The immunodominant T helper 2 (Th2) response elicited in BALB/cmice by the Leishmania LiP2a and LiP2b acidic ribosomal proteinscannot be reverted by strong Th1 inducers

S. Iborra,* D. R. Abánades, N. Parody,J. Carrión, R. M. Risueño,M. A. Pineda, P. Bonay, C. Alonsoand M. SotoCentro de Biología Molecular Severo Ochoa,

Departamento de Biología Molecular, Facultad de

Ciencias, Universidad Autónoma de Madrid,

Madrid, Spain

Summary

The search for disease-associated T helper 2 (Th2) Leishmania antigens andthe induction of a Th1 immune response to them using defined vaccinationprotocols is a potential strategy to induce protection against Leishmaniainfection. Leishmania infantum LiP2a and LiP2b acidic ribosomal protein(P proteins) have been described as prominent antigens during human andcanine visceral leishmaniasis. In this study we demonstrate that BALB/cmice infected with Leishmania major develop a Th2-like humoral responseagainst Leishmania LiP2a and LiP2b proteins and that the same response isinduced in BALB/c mice when the parasite P proteins are immunized asrecombinant molecules without adjuvant. The genetic immunization ofBALB/c mice with eukaryotic expression plasmids coding for these proteinswas unable to redirect the Th2-like response induced by these antigens, andonly the co-administration of the recombinant P proteins with CpG oligode-oxynucleotides (CpG ODN) promoted a mixed Th1/Th2 immune response.According to the preponderance of a Th2 or mixed Th1/Th2 responseselicited by the different regimens of immunization tested, no evidence ofprotection was observed in mice after challenge with L. major. Althoughalterations of the clinical outcome were not detected in mice presensitizedwith the P proteins, the enhanced IgG1 and interleukin (IL)-4 responseagainst total Leishmania antigens in these mice may indicate an exacerba-tion of the disease.

Keywords: adjuvants, antibody responses, CpG DNA, DNA vaccines,

Leishmania spp.

Accepted for publication 14 August 2007

Correspondence: M. Soto, Centro de Biología

Molecular Severo Ochoa, Departamento de

Biología Molecular, Facultad de Ciencias,

Universidad Autónoma de Madrid, 28049

Madrid, Spain.

E-mail: msoto@cbm.uam.es

*Present address: Unidad de Inmunología Viral,

Centro Nacional de Microbiología, Instituto de

Salud Carlos III, Crta. Pozuelo Km 2, 28220

Majadahonda, Madrid, Spain.

Introduction

Leishmaniases comprise several diseases caused by intracel-lular protozoan parasites belonging to the genus Leishmania.The clinical manifestations of leishmaniasis in humans rangefrom cutaneous leishmaniasis, which heal spontaneously, topotentially fatal visceral leishmaniasis [1]. Outcome of infec-tion is determined by interactions between the host immunesystem and the different parasite species, yet the pathogenesisof leishmaniasis remains unclear. A model of Leishmaniavirulence has been proposed involving two different groups ofparasite molecules [2,3]. One group consists of surface andsecreted products that are necessary for the establishment ofinfection as a prerequisite for virulence but that by themselvesdo not cause disease. The second group of parasite moleculesconsists of highly conserved, intracellular molecules referredto as ‘pathoantigens’. The inadequate humoral response

induced against these antigens is thought to result in immu-nopathology, due mainly to the adverse effects of immunecomplexes, particularly uveitis [4], lesions in the centralnervous system [5] or nephritis in dogs suffering visceralleishmaniasis [6–9] and in hamsters and mice infected withL. donovani [10,11]. In addition, immune complexes havebeen also involved in anaemia in hamsters infected withL. donovani [12]. Finally, it has been shown recently that bothin mice infected with L. major and in humans suffering vis-ceral leishmaniasis, the presence of IgG immune complexescorrelates with an inability to resolve infections. This effect,that relies on the induction of interleukin (IL)-10, demon-strates that the presence of immune complexes can be detri-mental to a host infected with this intracellular pathogen [13].

