Abstract
The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.
Leishmania major is an etiological agent of cutaneous leishmaniasis, a disease characterized by cutaneous lesions that can be self-resolving with life-long immunity or chronic when accompanied by defective cellular immune responses (24). The disease is prevalent in many tropical and subtropical regions of the world, where it is transmitted via the bite of an infected sand fly.
To date, there are no proven vaccines against any form of leishmaniasis; however, several approaches are being tested (reviewed in references 6, 8, 18, and 23). In brief, vaccines based on killed promastigotes with or without Mycobacterium bovis BCG have shown significant protection against L. braziliensis, L. mexicana, L. major, and L. donovani in humans. In addition, in experimental animal models, several other vaccine preparations are being tested. Examples include attenuated live parasites; subunit vaccines delivered by live carriers such as M. bovis BCG expressing the surface protease gp63 of L. major; vaccinia virus expressing the glycoprotein gp46/M2/PSA-2; gp63 expressed in attenuated Salmonella enterica serovar Typhimurium; purified recombinant or native proteins formulated with an adjuvant such as the Leishmania homologue of receptors for activated C kinase plus interleukin-12 (IL-12), gp46/M2/PSA-2, or protein dp72 plus Corynebacterium parvum, T-cell epitopes plus Poloxamer 407; and vaccination with DNA encoding gp63, leishmanial activated C kinase, or gp46/M2/PSA-2. In general, these vaccination protocols elicited partial protection against L. major.
Protection of mice against L. major infection depends on the ability to generate macrophage-activating Th1 responses resulting in the production of gamma interferon (IFN-γ) and low levels of IL-4 (28). Furthermore, there is considerable evidence that Th2-type responses and the production of IL-4 result in the inability to control disease or in disease exacerbation (9, 10). However, this Th1/Th2 paradigm is currently being expanded. New evidence suggests that IL-10 is an additional factor controlling susceptibility to L. major in BALB/c mice (21). In addition, the idea that IL-4 might act in concert with IL-13 to produce an additive effect on L. major susceptibility has also been reported (17). All these reports suggest that a cumulative effect of three disease-promoting cytokines, IL-4, IL-10, and IL-13, may be important to disease outcome.
The present study was aimed at evaluating the potential of soluble culture-derived Leishmania secreted and excreted exogenous antigens (L. major SEAgs) as vaccine candidates against L. major infections in the susceptible BALB/c mouse model. Recently, these exoantigens were used to develop serological assays that demonstrated very high degrees of sensitivity and specificity. These assays efficiently detected antileishmanial antibodies, both immunoglobulin M (IgM) and IgG, in visceral leishmaniasis patients (15, 25). These findings, supported by Western blot patient profiles, indicated that leishmanial exoantigens are excellent diagnostic markers and are highly immunogenic in the infected host. In addition, recent work by Webb et al. (30) showed that culture filtrate proteins of L. major promastigotes (produced with a different strain of parasite with different production techniques) administered to mice with Corynebacterium parvum protected against a subsequent challenge with parasites.
The adjuvant administered with an antigen influences the quantity and quality of the ensuing immune response to the antigen. Therefore, we injected BALB/c mice with L. major SEAgs alone or in conjunction with adjuvants currently approved or close to approval for use in humans and formulations that have shown promise in vaccine trials. Aluminum in the form of aluminum hydroxide, aluminum phosphate, or alum has been commonly used as an adjuvant in many vaccines licensed by the U.S. Food and Drug Administration (3). IL-12 is a critical component in the development of cell-mediated immunity and stimulates cell proliferation and IFN-γ secretion by T and natural killer cells (11). Importantly, IL-12 has the ability to promote the development of CD4 Th1 cells, which are necessary for protective immunity in leishmaniasis (11, 29). It has also been shown that adsorption of both antigen and IL-12 onto alum enhances immunostimulatory effects that promote both humoral and Th1 cytokine responses to human immunodeficiency virus infection in mice (12) and to L. major in rhesus monkeys (13). Montanide ISA 720, a metabolizable oil-based adjuvant, has also been tested in humans (2).
We demonstrate here that, unlike most Leishmania antigens, which have shown protection only when administered with adjuvants, L. major SEAgs are unusual in that a single vaccination with these antigens alone resulted in significant inhibition of lesion development in susceptible BALB/c mice following challenge infection with L. major.
MATERIALS AND METHODS
L. major parasites.
Metacyclic promastigotes of L. major strain LV 39 (RHO-SU-59-P) were used. Parasites were maintained as previously described (27), and metacyclics were isolated from stationary-phase cultures by negative selection with peanut agglutinin (Sigma-Aldrich) (26).
