Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

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Microbes and Infection xx (2010) 1e11www.elsevier.com/locate/micinf

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Original article

Vaccination with the Leishmania infantum ribosomal proteins inducesprotection in BALB/c mice against Leishmania chagasi and Leishmania

amazonensis challenge

Miguel A. Chavez-Fumagalli a,1, Mariana A.F. Costa b,1, Dulcilene M. Oliveira c,1, Laura Ramırez d,Lourena E. Costa e, Mariana C. Duarte e, Vivian T. Martins e, Jamil S. Oliveira b,

Carlos C. Olortegi b, Pedro Bonay d, Carlos Alonso d, Carlos A.P. Tavares b, Manuel Soto d,*,1,Eduardo A.F. Coelho b,e,1

aDepartamento de Bioquımica e Imunologia Universidade Federal de Minas Gerais, 31.270-901, Belo Horizonte, Minas Gerais, BrazilbDepartamento de Neurociencias, Universidade Federal de Minas Gerais, 31.270-901, Belo Horizonte, Minas Gerais, Brazil

cCentro de Biologıa Molecular Severo Ochoa, CSIC-UAM, Departamento de Biologıa Molecular. Universidad Autonoma de Madrid, 28049 Madrid, SpaindDepartamento de Farmacologia, Universidade Federal de Minas Gerais, 31.270-901, Belo Horizonte, Minas Gerais, Brazil

eDepartamento de Patologia Clınica, Coltec, Universidade Federal de Minas Gerais, 31.270-901, Belo Horizonte, Minas Gerais, Brazil

Received 15 February 2010; accepted 18 June 2010

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Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in SouthAmerica. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccinationusing Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasitespecies. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production ofIFN-g, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver andspleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathologyand a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production ofIFN-g by CD4þ and CD8þ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediatedIL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies weredetected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.� 2010 Published by Elsevier Masson SAS.

Keywords: Leishmania; Visceral leishmaniasis; Tegumentary leishmaniasis; BALB/c mice; Leishmania ribosomal proteins; Vaccine

* Corresponding author. Centro de Biologıa Molecular Severo Ochoa

(CSIC-UAM), Departamento de Biologıa Molecular, Nicolas Cabrera 1.

Campus de Cantoblanco, Universidad Autonoma de Madrid, 28049 Madrid,

Spain. Tel.: þ34 91 1964508; fax: þ34 91 1964420.

E-mail address: [email protected] (M. Soto).1 These authors contributed equally to this work.

1286-4579/$ - see front matter � 2010 Published by Elsevier Masson SAS.

doi:10.1016/j.micinf.2010.06.008

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c mice against Leishmania chagasi and Leishmania amazonensis challenge, Micr

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1. Introduction

Leishmaniasis are a group of vector-transmitted diseasesthat are endemic in 88 tropical and subtropical countries(http://www.who.int/en/). Many geographical regions areendemic for multiple Leishmania species. This is the case inSouth America, where leishmaniasis are caused by at leasteight different species of Leishmania, each one with differentvirulence and pathogenesis determinants and many of themdisplaying common areas of transmission [1]. New World

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leishmaniasis can be grouped into two broad clinical cate-gories: visceral leishmaniasis (VL) and American tegumentaryleishmaniasis (ATL). The latter category includes a variety offorms that are commonly referred to their characteristic clin-ical and pathologic features: cutaneous leishmaniasis (CL),mucocutaneous leishmaniasis (MCL), and diffuse cutaneousleishmaniasis (DCL). VL is the most severe form of thedisease, and in the New World it is caused by Leishmaniachagasi infection. Leishmania amazonensis is capable ofproducing VL, although infection with this parasite is alsoassociated with CL, MCL and DCL [1,2]. In this context, to beeffective as a vaccine against American leishmaniasis, itscomponents should be shared by different parasite species indifferent vertebrate hosts and the protective efficacy of thesevaccine candidates should be analyzed in models of experi-mental infection caused by different parasite species.

Experimental leishmaniasis animal models have beenemployed for testing several candidate antigens in vaccinetrials, although they may not be entirely predictive of howeffective these vaccine candidates will perform in humans.Using mice models of CL and VL, it has been demonstratedthat the main challenge for the development of an effectiveLeishmania vaccine is to find a formulation able to inducea Th1-type long-lasting immunity. This is primed by cytokineslike IFN-g, IL-12 and GM-CSF, produced by specific T cellsand/or antigens presenting cells, but also controlling thedisease associated, IL-4 mediated, humoral responses (mainlyin CL models) and IL-10 deactivating responses [3,4]. Fromthese experimental vaccine studies in mouse models, it is clearthat some parasite fractions [5e7] or specific single proteins[8] are able to induce protection against Leishmania infection.

The antigenicity of the LRP has been demonstrated in dogsnaturally infected by Leishmania infantum or L. chagasi [9]. Ina previous work, we demonstrated that a subcellular fractioncomposed of the Leishmania major ribosomal proteins (LRP)administered in combination with a Th1 adjuvant (CpG oli-godeoxynucleotides; CpG-ODN) was able to induce protec-tion against L. major infection in C57BL/6 resistant andBALB/c susceptible mice [5]. In both mice strains, protectionwas correlated to an LRP-specific production of IFN-g [5]. InBALB/c mice, protection was also correlated with a reductionof IL-4 and IL-10-mediated responses [5]. In this work wehave investigated the role of an LRP based vaccine in theBALB/c experimental model using two different parasitespecies: L. chagasi and L. amazonensis.