Effective primary immunity against L. major in mouserequires IL-12-dependent production of interferon (IFN)-gfrom CD4+ T cells [T helper 1 (Th1) response] and CD8+

Clinical and Experimental Immunology ORIGINAL ARTICLE doi:10.1111/j.1365-2249.2007.03501.x

375© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

T cells, which mediates nitric oxide (NO)-dependent killingby infected macrophages (reviewed in [14–16]). In contrast,susceptibility correlates with the dominance of an IL-4-driven Th2 response, as it has been observed in certainstrains, mainly in BALB/c mice. In recent years, severalrecombinant leishmanial antigens have been identified andtested as vaccine candidates [16]. Some of them were testedbecause they elicit primarily a Th1-type response inL. major-infected BALB/c mice, in spite of the stronglybiased Th2 responses mounted in these mice [17]. In clearcontrast, some other promising vaccine candidates werestrong inducers of Th2 responses in BALB/c mice infectedwith L. major. Thus, it was demonstrated first that BALB/cmice could be protected against L.major infection by simplyredirecting the early Th2 response induced against one singleantigen, the Leishmania homologue of the receptor for acti-vated C kinase (LACK) towards a Th1 response [18]. Thesame results were obtained when other Th2-inducing para-site proteins, such as the L. mexicana cysteine protease(CPB2·8) [19] or the parasite nucleosome forming histones[20], were administered with Th1-modulating adjuvants.Thus, redirecting the Th2 responses induced against someLeishmania epitopes towards a Th1 response is a promisingstrategy to induce protection against L. major infection [21].

The Leishmania P protein family, constituents of the largesubunit of ribosomes, comprises three members (namelyLiP0, LiP2a and LiP2b) and can be considered as potentimmunostimulatory proteins during the leishmaniasisprocess. They have been described as immunodominantantigens recognized by sera from both human and dogsinfected naturally with L. chagasi–L. infantum [22–24]. Also,administration in BALB/c mice of the L. infantum recombi-nant LiP2a protein (rLiP2a) or LiP0 (rLiP0), in the absenceof any added adjuvant, elicited IgG1 humoral responses[25,26]. Remarkably, immunization of the LiP0 in BALB/cmice as a DNA vaccine or as recombinant protein combinedwith CpG oligodeoxynucleotides (GpG ODN) redirect thisresponse towards a specific Th1 response that correlates withthe induction of partial protection after challenge withL. major [26,27].

In this paper we show that BALB/c mice infected withL. major exhibit a Th2 humoral response against the rLiP2aand rLiP2b proteins. Further, we analyse if DNA vaccinationor the use of CpG oligodeoxynucleotides (ODN) adjuvantredirect the Th2 responses induced by these antigens. Anincreased IgG2a–IFN-g response was observed only byco-administration of CpG ODN with the recombinant pro-teins in naive mice or in mice primed previously with Pproteins genes, suggesting that this adjuvant triggeredTh1-specific responses. Notwithstanding, the Th2 responseagainst the parasite LiP2a and LiP2b proteins was notinhibited, as the IgG1 response was also enhanced by thisadjuvant. Finally, we demonstrate that these mixed immuneresponses are not correlated with protection against L. majorinfection in BALB/c mice.

Materials and methods

Mouse strains and parasites

Female BALB/c mice were 6–8 weeks old (Harlan InterfaunaIbérica SA, Barcelona, Spain). L. major amastigotes (cloneWHOM/IR/-173) were obtained from popliteal lymph nodesfrom infected BALB/c mice and transformed to stationaryphase promastigotes by culturing at 26°C in Schneider’smedium (Gibco, BRL, Grand Island, NY, USA) plus 20% fetalcalf serum (FCS).

Plasmid constructs

The cDNAs coding for the LiP2a and LiP2b proteins wereobtained after EcoRI digestion of pUC8-22 and pUC8-26plasmids [28] and subcloned in pcDNA3 (Invitrogen,San Diego, CA, USA). Endotoxin-free plasmid DNA frompcDNA3-LiP2a and pcDNA3-LiP2b clones was isolated usingthe EndoFree Plasmid Giga kit (Qiagen, Hilden, Germany).

For expression of rLiP2a and rLiP2b proteins, their codingregions (CR) were polymerase chain reaction (PCR) ampli-fied and subcloned into the pQE30 expression vector(Qiagen). Primers employed were: sense, 5′-CGGGATCCATGCAGTACCTCGCCGCGTA-3′ (positions 1–20 of theP2a-CR); anti-sense, 5′-GCGTCGACTTAGTCAAAGAGACCGAAGC-3′ (reverse and complementary to positions302–318 of the P2a-CR) and sense, 5′-CGGGATCCATGTCCACCAAGTACCTCGC-3′ (positions 1–20 of theP2b-CR); anti-sense, 5′-CCCAAGCTTAGTCGAACAGACCGAAGC-3′ (reverse and complementary to positions 319–333 of the P2b-CR). BamHI and SalI restriction sites (under-lined) were included for cloning purposes.