L. major SEAgs, adjuvants, and mitogen.
L. major SEAgs were produced as previously described (25). Briefly, L. major parasites were initially grown in supplemented medium to the late log phase. The parasites were centrifuged and washed four times in serum-free medium (XOM, described in reference 15). Washed promastigotes were inoculated into XOM to give a final density of 108 cells/ml and incubated at 26°C for 72 h in roller bottles. Thereafter, the spent medium was harvested by centrifugation twice at 9,000 × g for 30 min, and the relative protein concentration of L. major SEAgs was estimated by measuring the optical density at 280 nm.
Alum (Rehydragel HPA; Reheis Inc., N.J.), recombinant murine IL-12 (rmIL-12 (Genetics Institute, Cambridge, Mass.), and Montanide ISA 720 (Seppic Inc.) were used as adjuvants. Concanavalin A was obtained from Miles Laboratories (Kankakee, Ill.) and used at a concentration of 1 μg/ml in cultures.
Immunization of BALB/c mice.
Young adult female BALB/c mice obtained from the National Cancer Institute (Bethesda, Md.) were used in all experiments. These experiments complied with all relevant federal guidelines and institutional policies. In pilot experiments we determined the optimal dose of L. major SEAgs and adjuvant and the most effective route of delivery for the antigens with and without adjuvant; 50 μg of L. major SEAgs was consistently protective against a challenge with L. major, and higher doses of L. major SEAgs did not improve protection. Protection was greatest when L. major SEAgs were delivered by a subcutaneous route as opposed to an intraperitoneal route. In all experiments presented here, L. major SEAgs were injected subcutaneously in the rump. When L. major SEAgs were injected with alum, 10 μg of alum was used; higher doses were not more protective than 10 μg. When IL-12 was used as the adjuvant, 1 μg of the cytokine was coinjected with L. major SEAgs because this dose was successfully used to immunize BALB/c mice with whole-cell lysates of L. major (1). When Montanide ISA 720 was used as the adjuvant, L. major SEAgs and Montanide ISA 720 were mixed in a 1:3 vol/vol ratio according to the manufacturer's directions. Groups of BALB/c mice were immunized once or twice by injecting the various combinations of L. major SEAgs with and without adjuvants subcutaneously. For single immunizations, mice were immunized and challenged 10 days later with metacyclic promastigotes. For double immunizations, mice were boosted with the same antigen with and without adjuvant 13 days after the initial immunization and challenged with parasites 13 days later.
Infection of mice and determination of parasite numbers in cutaneous lesions.
Control and immunized BALB/c mice were challenged in a hind footpad with 105 metacyclic promastigotes. Lesion development was followed by measuring the thickness of the infected footpad compared to the thickness of the same footpad prior to infection with a vernier caliper. At the times designated in the Results, duplicate mice were sacrificed to estimate the parasite burden in the footpads with a limiting dilution assay (14).
Cell cultures: cytokines and proliferation.
Lymphatic tissue (the spleen or the popliteal and inguinal lymph nodes that drained the footpad in mice infected with L. major) was harvested from duplicate mice of each experimental group just prior to infection (day 0) or at day 7, 10, 13, or 28 postinfection (see below for details). When spleen cells were used, mononuclear cells were purified with Ficoll gradients (Sigma, St. Louis, Mo.).
Mononuclear cells were adjusted to 2 × 106/ml in Dulbecco's modified Eagle's medium (16) containing 0.5% normal mouse serum (Harlan Bioproducts, Indianapolis, Ind.) and 5 × 10−5 M 2-mercaptoethanol (Sigma). Cells were stimulated in vitro (37°C in 5% CO2) with 50 μg of L. major SEAgs/ml, L. major (2 × 105/ml), or medium alone. Supernatants were collected after 48 or 72 h (see Results) of culture and tested by enzyme-linked immunosorbent assay (ELISA) for IL-4 and IFN-γ with commercial anticytokine antibody pairs (Becton Dickinson/Pharmingen, San Jose, Calif.) and protocols provided by the manufacturer. Alternatively, after 5 days, the cultures were pulsed with 1 μCi of [3H]thymidine (5 Ci/mmol; Amersham, Arlington Heights, Ill.) for 18 h and harvested, and cell proliferation was evaluated by liquid scintillation counting.
Preparation of parasite extracts.
