Vaccine 24 (2006) 5645–5652

Efficacy of vaccination with a combination of Leishmania amastigoteantigens and the lipid A-analogue ONO-4007 for immunoprophylaxis

and immunotherapy against Leishmania amazonensis infectionin a murine model of New World cutaneous leishmaniasis

Manuel Calvopina a,∗, Paola A. Barroso a, Jorge D. Marco a, Masataka Korenaga a,Philip J. Cooper b,c, Shigeo Nonaka d, Yoshihisa Hashiguchi a

a Department of Parasitology, Kochi Medical School, Kochi University, Kochi 783-8505, Japanb Laboratorio de Investigaciones, Hospital Pedro Vicente Maldonado, Pichincha Province, Ecuador

c St. George’s University of London, London, UKd Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Nishihara-cho, Okinawa 903-0215, Japan

Received 23 February 2006; accepted 2 March 2006Available online 29 March 2006

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Activation of innate immunity using adjuvants that activate Toll-like receptor 4 pathways have great potential for improving the protectionnduced by parasite vaccines. We investigated protective and therapeutic effects of a vaccine against leishmaniasis containing a combinationf an adjuvant synthetic lipid A-analogue, ONO-4007 and Leishmania amazonensis antigens. ONO-4007 was co-injected with soluble andembrane-enriched L. amazonensis-amastigote antigens into BALB/c mice that had either already been infected with 1 × 106 L. amazonensis

romastigotes (immunotherapy study) or before challenge with the same infectious dose (immunoprophylaxis study). Sixty percent of miceaccinated before infectious challenge controlled their Leishmania infections – defined by the absence of footpad-swelling and negativeeishmania cultures – compared to 0% of controls, and 40% of mice vaccinated after infection resolved their infections compared to 0% ofontrols. Protective immunity in both immunoprophylaxis and immunotherapy models was associated with increased protein production of IL-2 and IFN-�. These data suggest that vaccination with a combination of ONO-4007 and amastigote antigens of L. amazonensis may be usefulor the prevention and treatment of leishmaniasis, and that the protective immunity induced is associated with the production of type-1 cytokines.

2006 Elsevier Ltd. All rights reserved.

eywords: Leishmaniasis; Vaccine; ONO-4007; Adjuvants

. Introduction

New World cutaneous leishmaniasis (CL) is an importantnfection in many regions of the Americas and it causes sig-ificant disfigurement and long-term morbidity [1]. Cellularmmune mechanisms are critical for the healing of CL lesionsnd for protection against subsequent infectious exposuresn both humans and mice. The development of long-lastingmmunity following healing would suggest that the develop-ent of effective vaccines against CL is a realistic goal.

∗ Corresponding author. Tel.: +81 88 880 2417; fax: +81 88 880 2415.E-mail address: mcalvopina@hotmail.com (M. Calvopina).

Currently, the treatment of CL depends on the use of highlytoxic compounds (e.g., pentavalent antimonials and pentami-dine) and no new drugs have emerged over the past 50 yearsthat have gained widespread acceptance for the treatment ofCL. A growing problem with the use of current drugs is drugresistance [2] and there is an urgent need for the developmentof new therapies.

Vaccines against leishmaniasis hold considerable promiseand although several vaccine candidates have been evaluatedin murine models of CL including whole killed parasites,live-attenuated parasites, and recombinant and synthetic pep-tides [3,4], none have been licensed for human use [1].An alternative therapeutic approach for active infections is

264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2006.03.023

5646 M. Calvopina et al. / Vaccine 24 (2006) 5645–5652

immunotherapy and studies in animal models [5–7] and clin-ical trials in Venezuela and Brazil have shown high cure rateswhen whole-parasite antigens are injected in combinationwith either BCG [8–10] or meglumine antimoniate [11]. Fur-ther, a cocktail of recombinant leishmanial antigens in thelipid A-derived adjuvant, MPLR, have shown efficacy in thetreatment of mucosal leishmaniasis [12,13].