Leishmania antigens

The rLiP2a and rLiP2b were over-expressed in Escherichiacoli transformed with pQE-LiP2a or with pQE-LiP2b andpurified under native conditions onto Ni-nitrilotriacetic acidagarose columns (Qiagen). The recombinant proteins werepassed through a polymyxin-agarose column (Sigma, StLouis, MO, USA). Residual endotoxin content (< 12 pg/mgof recombinant protein) was measured by the Quantita-tive Chromogenic Limulus amebocyte assay QCL-1000(BioWhittaker, Walkersville, MD, USA). Purity of proteinswas assessed in a Coomassie blue-stained sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE)gel (Fig. 1a). Total soluble Leishmania antigen (SLA) ofL. major was prepared as described [27].

Expression of pcDNA3-LiP2a and pcDNA3-LiP2b inCOS-7 cells

To confirm that the DNA constructs were functional, COS-7cells were transfected as described [27] and analysed by

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376 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

immunofluorescence. Briefly, cells were fixed 24 h aftertransfection with 2% paraformaldehyde for 20 min, washedwith phosphate-buffered saline (PBS), blocked and perme-abilized with 1% bovine serum albumin plus 0·1% saponinin PBS for 1 h at room temperature, and incubated for 1 hwith sera from mice vaccinated with rLiP2a and rLiP2b.Afterwards, the coverslips were rinsed with PBS and incu-bated for 1 h with an Alexa488-conjugated goat anti-mouseIgG secondary antibody (Molecular Probes, Eugene, OR,USA). The coverslips were mounted with Mowiol (Calbio-chem, Darmstad, Germany) and visualized on a Zeiss Axio-vert 200 microscope (Fig. 1b–d).

Immunizations and parasite challenge

BALB/c mice were immunized following four regimens:recombinant protein inoculation adjuvated or not with CpGODN, genetic vaccination and a prime-boosting regimen.

Mice were immunized subcutaneously (s.c.) in the rightfootpad with 2 mg of rLiP2a or rLiP2b alone or plus 50 mgof CpG ODN (25 mg of each phosphorothioate-modifiedimmunostimulatory ODN containing CpG motifs asdescribed previously in [27]). Each group was boosted2 weeks later using the same regimen. Control mice receivedPBS or 50 mg CpG-ODN alone.

For genetic vaccinations mice were inoculated s.c. threetimes at 2-week intervals in the right footpad with eitherPBS, 100 mg of pcDNA3 (empty vector), 100 mg pcDNA3-LiP2a or 100 mg pcDNA3-LiP2b. Prime-boosted mice wereimmunized twice, 2 weeks apart, with 100 mg of pcDNA3-LiP2a or 100 mg pcDNA3-LiP2b and boosted 2 weeks laterwith 2 mg of rLiP2a or rLiP2b adjuvated with 50 mg of CpG

ODN. Control mice received two doses of pcDNA3 and wereboosted with 50 mg of CpG ODN.

Parasite challenge was carried out by s.c. inoculationwith 5 ¥ 104 stationary-phase promastigotes of L. majorinto the left footpad 4 weeks after the last inoculation. Theprogress of the infection was followed as described previ-ously [20].

Measurement of cytokines in supernatants

The release of IFN-g and IL-4 was measured in the superna-tants of splenocytes stimulated with rLiP2a or rLiP2b(3 mg/ml) or SLA (12 mg/ml), as described previously [27],using commercial enzyme-linked immunosorbent assay(ELISA) kits (Diaclone, Besançon, France).

Determination of antibody titres and isotypes

Serum samples were analysed for specific anti-rLiP2a, anti-rLiP2b or anti-SLA antibodies. ELISA assays were performedas described previously [27]. The reciprocal end-point titrewas determined by serial dilution of the sera, and wasdefined as the inverse of the highest serum dilution factorgiving an absorbance > 0·2. The following horseradishperoxidase-conjugated anti-mouse immunoglobulins(Nordic Immunological Laboratories, Tilburg, the Nether-lands): anti-IgM (1/2000), anti-IgG (1/2000), anti-IgG1(1/1000) and anti-IgG2a (1/500). Ortophenylene diaminedihydrochloride-OPD (Dako, A/S, Glostrup, Denmark) wasused as peroxidase substrate for ELISA assays. After 15 min,the reaction was stopped by addition of 100 ml of H2SO4 1 Mand the absorbance was read at 450 nm.

