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Infection and Immunity, September 2005, p. 5842-5852, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5842-5852.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vaccination with the Leishmania infantum Acidic Ribosomal P0 Protein plus CpG Oligodeoxynucleotides Induces Protection against Cutaneous Leishmaniasis in C57BL/6 Mice but Does Not Prevent Progressive Disease in BALB/c Mice

Salvador Iborra,1 Javier Carrión,1 Charles Anderson,2 Carlos Alonso,1 David Sacks,2 and Manuel Soto1*

Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, 28049 Madrid, Spain,1 Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208922

Received 15 February 2005/ Returned for modification 25 March 2005/ Accepted 15 April 2005


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ABSTRACT
 
We have examined the efficacy of the administration in mice of a molecularly defined vaccine based on the Leishmania infantum acidic ribosomal protein P0 (rLiP0). Two different challenge models of murine cutaneous leishmaniasis were used: (i) subcutaneous inoculation of L. major parasites in susceptible BALB/c mice (a model widely used for vaccination analysis) and (ii) the intradermal inoculation of a low infective dose in resistant C57BL/6 mice (a model that more accurately reproduces the L. major infection in natural reservoirs and in human hosts). First, we demonstrated that C57BL/6 mice vaccinated with LiP0-DNA or rLiP0 protein plus CpG oligodeoxynucleotides (ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load. This protection was associated with production of gamma interferon (IFN-{gamma}) in the dermal site. Secondly, we showed that immunization with rLiP0 plus CpG ODN is able to induce only partial protection in BALB/c, since these mice finally developed a progressive disease. Further, we demonstrated that LiP0 vaccination induces a Th1 immunological response in both strains of mice. In both cases, the antibodies against LiP0 were predominantly of the immunoglobulin G2a isotype, which was correlated with an rLiP0-stimulated production of IFN-{gamma} in draining lymph nodes. Finally, we demonstrated that LiP0 vaccination does not prevent the Th2 response induced by L. major infection in BALB/c mice. Taken together, these data indicate that the BALB/c model of cutaneous leishmaniasis may undervalue the potential efficacy of some vaccines based on defined proteins, making C57BL/6 a suitable alternative model to test vaccine candidates.


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INTRODUCTION
 
Vaccines for a variety of diseases caused by intracellular infections like tuberculosis, malaria and leishmaniasis require the induction and maintenance of cellular immune responses. Infection by Leishmania major, the main etiologic agent of zoonotic cutaneous leishmaniasis in the Old World, has been extensively used in mouse models to understand the requirements for effective vaccination. Despite the evidence of acquired immunity and resistance to reinfection in natural Leishmania hosts, suggesting that a vaccine is feasible, there is no vaccine for human leishmaniasis. The chemotherapy available at present for this disease is far from satisfactory and there is increasing resistance against those drugs (6).

To date, leishmanization is the most effective induction of protective immunity to prevent disfiguring cutaneous leishmaniasis (9, 17, 23). However, this active infection with live parasites has been restricted or abandoned completely due to several associated problems, such as the development of uncontrolled skin lesions and immunosuppression (12). Recent advances in the design of vaccines against leishmaniasis are based on molecularly defined antigens, the so-called second-generation vaccines (24). One of these molecules is the L. infantum acidic ribosomal P0 protein (rLiP0), a structural component of the large ribosome subunit that has been described as an immunodominant antigen recognized by sera from both patients and dogs infected with L. chagasi-L. infantum (31, 32). We have previously shown that DNA vaccination with this antigen partially protects BALB/c mice against L. major infection (16). In addition, the C-terminal region of the LiP0 is present in a multicomponent protein that when administered to dogs, using live BCG as adjuvant, confers protection against L. infantum infection (22).

Effective primary immunity against L. major in mouse is known to require interleukin 12 (IL-12)-dependent production of gamma interferon (IFN-{gamma}) from CD4+ T cells (Th1 response), which mediates nitric oxide (NO)-dependent killing by infected macrophages (recently reviewed in references 10, 27, and 29). Whereas C57BL/6 mice develop protective Th1 responses and control infection, susceptible BALB/c mice exhibit an IL-4-driven Th2 response produced by a restricted population of Vß8/V{alpha}4 CD4+ T cells and are unable to control infection. The genetic susceptibility of BALB/c mice to L. major may be prevented by treatment with IL-12 protein or neutralizing antibodies to IL-4 at the time of infection, which has been shown to shift the immune response to a Th1 profile (15, 28). The usefulness of this model for vaccine development relies on the possibility to modify the Th responses and to assess the correlation of these responses with functional and biological outcomes.

Although most studies involving vaccination against cutaneous leishmaniasis have been conducted in BALB/c mice, the healing lesions observed in C57BL/6 mice may provide a more relevant model of L. major infection in natural reservoirs and in human hosts. This infection model takes into account two main features of natural transmission: low dose (100 to 1,000 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). The analysis of adaptive immunity in this model confirmed a role for Th1 cells, and in addition revealed an essential requirement for CD8+ T cells (2).