The inclusion of adjuvants in vaccines for leishmaniasisis critical for efficacy against experimental murine leishma-niasis [14]. Recent research has focused on the identificationof adjuvants that stimulate cellular immunity, and those thatact through Toll-like receptor 4 (TLR4) have shown particu-lar promise [13,15]. ONO-4007 is a synthetic analogue oflipid A, the bioactive centre of lipopolysaccharide (LPS)[16] and has strong antitumour effects due to its capacityto enhance cellular immunity through the induction of highlevels of pro-inflammatory cytokines (IL-1a, IL-6, TNF-�),type-1 cytokines (IL-12 and IFN-�) and nitric oxide synthase(iNOs) [17–20] by the probable stimulation of TLR4 ligandsof the innate immune system [21,22]. The ability of ONO-4007 to enhance type-1 immunity and nitric oxide productionmay have potent antileishmanial effects and is supported byour previous observations [23] that have shown that ONO-4007 alone is able to inhibit the intracellular proliferation ofL. amazonensis and L. major amastigotes in macrophages.

Recent advances have been made towards understand-iminte�aac

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in RPMI-1640 medium (Nissui, Japan) supplemented with10% heat-inactivated fetal bovine serum (FBS), 2 mMl-glutamine, 100 units/ml of penicillin and 100 �g/ml ofstreptomycin. The murine macrophage cell line J774 (RikenBioresource Center, Tsukuba, Ibaraki, Japan) was culturedin the same medium and maintained in a 5% CO2–95% airmixture at 37 ◦C.

2.2. Amastigotes

Metacyclic promastigotes of L. amazonensis werereleased in 75 cm2 tissue culture flasks containing adherentJ774 macrophages at a ratio of 10:1 and maintained in a 5%CO2–95% air mixture at 34 ◦C as described previously [28].After 16 h of culture, the flasks were washed to remove freeparasites. Infected macrophages were collected in PBS andvortexed to release amastigotes that were isolated by cen-trifugation in 35% Percoll (Sigma, St. Louis, USA) in PBSat 3500 × g for 30 min, washed five times in sterile PBS pH7.2 and frozen at −80 ◦C until use.

2.3. Soluble (SLA) and membrane-enriched (MEA)antigens

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ng the host immune response in CL [24]. In particular, theechanisms involved in the pathogenesis of L. amazonensis

nfection appear to involve impaired activation of early immu-ity such as reduced expression of inflammatory cytokineshat are important for Th1-cell development and macrophageffector functions [25]. On the other hand, low levels of IFN-accompanied by the relative absence of IL-4 are typical of

ctive disease associated with this Leishmania species [26],nd protective immunity and healing are mediated by type-1ytokines [27].

Selected agonists of the innate immune response couldvercome early barriers to the development of protectivemmunity to L. amazonensis infection through the activationf pro-inflammatory and type-1 cytokines. To investigate theotential of ONO-4007 to enhance protective immunity to L.mazonensis, we vaccinated BALB/c mice with ONO-4007ombined with L. amazonensis-amastigote antigens beforenfectious challenge (immunoprophylaxis) and during activenfection (immunotherapy) with the same strain of L. ama-onensis, in an experimental model of New World CL.

. Materials and methods

.1. Parasites and macrophage culture

Leishmania amazonensis (MHOM/BR/73/M2269)as used to infect and challenge mice, and was used toroduce vaccine antigens for immunoprophylaxis andmmunotherapeutic studies. Promastigotes were cultured

SLA and MEA antigens were prepared from L. amazonen-is amastigotes, as described previously [29]. Briefly, frozentored amastigotes were resuspended in 1 ml of a cold solu-ion of 0.05 Tris–HCl, pH 7.8, in the presence of enzymenhibitors (100 U/ml aprotinin, 2 mM pepstatin A and 2 mMhenylmethylsulfonylfluoride; Sigma). Parasites were sub-ected to 10 freeze–thaw cycles consisting of immersion iniquid nitrogen and thawing at 35 ◦C. The suspension wasentrifuged at 25,000 × g for 1 h at 4 ◦C. The resulting pelletsere extracted in a detergent/enzyme inhibitor mixture (1:1,/v; 1.0% sodium dodecyl sulfate, SDS) for 14 h at 4 ◦C andentrifuged at 105,000 × g for 1 h at 4 ◦C to remove cellularebris. The supernatant from the first centrifugation (solublentigens) was pooled with the supernatant from the secondentrifugation (extracted membrane proteins). The proteinoncentration was determined using the Bradford methodBio-Rad Labs, CA, USA), and the final concentration wasdjusted to 250 �g of protein/50 �l and stored at −80 ◦C.