Fig. 1. (a) Purification of Leishmania infantum

rLiP2a and rLiP2b after expression in

Escherichia coli. Five mg of rLiP2a (lane 1) and

rLiP2b (lane 2) were electrophoresed

on linear 10–14% gradient sodium dodecyl

sulphate-polyacrylamide gel electrophoresis gel.

The Coomassie-blue staining of the gel is

shown. (b–d) Heterologous cytoplasmic

expression of Leishmania P proteins in COS-7

cells. Immunofluorescence of COS-7 cells

transfected transiently with pcDNA3-LiP2a (b)

and pcDNA3-LiP2b (c); 24 h after transfection

cells were fixed and immunostained with a

polyclonal anti-LiP2a (b) or anti-LiP2b

serum (c). As secondary antibody an

Alexa488-conjugated goat anti-mouse IgG was

employed. As a control, COS-7 cells transfected

with pcDNA3-LiP2a were incubated with

the sera from preimmune mice (d). Similar

results were obtained when cells transfected

with pcDNA-LiP2b were incubated with

the corresponding preimmune sera

(data not shown).

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Statistical analysis

Statistical analysis was performed by a Student’s t-test.Differences were considered significant when P < 0·05.

Results

Antigenicity of the Leishmania P proteins: effect of theDNA vaccines and the CpG ODN on the immuneresponses elicited by the Leishmania P proteins

Using the purified rLiP2a and rLiP2b His-tagged recombi-nant proteins (Fig. 1a), we detected the presence of anti-Leishmania P proteins antibodies in sera of BALB/c miceinfected with L. major. The L. infantum recombinant P pro-teins were employed as antigens for the ELISA assays becausethey were 100% conserved with their corresponding L. majororthologues (Accession no. Q4Q6R6 for LmP2a andQ4QF62 for LmP2b). The humoral response was predomi-nantly of the IgG1 isotype (the mean IgG1/IgG2a ratio was10·2 � 2·5 for rLiP2a and 8·6 � 2·5 for rLiP2b), althoughthe intensity of the anti-LiP2b response was somewhathigher than the anti-LiP2a response (Fig. 2a). Because theinduction of IgG1 and IgG2a antibodies is used as a markerof Th2-type and Th1-type immune responses, respectively[29], we may conclude that during L. major infection a Th2-like humoral response is induced against these antigens.

In a previous work we described that the intraperitoneal(i.p.) administration of the rLiP2a protein in the absence ofadjuvant elicited a predominant IgG1 humoral response[25]. Here we extend this result to BALB/c mice immunizeds.c. with either the rLiP2a or the rLiP2b recombinant pro-teins alone. A specific antibody response was detected 7 daysafter the first dose (Fig. 2b). A progressive switch from IgMto IgG antibodies was observed after the protein boosting.Although some IgG2a specific antibodies were detected, theIgG1 response was predominant after the second dose. TheIgG1/IgG2a ratio against rLiP2a and rLiP2b was 9·1 � 1·2and 20·3 � 2·1, respectively, demonstrating that BALB/cmice immunized with P proteins develop a specific Th2-likeresponse.

We analysed further if the use of CpG ODN or geneticadministration of the Leishmania P proteins counteracts theTh2 response induced by these antigens. For that purpose,eukaryotic expression vectors coding for LiP2a and LiP2bwere constructed and their expression was tested in COS-7cells (Fig. 1b–d). We followed three different inoculationregimens: recombinant protein inoculation adjuvated withCpG ODN, genetic vaccination and a prime-boostingregimen (see Materials and methods for details).

The humoral response elicited by these procedures isdepicted in Fig. 2c–d. Only the CpG motifs were able to altersignificantly the IgG1/IgG2a ratio driven by the proteinsalone. The IgG1/IgG2a ratio after administration of therLiP2a and rLiP2b proteins in the presence of CpG motifs

was 1·42 � 0·4 and 1·14 � 0·35, respectively. Notably, thegenetic immunization of the proteins modified the IgG1/IgG2a only slightly compared to inoculation of P proteinsalone, as the mean IgG1/IgG2a ratio was 4·9 � 1·4 for rLiP2aand 14·2 � 2·1 for rLiP2b. Mice primed with P2a or P2bgenes and boosted with the corresponding recombinantproteins plus CpG ODN exhibit a variable intensity inthe humoral response, but a similar IgG1/IgG2a ratio(1·39 � 0·45 for LiP2a and 2·3 � 0·51 for LiP2b).