Several vaccine studies have demonstrated that long-lasting cellular responses and protection against L. major may be achieved by genetic vaccination, or by coinoculation of Leishmania antigen plus immunostimulatory CpG oligodeoxynucleotides (ODN) (11, 25). It has been shown also that CpG ODN trigger a therapeutic response in BALB/c mice infected with L. major (35) or L. donovani (7). Finally, it has been demonstrated that the pathogenicity of leishmanization may be reduced by the coinjection of the parasite with CpG ODN (21). These effects probably rely on their ability to induce both innate and adaptive cellular immune responses. These ODN stimulate macrophages and dendritic cells (DC) to synthesize several cytokines including IL-12, IL-18, tumor necrosis factor alpha (TNF-{alpha}), IFN-{alpha}, IFN-ß, and IFN-{gamma} and to upregulate costimulatory molecules such as CD40 and major histocompatibility complex class II (MHC-II) (13, 18, 33). Moreover, CpG ODN may activate DC, leading to the presentation of soluble protein to class I-restricted T cells and the induction of cytotoxic T-lymphocyte responses (4, 8, 34).

In the present paper, we have evaluated the vaccination with the LiP0 protein using very different challenge systems: high-dose inocula in the footpad of BALB/c mice (widely used in cutaneous leishmaniasis vaccine assays) and low dose in the ear of C57BL/6 mice (a model that more closely mimics the human disease in terms of route and dose infection). We demonstrate that C57BL/6 mice inoculated with LiP0 plus CpG ODN were protected against cutaneous leishmaniasis, showing a reduction of the parasite number in the inoculation site, and a moderation of dermal pathology. In contrast, BALB/c mice vaccinated in the same fashion were only partially protected against L. major challenge, developing delayed but ultimately progressive, nonhealing lesions. We show that although LiP0 vaccine induces a specific Th1 response in both strains it does not abrogate the Th2 response induced by L. major challenge in BALB/c mice, which may account for its different efficacy in these two models.


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MATERIALS AND METHODS
 
Mouse strains and parasites. Female BALB/c and C57BL/6 mice were 6 to 8 weeks old at the onset of experiments and were purchased from Harlan Interfauna Ibérica S.A. (Barcelona, Spain). L. major parasites (clone WHOM/IR/173) and clone V1 (MHOM/IL/80/Friedlin) were kept in a virulent state by passage in BALB/c mice. The L. major amastigotes were obtained from popliteal lymph nodes, and after transformation from amastigote to promastigote form, parasites were cultured and harvested at late stationary phase. Promastigotes of both clones were cultured at 26°C in Schneider's medium (Gibco BRL) supplemented with 20% fetal calf serum (FCS). Infective-stage promastigotes (metacyclics) of L. major (clone V1) were isolated from stationary cultures by negative selection using peanut agglutinin (Vector Laboratories, Burlingame, CA).

Plasmids, ODN, and leishmanial antigens. The plasmids used in this study were pcDNA3 (empty vector) and pcDNA3 encoding the Leishmania infantum P0 ribosomal protein (pcDNA3-LiP0) constructed as described previously (16). Endotoxin-free plasmid DNA was isolated using the EndoFree Plasmid Giga kit (QIAGEN, Hilden, Germany). Plasmids used in this study were all suspended in sterile phosphate-buffered saline (PBS).

Phosphorothioate-modified ODN sequences containing CpG motifs were synthesized by Isogen (The Netherlands). The sequences of the immunostimulatory ODN (5' to 3') were TCAACGTTGA and GCTAGCGTTAGCGT. The his-tagged rLiP0 was produced in Escherichia coli M15 transformed with pQE-LiP0 and purified as previously described (16). The recombinant protein was further passed through a polymyxin-agarose column (Sigma, St. Louis, Mo.) to eliminate endotoxins. The lipopolysacharide content was measured by the Quantitative Chromogenic Limulus Amebocyte Assay QCL-1000 (BioWhittaker, Walkersville, Md.), showing that preparations were essentially free of endotoxin (<12 pg µg–1 of rLiP0).

A library of overlapping peptides covering the whole LiP0 protein sequence was synthesized by the simultaneous multiple-peptide solid phase synthetic method using a polyamide resin and FMOC chemistry as described before (32). ALM (autoclaved L. major) is prepared from freeze-thaw lysates of autoclaved L. major promastigotes bound to aluminum hydroxide, prepared as previously described (14).

Total proteins of L. major (soluble Leishmania antigen [SLA]) were prepared by three freezing and thawing cycles of stationary promastigotes of L. major suspended in PBS. After cell lysis, soluble antigens were separated from the insoluble fraction by centrifugation for 15 min at 12,000 x g using a microcentrifuge.

Immunizations and parasite challenge. C57BL/6 mice were injected subcutaneously (s.c.) in the footpad with either PBS, 100 µg of pcDNA3 (empty vector) or pcDNA3-LiP0. Each group was inoculated three times at 2-week intervals. Additional groups were immunized with either 10 µg of rLiP0 plus 50 µg of CpG ODN (25 µg of each immunostimulatory ODN), 50 µg of ALM plus CpG ODN (50 µg), CpG ODN (50 µg) adjuvant alone, or PBS. These mice were boosted two weeks later with the same immunization regimen. The infection was performed 4 weeks after the last vaccination by intradermal (i.d.) inoculation of 1,000 metacyclic promastigotes of L. major (clone V1) into the dermis of both ears of the mouse. The evolution of the infection was monitored by measuring the diameter of the induration of the ear lesion with a metric caliper.