.4. Adjuvant

ONO-4007 was kindly donated by the Ono Pharmaceuti-al Co., Osaka, Japan. The drug was provided in ampoulesontaining 100 mg of ONO-4007 dissolved in 2 ml of 55%thanol. It was reconstituted at 1 mg/0.1 ml in 5% glucose.

.5. Mice vaccination

Eight to 10 weeks old female BALB/c mice were obtainedrom Japan SLC (Hamamatsu, Japan). Thirty-four mice weresed each for immunoprophylaxis and immunotherapy stud-

M. Calvopina et al. / Vaccine 24 (2006) 5645–5652 5647

ies in two separate experiments: in each study, 8 were injectedwith saline alone (Group A); 8 were injected with 250 �gSLA-MEA alone (Group B); 8 were injected with 50 mg/kgONO-4007 alone (Group C); and 10 mice were injected intra-dermally with 250 �g SLA-MEA Leishmania antigen plus50 mg/kg ONO-4007 (Group D). Mice were maintained in aspecific-pathogen-free facility and were provided with foodand water ad libitum. All experiments were carried out fol-lowing protocols approved by the Institutional Animal Careand Use Committee of Kochi University.

2.6. Immunotherapeutic study

Prior to infection, mice were randomly assigned to the fourintervention groups. All mice were inoculated intradermallywith a suspension of 1 × 106 L. amazonensis metacyclic pro-mastigotes (in 0.1 ml of PBS) into the dorsal side of theright hind footpad. Three weeks after infection, mice werevaccinated seven times at 3-day intervals, in the left foot-pad, according to their pre-assigned vaccination group. Ina separate experiment, five mice were treated with 250 �gSLA/MEA Leishmania antigen plus 50 mg/kg ONO-4007just 1 week after receiving 1 × 106 L. amazonensis meta-cyclic promastigotes.

2.7. Immunoprophylaxis study

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immunoprophylaxis study (except four of Group D that wereused to asses the longevity of protection). In a further exper-iment to measure IL-12p-70, three mice from each subgroupwere sacrificed at 24 h after receiving the final vaccine dose.Draining popliteal and inguinal lymph nodes were removedfrom each mouse. The lymph nodes were combined and thecells were disassociated with a cell strainer homogenizerand resuspended in RPMI-1640 medium (Nissui, Japan) sup-plemented with 5% FBS, penicillin 100 U/ml, streptomycin100 �g/ml, 2 mM l-glutamine, 0.05 mM 2-mercaptoethanol,15 mM HEPES and 0.1 mM sodium pyruvate, at a density of106 cells/ml in a final volume of 200 �l in U-bottom 96-wellplates (Nunc, Denmark). The cells were incubated in mediumonly (unstimulated), or were stimulated with 50 �g/ml ofSLA-MEA L. amazonensis-antigens. Supernatants were col-lected at 72 h and stored at −80 ◦C until analysis.

2.10. Cytokine ELISA assay

Concentrations of IL-12p70, IFN-� and IL-4 in cul-ture supernatants were measured by sandwich ELISA usingcommercially available antibody pairs (eBioscience, USA).Briefly, Maxisorp 96-well plates (Nunc, Denmark) werecoated overnight with the capture antibody, blocked withPBS/10% BSA, and incubated with the culture supernatantsand recombinant cytokine standards. After incubation withtwa4cc

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Mice were randomly assigned to vaccination groups andaccinated as described for the therapeutic study. One dayfter the final vaccination, all mice were challenged intra-ermally with a suspension of 1 × 106 L. amazonensis meta-yclic promastigotes (in 0.1 ml of PBS) into the dorsal sidef the right hind footpad. To assess the longevity of protec-ion, four successfully immunized mice (i.e., no evidence ofutaneous lesions and parasites in cultures at 5 weeks post-nfection) were re-challenged at 2 months post-vaccinationgain with 1 × 106 L. amazonensis promastigotes.