To determine whether there was a correlation between theswitch of the IgG1/IgG2a ratio and the cytokine milieu elic-ited by these immunizations, we analysed the IFN-g produc-tion in splenocytes cultures established from vaccinated miceand stimulated with the recombinant P proteins. A specificproduction of IFN-g was observed in all the groups of vac-cinated mice after 48 h of stimulation with the correspond-ing protein, rLiP2a (Fig. 3a) or rLiP2b (Fig. 3b), althoughco-administration of CpG ODN (recombinant proteins plusCpG ODN and prime-boost groups) leads to a significantincrease in IFN-g production specific for both P proteins.No IL-4 was detected in these assays.

The course of L. major infection in vaccinated mice

We then analysed whether the Th1-like response triggered byCpG ODN adjuvant against P proteins was able to alter thecourse of a subsequent L. major challenge. The results indi-cated that the mixed Th1/Th2 immune response induced bythe administration of the recombinant proteins plus CpGODN (Fig. 4a) and the prime-boost regimen (Fig. 4b) didnot have any influence in the time–course of the inflamma-tion of the footpad due to L. major infection, nor did theinoculation of these antigens as DNA vaccines alter thefootpad swelling due to L. major infection (Fig. 4c). The lackof protection was confirmed by the observation that asimilar parasite load was found in the DLN of the controland of the vaccinated mice (data not shown).

Mice presensitized with P proteins exhibit a mixed Th1/Th2 response against the LiP2a and LiP2b proteins and anenhanced Th2 response against the whole soluble antigensof the parasite.

Humoral and cellular responses against the LiP2a andLiP2b antigens were analysed in mice infected with L. majorin order to determine the immunological parameters asso-ciated with the lack of protection. After L. major infection,those mice immunized with the P proteins showed anenhanced ability to recognize the recombinant rLiP2a(Fig. 5a) and rLiP2b (Fig. 5b) proteins as the titre for bothIgG1 and IgG2a, being higher in the vaccinated mice than intheir corresponding controls. No statistical differencesbetween the two isotypes were detected. In order to analysethe antigen-driven cellular responses, splenocytes from dif-ferent mice groups were stimulated in vitro with eitherrLiP2a or rLiP2b and the presence of IFN-g and IL-4 in the

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378 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

supernatants was assessed. The specific production of IFN-gand IL-4 was higher in the groups of vaccinated mice relativeto control mice after 48 h of stimulation with the corre-sponding protein, rLiP2a (Fig. 5c,e) or rLiP2b (Fig. 5d,f).

Although no alterations in the time–course of L. majorinfection were observed in mice vaccinated with Pproteins, we determined whether the immunization withthese antigens alters the IL-4 driven Th2 response that leadsto susceptibility in BALB/c mice; the titre of the IgG1 and

IgG2a antibodies against the soluble Leishmania antigens(SLA) was determined 7 weeks after challenge in the differ-ent groups of mice (Fig. 6a). Notably, the ratio IgG1/IgG2awas higher in mice vaccinated with either rLiP2a or rLiP2b,especially in the prime-boost regimen. This suggests thatthe cytokine milieu influenced by the administration of theP proteins may have favoured a Th2 response to the wholeparasite. In agreement with this finding, it was observedthat the in vitro production of IFN-g specific for SLA was

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Fig. 2. (a) Leishmania P proteins induced a predominant T helper 2-like response in L. major-infected BALB/c mice. Analysis of the anti-LiP2a and

anti-LiP2b humoral response in BALB/c mice infected with L. major. Six naive BALB/c mice were inoculated with 5 ¥ 104 stationary-phase L. major

promastigotes into the left footpad. Sera were obtained after 8 weeks of infection and tested individually by enzyme-linked immunosorbent assay

(ELISA) against the recombinant rLiP2a and rLiP2b P proteins. Reciprocal end-point titres for IgG1 and IgG2a antibodies subclasses were

determined as indicated in Materials and methods. (b) Analysis of the specific humoral response induced in BALB/c mice immunized with the

LiP2a and LiP2b acidic ribosomal proteins without adjuvants. Six mice were immunized twice in the right footpad with 2 mg of rLiP2a or rLiP2b.