L. infantum LRP extract was administered with saponin, anadjuvant capable of inducing Th1 immune responses and theproduction of cytotoxic T lymphocytes [10]. Saponin has beenemployed for the induction of immune responses in mice anddogs combined with Leishmania extracts [11,12] or recombi-nant proteins [13,14]. We show that the Th1 immune responseinduced by the co-inoculation of LRP plus saponin confersprotection against the challenge with L. chagasi and L. ama-zonensis parasites in BALB/c mice. In both models, protectioncorrelates to an LRP-specific and IL-12-dependent IFN-gproduction mediated by CD4þ and CD8þ T cells anda diminished production of parasite-specific IL-4 and IL-10.

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Thus, we present evidence that these evolutionary conservedproteins combined with Th1-type adjuvants may be relevant incomposing an effective pan-Leishmania vaccine.

2. Materials and methods

2.1. Mice and parasites

The Animal Use Committee of the Federal University ofMinas Gerais (CETEA) (Brazil) approved experimentalprotocols. Female BALB/c mice (4e6 weeks old) werepurchased from Institute of Biological Sciences, ICB,Federal University of Minas Gerais, Belo Horizonte, Brazil.L. chagasi (MOM/BR/1970/BH46), L. amazonensis (IFLA/BR/1967/PH-8) and L. infantum (MCAN/ES/1996/BCN/150/MON-1) parasites were grown at 24 �C in Schneider’s(Sigma, St. Louis, MO, USA) medium supplemented with20% heat-inactivated fetal bovine serum (FBS) (Sigma),20 mM L-glutamine, 200 U/ml penicillin, 100 mg/ml strep-tomycin and 50 mg/ml gentamicin, pH 7.4.

2.2. Antigen preparation

Soluble Leishmania antigenic (SLA) extract was preparedfrom stationary-phase promastigotes of L. chagasi andL. amazonensis growing in liquid culture, as previouslydescribed [15]. LRP was prepared from logarithmic phasepromastigotes of L. infantum, as previously described [5].

2.3. Immunization, challenge infection, cutaneous lesiondevelopment and parasite quantitation

Mice (n ¼ 16 per group) were subcutaneously immunizedin their left hind footpads with 12 mg of LRP associated with25 mg of saponin (Quillaja saponaria bark saponin) (Sigma),saponin alone or received saline. Three doses were adminis-tered in 2-week intervals. Four weeks after the final inocula-tion, eight animals per group were sacrificed for the analysis ofthe immune response elicited by vaccination. At the same timethe remaining animals were s.c. infected, into the right hindfootpad, with 105 stationary-phase promastigotes of L. chagasi(n ¼ 4, per group) or with 106 stationary-phase promastigotesof L. amazonensis (n ¼ 4 per group).

In mice infected with L. amazonensis, the course of thedisease was monitored at weekly intervals by measuringfootpad thickness with a metric calliper and expressed as theincrease in thickness of the infected footpad compared to theuninfected footpad. At week eight post-challenge all animalswere sacrificed and their spleens were harvested for parasitequantification and immunological analysis. Additionally,livers (in mice infected with L. chagasi) and infected skinfragments (in mice infected with L. amazonensis) werecollected for parasite quantification. Sera samples were alsocollected before and after challenge for immunologicalanalysis.

The number of parasites in the different tissues wasdetermined by a limiting-dilution assay [16].

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Splenocyte cultures and cytokine assays were performedbefore and after challenge as described previously [15].Briefly, single cell preparations from spleen tissue were platedin duplicate in 24-well plates (Nunc, Nunclon�, Roskilde,Denmark) at 2 � 106 cells/ml. Cells were incubated in DMEMmedium alone (background control) or separately stimulatedwith SLA (50 mg/ml) or LRP (12 mg/ml) at 37 �C in 5% CO2

for 48 h. IFN-g, IL-4, IL-10, IL-12 and GM-CSF supernatantswere assessed by sandwich ELISA using monoclonal anti-bodies specific for mouse cytokines (capture and detection)provided in commercial kits (Pharmingen, San Diego, CA,USA), following manufacturer’s instructions. When indicated,and in order to block IL-12, CD4þ and CD8þ mediated T cellcytokine release, spleen cells stimulated with LRP (12 mg/ml)were incubated in the presence of 5 mg/ml of monoclonalantibody (mAb) against either mouse IL-12 (C17.8), CD4 (GK1.5) or mouse CD8 (53-6.7). Appropriate isotype-matchedcontrols: rat IgG2a (R35-95) and rat IgG2b (95-1) were alsoemployed in the assays. Antibodies (no azide/lowendotoxin�) were purchased from BD (Pharmingen).

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Release of nitrite was determined in unstimulated or SLA-stimulated splenocyte cultures established at week 8 post-challenge. Following incubation for 48 h, 100 ml of culturesupernatant was mixed with an equal volume of Griess reagent(Sigma). After an incubation of 10 min at room temperature,nitrite concentration was calculated using a standard curve ofknown concentrations. Data are expressed as mM per2 � 106 cells per 48 h.