.8. Infection cure, footpad-swelling and parasiteulture

The progress of infection was assessed by measuring thehickness of the infected footpad weekly using a dial microm-ter (Mitutoyo Co., Japan) as described previously [30].easurement of footpad-swelling is the standard method for

ssessing susceptibility to Leishmania disease in mice [31].ecause absence of footpad-swelling may not reflect ster-

le immunity, we obtained tissue by needle aspiration fromhe infected footpad at the end of follow-up and culturedor Leishmania parasites in USMARU biphasic medium asescribed previously [32].

.9. Lymph node cell suspension and culture

All animals were euthanized 8 weeks post-infection forhe immunotherapy study and 5 weeks after challenge for the

he biotinylated detection antibody, plates were incubatedith avidin-conjugated alkaline phosphatase, followed by

ddition of substrate. Mean absorbance was measured at50 nm using a spectrophotometer (Thermomax, USA) andytokine concentrations were determined from standardurves generated from recombinant cytokines.

.11. Statistical analysis

Statistical differences between vaccination groups weressessed using one way analysis of variance for comparisonsf measurements of footpad-swelling and cytokine levels.ost hoc analyses in which vaccination groups were com-ared were adjusted using the Bonferroni correction. Allnalyses were performed in SPSS 11.0. p-Values of less than.05 were considered statistically significant.

. Results

.1. Immunotherapy study

.1.1. ONO-4007 alone or in combination with SLA-EA antigens has therapeutic potential for the treatment

f leishmaniasisMean footpad-swellings for the four vaccination groups

re shown in Fig. 1. Six of 10 (60%) mice treated with theombination (Group D) had no footpad-swelling by 8 weeksf follow-up although 2/6 remained culture positive for Leish-ania parasites (i.e., cure was 40%). Footpad-swelling was

5648 M. Calvopina et al. / Vaccine 24 (2006) 5645–5652

Fig. 1. Development of swelling of right footpads in the four groups inthe immunotherapy study. Vaccination groups were: saline (A), SLA/MEALeishmania amastigote antigens only (B), ONO-4007 alone (C) and ONO-4007 plus SLA/MEA Leishmania amastigote antigens (D). Each line repre-sents the mean footpad-swelling of each group, and bars show the standarderror of the means (S.E.M.) from two independent experiments. Arrowsrepresent vaccination time points after inoculation in the right hind foot-pad with 1 × 106 metacyclic L. amazonensis promastigotes (0 = infectiontime). Footpad-swelling was measured weekly for up to 8 weeks post-infection. Symbols: (*) Statistical difference between Groups C vs. GroupsA (p < 0.0001) and Groups C vs. Groups B (p < 0.0001); (+) statistical differ-ence between Groups D vs. Groups A (p < 0.0001) and Groups D vs. GroupsB (p < 0.0001); (#) statistical difference between Groups D vs. Groups C(p = 0.069).

reduced (p < 0.0001) in mice injected with the combinationor with ONO-4007 alone compared to control-saline (GroupA) and Leishmania antigen (Group B) vaccinated groups.Mice vaccinated with antigens alone had no evidence ofreduced swelling compared to control-saline mice. Footpad-swelling was not significantly different (p = 0.069) betweenmice receiving the combination (Group D) and ONO-4007alone (Group C) however, only one mouse was negative forparasite culture in Group C compared to four in Group D. Allmice receiving saline or Leishmania-antigens alone devel-oped progressive footpad-swelling that had become ulcer-ative by the end of follow-up. In a separate uncontrolledexperiment of mice treated with the combination 1 week afterinfection (rather than 3 weeks), healing with parasite-negativecultures was observed in 4/5 (80%) mice (data not shown).