Sera were obtained and assayed individually by ELISA 7 days after the first immunization (white bars) or 7 days after the second immunization

(solid bars). Reciprocal end-point titre was determined for IgM, IgG, IgG1 and IgG2a antibodies subclasses. No reactivity was detected in

the preimmune sera. (c–d) Immune responses elicited by the genetic inoculation of parasite P proteins. BALB/c mice (six mice per group)

were immunized with the recombinant rLiP2a or rLiP2b proteins alone and adjuvated with CpG oligodeoxynucleotides (ODN), by genetic

vaccination and by prime-boosting regimen, as indicated in Materials and methods. As control, mice were vaccinated in the same manner with

phosphate-bufered saline, with CpG ODN alone, with the empty pcDNA3 plasmid and with the empty plasmid and CpG ODN. For measurement

of the specific anti-LiP2a (b) or anti-LiP2b (c) humoral responses sera were individually assayed by ELISA 7 days after the last immunization.

The reciprocal end-point titre is shown. None of the preimmune or the control sera showed reactivity against the corresponding antigens.

Antigenicity of Leishmania Proteins

379© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

similar in splenocytes from all of the mice groups (Fig. 6b),whereas the level of IL-4 was significantly higher insplenocytes from immunized mice than in controls(Fig. 6c), confirming that presensitization with P proteinsenhanced the Th2 response of the BALB/c mice againstL. major.

Discussion

Upon infection with L. major, mice of the resistant pheno-type clearly develop a dominant Th1 immune response tothe parasite antigens, whereas BALB/c mice develop a typicalTh2 response and succumb to infection. This association ofresistance and susceptibility with the emergence of the twophenotypically distinct subsets of CD4+ T cells has been themost striking concept arising from the murine model ofcutaneous leishmaniasis (reviewed in [14–16]). Those Leish-mania antigens that predominantly stimulate Th1 responses

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stimulated in vitro with the rLiP2a (a) and rLiP2b (b) P proteins.

Groups of mice described in Fig. 2 were killed 4 weeks after the last

immunization, and their splenocytes were obtained and cultured in

vitro for 48 h in the presence of the corresponding rLiP2a or rLiP2b

soluble proteins or medium alone. The supernatants were harvested

and assayed individually for IFN-g production by enzyme-linked

immunosorbent assay.

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Fig. 4. Course of Leishmania major infection in BALB/c vaccinated

mice. Mice (six per group) were immunized twice s. c. with the

recombinant rLiP2a or rLiP2b proteins adjuvated with CpG

oligodeoxynucleotides (ODN) (a), primed twice with the recombinant

plasmid and boosted with the rLiP2a or rLiP2b adjuvated with CpG

ODN (b), or three times with the pcDNA3-LiP2a or pcDNA3-LiP2b

eukaryotic expression plasmids (c), as indicated in Materials and

methods. As control, mice were vaccinated in the same manner with

CpG ODN alone (a), with the empty plasmid and CpG ODN (b) and

with the empty pcDNA3 plasmid (c). For comparison, unvaccinated

groups injected with phosphate-buffered saline were included (a–c).

One month after the last immunization, mice were infected in the left

hind footpad with 5 ¥ 104 L. major stationary-phase promastigotes.

Footpad swelling is given as the difference of thickness between the

infected and the uninfected contralateral footpad. No significant

differences were found between vaccinated mice and their

corresponding controls in any group.

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380 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

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Ant

ibod

y tit

re

IgG1IgG2a

Ant

ibod

y tit

re

IgG1IgG2a

(a) (b)

(c) (d)

(e) (f)

0

0·5

1

1·5

2

2·5

3

3·5

IFN

-γ (

ng/m

l)

LiP2a

Medium

0

0·5

1

1·5

2

2·5

3

IFN

-γ (

ng/m

l)

rLiP2b

Medium

0

5

10

15

20

25

30

35

40

IL-4

(ng

/ml)

LiP2a

Medium

0

10

20

30

40

50

60

IL-4

(pg

/ml)

rLiP2bMedium

Fig. 5. Analysis of the immune response against the acidic ribosomal proteins in vaccinated and control mice after Leishmania major challenge.

Serum samples from different groups of vaccinated and control mice were obtained 7 weeks after challenge and the reciprocal end-point titres for

IgG1 and IgG2a antibodies against rLiP2a (a) and rLiP2b (b) were determined by enzyme-linked immunosorbent assay (ELISA). At the same time

mice were killed and their splenocytes were obtained and cultured in vitro in complete medium with or without the recombinant proteins. The

supernatants were harvested and the specific rLiP2a-driven (c) or rLiP2b-driven (d) production of interferon-g, and the specific rLiP2a-driven

(e) or rLiP2b-driven (f) production of interleukin-4 was assayed by ELISA.