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Fig. 1. Cytokine production and antibody response induced by vaccination in

BALB/c mice. Spleen cells obtained from mice four weeks after the last

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LRP- and SLA-specific IgG, IgG1 and IgG2a antibodieswere measured by ELISA, as described elsewhere [15].Peroxidase-labeled antibodies specific to mouse IgG, andmouse IgG1 or IgG2a isotypes (Sigma) were diluted at 1:5000and incubated for 1 h at 37 �C, then incubated with H2O2 ando-phenylenediamine. Optical densities were read at 492 nm ina spectrophotometer.

vaccine dose were cultured in vitro and non-stimulated (medium; background

control) or stimulated with LRP (12 mg/ml) for 48 h at 37 �C, 5% CO2. IFN-g,

IL-12, GM-CSF, IL-4 and IL-10 levels were assessed by ELISA in culture

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supernatants. Each bar represents the mean �SD of data from eight individual

mice. Differences in the IFN-g, IL-12 and GM-CSF levels between LRP plus

saponin and the other two groups (saline and saponin) were statistically

significant (**P < 0.005) (A). Serum samples obtained from mice four weeks

after the last vaccine dose were individually tested by ELISA to determine the

presence of LRP-specific IgG, IgG1 and IgG2a antibodies. Each bar represents

the mean �standard deviation (SD) of data from four individual mice.

Differences in the LRP-specific IgG (**P < 0.005), IgG1 (*P < 0.05) and

IgG2a (**P < 0.005) reactivity between the LRP plus saponin group and

control (saline and saponin) groups were statistically significant (B). Data

shown are representative of two independent experiments with similar results.

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The statistical analysis was made using the GraphPad Prismsoftware (version 4 for Windows). All data comparisons weretested for significance by means of the one-way analysis ofvariance (ANOVA) using the Bonferroni’s post-test formultiple comparisons of groups. P values <0.05 wereconsidered statistically significant (*P < 0.05, **P < 0.005).Data are representative of two independent experiments withsimilar results.

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c mice against Leishmania chagasi and Leishmania amazonensis challenge, Micr

3. Results

3.1. Immunogenicity of the LRP combined with saponinin BALB/c mice

The immunogenicity of the Leishmania ribosomal proteinswas evaluated in BALB/c mice after administration of the LRPin the presence of saponin. Following in vitro stimulation withLRP, spleen cells from the LRP plus saponin vaccinated miceproduced significantly higher levels of IFN-g, IL-12 and GM-CSF than those secreted by spleen cells from control mice,namely, saline and saponin groups (Fig. 1A). No increase in

ith the Leishmania infantum ribosomal proteins induces protection in BALB/

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IL-4 and IL-10 production was observed after stimulation withLRP in any experimental groups. In addition, mice vaccinatedwith LRP plus saponin showed a specific anti-LRP humoralresponse that was predominantly of the IgG2a isotype(Fig. 1B).

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3.2. Immunization with LRP plus saponin protectsBALB/c mice against L. chagasi and L. amazonensischallenge

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Fig. 2. Protection in BALB/c mice against L. chagasi infection by immuni-

zation with LRP plus saponin. Mice vaccinated with saponin or LRP plus

saponin or inoculated with saline were s.c. challenged with 105 promastigotes

of L. chagasi. The number of parasites in the liver and spleen was measured

eight weeks after infection. Mean �standard deviation (SD) of four mice in

each group is shown. Differences in the parasite load in the liver

(**P < 0.005) and spleen (*P < 0.05) between LRP plus saponin and control

(saline and adjuvant) groups were statistically significant (A). Determination

of the amounts of nitrite in the supernatants of splenic cell cultures established

from the three groups of mice, eight weeks after infection. Single cell

suspensions were obtained from spleen of mice and were non-stimulated

(medium; background control) or stimulated with SLA (50 mg/ml). Mean �SD

of nitrite levels determined in four individual mice per group is shown.

Differences in nitrite levels between LRP plus saponin and saline and saponin

groups were statistically significant (**P < 0.005) (B). Data shown are

representative of two independent experiments with similar results.

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In order to evaluate the protective efficacy of LRP plussaponin vaccination against different forms of murine leish-maniasis, immunized mice were separately challenged bysubcutaneous injection of two different Leishmania species.For the VL model, mice were infected with 105 stationary-phase promastigotes of L. chagasi. Eight weeks after chal-lenge, the parasite number in spleen and liver of infected micewas determined. Significant reduction of parasites was seen inmice immunized with LRP plus saponin as compared withthose that received saline or adjuvant alone (Fig. 2A). Theimmunized mice showed a 6.7-log and 4.7-log reduction in thenumber of parasites in liver and a 12.3-log and 9.3-logreduction in spleen as compared with saline and adjuvantgroups, respectively.

To determine the influence of the immunization with LRPplus saponin on L. chagasi specific killing effector function inthe spleen of infected mice, nitrite was assayed as an indicatorof nitric oxide (NO) production in macrophages. The nitriteproduction in splenocyte supernatants was significantly higherin mice vaccinated with LRP plus saponin after stimulationwith L. chagasi SLA when compared with control groups thatproduced minimum amounts of this product (Fig. 2B).

