated for 14 h with 2 μg/ml anti-CD28 (eBioscience, San Diego, CA) and anti-CD49d (eBioscience) plus 20 μg/ml Leish-111f, a pool of 15-mer peptides overlapping by 10 amino acids covering the entire length of the Leish-111f protein sequence (1 μg/ml/peptide final concentration), phorbol myristate acetate/ionomycin, or no antigen (as a negative control) in complete medium at 37°C. After 2 h of incubation at 37°C, brefeldin A (GolgiPlug; BD Biosciences, San Jose, CA) was added to the wells, and the incubation was resumed for an additional 12 h at 37°C. Cells were blocked with anti-CD16/32 (eBioscience) diluted 1:50 in 50 μl and then stained with Alex Fluor 700-anti-CD3 (eBioscience), PerCP-anti-CD4 (BD Biosciences), and phycoerythrin (PE)-anti-CD8 (BD Biosciences). Then cells were fixed using a Cytofix/Cytoperm kit (BD Biosciences). Intracellular staining was then performed according to the BD Biosciences protocol. Cells were blocked with anti-CD16/32 and then intracellularly stained with fluorescein isothiocyanate (FITC)-anti-tumor necrosis factor (TNF) (eBioscience), Pacific Blue-anti-IL-2 (eBioscience), and PE-Cy7-anti-IFN-γ (BD Biosciences). Cells were analyzed with an LSRII fluorescence-activated cell sorter (FACS) machine (BD Biosciences) and DIVA software to quantify CD4⁺ T cells producing IFN-γ, IL-2, and TNF.

Statistical methods.

The efficacy of the Leish-111f+MPL-SE vaccine was compared to saline by using the one-tailed t test. A P value of <0.05 was considered significant.

RESULTS

Antibody responses induced by the Leish-111f+MPL-SE vaccine.

To evaluate the humoral response induced by the Leish-111f+MPL-SE vaccine, mice were injected with the vaccine or controls three times at 3-week intervals. Two weeks after the last immunization, IgG1 and IgG2a levels against Leish-111f and its individual components were measured (Fig. 1A to D). Significant differences in IgG antibodies were observed between vaccine groups (P < 0.05, analysis of variance). As expected, before infection, control mice that received saline or adjuvant alone had very low total specific IgG levels (data not shown).

FIG. 1.

Antibody responses induced by the Leish-111f+MPL-SE vaccine. Groups of eight C57BL/6 mice were immunized three times 3 weeks apart by subcutaneous injection of 10 μg of Leish-111f with 20 μg of MPL-SE adjuvant or saline. Serum samples were collected 2 weeks after the last injection and analyzed by ELISA for the presence of anti-Leish-111f (D), anti-LmSTI1 (C), anti-TSA (A), and anti-LeIF (B) IgG1 and IgG2a. Each point represents the mean and standard error of the means of data from individual mice. This experiment was repeated with similar results.

To examine whether the humoral response induced by the vaccine was long-lived, we then enumerated by ELISPOT the number of Leish-111F-specific plasma cells in the BM induced by the vaccine (44). We did this by measuring both Leish-111f-specific IgG1-and IgG2a-secreting plasma cells. Significant differences in IgG ASCs were observed between the vaccine and adjuvant-alone controls (Fig. 2).

FIG. 2.

Antibody-secreting cells induced by the Leish-111f+MPL-SE vaccine. Groups of eight C57BL/6 mice were immunized three times 3 weeks apart by subcutaneous injection of 10 μg of Leish-111f with 20 μg of MPL-SE adjuvant or saline. BM IgG1- and IgG2a-secreting cells were enumerated by an antigen-specific ELISPOT assay two weeks after the last injection. Each bar represents the mean and standard error of the means of data from individual mice.

T-cell responses induced by the Leish-111f+MPL-SE vaccine.