BALB/c mice were s.c. inoculated in the right footpad with either 0.5, 2, or 10 µg of rLiP0 plus 50 µg of CpG ODN (25 µg of each immunostimulatory ODN), CpG ODN (50 µg) adjuvant alone or PBS. Each group was boosted 2 weeks later using the same regimen. Parasite challenge was carried out by s.c. inoculation with 5 x 104 stationary-phase promastigotes of L. major (clone WHOM/IR/-173) into the left (untreated) footpad four weeks after the last inoculation. The progress of the infection was followed by measuring the thickness with a metric caliper. The contralateral footpad of each animal represented the control value, and the swelling was calculated as follows: thickness of the left footpad minus thickness of the right footpad. The animals were euthanized when the lesions became necrotic.

Parasite quantitation. The number of parasites was determined in ear and footpad by limiting dilution assay (3). Briefly, footpad sections and ears were recovered from infected BALB/c and C57BL/6 mice, respectively. The ventral and dorsal sheets of the infected ears were separated. The footpad sections and ear sheets were deposited in Dulbecco's modified Eagle medium containing Liberase CI enzyme blend (50 µg ml–1). After 2 h of incubation at 37°C, the tissues were cut into small pieces, homogenized and filtered using a cell strainer (70-µm pore size). The homogenized tissue was serially diluted in a 96-well flat-bottomed microtiter plate containing Schneider's medium plus 20% FCS or biphasic medium, prepared using 50 µl of NNN medium containing 30% defibrinated rabbit blood and overlaid with 50 µl of M199/S as previously described (21). The number of viable parasites was determined from the highest dilution at which promastigotes could be grown up to 7 days of incubation at 26°C. The number of parasites was also determined in the local draining lymph nodes (DLN) of infected ears (retromaxillar) and footpad (popliteal). The lymph nodes were recovered, mechanically dissociated and then serially diluted as above. In the ear and LN the parasite load is expressed as the number of parasites in the whole organ. In the footpad the parasite load is expressed as the number of parasites per mg tissue.

Measurement of cytokines in supernatants. Spleens and lymph nodes were removed aseptically from mice after cervical dislocation. Splenocytes and draining lymph node cell (LNC) suspensions were seeded in complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, and 10 mM 2-mercaptoethanol). Next, 3 x 106 cells were seeded in 48-well plates during 72 h at 37°C in the presence of rLiP0 (3 µg ml–1) or SLA (12 µg ml–1). The release of IFN-{gamma} and IL-4 was measured in the supernatants of splenocytes and LNC cultures, and was determined by commercial enzyme-linked immunosorbent assay (ELISA) kits (Diaclone, Besançon, France). In parallel, LNCs stimulated with rLiP0 were incubated in the presence of 10 µg ml–1 of monoclonal antibody (MAb) against either mouse CD4 (GK 1.5), mouse IL-12 (C17.8), or mouse CD8 (53-6.7). Appropriate isotype-matched controls were also analyzed in the assay. The antibodies (no azide/low endotoxin) were purchased from BD (PharMingen).

When bone marrow-derived dendritic cells (BMDDC) were used as antigen- presenting cells they were pulsed for 24 h with L. major amastigotes, at a parasite to cell ratio of 5:1. Afterwards, dermal cell suspensions from infected mice were stimulated with L. major-infected or uninfected BMDDC (2 x 105) in complete RPMI medium. Then IFN-{gamma} production was analyzed in 48-h supernatants as described above. For T-cell epitope determination, BMDDC (2 x 105) were pulsed for 16 h with either rLiP0 (12 µg ml–1) or each peptide (15 µg ml–1) covering the whole LiP0 sequence in U-bottom plates. Aferwards, BMDDC were harvested, washed and used to stimulate splenocytes from LiP0-vaccinated mice. Cytokine production was analyzed in supernatants after 72 h.

Intracytoplasmic staining of IFN-{gamma}. For the analysis of the frequency of T-cell-producing IFN-{gamma}, single-cell suspensions from four ears were cultured in a 24-well tissue plate. Subsequently, the dermal cells were stimulated for 16 h with L. major-infected BMDDC, as described before. Then, brefeldin A (10 µg ml–1) reagent was added to the culture and further incubated for an additional period of 6 h. Afterwards, cells were harvested, washed twice in PBS with 1% FCS and incubated with an anti-FC{gamma} III/II receptor (FcBlock) prior to staining. Then cells were stained for the surface markers TCRß (H57-597; Cy-chrome labeled) and either CD4 (GK1.5; fluorescein isothiocyanate-labeled) or CD8 (53-6.7, fluorescein isothiocyanate-conjugated) for 30 min on ice. Cells were then washed twice and fixed for 20 min in Cytofix/Cytoperm buffer. Next, cells were washed twice and incubated with phycoerythrin (PE)-conjugated rat anti-mouse IFN-{gamma} (XGM.1.2) for 30 min at 4°C. Finally cells were washed twice and analyzed on a FACSCalibur flow cytometer. The specificity of the anti-cytokine MAb was tested by both negative staining of nonpermeabilized cells and a PE-conjugated isotype-matched control (R3-34). For each sample, at least 100,000 cells were analyzed. The data were collected and analyzed using FACScalibur and CELLQuest software. The frequency of CD4+ and CD8+ T cells was determined by gating on TCRß+cells. All reagents and conjugate monoclonal antibodies were purchased from Becton Dickinson (PharMingen, San Diego, CA).