For a model of CL, mice were infected by a high inoculum(106 stationary-phase promastigotes) of L. amazonensis. Thedevelopment of dermal lesion was evaluated by measuringfootpad swelling (Fig. 3A) and parasite loads (Fig. 3B) in theinfected footpads and spleens of the mice. Animals immunizedwith LRP plus saponin displayed significant reductions in thefootpad swelling, as compared to saline and saponin groups(30% and 60%, respectively) (Fig. 3A). In addition, immu-nized mice showed a 4.5-log and 3.2-log reduction in thenumber of parasites in spleen as compared with saline andadjuvant groups, respectively (Fig. 3B). Correlating to thelower parasite burden, nitrite production in splenocyte super-natants was significantly higher in mice vaccinated with LRPplus saponin than saline or saponin groups, after stimulationwith L. amazonensis SLA (Fig. 3C).

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3.3. Characterization of the cellular response elicitedafter infection

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To determine the immunological parameters associatedwith protection induced in the mice by LRP plus saponin, theproduction of different cytokines in the supernatants of spleencell cultures, established from the different mice groups andstimulated with L. chagasi or L. amazonensis SLA and L.infantum LRP after challenge was determined.

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After L. chagasi infection, the spleen cells from micevaccinated with LRP plus saponin produced higher levels ofSLA- and LRP-specific IFN-g than those secreted by spleencells from both control mice groups at week eight after chal-lenge (Fig. 4A). This data correlates well with the nitriteproduction pattern shown in Fig. 2B, since this killing effectormolecule against leishmaniasis is produced by IFN-g stimu-lated macrophages [17]. The contribution of CD4þ and CD8þ

T cells and the dependence on IL-12 to the LRP- and SLA-specific production of IFN-g in the spleen of the mice vacci-nated with LRP plus saponin and infected were also analyzed.Production of IFN-g was completely suppressed by anti-IL-12or anti-CD4 monoclonal antibodies. The addition of anti-CD8

ith the Leishmania infantum ribosomal proteins induces protection in BALB/

ob Infect (2010), doi:10.1016/j.micinf.2010.06.008

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Fig. 3. Protection in BALB/c mice against L. amazonensis infection by immu-

nization with LRP plus saponin. Mice vaccinated saponin or LRP plus saponin or

inoculated with saline were s.c. challenged with 106 stationary-phase promasti-

gotes of L. amazonensis. Mean�SD of four mice in each group is shown. Lesion

development in the infected groups was monitored weekly until eight weeks after

infection (A) Differences in the footpad swelling between LRP plus saponin and

control (saline and adjuvant) groups were statistically significant at week eight

after infection (*P< 0.05). Parasite burden determination in the infected footpads

and spleens was analyzed eight weeks after infection (B). Differences in the

parasite loads between LRP plus saponin and control (saline and adjuvant) groups

were statistically significant (**P< 0.005).Determination of the amount of nitrite

in the supernatants of splenic cell cultures established from the three groups of

mice, eight weeks post-infection. Single cell suspensions were obtained from

spleen of mice and were non-stimulated (medium: background control) or stim-

ulatedwithSLA(50mg/ml) (C).Themean�SDofnitrite levels determined in four

individualmice per group is shown. Differences in nitrite levels between LRP plus

saponin and control groups were statistically significant (**P < 0.005). Data

shown are representative of two independent experiments with similar results.

5M.A. Chavez-Fumagalli et al. / Microbes and Infection xx (2010) 1e11

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antibodies to the spleen cell cultures also induced a significantreduction in the amount of this cytokine in the supernatants,although levels were significantly higher than those observedin anti-IL-12 or anti-CD4 treated cultures (Fig. 4A). Theobservation that anti-CD4 and anti-CD8 inhibited productionof IFN-g is providing an indirect assessment of CD8þ andCD4þ T cell production of this cytokine, respectively. Theability of anti-CD4 antibody to completely inhibit productionof IFN-g should be indicating that CD4þ T cells wereproviding a helper function for class I-dependent secretion ofIFN-g as it was previously reported in [18]. Altogether, ourdata may be indicating that co-inoculation of saponin and theLRP antigen induced both MHC class I and class II-restrictedresponses.

The IL-12 and GM-CSF production following in vitrostimulation with LRP or SLA was also analyzed eight weeksafter L. chagasi challenge. Spleen cells from mice immunizedwith LRP plus saponin produced significantly higher levels ofthese cytokines after stimulation with both antigen prepara-tions relative to control groups (Fig. 4B and C for IL-12 andGM-CSF, respectively). Finally, the SLA- or LRP-drivenproduction of IL-4 (Fig. 4C) and IL-10 (Fig. 4D) was alsoanalyzed after L. chagasi challenge. The LRP extract did notinduce the production of these cytokines by the spleen cells ofthe three mice groups. Moreover, the SLA-specific productionof IL-4 and especially IL-10 cytokines observed in the controlgroups was absent in the LRP plus saponin vaccinated mice.