Antigenicity of Leishmania Proteins

381© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

Fig. 6. Analysis of the immune response

against total parasite proteins induced by the

Leishmania major challenge in vaccinated and

control mice. (a) Analysis of the humoral

response against soluble Leishmania antigen

(SLA) from the different regimens of

vaccination and the corresponding controls.

Serum samples were obtained 7 weeks after

challenge and the reciprocal end-point titre for

IgG1 and IgG2a antibodies against SLA was

determined individually by enzyme-linked

immunosorbent assay (ELISA). The ratio

between IgG1 and IgG2a titres for the different

groups has been also included. Seven weeks

after infection animals from the different

regimens of vaccination and the corresponding

controls were killed and their splenocytes were

obtained and cultured in vitro in complete

medium with or without SLA. The supernatants

were harvested and assayed for interferon-g(b) and interleukin-4 (c) by ELISA. *P < 0·05;

**P < 0·02.

(a)

0

50

100

150

200

250

300

IL-4

(pg

/ml)

(b)

(c)

0

1000

2000

3000

4000

5000

6000

7000

IFN

-γ (p

g/m

l)

SLA

Medium

SLA

Medium **

**

*

*

0

5 000

10 000

15 000

20 000

25 000

30 000

35 000

40 000

Saline

CpG

pcDNA3

pcDNA3

+ CpG

rLiP

2a +

CpG

pcDNA3-

LiP2a

Prime-

boos

t LiP

2a

rLiP

2b +

CpG

pcDNA3-

LiP2b

Prime-

boos

t LiP

2b

Saline

CpG

pcDNA3

pcDNA3

+ CpG

rLiP

2a +

CpG

pcDNA3-

LiP2a

Prime-

boos

t LiP

2a

rLiP

2b +

CpG

pcDNA3-

LiP2b

Prime-

boos

t LiP

2b

Saline

CpG

pcDNA3

pcDNA3

+ CpG

rLiP

2a +

CpG

pcDNA3-

LiP2a

Prime-

boos

t LiP

2a

rLiP

2b +

CpG

pcDNA3-

LiP2b

Prime-

boos

t LiP

2b

Ser

a tit

re

IgG1

IgG2a

3·24 2·67 2·66 1·87 13·4 7·34 16·4 3·7 7·3 32

IGg1/IgG2a

S. Iborra et al.

382 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

in patient cells or spleen and lymph node cells from miceinfected with L. major have been commonly accepted aspromising vaccine candidates. In contrast, antigens thatstimulate predominantly a Th2 response have been consid-ered as ‘pathoantigens’ because the response induced againstthem is thought to result in immunopathology [2,3]. Para-doxically, several leishmanial antigens against which a Th1response is developed during the infection are not necessar-ily protective, whereas redirecting the Th2 responses inducedagainst some Leishmania epitopes towards a Th1 responsemay induce a substantial protection against infection(recently reviewed in [21]).

In this study we show that a humoral response is inducedagainst the LiP2a and LiP2b proteins in BALB/c miceinfected experimentally with L. major. These proteins havebeen also described as antigens in dogs [24] and humans[30] infected naturally with L. infantum and in hamster [31]and mouse (N. Parody, laboratory results) infected experi-mentally with L. infantum. In addition, antibodies againstribosomal P proteins have been found in sera from patientswith infectious diseases other than leishmaniasis, such asChagas’ disease [32], patients with fungal allergies [33,34]and patients having autoimmune processes such as systemiclupus erythematosus [35]. Interestingly, it has been reportedthat these anti-P antibodies are implicated in the immuno-pathology of these diseases [36–41]. Moreover, our dataindicate that the anti-Leishmania P2a and P2b antibodiesinduced in infected BALB/c mice were predominantly of theIgG1 isotype. Thus, we may conclude that during the murineinfection a Th2-like response against these antigens isinduced.