Determination of antibody titers and isotypes. Serum samples were analyzed for specific anti-LiP0 antibodies. Briefly, standard ELISA plates were coated overnight at room temperature with 100 µl of rLiP0 (2 µg ml–1 in PBS). The titer was determined by serial dilution of the sera, and was defined as the inverse of the highest serum dilution factor giving an absorbance of >0.2.

The Falcon assay screening test-ELISA (FAST-ELISA; Becton Dickinson) was used instead of the standard ELISA for the determination of the reactivity against the synthetic peptides covering the LiP0 sequence. The lids were coated with 100 µl of each synthetic peptide (100 µg ml–1) as described before (32). The isotype-specific analyses were done with the following horseradish peroxidase-conjugated anti-mouse immunoglobulins (Igs) (Nordic Immunological Laboratories, Tilburg, The Netherlands): anti-IgG (1:2,000), anti-IgG1 (1:1,000) and anti-IgG2a (1:500). Orthophenylene diamine dihydrochloride (OPD; Dako A/S, Glostrup, Denmark) was used as peroxidase substrate. After 15 min, the reaction was stopped with the addition of 100 µl of H2SO4 (1 M) and the absorbance was read at 450 nm.

Statistical analysis. Statistical analysis was performed by Student's t test. Differences were considered significant when P < 0.05.


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RESULTS
 
Vaccination with LiP0-DNA or rLiP0 plus CpG ODN confers protection against dermal pathology due to L. major challenge in C57BL/6 mice. We have previously described that vaccination with LiP0 DNA (pcDNA3-LiP0) partially protects against L. major infection in BALB/c mice. Here, this antigen has been tested, either as a DNA vaccine or as recombinant protein (rLiP0) adjuvanted with CpG ODN, using low-dose intradermal challenge in C57BL/6 mice.

Mice inoculated with empty vector (pcDNA3) or CpG ODN alone, and challenged 4 weeks later with 1,000 metacyclic promastigotes in the ear dermis, developed lesions similar in size and duration to unvaccinated mice (PBS alone) (Fig. 1). These lesions appeared at week 4, reached a peak at 6 to 7 weeks, and were almost completely healed at 14 to 16 weeks. In contrast, mice vaccinated with either LiP0-DNA or rLiP0 (10 µg) plus CpG ODN were protected against the development of dermal lesions since little or no pathology was observed (particularly in the rLiP0 plus CpG ODN-vaccinated mice). Since the number of parasites in the infected site peaks just before the development of lesion (1), we determined the parasite load in the ear and in the local draining lymph node (retromaxillar) at week 4. The number of parasites in the ear (Fig. 2A) and in the DLN (Fig. 2B) was not significantly different between unvaccinated mice (PBS group) and mice vaccinated with CpG ODN alone or control DNA. In contrast, mice vaccinated with LiP0-DNA had a ~40- to 50-fold reduction in the parasite load in the ear skin (1.5 x 105 parasites for LiP0-DNA versus 6.6 x 106 parasites for control DNA), whereas mice vaccinated with rLiP0 plus CpG had a ~150-fold reduction (4.2 x 104 parasites for rLiP0 plus CpG versus 6.4 x 106 parasites in CpG mice). Similarly, the parasite number in the DLN was reduced ~25-fold in mice vaccinated with rLiP0 plus CpG (1.1 x 104 parasites) compared with mice vaccinated with CpG (2.7 x 105), and ~40-fold in mice vaccinated with LiP0-DNA (1.9 x 104 parasites) compared with mice vaccinated with empty vector (8 x 105 parasites). (Fig. 2B). The highest reduction in the number of parasites and in dermal pathology was observed in ALM plus CpG ODN-inoculated mice, a vaccine that has been previously observed to confer an excellent protection in this model of infection (25). After healing, in control mice (16 weeks after challenge) a reduction in the number of parasites was observed in all of the groups, although differences between vaccinated and control mice still remained. Thus, the reduced pathology observed in LiP0-vaccinated mice correlates well with a reduction in the parasite burden in the skin and in the local DLN.



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  		FIG. 1. Course of L. major infection in C57BL/6 vaccinated mice. Mice (eight per group) were vaccinated s.c. with three doses of either 100 µg of pcDNA3 or 100 µg of pcDNA3-LiP0, and twice with 10 µg of rLiP0 plus 50 µg of CpG ODN, 50 µg of ALM plus 50 µg of CpG ODN, 50 µg of CpG ODN alone or PBS. 4 weeks after the last inoculation, mice were infected by i.d. inoculation into the ear with 1,000 metacyclic promastigotes of L. major. Values represent the mean lesion diameter + standard deviation (SD). *, P < 0.001 significant decrease for ALM or rLiP0 plus CpG ODN compared with CpG ODN alone, and pcDNA3-LiP0 compared with pcDNA3. The experiment was repeated with similar results.



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  		FIG. 2. Parasite burden in the ear dermis (A) and in the local draining lymph nodes (B) in C57BL/6 mice quantitated at 4 weeks postinfection (white bars) and 16 weeks postinfection (grey bars). Results are expressed as the mean ± SD of eight ears and draining lymph nodes. *, P < 0.05, significant decrease for rLiP0 plus CpG ODN or ALM plus CpG ODN compared with CpG ODN alone, and pcDNA3-LiP0 compared with pcDNA3. Results represent the mean and standard deviation. The experiment was repeated with virtually the same results.