Similar cytokines production profiles were observed eightweeks after L. amazonensis challenge infection. LRP plussaponin vaccinated mice spleen cells produced higher levels ofIFN-g (Fig. 5A), IL-12 (Fig. 5B) and GM-CSF (Fig. 5C)cytokines after in vitro stimulation with L. amazonensis SLAand LRP compared to saline and saponin mice groups. Asobserved in L. chagasi infected mice, the SLA- and LRP-specific secretion of IFN-g was inhibited by anti-IL-12 or anti-CD4 monoclonal antibodies whereas treatment with anti-CD8antibodies only partially reduced the level of this cytokine inthe culture supernatants (Fig. 5A). The L. amazonensis SLA-specific production of IL-4 (Fig. 5D) and IL-10 (Fig. 5E)detected in spleen cells from control mice (saline and saponingroups) were significantly reduced in LRP plus saponinvaccinated mice. Remarkably, the level of SLA-induced IL-4was higher in control mice infected with L. amazonensis(Fig. 5D) than control mice infected with L. chagasi (Fig. 4D).

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3.4. Characterization of the anti-SLA humoral responseafter infection

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In BALB/c mice, the IL-4-dependent production of hightiters of antibodies is associated with disease progression dueto L. amazonensis [19] infection, but it is not certified in theL. chagasi murine model [20]. We analyzed the humoralresponses elicited against SLA after infection in the differentmice groups to compare the global anti-Leishmania antigenshumoral response induced by infection with both Leishmaniaspecies. Very low levels of anti-SLA antibodies were observedin the sera of all mice groups infected with L. chagasi

ith the Leishmania infantum ribosomal proteins induces protection in BALB/

ob Infect (2010), doi:10.1016/j.micinf.2010.06.008

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1000

1500

2000

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IFN

-γ (p

g/m

l)

Saline SaponinLRP + saponin

Control α-CD4 α-CD8 α-IL12

MediumLRPSLA

**A

**

**

Fig. 4. Production of cytokines by spleen cells after L. chagasi challenge infection. Single cell suspensions were obtained from spleen of mice, eight weeks after

infection. Cells were non-stimulated (medium; background control) or separately stimulated with L. infantum LRP (12 mg/ml) or L. chagasi SLA (50 mg/ml) for

48 h at 37 �C, 5% CO2. IFN-g (A) IL-12 (B), GM-CSF (C), IL-4 (D) and IL-10 (E) levels were measured in culture supernatants by ELISA. Mean �SD of cytokine

levels determined in four individual mice per group is shown. Differences in the levels of IFN-g, IL-12 and GM-CSF between LRP plus saponin group and saline or

saponin groups for SLA or LRP stimulus were statistically significant (**P < 0.005). Differences in the SLA-specific levels of IL-4 and IL-10 between LRP plus

saponin group and saline or saponin groups were statistically significant (**P < 0.005). In panel A, the analysis of the involvement of IL-12 and T cells in the

production of IFN-g in mice vaccinated with LRP plus saponin is also shown. The level of IFN-g was measured by ELISA in the supernatants of spleen cell

cultures stimulated as above in the absence (control) or in the presence of either anti-IL-12, anti-CD4 or anti-CD8 monoclonal antibodies. When isotype-matched

control antibodies were employed, the level of IFN-g was similar to that observed in the absence of antibodies (data not shown). Differences between untreated

cells and cultures treated with anti-CD4, anti-CD8 and anti-IL-12 monoclonal antibodies were statistically significant for both, SLA- and LRP-stimulated cells

(**P < 0.005). Differences in the SLA- or LRP-specific IFN-g production between anti-CD8 and anti-CD4 or anti-IL-12 treated cells were statistically significant

(*P < 0.05). Data shown are representative of two independent experiments with similar results.

6 M.A. Chavez-Fumagalli et al. / Microbes and Infection xx (2010) 1e11

Please cite this article in press as: Miguel A. Chavez-Fumagalli et al., Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/

c mice against Leishmania chagasi and Leishmania amazonensis challenge, Microb Infect (2010), doi:10.1016/j.micinf.2010.06.008

MICINF3610_proof ■ 13 July 2010 ■ 6/11

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B C

DE

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* *

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l)

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IL-4

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ml)

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0 (p

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****

0

200

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800

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2 (p

g/m

l)

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Saline Saponin LRP+ saponin

** **

0

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100

150

200

250

300

350

400

450

Saline Saponin LRP+ saponin

GM

-CSF

(pg/

ml)

MediumLRPSLA

****

Fig. 5. Production of cytokines by spleen cells of BALB/c mice after L. amazonensis challenge infection. Single cell suspensions were obtained from spleen of

mice, eight weeks after L. amazonensis infection. Cells were non-stimulated (Medium; background control) or separately stimulated with L. infantum LRP (12 mg/

ml) or L. chagasi SLA (50 mg/ml) for 48 h at 37 �C, 5% CO2. IFN-g (A) IL-12 (B), GM-CSF (C), IL-4 (D) and IL-10 (E) levels were measured in culture

supernatants by ELISA. Mean �SD of cytokine levels determined in four individual mice per group is shown. Differences in the levels of IFN-g, IL-12 and GM-