Stationary-phase L. major promastigotes were collected, centrifuged, washed three times, and resuspended in phosphate-buffered saline (PBS) to a final concentration of 5 × 108 parasites/ml in the presence of protease inhibitor cocktail set III (Calbiochem, San Diego, Calif.) and frozen at −20°C. The protease inhibitor cocktail contained AEBSF hydrochloride (2 mM final concentration), aprotinin (1.6 μM), bestatin (100 μM), E-64 (30 μM), leupeptin hemisulfate (40 μM), and pepstatin A (20 μM). The parasites were subjected to three cycles of freezing and thawing and centrifuged at 13,800 × g at 4°C for 10 min. The pellets were then extracted with Triton X-100 (1% in PBS) with protease inhibitors and centrifuged again at 13,800 × g at 4°C for 10 min to collect the Triton X-100 fractions that were used in the Western blot analyses.
SDS-PAGE.
To obtain a silver-stained profile of the main constituents of the L. major SEAgs, 200 μl of the preparation (1,292 μg/ml) was precipitated with 3 volumes of cold acetone and kept at −20°C for 24 h. The precipitate was collected by centrifugation at 13,800 × g at 4°C for 10 min, drained, and left to dry. The precipitate was then resuspended in 20 μl of PBS and mixed with 3 μl of 0.5 M dithiothreitol and 7.5 μl of 4× LDS buffer (Invitrogen, Carlsbad, Calif.). The mixture was incubated 5 min at 90°C and then 5 min on ice before the electrophoresis; 10 μl of the mixture was loaded into a precast sodium dodecyl sulfate (SDS)-4 to 12% polyacrylamide gel electrophoresis (PAGE) gel (Invitrogen) and run in morpholineethanesulfonic acid (MES) buffer at 200 V for 35 min. The gel was fixed and silver stained following the recommendations of the manufacturer of silverXpress (Invitrogen). As molecular weight standards, a prestained rainbow protein mix (Amersham Biosciences, Piscataway, N.J.) was used.
Western blot analysis.
The proteins of the L. major SEAgs and the Triton X-100 extract of L. major promastigotes were electrophoretically separated as indicated above and then transferred to nitrocellulose paper (Bio-Rad, Hercules, Calif.) in Tris-HCl-glycine-methanol buffer at 100 V for 1 h. The membrane was blocked with 1% milk in PBS for 1 h and probed with the following antibodies against Leishmania components for 1 h at room temperature: mouse monoclonal antibody 235 against gp63 (gift of R. McMaster, University British Columbia, Vancouver, Canada) diluted 1:1,000 in PBS; mouse monoclonal antibody 79.3 against lipophosphoglycan (gift of S. Turco, University of Kentucky, Lexington) diluted 1:1,000; and rabbit polyclonal anti-gp46 (gift of D. McMahon-Pratt, Yale University, New Haven, Conn.) diluted 1:100. Controls consisted of normal mouse and normal rabbit sera. The membrane strips were then washed three times with PBS containing 0.2% Tween 20 (PBS-T) and incubated with a 1:1,000 dilution of goat antibodies specific for mouse IgG (heavy and light chains) or rabbit IgG (heavy and light chains) conjugated to alkaline phosphatase (KPL, Gaithersburg, Md.). After 1 h, the membranes were washed three times with PBS-T and then were incubated with the phosphatase substrate nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) (KPL) for 15 min at room temperature in the dark.
Image analysis.
The images of the Western blots were scanned and processed with Adobe Photoshop 7.0 software. The molecular weights of the bands were calculated with Alpha Imager 2200 software.
Statistical analyses.
Statistical analyses were performed with Sigma Stat (SPSS, Chicago, Ill.). Data for lesion progression were analyzed with analysis of variance for group comparisons and t tests for other analyses. A P value of <0.05 was considered significant.
Groups of two mice each were used for in vitro analyses; experiments that monitored lesion development consisted of groups of four to six mice each. All experiments were performed independently at least twice.
RESULTS
L. major SEAgs prime specific cells in susceptible BALB/c mice.
Our first goal was to assess the immunogenicity of L. major SEAgs in L. major-susceptible BALB/c mice. To achieve this, BALB/c mice were injected subcutaneously with 50 μg of L. major SEAgs in the rump. Ten days later, immunized mice or their controls were sacrificed, and their spleen cells were collected and pooled for lymphocyte proliferation assays. Mononuclear cell suspensions (2 × 106/ml) were stimulated with medium alone (negative control), 2 × 105 live L. major/ml, or 50 μg of L. major SEAgs/ml. After 5 days, the cultures were pulsed with 1 μCi of [3H]thymidine for 18 h and harvested, and the degree of proliferation was assessed by liquid scintillation counting.