Protection against L. major infection in LiP0-vaccinated C57BL/6 mice is correlated with a specific Th1 response. Immunity to leishmaniasis is known to depend on a Th1-type response against the parasite. To test whether LiP0 administration is able to induce a Th1 response against L. major, IFN-{gamma} production was analyzed 4 weeks after challenge. In addition, we evaluated the frequency of IFN-{gamma}-producing T cells in the infected ears by flow cytometry. Dermal cell suspensions from ears and retromaxillar DLN cells were in vitro restimulated with uninfected or L. major-infected BMDDC and supernatants were assayed 48 h later. Both dermal cell suspensions (Fig. 3A) and DLN cells (Fig. 3B) from mice vaccinated with either LiP0-DNA or rLiP0 plus CpG ODN secreted higher levels of IFN-{gamma} compared with cells from control mice. The highest levels of this cytokine were found in the supernatants of cells from mice vaccinated with ALM plus CpG ODN. As shown in Fig. 3C, the frequency of CD4+ and CD8+ T cells producing IFN-{gamma} in LiP0-vaccinated mice was substantially higher after stimulation with L. major-infected BMDDC compared to uninfected BMDDC stimulation. The increase was more accentuated in CD4+ (~3.5-fold increase in both LiP0-vaccinated groups) than in CD8+ T cells (~2.8-fold increase). In contrast, BMDDC pulsed with L. major stimulated only a low frequency of T cells in mice immunized with either empty DNA or CpG alone (<2-fold increase). Thus, these data are a direct support that protection against L. major infection in LiP0-vaccinated mice is correlated with a specific Th1 response.



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  		FIG. 3. Leishmania-specific production of IFN-{gamma} after infection with L. major. C57BL/6 mice were vaccinated as described above and then challenged with 1,000 metacyclic promastigotes of L. major. Dermal cell suspensions from the pooled infected ears (A) and DLN cells (B) were obtained 4 weeks after infection from C57BL/6 mice (four mice per group, eight ears and eight DLN). The cultures were stimulated with either uninfected or L. major-infected BMDDC. IFN-{gamma} levels were measured in culture supernatants 48 h later by ELISA. Data are the mean concentrations of triplicate assays + SD. (C) The intracellular production of IFN-{gamma} by CD4+ and CD8+ T cells in C57BL/6 mice was assessed in the dermal cells of mice vaccinated with pcDNA3-LiP0 and rLiP0 + CpG and their corresponding controls (empty DNA vector and CpG, respectively). After 24 h of in vitro restimulation with uninfected BMDDC or infected BMDDC, cells were processed for flow cytometry. Analyses are gated on TCRß+ cells. Numbers represent the percentage of CD4+ or CD8+ T cells positive for IFN-{gamma}.

Inoculation of rLiP0 plus CpG ODN induces partial protection against L. major infection in BALB/c mice. Taking into account that rLiP0 plus CpG ODN confers a protective response that almost completely abrogates cutaneous leishmaniasis in C57BL/6 mice, we decided to assess if this vaccine is also effective in the BALB/c model of infection. Mice were vaccinated with CpG ODN or with rLiP0 (10, 2, or 0.5 µg) plus CpG ODN. In all cases, IgG2a antibodies specific for LiP0 were detected (data not shown), suggesting that each one of these formulations is able to induce a Th1-type immune response. Subsequently, mice were challenged with 5 x 104 L. major stationary promastigotes 4 weeks after the last inoculation. Mice of all of the groups developed a progressive footpad swelling and ulcerative and necrotic lesions, and consequently were euthanized at week 8 of infection. Interestingly, a delay in the footpad swelling was observed in mice immunized with 10 µg of rLiP0 plus CpG ODN (Fig. 4A). The protection was rLiP0 dose dependent, since reduction of inflammation and pathology was only patent in mice inoculated with 10 µg of rLiP0 protein and no significative protection was observed in mice inoculated with either 2 or 0.5 µg. When parasite load was analyzed at week 4 after infection, also a partial protection was detected (Fig. 4B and Fig. 4C). Mice vaccinated with 10 µg of rLiP0 plus CpG ODN showed a ~150-fold and a ~100-fold reduction in the parasite number in the footpad and in the popliteal lymph node, respectively (compared with CpG-vaccinated group). Parasite load in the footpad and in the DLN increased with time in the vaccinated groups and, at 8 weeks after infection, only slight differences were observed between mice vaccinated with 10 µg of rLiP0 plus CpG ODN and control mice (Fig. 4B and 4C).



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  		FIG. 4. Course of L. major infection in BALB/c vaccinated mice. (A) BALB/c mice (six per group) were immunized s.c. twice with either PBS, 50 µg CpG ODN or rLiP0 (0.5, 2, or 10 µg) plus 50 µg of CpG ODN. One month after the last immunization, the animals were infected in the left hind footpad with 5 x 104 L. major promastigotes. Footpad swelling is given as the difference of thickness between the infected and the uninfected contralateral footpad. Four weeks after challenge, differences between controls (CpG ODN) and vaccinated mice (10 µg of rLiP0 plus CpG ODN) were found to be significant (*, P < 0.001). At weeks 4 (white bars) and 8 after infection (grey bars), the number of viable parasites present in the footpad per mg (B) and total parasites in the popliteal lymph node of the infected leg (C) were determined by limiting dilution. Results represent the mean and standard deviation representative of three independent experiments (*, P < 0.05).