The data presented here demonstrate that the parasiteP proteins behave as strong immunogens, as they elicithumoral responses when immunized s.c. in the footpads ofBALB/c mice in the absence of any adjuvant. The progressiveswitch from IgM to IgG antibodies after the protein boostingcan be taken as an indication that the immunization withthese proteins elicited a T cell-dependent immune response.Concomitantly with specific humoral response obtainedduring infection with L. major, there is a switch of antibodyclasses to the IgG1 isotype after the second administration ofboth recombinant proteins. Thus, we conclude that theadministration of these proteins induces a Th2-like humoralresponse in the BALB/c mice.

Strikingly, the genetic administration of these antigens asDNA vaccines, a general strategy employed to promote a Th1type response, induce only slight changes in the Th2-likeresponse against Leishmania P proteins. As far as we areconcerned, only few examples of antigens that generate adominant Th2-type response in mice when used in geneticimmunizations have been described, including the serinerepeat antigen (SERA) from Plasmodium falciparum [42]and the 45 W antigen from Taenia ovis [43]. Also, DNAimmunization of the Leishmania meta 1 antigen, a proteinover-expressed in metacyclic promastigotes, induced a

predominant Th2-type of response that correlated with lackof protection after Leishmania infection [44]. Data presentedhere demonstrate that it is only by co-administration of theCpG ODN adjuvant, both administered with the recombi-nant rLiP2a and rLiP2b proteins or in a prime-boostregimen, that a balanced IgG2a/IgG1 ratio is observed con-comitantly with higher levels of IFN-g induced in vitro.These data indicate that the use of CpG ODN adjuvantinduced Th1 responses against Leishmania P proteins,although these immunization protocols were not able toinhibit completely the Th2-like response induced againstthese antigens. The potential of CpG ODN to suppress Th2responses in mouse has been demonstrated widely in para-sitic diseases [45–47] and allergy disorders [48], but in somereports it has also been demonstrated that CpG ODN mayfail to down-regulate neonatally Th2 triggered responses[49] or ongoing IgE responses [50].

Taking into account the antigenicity of the LiP2a andLiP2b proteins, and the correlation of Th2 and Th1responses with susceptibility and resistance to L. major infec-tion in BALB/c mice, we analysed the effects of the differentinoculation regimens in the course of L. major infection. Asindicated above, genetic immunization of some definedLeishmania antigens that induce Th2 responses during infec-tion protects BALB/c mice against cutaneous leishmaniasis.It has also been described that immunization with aquimeric protein (Q protein) containing the LiP2a andLiP2b, engineered to have deletions of the carboxyl-terminalregions and the antigenic regions of the H2A and the LiP0Leishmania proteins [51] mixed with live bacille Calmette–Guérin (BCG), confers protection to dogs challenged withL. infantum [52]. In addition, co-administration of Qprotein mixed with CpG ODN resulted in a reduction of theparasite burden of BALB/c mice infected with L. infantum[53]. However, no differences in the clinical outcomes ofinfection after challenge with L. major were observed in anyof the groups of mice employed in this work. No protectionwas observed in mice vaccinated with the rLiP2a or rLiP2bantigens mixed with CpG (recombinant proteins plus CpGODN and prime-boost), in spite of the specific Th1-likeresponse elicited against the P proteins. This lack of protec-tion correlates with the induction of mixed Th1/Th2responses against the P proteins in all the vaccinated micegroups after infection. As it has been suggested that theinduction of a Th2 response simultaneous to a Th1 couldabrogate the protective function of this response in this par-ticular model of infection [26,54], this may explain the lackof protection of the mixed Th1/Th2 induced by the inocu-lation of the P proteins with CpG ODN. In fact, we havedescribed that the IgG1 and the IL-4 response against thewhole parasite is enhanced in those mice presensitized withthe acidic ribosomal proteins. A slight exacerbation of thedisease may be expected due to this enhancement and, prob-ably, due to the pre-existing immune response against Leish-mania P proteins induced by the vaccination. However, this

Antigenicity of Leishmania Proteins

383© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 375–385

enhancement seems not to be enough to change the clinicalfeatures of the disease in the BALB/c, otherwise a highlysusceptible strain of mice.

Given that immunomodulation of the Th2 responseinduced against Leishmania antigens towards a Th1-type hasbeen demonstrated to be a potential strategy for the devel-opment of vaccines against leishmaniasis, the existence ofcertain parasite antigens inducing Th2 responses whichcannot be redirected fully by the usual Th1 inducers shouldbe taken into account for the design of a molecularly definedvaccine against this disease.

Acknowledgements

This work was supported by grant PI050973 from FIS. Aninstitutional grant from Fundación Ramón Areces is alsoacknowledged.

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