3.2. Immunoprophylaxis study

3.2.1. Protective immunity is induced after vaccinationwith ONO-4007 plus Leishmania SLA-MEA antigens butnot with the antigens alone

Mean footpad-swelling in the four groups after infectiouschallenge is shown in Fig. 2. No footpad-swelling or lesionsof leishmaniasis were observed in 7/10 of mice vaccinatedwith the combination (Group D), but 1/7 of the protected micecontinued to have positive Leishmania cultures (i.e., protec-t(sfi

Fig. 2. Development of swelling of right footpads in the four groups in theimmunoprophylaxis study. Vaccination groups were: saline (A), SLA/MEALeishmania amastigote antigens only (B), ONO-4007 alone (C) and ONO-4007 plus SLA/MEA Leishmania amastigote antigens (D). Each line repre-sents the mean footpad-swelling of each vaccination group, and bars showthe standard error of the means (S.E.M.) from two independent experiments.Mice vaccinated with the respective compound were infected with 1 × 106

metacyclic L. amazonensis promastigotes (0 = challenge time) in the righthind footpad 3 weeks after the seventh dose. Footpad-swelling was measuredweekly for up to 5 weeks post-challenge. Symbols: (*) Statistical differencebetween Groups C vs. Groups A (p < 0.0001) and Groups C vs. GroupsB (p < 0.0001); (+) statistical difference between Groups D vs. Groups A(p < 0.0001) and Groups D vs. Groups B (p < 0.0001); (#) statistical differ-ence between Groups D vs. Groups C (p < 0.001).

alone as vaccine candidates [33]. There was significant differ-ence of footpad-swelling between mice receiving the vaccinecombination (p < 0.0001) or ONO-4007 alone (p < 0.0001)compared to either saline or antigen groups. However, in con-trast to the immunotherapy study, mice receiving the vaccinecombination (Group D) had significantly reduced footpad-swelling (p < 0.001) compare to those receiving ONO-4007alone (Group C, in which none was protected). To exam-ine the longevity of protection, four protected BALB/c micevaccinated with the combination were re-challenged after 2months; none of the mice developed footpad-swelling by 4weeks after re-challenge and parasite cultures were negative(data not shown).

3.3. Cytokine correlates of protective immunity

3.3.1. IL-12p70 and IFN-γ are produced by ex vivolymph node cell cultures after vaccination withONO-4007 alone or in combination with Leishmaniaamastigote antigens

Lymph node cells were cultured from mice in boththerapeutic and prophylactic studies. Only cultures stimu-lated with Leishmania antigen from mice vaccinated withONO-4007 alone or with the combination (Fig. 3A and B)showed elevated IL-12p70 when animals were sacrificed 24 hafter receiving the seventh vaccine dose. IL-12p70 was notdwtI

ion was 60%). None of the mice injected with antigens aloneGroup B) or saline (Group A) was protected from footpad-welling and all were positive for Leishmania cultures, con-rming previous studies regarding the inefficacy of antigens

etectable from cultures of mice euthanized after 5 and 8eeks. In both studies, at the end of follow-up (8 weeks for the

herapeutic and 5 weeks for the prophylaxis studies), greatestFN-� responses (3.7-fold higher compared to saline-injected

M. Calvopina et al. / Vaccine 24 (2006) 5645–5652 5649

Fig. 3. Production of cytokines by lymphocytes by ex vivo lymph nodecell cultures in the immunotherapy (A) and immunoprophylaxis (B) studies.Columns are: IFN-� (clear), IL-4 (shaded) and IL-12 (hashed bars). Sym-bols show comparisons made between groups for each cytokine: (*, IFN-�)for Group D vs. Group A (p < 0.0001), Group C vs. Group A (p < 0.0001 inGroup A and 0.281 in Group B); (+, IL-4) for Group B vs. Groups A, C andD (p < 0.0001); (#, IL-12) for Group D vs. Groups A, B and C (p < 0.0001).These results are representative of two separate experiments with four to fivemice per group. Bars on columns represent S.E.M.s. SLA/MEA, L. amazo-nensis soluble (SLA) and membrane-enriched (MEA) amastigote antigens.nd, none detected.