To evaluate cellular immune responses at the single-cell level, we analyzed, by ELISPOT and multiparameter flow cytometry, T-cell phenotypes after immunization with this vaccine. Based on the recall conditions employed for these in vitro studies, the Leish-111f+MPL-SE vaccine did not appear to significantly affect the overall total number of CD8⁺ T cells. On the contrary, the vaccine increased the number of IFN-γ-producing CD4⁺ T cells by 3.6% ± 1.2% or 12% ± 2.3%, depending on the experiment and in vitro recall conditions (Fig. 3D and Fig. 4B and C), characteristic of the induction of a Th1-type immune response. IFN-γ and TNF have been found to be involved in resistance to infection in murine VL (24, 26), while IL-10 correlates with susceptibility (20). Based on the important roles of these cytokines, a more comprehensive analysis of the levels of IFN-γ, IL-2, IL-10, and TNF produced by cells of Leish-111f+MPL-SE-vaccinated and nonvaccinated mice in response to the Leish-111f antigen was performed before infection (Fig. 3 and Fig. 4). Before infection, mice given the Leish-111f+MPL-SE vaccine produced higher levels of IFN-γ (20.9 ± 2.2 ng/ml) in response to in vitro recall with Leish-111f than mice injected with either saline or MPL-SE alone (Fig. 3A). As shown in Fig. 3D and Fig. 4, intracellular cytokine staining/multiparameter flow cytometry indicated that the frequency of IFN-γ-secreting CD4⁺ cells (15.6% ± 6.5% or 3.6% ± 1.8%, depending on the intracellular cytokine staining protocol) correlated well with the levels of IFN-γ observed by ELISA.

FIG. 3.

T-cell responses induced by the Leish-111f+MPL-SE vaccine. Groups of three C57BL/6 mice were immunized subcutaneously in the right rear footpad and at the base of the tail with 10 μg of Leish-111f formulated with 20 μg of MPL-SE. Mice were boosted twice 3 weeks apart, and 10 days later, spleens were removed, and splenocytes were stimulated in vitro with Leish-111f, TSA, LmSTI1, LeIF, and soluble Leishmania antigen (all at 10 μg/ml), concanavalin A (1 μg/ml), or medium alone. The elicitation levels of IFN-γ (A), IL-4 (B), and IL-10 (C) were assessed by sandwich ELISA using supernatants removed after 72 h of in vitro incubation. Cells were pooled from three mice per group. Each bar represents the means and standard errors of triplicate wells. Data shown are the means of duplicate wells. This experiment was repeated with similar results. (D, E, and F) For intracellular cytokine staining, splenocytes were stimulated overnight with 30 μg/ml Leish-111f as antigen (or without antigen as a negative control). Cells were then pulsed with GolgiStop for 4 to 6 h, washed, permeabilized, and stained using either a cocktail containing anti-CD3 FITC, anti-IFN-γ PE, and anti-CD4 PE-Cy5 or a second cocktail containing anti-TNF-α FITC, anti-CD4 PE, and anti-CD3 PE-Cy5. FACS data were collected on a BD FACScalibur flow cytometer and analyzed using CELLQUEST software to quantify CD4⁺ T cells producing IFN-γ and TNF.

FIG. 4.

Flow cytometric analysis of Leish-111f-specific T-cells. (A) Representative plots from flow cytometry data acquired. (B) IFN-γ, IL-2, and TNF production by CD4⁺ and CD8⁺ T cells in response to in vitro stimulation with medium or Leish-111f. Splenocytes were purified from mice that were injected with saline or Leish-111f+MPL-SE and were incubated in the presence of anti-CD28 and anti-CD49d with the addition of medium or Leish-111f. Cytokine production was analyzed by flow cytometry. (C) Single-cell analysis of Leish-111f-specific CD4⁺ and CD8⁺ T cells producing single, double, or triple Th1-type cytokines.

In addition, there was a modest increase in both IL-2 (9.6% ± 1.4%) and TNF (15.6% ± 5.3%)-producing CD4⁺ T cells observed for mice immunized with the Leish-111f+MPL-SE vaccine, while lower levels of TNF were produced in saline-injected mice.

In contrast to the robust Th1-type immune responses observed with the Leish-111f+MPL-SE vaccine, the levels of Th2 or inhibitory cytokines such as IL-4 were low (Fig. 3B). The amounts of Leish-111f-specific IL-10 produced by splenocytes before challenge varied from 830 to 2,030 ng/ml in mice immunized with the Leish-111f+MPL-SE vaccine to 0 and 160 ng/ml in those receiving saline and MPL-SE, respectively (Fig. 3C). In murine studies, both IFN-γ and TNF are implicated in the macrophage killing of intracellular L. donovani organisms, through the upregulation of inducible NO synthase and the production of nitrite oxide (23), while IL-2 may drive the expansion of these T cells. Nitric oxide has been demonstrated to be critical for the leishmanicidal activity of murine macrophages (17) and has also been shown to induce a Th1 response (28). However, IL-10 is known to down-regulate macrophage NO production (20). To further determine the ongoing immune response dynamics in the vaccinated versus the control mice, the induction of NO/nitrite was examined. Differences in the amounts of this antimicrobial agent were not observed between immunized and control mice (range, 5 to 13 μM in both groups). Therefore, unlike our observations for the levels of IFN-γ and at variance with published results (15, 17, 28), the vaccinated mice did not demonstrate higher levels of nitric oxide induction (Fig. 5).