A specific Th1 response is induced after administration of rLiP0 plus CpG ODN in both C57BL/6 and BALB/c mice. For a better understanding of the distinct protective capacity of the LiP0 vaccine in the two infection models, we assessed the immune responses against this antigen in both C57BL/6 and BALB/c mice. We have previously described that rLiP0 administration in BALB/c mice is able to induce a specific IgG response in the absence of adjuvant, with a preponderance of the IgG1 isotype (16). Here, our data show that after vaccination with 10 µg of rLiP0 plus CpG ODN, the anti-LiP0 response was predominantly of the IgG2a isotype in both C57BL/6 and BALB/c mice (Fig. 5A). Since the induction of IgG1 and IgG2a antibodies is used as a marker of Th2-type and Th1-type immune responses, respectively (5), we may conclude that CpG ODN adjuvant is able to switch the immune response against LiP0 from a Th2 to a Th1 response.



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  		FIG. 5. Analysis of the specific humoral response induced in BALB/c and C57BL/6 mice immunized with 10 µg of rLiP0 plus CpG ODN. One month after the second inoculation, mice (n = 6) were bled and sera were tested by ELISA for specific anti-LiP0 antibody responses of both IgG1 and IgG2a isotypes (A). Each sample was assayed separately, and the titer was determined as indicated in the Materials and Methods section. The reactivity of these sera against the synthetic peptides covering the LiP0 sequence was analyzed by FAST-ELISA (B). The optical density at 450 nm (OD450) values represent the reactivities obtained with the pooled sera diluted 1:500 using as second antibody a horseradish peroxidase-conjugated anti-IgG at 1:2,000 dilution. None of the preimmune, the PBS or the CpG ODN control sera showed reactivity against either the recombinant protein or the synthetic peptides (not shown).

The B-cell epitope distribution was analyzed by screening a collection of 22 synthetic peptides of 20 amino acids, overlapping by five amino acids, covering the entire sequence of the protein with the sera from mice vaccinated with LiP0. As shown in Fig. 5B, sera from both mouse strains exhibited a high reactivity against the C-terminal sequence shared by peptides P21 and P22 that are almost identical. This C-terminal sequence (AAKEREPEESDEDDFGMG) is the unique linear antigenic determinant in the LiP0 protein recognized by sera from dogs suffering viscerocutaneous leishmaniasis (32). In addition, sera from vaccinated mice identified some other linear antigenic B-cell determinants that are either recognized by both strains (peptide P5) or only for one of them (peptide P9 by the sera from C57BL/6 while peptides P6 and P14 by the sera from BALB/c mice).

In addition, we have analyzed the cytokine production induced by LiP0 vaccination in both strains of mice. After in vitro stimulation with rLiP0, DLN cells from rLiP0 + CpG ODN- vaccinated mice secreted higher levels of IFN-{gamma} than controls (Fig. 6A). No increase in IL-4 production was observed after stimulation with rLiP0 for all of the groups (data not shown). Notably, mice vaccinated with CpG ODN secreted also higher levels of IFN-{gamma} than mice inoculated with PBS, suggesting that the effect of the adjuvant is still patent at week 4 after vaccination. The contribution of CD4+ and CD8+ T cells and the dependence on IL-12 to the rLiP0-specific production of IFN-{gamma} was also analyzed in vitro. As shown in Fig. 6B, cytokine synthesis induced by rLiP0 was completely inhibited by anti-IL-12 or anti-CD4 monoclonal antibodies, independently of the strain. The addition of anti-CD8 antibodies partially reduced the levels of this cytokine in the supernatants of DLN from both strains. Thus, in both strains of mice, vaccination with rLiP0 and CpG ODN induced an IL-12-dependent specific IFN-{gamma} response that is mainly mediated by CD4+ T cells, but CD8+ T cells also contribute to this response.



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  		FIG. 6. Cytokine production by draining LNCs of LiP0-vaccinated mice. Mice (three per group) were immunized twice s.c. with either PBS, 50 µg CpG ODN or 10 µg of rLiP0 plus 50 µg CpG ODN. One month after the last immunization, the animals were euthanized, and their LNCs were obtained and cultured in vitro for 3 days in the presence of rLiP0 or medium alone (panel A). In parallel, DLN cultures were stimulated with rLiP0 in the presence of either anti-IL-12, anti-CD4 or anti-CD8 monoclonal antibodies (panel B). In both cases, the supernatants were harvested and assayed for cytokine production. The levels of IFN-{gamma} were assessed by ELISA after 72 h of incubation. Data correspond to one representative experiment of two independent experiments with similar results. Differences in IFN-{gamma} production between treatment with anti-CD8 monoclonal antibodies and treatment with control antibodies were statistically significant (*, P < 0.05).