The results showed that spleen cells from L. major SEAg-immunized mice proliferated when restimulated with either L. major or L. major SEAgs in vitro (Fig. 1). Stimulation of cells from L. major SEAg-immunized mice with either L. major or L. major SEAgs was significantly higher than in those from the controls (P < 0.05), suggesting recall proliferative responses. Some cultures were also stimulated with concanavalin A as an internal control for cell viability. In these cultures, the degree of proliferation of cells from all groups was equivalent (data not shown).
FIG. 1.
L. major SEAgs prime specific cells in susceptible BALB/c mice. BALB/c mice were immunized with 50 μg of L. major SEAgs alone subcutaneously in the rump. Ten days later, spleen cells from duplicate immunized and control (injected with vehicle used to inject L. major SEAgs) mice were collected and pooled for lymphocyte proliferation assays. Mononuclear cell suspensions adjusted to 2 × 106/ml were prepared as described in Materials and Methods. These cells were stimulated in vitro with 2 × 105 L. major/ml or 50 μg of L. major SEAgs/ml. After 5 days, the cultures were assessed for proliferation as described in Materials and Methods.
L. major SEAgs stimulate protective immunity against a subsequent challenge with L. major in susceptible BALB/c mice.
Our second goal was to assess the ability of L. major SEAgs to protect against a subsequent challenge with L. major in susceptible BALB/c mice. BALB/c mice were injected subcutaneously with 50 μg of L. major SEAgs, and 10 days later the immunized mice together with controls were challenged with 105 metacyclic LV 39 promastigotes subcutaneously in one hind footpad. Lesion sizes in mice were monitored by measuring the increase in size of the infected footpad. As shown in Fig. 2, one injection of 50 μg of L. major SEAgs alone without adjuvant induced significant protection (P < 0.05) against a subsequent challenge with L. major. This finding was corroborated by lesion parasite burden analyses (Table 1).
FIG. 2.
L. major SEAgs stimulate protective immunity against a subsequent challenge with L. major in susceptible BALB/c mice. BALB/c mice (n = 6) were immunized with 50 μg of L. major SEAgs alone subcutaneously in the rump. After 10 days, these mice and control mice were challenged in the left hind footpad with 105 L. major metacyclic promastigotes. Lesion sizes in mice (thickness of the infected footpad minus the thickness of the footpad prior to infection) were measured with a vernier caliper. Lesion sizes are given as means ± standard error. Differences between lesion sizes became significant (P > 0.05) by day 7 of infection.
TABLE 1.
Parasite burden in lesions of BALB/c mice immunized once with SEAgsa
| Expt | No. of L. major (106)/footpad (95% confidence limits)
|
Reduction in parasite burden (-fold) | |
|---|---|---|---|
| Control | Vaccinated | ||
| I | 9.18 (5.04-13.33) | 0.83 (0.41-1.25) | 11.04 |
| II | 5.30 (1.60-8.90) | 0.20 (0.03-0.30) | 26.50 |
| III | 7.25 (2.38-9.70) | 0.21 (0.04-0.38) | 34.50 |
Mice were treated as described in the legend to Fig. 2. Twenty-eight days postinfection, duplicate mice were killed, and the parasite burdens in their footpads were analyzed with the limiting dilution assay described in Materials and Methods. The table presents three replicate experiments.
Vaccination with L. major SEAgs plus rmIL-12 in alum also proved to be protective but no more protective than L. major SEAgs alone.
Protection with L. major SEAgs alone prompted us to evaluate the effects of coinjecting these antigens with adjuvants. We tested the conventional adjuvants alum, rmIL-12, and Montanide ISA 720. Alum is currently the only Food and Drug Administration-approved adjuvant in clinical use in humans. The other adjuvants, rmIL-12 and Montanide ISA 720, have shown good results in several primate and human vaccination studies.
The results showed that coinjecting L. major SEAgs together with alum, rmIL-12, or Montanide ISA 720 did not increase protection. The only adjuvant system that equalled the protection seen with L. major SEAgs alone was IL-12 plus alum (Fig. 3). This conclusion was corroborated by the fact that similar numbers of parasites were present in the lesions of mice treated with L. major SEAgs alone or L. major SEAgs plus IL-12 plus alum (Table 2). However, interestingly, immunization with L. major SEAgs or L. major SEAgs plus IL-12 plus alum led to the induction of dissimilar levels of the Th1-associated cytokine IFN-γ, with L. major SEAgs plus IL-12 plus alum-treated mice producing approximately 10-fold less IFN-γ when cells from the mice were restimulated with L. major parasites in vitro (Table 3).