FIG. 5.

NO production by the Leish-111f+MPL-SE vaccine. Groups of three C57BL/6 mice were immunized subcutaneously in the right rear footpad and at the base of the tail with 10 μg of Leish-111f formulated with 20 μg of MPL-SE. Mice were boosted twice 3 weeks apart, and 10 days later, spleens were removed, and splenocytes were stimulated in vitro with Leish-111f (at 10 μg/ml) or with medium alone. The elicitation of NO, quantified by the accumulation of nitrite in the culture medium, was measured 48 h later. Data represent the means ± standard deviations of two experiments.

To further investigate cellular responses induced by rLeish-111f+MPL-SE vaccination, flow cytometric analysis of Th1-type cytokine production by CD4⁺ and CD8⁺ T cells was performed. Spleen cells, which were harvested after in vitro cultivation with or without Leish-111f protein or a pool of 15-mer peptides, were gated based on forward and side scatter first and then CD3 expression (Fig. 4). CD4⁺ or CD8⁺ cells were further gated from the CD3⁺ population. Those populations were analyzed for the frequencies of cells producing TNF, IL-2, or IFN-γ. FACS data show that antigen-specific CD4⁺ and CD8⁺ cells were induced by rLeish-111f+MPL-SE vaccination (Fig. 3D to F and Fig. 4A to C). Significant levels of CD4⁺ cells producing TNF, IL-2, or IFN-γ in stimulation with rLeish-111f were found in mice immunized with Leish-111f+MPL-SE. Low levels of cytokine-producing Leish-111f-specific CD8⁺ cells were found in mice immunized with rLeish-111f+MPL-SE. We further divided the CD4⁺ and CD8⁺ cells into seven distinct populations based on expression patterns of TNF, IL-2, or IFN-γ at a single-cell level and found that many antigen-specific T cells producing these multiple cytokines were induced by rLeish-111f+MPL-SE (Fig. 4C). The majority of Leish-111f-specific CD4⁺ T cells produced IL-2 and TNF or all three cytokines (IFN-γ, IL-2, and TNF). Leish-111f-specific CD8⁺ T cells expressing all three of these cytokines were not produced by the immunization of Leish-111f+MPL-SE. Coexpression of IFN-γ and IL-10 (by regulatory T cells) was not evaluated.

Efficacy of the GMP material in the murine model of visceral leishmaniasis.

Initial mouse studies in the CL and ML mouse models were undertaken with laboratory-grade Leish-111f protein. After GMP material was produced, additional studies were undertaken to demonstrate that the GMP fusion would still protect in these models as well as in the VL mouse model. In these studies, mice were challenged with live L. infantum organisms after a three-dose immunization schedule with the Leish-111f+MPL-SE vaccine. Immunized mice were challenged by intravenous injection of 5 × 10⁶ L. infantum promastigotes, and the parasite burdens in the spleens and livers were evaluated 1 month after infection. Figure 6A shows that control mice presented a high parasite burden in the liver at 5 weeks postchallenge, whereas mice immunized with the Leish-111f+MPL-SE vaccine had significantly lower parasite loads, with a 91.7% reduction (P < 0.05 in the spleen [one-tailed t test]). No protection was induced by the adjuvant-alone control (data not shown).

FIG. 6.

Protection against L. infantum infection in mice (A) and hamsters (B) immunized with the Leish-111f+MPL-SE vaccine. (A) Groups of five C57BL/6 mice were immunized subcutaneously in the right rear footpad and at the base of the tail with 10 μg of Leish-111f formulated with 20 μg of MPL-SE. Mice were boosted twice 3 weeks apart and challenged with L. infantum parasites 30 days later. At 7, 14, and 35 days later, livers and spleens were removed, and the liver parasite burden was evaluated. Means and standard deviations of parasite burdens in the livers of five mice are expressed in log₁₀. *, P < 0.05 by unpaired t test compared with saline. These data represent four independent experiments with similar results. (B) Five hamsters per group were injected three times 21 days apart with 10 μg of Leish-111f formulated with MPL-SE or saline or with MPL-SE alone through the intramuscular route. All the hamsters were challenged 1 month after the last injection with 5 × 10⁶ live promastigotes through the intracardiac route. The organ parasite burden was determined by counting 25 consecutive microscopic fields (magnification of ×40) per section of spleens and livers. Means and standard deviations of parasite burdens in the spleens and livers of five hamsters are expressed in log₁₀. *, P < 0.05 by unpaired t test compared with saline.

Protection against L. infantum infection in hamsters.