CSF between LRP plus saponin group and saline or saponin groups for SLA or LRP stimulus were statistically significant (**P < 0.005). Differences in the SLA-

specific levels of IL-4 and IL-10 between LRP plus saponin group and saline or saponin groups were statistically significant (**P < 0.005). In panel A, the analysis

of the involvement of IL-12 and T cells in the production of IFN-g in mice vaccinated with LRP plus saponin is also shown. The level of IFN-g was measured by

ELISA in the supernatants of spleen cell cultures stimulated as above in the absence (control) or in the presence of either anti-IL-12, anti-CD4 or anti-CD8

monoclonal antibodies. When isotype-matched control antibodies were employed, the level of IFN-g was similar to that observed in the absence of antibodies (data

not shown). Differences between untreated cells and cultures treated with anti-CD4, anti-CD8 and anti-IL-12 monoclonal antibodies were statistically significant

for both, SLA- and LRP-stimulated cells (**P < 0.005). Differences in the SLA- or LRP-specific IFN-g production between anti-CD8 and anti-CD4 or anti-IL-12

treated cells were statistically significant (*P < 0.05). Data shown are representative of two independent experiments with similar results.

7M.A. Chavez-Fumagalli et al. / Microbes and Infection xx (2010) 1e11

Please cite this article in press as: Miguel A. Chavez-Fumagalli et al., Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/

c mice against Leishmania chagasi and Leishmania amazonensis challenge, Microb Infect (2010), doi:10.1016/j.micinf.2010.06.008

MICINF3610_proof ■ 13 July 2010 ■ 7/11

781782783784785786787788789790791792793794795796797798799800801802803804805806807808809810811812813814815816817818819820821822823824825826827828829830831832833834835836837838839840841842843844845

846847848849850851852853854855856857858859860861862863864865866867868869870871872873874875876877878879880881882883884885886887888889890891892893894895896897898899900901902903904905906907908909910

0

0.05

0.1

0.15

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IgG IgG1 IgG2a

O.D

. (49

2 nm

)

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IgG IgG1 IgG2a

O.D

. (49

2 nm

)

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*

Saline

Saponin

LRP + saponin

A

B

0.6

*

Fig. 6. Antibody response against SLA after Leishmania infection. Serum

sampleswere obtained frommice infectedwithL. chagasi (A) orL. amazonensis

(B), eight weeks after challenge. Sera were individually tested by ELISA to

determine the presence of L. chagasi SLA-specific (A) or L. amazonensis SLA-

specific (B) IgG, IgG1 and IgG2a antibodies. Each bar represents themean�SD

of data from four individual mice per group. After L. chagasi infection, only

differences in the SLA-specific IgG reactivity between saline and LRP plus

saponin groups were found to be statistically significant (*P < 0.05). After

L. amazonensis infection, differences in the SLA-specific IgG reactivity between

saline and LRP plus saponin groups were found to be statistically significant

(*P< 0.05).Also,IgG1 (**P< 0.005) and IgG2a (*P< 0.05) reactivity between

protected mice and control (saline and saponin) groups were statistically

significant. Data shown are representative of two independent experiments with

similar results.

8 M.A. Chavez-Fumagalli et al. / Microbes and Infection xx (2010) 1e11

MICINF3610_proof ■ 13 July 2010 ■ 8/11

911912913914915916917918919920921922923924925926927928929930931932933934935936937938939940941942943944945946947948949950951952953954955956957958959960961962963964965966967968969970971972973974975

976977978979980981982983984985986987988989990991992993994995996997998999

10001001100210031004100510061007100810091010101110121013101410151016101710181019102010211022102310241025102610271028102910301031103210331034103510361037103810391040

(Fig. 6A). We only found significant differences in the level oftotal IgG antibodies between saline and LRP plus saponinvaccinated mice, being lower in this last group. Similar levelsof SLA-specific IgG1 and IgG2a antibodies were found in thethree groups, the anti-SLA IgG2a antibodies being slightlyhigher in the LRP plus saponin vaccinated mice.

On the contrary, mice infected with L. amazonensis showedhigh levels of anti-SLA antibodies. Results shown in Fig. 6Bindicate that immunization with LRP plus saponin conditionedthe global Leishmania-specific humoral response induced byL. amazonensis, since antibodies produced by parasite-chal-lenged mice vaccinated with LRP plus saponin were mainly ofthe IgG2a isotype, being the anti-SLA reactivity of the IgG1isotype antibodies significantly lower than those detected inthe other two groups.

Please cite this article in press as: Miguel A. Chavez-Fumagalli et al., Vaccination w

c mice against Leishmania chagasi and Leishmania amazonensis challenge, Micr

4. Discussion

The development of vaccines against leishmaniasis requiresthe definition of potential candidates to be capable of inducingprotective responses against different Leishmania species. Inthis way, many conserved intracellular Leishmania proteinslike histones, ribosomal proteins, heat shock proteins, cisteine-proteinases and microtubule related proteins have been iden-tified as antigenic and/or immunogenic in individuals sufferingfrom different clinical forms of leishmaniasis [21,22]. Amongthem, Leishmania ribosomes can be considered as immuno-logically relevant particles during infection because some oftheir protein constituents are antigenic in human and dogsnaturally infected with different Leishmania species [9,21,23].In addition, some ribosomal proteins have been related to cellactivities and cytokine release after infection in murine modelsresulting in dysfunction of the host immune system [21,24,25].On the other hand, the administration of defined ribosomalantigens [26] or total parasite ribosomal proteins [5] using Th1promoting adjuvants was able to induce protective responsesin models of murine CL.