FIG. 3.
L. major SEAgs vaccinated as effectively in the presence or absence of adjuvants. Ten days after vaccination, mice were challenged with 105 L. major metacyclic promastigotes. Lesion sizes were followed as described in the legend to Fig. 2.
TABLE 2.
Parasite burden in lesions of BALB/c mice immunized with SEAgs with and without adjuvantsa
| Treatment | No. of L. major (106)/footpad (95% confidence limits) | Reduction in parasite burden (-fold) |
|---|---|---|
| None (control) | 5.3 (1.6-8.9) | |
| SEAgs + 10 μg of alum | 2.9 (0.1-0.5) | 1.8 |
| SEAgs + rmIL-12 | 5.0 (0.1-0.8) | 1.1 |
| SEAgs + Montanide ISA 720 | 3.3 (0.7-5.8) | 1.6 |
| SEAgs + rmIL-12 + alum | 0.2 (0.1-0.2) | 26.5 |
| SEAgs alone | 0.2 (0.04-0.4) | 25.3 |
BALB/c mice were immunized with 50 μg of SEAgs alone or coinjected with alum, rmIL-12, rmIL-12 coadsorbed in alum, or Montanide ISA 720. Ten days later, mice were challenged in the left hind footpad with 105 L. major metacyclic promastigotes. At 28 days postinfection, duplicate mice were killed, and the parasite burdens in their footpads were analyzed with the limiting dilution assay described in Materials and Methods.
TABLE 3.
Cytokines produced by BALB/c mice vaccinated with SEAgsa
| Treatment | Stimulus | Cytokine concn (pg/ml ± SE)
|
|
|---|---|---|---|
| IFN-γ | IL-4 | ||
| Control | L. major | 62 ± 0 | 19.1 ± 1.8 |
| SEAgs | 0 ± 0 | 7.6 ± 0.6 | |
| SEAgs alone | L. major | 683 ± 52 | 1.2 ± 0.9 |
| SEAgs | 828 ± 52 | 10.2 ± 0.9 | |
| SEAgs + rmIL-12 + alum | L. major | 42 ± 1 | 1.3 ± 0.1 |
| SEAgs | 442 ± 11 | 9.0 ± 1.7 | |
Mice were vaccinated as described in the legend to Fig. 2. Cells were harvested from the draining lymph nodes (inguinal and popliteal) 7 days postinfection. Cells were adjusted to 2 × 106/ml and stimulated with either 50 μg of SEAgs or 2 × 105 L. major/ml. Culture supernatants were collected 48 h later, and the concentrations of IFN-γ and IL-4 were determined by ELISA as described in Materials and Methods.
It should be mentioned that we also immunized mice twice with either L. major SEAgs plus alum or plus IL-12 or plus Montanide ISA 720 and then challenged the mice with L. major. This engendered low levels of protection (data not shown) similar to the levels of protection seen in Fig. 3. We therefore concluded that the only two systems worth exploring further were immunization with L. major SEAgs alone and immunization with L. major SEAgs plus IL-12 plus alum.
Double immunizations with the L. major SEAgs alone proved to be more protective than double immunizations with L. major SEAgs plus IL-12 plus alum.
Groups of BALB/c mice were injected subcutaneously with L. major SEAgs alone or L. major SEAgs plus IL-12 plus alum and 13 days later (7) subcutaneously again with the same preparations. Thirteen days after the second immunization, duplicate vaccinated and control mice were sacrificed, and their spleen cells were collected and pooled for lymphocyte proliferation assays. Mononuclear cells were stimulated in vitro with L. major, L. major SEAgs, or medium alone. Five days later, the degree of proliferation of the cells was assessed as described in Materials and Methods. The results showed that mononuclear cells harvested from mice immunized twice with L. major SEAgs or L. major SEAgs plus rmIL-12 plus alum showed enhanced proliferative responses compared to those from mice given a single immunization with L. major SEAgs (compare Fig. 1 and 4).
FIG. 4.
Proliferation of cells from doubly-immunized mice. BALB/c mice were doubly immunized as described in Materials and Methods with L. major SEAgs alone or L. major SEAgs formulated with rmIL-12 coadsorbed in alum. Thirteen days after the second injection, spleen cells were collected and pooled for lymphocyte proliferation assays. Single-cell suspensions adjusted to 2 × 106/ml were prepared as described in Materials and Methods. The spleen cells were stimulated in vitro with 2 × 105 L. major/ml, 50 μg of L. major SEAgs/ml, or medium alone. After 5 days, the cultures were pulsed with 1 μCi of [3H]thymidine for 18 h, harvested, and counted in a liquid scintillation counter.