For evaluating protection against challenge in bigger rodents, LVG golden Syrian hamsters were challenged with live L. infantum parasites after a three-dose immunization schedule with the Leish-111f+MPL-SE vaccine, and the parasite burdens in the spleens and livers were evaluated 1 month after infection (Fig. 6). Figure 6B shows that control hamsters presented a high parasite burden, whereas animals immunized with the Leish-111f+MPL-SE vaccine had significantly lower parasite loads, with a 99.6% reduction (P < 0.05 in the spleen [one-tailed t test]).

DISCUSSION

We have chosen to develop a Leishmania vaccine antigen, Leish-111f, that is a fusion of three distinct, conserved Leishmania antigens. These three antigens (TSA, LmSTI1, and LeIF) were selected for the development of a subunit vaccine based on their demonstrated abilities to induce at least partial protection in the BALB/c mouse model of L. major challenge in either prophylactic (TSA and LmSTI1) or therapeutic (LeIF) applications (40, 41, 48, 50). All three antigens are present in both the amastigote and the promastigote forms of the parasite and are highly conserved among Leishmania spp, a requisite for ensuring cross-species protection. In addition to simplifying the manufacturing process, immunization with a single antigen may ensure equivalent uptake of the components by individual antigen-presenting cells and, in turn, generate an immune response that is broadly specific for all the immunogenic epitopes spanning the polyprotein. The Leish-111f polyprotein-plus-MPL-SE formulation has previously been assessed for immunogenicity and vaccine efficacy using the L. major and L. amazonensis challenge models with BALB/c and C57BL/6 mice, respectively (7, 9, 31, 43). The results of the prior studies showed that all mice appeared healthy and that no observable toxicity occurred for the duration of the experiments. A Th1 phenotypic response, characterized by in vitro lymphocyte proliferation, IFN-γ production, and IgG2a antibodies, was observed with little if any IL-4 production.

Moreover, the vaccine formulation conferred immunity against subcutaneous challenge with L. major for at least 3 months. In order to feel comfortable with our cyclic GMP product prior to going into human trials, we wanted to be assured that the changes we had made to achieve manufacturability and regulatory compliance did not interfere with the product's primary function, protection against leishmaniasis. The findings in this report extend earlier studies and demonstrate (i) that Leish-111f+MPL-SE, when given prophylactically, controls infections by the L. donovani complex, a causative agent of VL in humans and dogs, and (ii) that the prophylactic effect was substantially better than with any previously reported vaccine candidate. Prior publications report twofold reductions in parasite burdens after immunization with candidate vaccines (1, 14, 34, 36, 46). Protection against challenge with L. infantum in Leish-111f+MPL-SE-immunized animals appears to be associated with a Th1-type immune response. Our previous studies demonstrated that MPL-SE clearly augments the Leish-111f-specific Th1 response in BALB/c mice. Immunizing with Leish-111f+MPL-SE produced a robust antibody (IgG1 and IgG2a) response to two of the three component antigens and resulted in the activation and/or proliferation of Leish-111f-specific cells producing and releasing IFN-γ, IL-2, and TNF, which are known to be associated with protection against leishmaniasis (21, 25, 26, 27, 45). TNF acts in synergy with IFN-γ in killing Leishmania parasites (16, 18). Thus, the induction of Leish-111f-specific T cells capable of producing multiple cytokines upon antigen recall is likely to be more beneficial for the control of Leishmania infection than those producing single cytokines.

In contrast to other reports, NO production was not increased after immunization, despite increases in IFN-γ and TNF. Leish-111f-specific IL-10 production, which is reported to be important for the cellular immune depression that accompanies active VL disease, as well as that of IL-4/IL-13 promoting susceptibility to Leishmania infections, was moderate and did not cause vaccine failure. In conclusion, this study demonstrated that immunization with a vaccine containing Leish-111f+MPL-SE is highly immunogenic and confers a significant degree of protection against murine L. infantum infection in a prophylactic setting. Immunizing with Leish-111f+MPL-SE resulted in high levels of IFN-γ, TNF, and IL-2, producing effector T cells upon in vitro recall and protection against L. infantum challenge. This observation prompts a question about the biologically effective amounts of IFN-γ, TNF, and IL-2 and the anatomical localization of these cytokines that are required to induce protection against VL. Although additional effector mechanisms may be involved, these results suggest that high levels of T cells producing all three of these cytokines may be required for protection against visceral disease. Future studies will explore the use of this vaccine approach for a composite/multicomponent strategy against VL, as well as the ability of the vaccine to elicit central and effector memory based on CCR7 expression.