The purpose of this study was to analyze whether a prepa-ration of L. infantum ribosomal proteins administered incombination with saponin was protective against L. chagasiand L. amazonensis challenge infection in BALB/c mice. Weshow that immunization with LRP extract obtained fromL. infantum plus saponin was able to induce a predominantTh1 immune response against parasite ribosomal proteins.Vaccinated mice showed an in vitro LRP-specific productionof IFN-g and IL-12 combined with the presence of LRP-specific antibodies that were mainly of the IgG2a isotype,a humoral marker of Th1-type mediated responses [27]. Inaddition, very low levels of LRP-specific IL-4 or IL-10 wereobserved in the vaccinated mice.

After L. chagasi infection LRP plus saponin immunizedmice displayed significant reduction of parasites in the spleenas well as in the liver when compared with mice vaccinatedwith the adjuvant alone or that received the saline solutionemployed in the vaccine formulation. The evaluation ofparasite burden in both organs is an important marker ofvaccine efficacy against VL, in view of the fact that an organ-specific immune response was observed after infection withviscerotropic Leishmania species following intravenous,intradermal or subcutaneous challenge in mice [20]. Duringthe early stages of visceral infection in BALB/c mice, para-sites multiply in large numbers in the liver, and afterwards thehepatic parasite load tends to decrease while parasitism in thespleen tends to increase. Thus, the liver can be considered anindicator of the initial multiplication of parasites and spleen asa reservoir for these microorganisms [28]. A reduction ofparasite burdens was also observed in LRP plus saponinvaccinated mice compared with both control groups after L.amazonensis challenge. The decrease in the number of para-sites found in the footpad was accompanied by a reduction ofthe dermal inflammatory lesions. Contrary to the lack of CLlesions found in BALB/c mice vaccinated with LRP plus CpG-ODN after L. major infection [5,29], mice vaccinated with

ith the Leishmania infantum ribosomal proteins induces protection in BALB/

ob Infect (2010), doi:10.1016/j.micinf.2010.06.008

9M.A. Chavez-Fumagalli et al. / Microbes and Infection xx (2010) 1e11

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LRP plus saponin showed some footpad inflammation.Differences may be due to the different model of CL, sinceL. amazonensis parasites are more pathogenic than L. major[19]. Alternatively, the use of saponin instead CpG-ODN asadjuvant may be also implicated in the different degree ofprotection observed.

In both experimental models employed in this work, thedecrease in number of parasites in the spleen from the LRPplus saponin vaccinated mice was correlated to the generationof NO, a molecule produced in activated macrophages that iscritical to the control of parasite replication. The SLA-dependent production of NO observed in the spleen of theprotected mice was correlated to an SLA-specific productionof IFN-g, a key cytokine implicated in the acquired immunityagainst infection with Leishmania species [28,30]. Our resultsshow that IFN-g was detectable in the supernatant of spleencells established from the LRP plus saponin vaccinated miceafter infection with both parasite species, and was rarelyundetected in cultures established from control groups after invitro re-stimulation. Both CD4þ and CD8þ T cells contributeto the production of IFN-g in the protected mice. The vaccine-induced activation of both cell subsets may be important forthe parasite killing observed in the spleen of protected mice,since the presence of both, antigen-specific Th1 responses andCD8þ T cell responses, was related to protection againstLeishmania donovani in mice vaccinated with a purifiedpreparation of the promastigote surface proteins gp63 [31] ordp72 [32]. This was also observed in mice immunized withhybrid cell vaccines composed of macrophages expressing thekinetoplastid membrane protein KMP-11 [33] and in micevaccinated with the recombinant hydrophilic acylated surfaceprotein B1 [34]. In addition, the generation of Leishmania-specific CD4þ and CD8þ T cell responses have been corre-lated with control of infection in asymptomatic subjectsinfected with L. infantum [35]. In the protected mice, the IFN-g production was IL-12-dependent and a parasite dependentproduction of IL-12 by spleen cells was detected after stim-ulation with parasite proteins. This cytokine has a central rolein determining initial and late resistance to Leishmaniainfection [30,36] and has been also related with the generationof protective immunity against L. infantum in BALB/c micevaccinated with membrane [37] or secreted [7] parasite frac-tions and in hamsters protected against the development of VLby vaccination with recombinant KMP-11 [38]. The protectiverole of IL-12 in VL may explain its ability to enhance themacrophage leishmanicidal activity [39,40].