Thirteen days after the second immunization, immunized mice together with their controls were challenged with L. major in a hind footpad. Immunizing twice with L. major SEAgs if anything was more protective than immunizing once (compare Fig. 2 and 5); e.g., after a single immunization, lesion size was approximately 1 mm at 28 days postinfection, whereas after two immunizations, lesions were almost nonexistent at 28 days. Curiously, in sharp contrast to immunizing twice with L. major SEAgs alone, immunizing twice with L. major SEAgs plus IL-12 plus alum induced little if any protection (Fig. 5). The same result was seen when the parasite burden within the lesions was assessed; the only group that showed a substantial decrease in parasite burden was the group immunized with L. major SEAgs alone (Table 4), a 53-fold reduction at day 28 and a 125-fold reduction at day 38 postinfection.
FIG. 5.
Immunizing twice with L. major SEAgs is highly protective for BALB/c mice. Mice were doubly immunized with either L. major SEAgs alone or L. major SEAgs plus rmIL-12 plus alum as described in Materials and Methods. Thirteen days later, the mice were challenged with 105 L. major metacyclic promastigotes in one hind footpad. Lesion sizes in mice were measured with a vernier caliper every 3 days for a total of 38 days. Groups consisted of 10 mice each, of which 4 were used to evaluate cytokine and parasite loads on days 28 and 38. Differences between lesion sizes in control mice versus mice immunized with L. major SEAgs became significant (P > 0.05) by day 17 of infection.
TABLE 4.
Parasite burden in doubly-immunized micea
| Treatment | Day postinfection | No. of L. major (106)/footpad (95% confidence limits) | Change in parasite burden (±fold) |
|---|---|---|---|
| Control | 28 | 1.3 (0.5-2.1) | |
| 38 | 5.0 (1.4-8.6) | ||
| rmIL-12 + alum | 28 | 1.3 (0.8-1.8) | +1.01 |
| 38 | 9.2 (0.5-13.3) | +1.8 | |
| SEAgs | 28 | 0.02 (0.01-0.04) | −53.2 |
| 38 | 0.04 (0.01-0.07) | −124.8 | |
| SEAgs + rmIL-12 + alum | 28 | 0.61 (0.04-0.9) | −2.1 |
| 38 | 8.01 (2.4-13.6) | +1.6 |
Mice were immunized as described in Materials and Methods. On the indicated days postinfection, duplicate mice were killed, and the parasite burdens in their footpads were analyzed as described in Materials and Methods.
Vaccination with L. major SEAgs induces a mixed Th1/Th2-type cytokine response.
In order to dissect the nature of the immune response induced by immunization with either L. major SEAgs alone or L. major SEAgs plus IL-12 plus alum, we determined the cytokines elicited by these immunization approaches. Thirteen days after the second immunization (but prior to challenge with L. major), spleens were removed, mononuclear cell suspensions were prepared, and the cells were stimulated in vitro with L. major SEAgs or L. major. We found that, in general, cells from mice immunized with L. major SEAgs plus rmIL-12 plus alum produced more cytokines (IFN-γ, IL-4, and IL-10) than cells from mice immunized with L. major SEAgs alone (Fig. 6). This was true whether the spleen cells from immunized mice were restimulated in vitro with L. major SEAgs or with L. major parasites (Fig. 6). Therefore, immunization with L. major SEAgs induced a mixed Th1/Th2-type cytokine response.
FIG. 6.
Cytokines produced by BALB/c mice doubly vaccinated with L. major SEAgs: profile prior to challenge with L. major. Mice were immunized twice with L. major SEAgs alone or L. major SEAgs plus rmIL-12 plus alum (see Materials and Methods). Thirteen days after the second immunization, mononuclear cells from the animals were restimulated in vitro with L. major SEAgs (50 μg/ml) or with L. major parasites (2 × 105/ml). Three days later, the supernatants of the cultures were harvested and tested by ELISA for IFN-γ (A), IL-4 (B), and IL-10 (C). The data depict mean cytokine production ± standard error.