Finally, we mapped the epitopes in the LiP0 antigen responsible for the IFN-{gamma} synthesis. Splenocytes from the LiP0-vaccinated mice were stimulated with BMDDC pulsed with rLiP0 protein or the collection of synthetic peptides. Interestingly, spleen cells from C57BL/6 mice secreted higher levels of IFN-{gamma} after stimulation with rLiP0 than those from BALB/c (Fig. 7). Moreover, splenocytes from C57BL/6 mice only secreted IFN-{gamma} in response to one peptide (P13), containing the sequence LLQKLNISPFYYQVNVLSVW, whereas this peptide did not induce cytokine synthesis either in splenocytes from control mice (CpG ODN) or in splenocytes from vaccinated BALB/c mice. In this strain, IFN-{gamma} synthesis was only slightly induced by BMDDC pulsed with two peptides (P1 and P4). Thus, these results indicate that IFN-{gamma}-producing T cells from vaccinated mice recognized different antigenic determinants in the LiP0 antigen, depending on the mouse strain.



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  		FIG. 7. Spleen cell IFN-{gamma} response to LiP0 and its synthetic peptides. Splenocytes from BALB/c and C57BL/6 mice vaccinated with 10 µg of rLiP0 plus 50 µg CpG ODN were stimulated with BMDDC pulsed with whole rLiP0 or the synthetic peptides covering the LiP0 sequence. The levels of IFN-{gamma} were assessed by ELISA after 72 h of incubation. Data correspond to one representative experiment of two independent experiments with similar results.

Vaccination with rLiP0 plus CpG ODN does not prevent a Th2 response in BALB/c mice infected with L. major. Since BALB/c mice vaccinated with LiP0 were partially protected against L. major infection, we decided to analyze if this vaccine reversed the Th2 response induced by the parasite. First, we analyzed the humoral response elicited against the LiP0 antigen in both strains of vaccinated mice 8 weeks after challenge. As shown in Fig. 8A, the antibodies against LiP0 were still predominantly IgG2a in C57BL/6 mice. In contrast, a mixed IgG1/IgG2a anti-LiP0 response was observed in BALB/c mice, indicating that the L. major infection induced a Th2 response against LiP0 in these mice. Second, we analyzed the IL-4 response 4 and 8 weeks after challenge. Splenocytes from control and vaccinated mice were in vitro re-stimulated with rLiP0 and SLA. No IL-4 was detected in C57BL/6 mice (data not shown). In addition, no IL-4 production could be detected in response to rLiP0 stimulation in BALB/c mice, whereas SLA induced IL-4 synthesis in both control and vaccinated mice (Fig. 8B). Concomitant with the early protection observed, at 4 weeks after infection the levels of IL-4 were lower in the vaccinated mice than in controls. Nevertheless, 8 weeks after challenge there was not a significant difference between these groups of mice. These data demonstrate that vaccination with rLiP0 plus CpG ODN is not enough to prevent the Th2 response induced by L. major infection in BALB/c mice.



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  		FIG. 8. Analysis of the Th2 response after L. major infection. (A) Eight weeks after parasite challenge vaccinated BALB/c and C57BL/6 mice (n = 5) were bled and sera were tested by ELISA for specific anti-LiP0 antibody responses of both IgG1 and IgG2a isotypes. The titers of the anti-rLiP0 antibodies in the BALB/c CpG control mice at week 8 after challenge were 1/2,700 and 1/900 for IgG1 and IgG2a, respectively (not shown). (B) At weeks 4 and 8 after the L. major challenge, the animals were euthanized, and their splenocytes were obtained and cultured in vitro for 3 days in the presence of rLiP0 or SLA. The supernatants were harvested and assayed for IL-4 by ELISA.


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DISCUSSION
 
In this study, we have evaluated the protective efficacy of a vaccine based on a molecularly defined antigen, the L. infantum acidic ribosomal protein P0, in two different models of infection, using two different mouse strains, C57BL/6 and BALB/c. First, the P0 protein has been tested in a C57BL/6 mouse model of L. major infection that more accurately mimics the pathology of the human cutaneous disease and the low-dose intradermal inoculation associated with sand fly challenge (1). The development of dermal lesions in this model occurs only after a prolonged silent phase of parasite replication in the skin and is dependent on and coincident with the onset of acquired immunity and the killing of parasites in the inoculation site. Using this challenge model, the criteria for vaccine efficacy may be more rigorously defined as an absence of cutaneous lesions. We have demonstrated that inoculation of the LiP0 antigen either as a DNA vaccine (pcDNA3-LiP0) without adjuvant or as a recombinant protein (rLiP0) with CpG ODN as adjuvant conferred substantial protection against dermal pathology due to L. major infection. This immunity resulted in some cases in a complete absence of dermal lesions, especially in mice vaccinated with rLiP0 plus CpG ODN. As expected, this moderate dermal pathology correlates with a substantial reduction in the parasite number in the infected ear and in the draining lymph node. Thus, our results indicate that vaccination with this single antigen is enough to confer notable protection. This protection is comparable to that displayed by mice vaccinated with ALM plus CpG ODN, and a multicomponent vaccine (composed of LACK, LmSTI1 and thiol-specific antioxidant protein) also tested in this model (19, 20, 25).

In terms of immune correlates of protection, it is well established that a Th1 response is necessary to mediate macrophage nitric oxide production required for parasite killing (reviewed in references 10, 27, and 29). This is consistent with our findings that dermal cells and DLN cells from vaccinated C57BL/6 mice produced larger quantities of IFN-{gamma} than controls at 4 weeks of infection. Moreover, the frequency of IFN-{gamma}-producing T cells was substantially higher in vaccinated mice. All these data indicate that immunization with the LiP0 vaccines induces a Th1 response that is correlated with protection against L. major infection.