Altogether, our data indicate that immunization with LRPplus saponin primed BALB/c mice for an LRP-specific Th1immune response that was maintained after L. chagasi andL. amazonensis challenge, protecting vaccinated mice againstdevelopment of VL and CL. These data are in agreement withthose reported by Iborra et al. [5], demonstrating that theparasite Th1-specific immune response elicited after immu-nization with LRP extracts combined with CpG-ODN wasable to induce protection in BALB/c and C57BL/6 miceagainst L. major infection. In BALB/c mice vaccinated withLRP plus CpG-ODN, protection against the development of

Please cite this article in press as: Miguel A. Chavez-Fumagalli et al., Vaccination w

c mice against Leishmania chagasi and Leishmania amazonensis challenge, Micr

CL was also correlated with a decrease of parasite-specific IL-4 and IL-10-mediated responses [5]. The decrease of thesecytokine responses has also been related to the induction ofvaccine-mediated protection against L. chagasi infection inBALB/c mice immunized with an A2 based DNA vaccine [41]and against L. amazonensis infection in the same mice strainafter immunization with the L. donovani recombinant A2protein administered with IL-12 [15]. Our results show thatmice vaccinated with LRP plus saponin showed a similarcontrol of these responses after L. chagasi and L. amazonensisinfection. Thus, a significant decrease was observed in the invitro production of SLA-specific IL-4 by spleen cells of thevaccinated mice after infection with both parasite species,when compared with both control mice groups. In L. ama-zonensis infected mice, the high levels of SLA-specific IL-4production observed in control groups was correlated to thegeneration of high levels of anti-Leishmania antibodies. Thus,mice that received saline or saponin showed higher anti-SLAIgG1 antibody levels in comparison to IgG2a levels, whereasin the case of mice immunized with LRP plus saponin, theparasite-specific antibodies were mainly of the IgG2a isotype.The presence of high parasite-specific Th2 mediated humoralresponses observed in control animals may have alsocontributed to disease progression, since Leishmania infectionwas impaired in the absence of circulating antibodies or inmice lacking the Fc receptors’ common-g chain [42].Furthermore, H-2q syngeneic high- and low-antibodyresponder mice (Biozzi mice) were shown to be susceptibleand resistant, respectively, to L. amazonensis infection [43].

Our results also show that protection against CL and VLprogression in BALB/c mice was also associated witha remarkable, significant decrease of the IL-10-mediatedimmune responses. Very low levels of parasite-specific IL-10production were detected after stimulation of the spleen cellsfrom vaccinated mice eight weeks post-infection with bothparasite species. However, spleen cells from control micegroups showed a significantly higher production of this cyto-kine. Regarding VL model, control of the parasite-mediatedIL-10 responses in the vaccinated mice may be critical forprotection, since IL-10 is considered as the most critical factorfor VL progression after infection with viscerotropic Leish-mania species [17,20] and IL-10 deficient BALB/c mice [44]or mice treated with an anti-IL-10 receptor antibody [45] areresistant to L. donovani infection. In addition, IL-10 has beenalso associated with CL disease due to L. amazonensisinfection, since IL-10 deficient BALB/c mice had lowernumber of parasites in lesions than wild-type mice [46].

Finally, our results show that the spleen cells from pro-tected mice produced higher levels of parasite-specific GM-CSF than controls, a cytokine related with macrophageactivation and resistance against different intracellular patho-gens including L. major [47] and L. donovani [48] in murinemodels. In addition, it has been shown that the immunizationof human volunteers with a crude Leishmania preparationusing GM-CSF as adjuvant induced parasite-specific Th1responses [49] and that administration of a therapeutic vaccinecontaining four Leishmania antigens combined with GM-CSF

ith the Leishmania infantum ribosomal proteins induces protection in BALB/

ob Infect (2010), doi:10.1016/j.micinf.2010.06.008

10 M.A. Chavez-Fumagalli et al. / Microbes and Infection xx (2010) 1e11

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11711172117311741175117611771178117911801181118211831184118511861187118811891190119111921193119411951196119711981199120012011202120312041205120612071208120912101211121212131214121512161217121812191220122112221223122412251226122712281229123012311232123312341235

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was correlated with resolution of mucosal lesions presentedin an antimonial-refractory mucocutaneous leishmaniasispatient [50].

Results presented herein together with our previouslyreported data [5] indicate that Leishmania ribosomal proteinscombined with Th1 adjuvants, like saponin, an adjuvant usedin veterinary procedures can be used for the development ofeffective vaccines against leishmaniasis caused by viscero-tropic and cutaneous Leishmania species. Taken together, ourdata confirm that high IFN-g and IL-12 and low IL-4 and IL-10 levels are required for protection of BALB/c mice againstL. chagasi, L. amazonensis and L. major. The association ofLRP extracts or their defined ribosomal constituents with Th1adjuvant systems might achieve these immune responses forthe development of a pan-Leishmania vaccine.

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Acknowledgments

MACF, MAFC and DMO are fellows of CAPES, Brazil.We would like to thank the financial support from Fundacao deAmparo a Pesquisa do Estado de Minas Gerais (FAPEMIG;CBB-APQ-01322-08) and Conselho Nacional de Desenvolvi-mento Cientıfico e Tecnologico (CNPq; APQ-577483/2008-0),Brazil. The study was also supported by grants from Labo-ratorios LETI S.L., from Ministerio de Ciencia e InnovacionFIS/PI080101 and from the Instituto de Salud Carlos III withinthe Network of Tropical Diseases Research (RICET RD06/0021/0008), Spain. An institutional grant from FundacionRamon Areces for the CBMSO is also acknowledged.

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