Next, we determined the cytokines produced after immunized mice were challenged with L. major. Twenty-eight days after immunized mice were challenged (a time when mice immunized with L. major SEAgs alone were resisting infection but mice immunized with L. major SEAgs plus rmIL-12 plus alum were not resisting infection; Fig. 5), cells from the mice were again restimulated in vitro with either L. major SEAgs or L. major parasites. As can be seen in Fig. 7, mice immunized with L. major SEAgs alone produced IFN-γ but low or undetectable levels of IL-4 and IL-10. In contrast, mice immunized with L. major SEAgs plus rmIL-12 plus alum in general produced much larger amounts of IL-4 and IL-10 and did not produce IFN-γ when restimulated in vitro with L. major parasites. Thus, at this time of infection, mice vaccinated with L. major SEAgs alone produced more type 1 cytokines, while mice vaccinated with L. major SEAgs plus rmIL-12 plus alum produced more type 2 cytokines (Fig. 7).
FIG. 7.
Cytokines produced by BALB/c mice doubly vaccinated with L. major SEAgs, profile after challenge with L. major. The protocol was identical to that for Fig. 6 except that the experiments were conducted 28 days after challenge with 105 L. major.
L. major SEAgs contain at least the leishmanial surface antigens lipophosphoglycan and gp46.
In an effort to characterize L. major SEAgs (since exo antigens of L. donovani and L. mexicana have been described before [15, 25] but not those of L. major), we silver-stained SDS-PAGE gels of the antigens and probed Western blots of the antigens with antibodies against common surface components of L. major. These analyses revealed that the L. major SEAgs contained approximately 10 to 12 major components by SDS-PAGE analysis (Fig. 8A) and that among these are the surface molecules lipophosphoglycan and gp46 (Fig. 8B). On the other hand, gp63 was not detectable in the L. major SEAgs (Fig. 8B).
FIG. 8.
SDS-PAGE and Western blot analyses of L. major SEAgs. (A) Silver stain of electrophoretically separated L. major SEAgs (Ex). (B) Western blot analysis of Triton X-100 extracts of L. major promastigotes (Tx) and L. major SEAgs (Ex) probed with antibodies against lipophosphoglycan (lane 1), gp46 (lane 2), or gp63 (lane 3).
DISCUSSION
In the present study, we investigated the potential of L. major culture-derived exoantigens (L. major SEAgs) alone and in conjunction with various adjuvants to elicit a protective immune response against challenge with the parasite in susceptible BALB/c mice. Recently, leishmanial exoantigens were used to develop ELISAs, which have very high sensitivity and specificity for antileishmanial antibodies (both IgM and IgG) in patients and in dogs infected with visceral leishmaniasis (15, 25). These findings suggested that exoantigens are highly immunogenic in these two hosts and thus might be good vaccine candidates.
We found that L. major SEAgs stimulated cells in BALB/c mice injected with the antigens and that L. major SEAgs also induced a recall response, since cells taken from mice injected twice with L. major SEAgs proliferated more in vitro when restimulated with L. major SEAgs than cells taken from mice injected only once with L. major SEAgs (compare Fig. 1 and 4). Moreover, a single injection of L. major SEAgs (in the absence of adjuvant) induced significant resistance to a subsequent challenge with L. major, with respect to both lesion size (Fig. 2) and parasite burden (Table 1).
The fact that no adjuvant was required for L. major SEAgs to induce protection was not expected, but this showed that L. major SEAgs are highly immunogenic in mice, as they have been shown to be in humans and dogs (15, 25). Since L. major SEAgs contain substantial amounts of lipophosphoglycan (Fig. 8) and lipophosphoglycan has been shown to have adjuvant-like activities (19, 31) which lead to enhanced cell proliferation and cytokine production, it is tempting to speculate that the immunogenicity of L. major SEAgs is due to their lipophosphoglycan content (Fig. 8). Thus, lipophosphoglycan, and perhaps other components of L. major SEAgs that have adjuvant qualities, may be responsible for the ability of L. major SEAgs to stimulate protection against L. major challenge in the absence of adjuvant.
Although stimulating mice once with either L. major SEAgs alone or L. major SEAgs in conjunction with IL-12 and alum (the only adjuvant that induced protection with L. major SEAgs) led to similar degrees of protection (Fig. 3), stimulating mice twice with either of these preparations led to different outcomes of infection. Double injections of L. major SEAgs alone induced greater protection against challenge with L. major, but unexpectedly, double injections of L. major SEAgs plus IL-12 plus alum induced no protection (Fig. 5).