Other models, based on a high-dose s.c. inoculum, mainly in BALB/c mice, induce rapidly evolving lesions that are formed as a consequence of large numbers of infected macrophages in the inoculation site. In view of the protection conferred to C57BL/6 mice by the LiP0-based vaccine, we wanted to test the efficacy of the vaccine in this model in which the outcome of the disease is clearly correlated with a strong Th2 response induced by infection. We have previously seen that BALB/c mice vaccinated with LiP0-DNA were partially protected against L. major challenge with high-dose s.c. inocula (16). Here we show that under similar conditions the administration of rLiP0 plus CpG ODN also induced partial protection against L. major infection. The delay in footpad swelling correlates with a reduction in the parasite load in the dermal site and the DLN at 4 weeks after infection. However, these mice ultimately developed nonhealing lesions and did not control infection.

Differences in the vaccination efficacy are not related to great differences in the specific immune responses generated in BALB/c and C57BL/6 mice. We have determined that in both cases the humoral response against the LiP0 protein was Th1-like. The antibodies against LiP0 were predominantly of the IgG2a isotype in both strains. Also, we observed that the C-terminal AAKEEPEESDEDDFGMG, which is the main antigenic determinant recognized by the sera of dogs infected with L. infantum, is also the main antigenic determinant recognized by the sera from vaccinated mice of both strains. In addition we have found two other B-cell epitopes that are specific for BALB/c or C57BL/6 mice and one that is equally recognized by both strains. Overall, we conclude that the humoral immune response induced by the vaccine is roughly equivalent in both strains of mice. The in vitro analysis of the cellular responses confirmed that a specific Th1-like immune response was induced by LiP0 administration in both strains. DLN cells from vaccinated mice secreted higher levels of IFN-{gamma} than controls following in vitro stimulation with rLiP0. In contrast, no IL-4 could be detected in these cultures. In both strains of mice, this IFN-{gamma} response was found to be IL-12 dependent and completely inhibited by anti-CD4 antibodies, whereas it was only partially inhibited by anti-CD8 antibodies. Finally, a single antigenic determinant in peptide P13 (containing the sequence LLQKLNISPFYYQVNVLSVW) stimulated IFN-{gamma} synthesis in splenocytes from C57BL/6 mice vaccinated with rLiP0, at the same level as the whole protein. In contrast, this epitope was not recognized by T cells from immunized BALB/c mice, and only a slight IFN-{gamma} response could be detected with BMDDC pulsed with peptides P1 and P4.

While the administration of rLiP0 plus CpG ODN induced a specific Th1 response in both BALB/c and C57BL/6 mice, this vaccine conferred a robust protection against L. major infection in C57BL/6 mice but did not prevent progressive disease in BALB/c mice. In these mice, the immune response generated by LiP0 administration (effector and/or memory T cells) may be compromised by the same conditions that establish their strong Th2 bias to L. major infection that is observed in naïve mice. In fact, BALB/c mice vaccinated with LiP0 still developed a Th2 response after L. major challenge, as evidenced by the increase in the titer of anti-LiP0 IgG1 antibodies after infection. Moreover, at 8 weeks postinfection splenocytes from control and vaccinated mice produced similar levels of IL-4 following in vitro stimulation with SLA. Thus, the anti-LiP0 Th1 response induced by the vaccine was not able to prevent the development of a Th2 predominant response against other parasite antigens following infection. This result is consistent with previous findings indicating that vaccines inducing a mixed Th1/Th2 response may be not protective against cutaneous leishmaniasis in BALB/c mice (26, 30).

In summary, the natural challenge model has revealed that LiP0 vaccines have the capacity to almost completely protect mice against dermal leishmaniasis. In contrast, these vaccines were not able to abrogate the Th2 response induced by L. major challenge in BALB/c mice and in consequence these mice succumbed to progressive disease. In fact, prior reports suggest that, if the criterion for vaccine efficacy is defined as an absence of progressive disease, some vaccines based on molecularly defined antigens tested in the BALB/c mice are not completely sastifactory (see reference 27 and references within). Among other possibilities, the different efficacies of these vaccine candidates may rely on their capacity not only to induce Th1 responses but to prevent the IL-4-driven Th2 response induced by L. major infection that leads to progressive disease. Taking into account that an IL-4-driven, polarized Th2 response is not clearly associated with the non-healing forms of leishmaniasis in humans (26), we conclude that the BALB/c model of cutaneous leishmaniasis may undervalue the potential of some vaccine candidates. So far as we are aware, there are only three studies that have tested vaccine candidates in the ear model of infection in C57BL/6 mice (19, 20, 25). Thus, we think that further studies in this suitable model will contribute to better characterize the protective efficacy of potential molecularly defined vaccines against this disease.


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ACKNOWLEDGMENTS
 
This work was supported by grant BIO2002-04049-C02-C1 from Ministerio de Ciencia y Tecnología from Spain. Also, an institutional grant from Fundación Ramón Areces is acknowledged.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, 28049 Madrid, Spain. Phone: (34-91) 4978454. Fax: (34-91) 4974799. E-mail: msoto{at}cbm.uam.es. Back

Editor: W. A. Petri, Jr.


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Infection and Immunity, September 2005, p. 5842-5852, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5842-5852.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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