In an effort to define the mechanism underlying the inability of double injections of L. major SEAgs plus IL-12 plus alum to induce protection against L. major challenge in BALB/c mice, we analyzed the cytokines produced by mice treated with either L. major SEAgs alone or L. major SEAgs plus IL-12 plus alum. All cytokine data are contained in Table 3 and Fig. 6 and 7. The only clear difference in Table 3 (mice immunized once with either L. major SEAgs or L. major SEAgs plus IL-12 plus alum) is that the antigen plus IL-12 plus alum-treated mice produced less IFN-γ, especially when challenged with L. major parasites in vitro. Thus, even after a single injection of L. major SEAgs plus IL-12 plus alum, mice were less able to produce IFN-γ; however, this amount of IFN-γ must have been sufficient, since the mice were protected from infection.
When cells from mice injected twice with L. major SEAgs plus IL-12 plus alum were harvested before the animals were infected with L. major parasites (Fig. 6), the cells produced more of all cytokines in vitro, but especially IFN-γ and IL-10. When cells were harvested from the mice after infection with L. major (Fig. 7), they continued to produce more IL-10 whether they were stimulated with L. major SEAgs or with L. major parasites. In contrast, cells from L. major SEAgs plus IL-12 plus alum-treated mice no longer produced more IFN-γ when stimulated with L. major SEAgs, and the cells produced nearly undetectable levels of IFN-γ when they were stimulated with L. major parasites (a result similar to that shown in Table 3).
Therefore, cells from mice vaccinated twice with L. major SEAgs plusIL-12 plus alum tended to produce less IFN-γ (especially when the cells were harvested from infected mice and the cells were restimulated in vitro with L. major parasites), but more IL-10. IL-10 is known to favor the survival of L. major in mice. Indeed, it has been shown by Sacks and colleagues (4, 5) that resistant mice harbor a low number of parasites in their tissues for their lifetime unless IL-10 is neutralized in the mice, which leads to complete destruction of L. major. That is, survival of L. major in mice is determined by the interplay that occurs between IFN-γ (which induces parasite destruction) and IL-10 (which favors parasite survival). If one or the other cytokine is overproduced, the parasite will or will not be destroyed, respectively.
IL-12 has been shown to induce both IFN-γ and IL-10 in mice injected with the cytokine (20). Thus, it is possible that in mice treated with L. major SEAgs plus IL-12 plus alum, the IL-12 present in the inoculum induced high levels of not only IFN-γ but also IL-10. In fact, this was the case, since mice treated with L. major SEAgs plus IL-12 plus alum were producing high levels of both IFN-γ (although cells from these animals did not produce IFN-γ when restimulated with L. major in vitro; Fig. 7) and IL-10 prior to infection with L. major (Fig. 6) and after infection (Fig. 7) with the parasite. In contrast, mice vaccinated with L. major SEAgs alone produced less IL-10 both before and after infection (in some cases as much as 10-fold less; Fig. 6 and 7). Thus, the ability of double injections of L. major SEAgs plus IL-12 plus alum to stimulate IL-10 production (Fig. 6 and 7) may be the reason it was unable to induce protection in treated mice.
It is important to mention that Noormohammadi et al. (22) recently made observations that may have direct bearing on the results presented here. These authors were studying the effect of including IL-12 DNA on the immunogenicity of gp46/M2/PSA-2 DNA for mice. While gp46/M2/PSA-2 DNA alone induced protection against a subsequent challenge with L. major, coadministering gp46/M2/PSA-2 and IL-12 DNA abolished protection. Since gp46/M2/PSA-2 is a component of L. major SEAgs (Fig. 8), it is possible that these authors observed a phenomenon similar to that reported here.
Taken as a whole, the results presented here confirm that L. major SEAgs are highly immunogenic and that they can elicit significant protection against challenge with L. major in susceptible BALB/c mice. Indeed, Webb et al. reported that injecting mice with leishmanial culture filtrate proteins plus Corynebacterium parvum also induced a mixed type 1-type 2 response and resistance to infection with L. major (30). Thus, a different form of the excreted and secreted antigens of Leishmania spp. also appears to be immunogenic and capable of inducing protection against challenge with L. major. Perhaps the most interesting attribute of L. major SEAgs is their ability to elicit protection in the absence of any adjuvant. Therefore, L. major SEAgs may be useful not only as a diagnostic tool but also in the development of vaccines against Old World cutaneous leishmaniasis.
Acknowledgments
We thank Karen Collins for assisting with antigen production. We acknowledge the technical assistance of Jeremy Jones, Leanna Nosbisch, Sheryl Carter, and Jeanette V. Bishop and the helpful discussions of R. Dean Gillespie.
The Medical Research and Materiel Command, Military Infectious Diseases Research Program, contract number DAMD17-01-P-0237, and NIH grants AI 27511 and 29955 funded this work.
Editor: W. A. Petri, Jr.
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