mice) were observed in the groups receiving the combina-tion (Group D). Mice injected with ONO-4007 alone hadalso higher values of IFN-� (3- and 1.5-fold greater thansaline-injected mice in the therapeutic and prophylaxis stud-ies, respectively). Surprisingly, in both studies, control micereceiving saline (Group A) showed significant levels of IFN-�that were greater than those observed in mice vaccinated withantigens alone (immunotherapy, p < 0.0001; immunoprophy-laxis, p = 0.044). Levels of IL-4 appeared to be inversely asso-ciated with IFN-� production in both studies. IL-4 productionwas significantly greater in mice receiving Leishmania anti-gens (Group B; p < 0.0001) than all other vaccinated groups,indicating that this Th2 cytokine was enhanced by the admin-istration of antigens. Furthermore, IL-4 production in saline

(Group A) was significantly higher (p < 0.05) compared tothose receiving ONO-4007 alone (Group C) or in combi-nation with Leishmania antigens (Group D), indicating thatinjecting ONO-4007 may suppress the production of IL-4.

4. Discussion

There is an urgent need for the development of effec-tive prophylactic vaccines and new therapies for leishma-niasis. Our data indicate that a vaccine that incorporatesthe novel lipid A-analogue ONO-4007 as an adjuvant forL. amazonensis-soluble and membrane-enriched amastigote-antigens induced high rates of protection against subsequentchallenge and during active infections with the same strain,and that the immunity generated was associated with the pro-duction of type-1 cytokines (IL-12 and IFN-�). Moreover,clinically healed lesions in most mice did not harbor liveLeishmania parasites as demonstrated by negative cultures.Our results are consistent with previous studies showing thatprotection and healing is feasible against L. amazonensisinfection and that resistance to infection and healing of activeinfections are associated with an enhanced type-1 cytokineimmune response [34–36].

In the present study, we used antigens derived from thepathogenic intracellular amastigote stage. In leishmaniasis,fcidtagedoltb[a1a

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or an immunoprotective vaccine to be effective, the vac-ine must be able to direct the host immune response againstntracellular amastigotes in affected tissues, as promastigote-erived vaccines tend to have low efficacies [37,38]. Fur-hermore, we used a combination of membrane and solublentigens to generate protective immunity. Membrane anti-ens may be particularly important as these antigens are mostxposed to host immunity. Immune protection may be depen-ent upon antigen accessibility and molecules expressedn the parasite surface membrane or that are secreted areikely to be important. For this reason, it has been proposedhat candidate vaccine antigens for future vaccines shoulde abundant and preferably secreted and surface-exposed39]. Importantly, the addition of ONO-4007 to amastigote-ntigens appeared to overcome the early suppression of type-cytokines that is typical of L. amazonensis infection [25]

s demonstrated by the stimulation of IL-12 and IFN-�.The protective and therapeutic effects in the present stud-

es were associated with expression of high levels of IFN-�nd the early expression of IL-12. We have shown previ-usly that ONO-4007 has activity against L. major and L.mazonensis associated with the capacity of ONO-4007 tonduce the production of high levels of TNF-� [23]. BothNF-� and IFN-� are important mediators in the induc-

ion of NO synthase in macrophages [40]. Previous studiesave demonstrated that the leishmanicidal activity of TNF-�s enhanced in the presence of IFN-� and or LPS [41,42].lthough, Qi et al. [43] have reported that stimulation ofurine macrophages by IFN-� can enhance the replication

f L. amazonensis amastigotes, however, they also show that

5650 M. Calvopina et al. / Vaccine 24 (2006) 5645–5652

amastigote replication can be controlled if macrophages arestimulated with either higher concentrations of IFN-� or arestimulated with a combination of IFN-� and LPS. Our find-ings are consistent with these observations, suggesting thathigher levels of IFN-� are strictly necessary to provide pro-tection against L. amazonensis challenge and to promotehealing of active lesions. LPS and other lipid A derivativesactivate innate immunity through stimulation of the TLR4pathway [21,22] as appears to be true for ONO-4007 andother lipid A-analogues that have shown efficacy as vac-cine adjuvants [44,45]. The early activation of TLR4 maybe important for the generation of a robust, long-lasting,and appropriate adaptive immune response against Leishma-nia parasites, particularly against L. amazonensis in whichthere is an impaired expression of inflammatory cytokinesand chemokines at early stages of infection [25].

An alternative explanation for the efficacy of our vaccinecombination could be through the ability of ONO-4007 toblock the secretion of transforming growth factor-beta (TGF-�) [19,20,46] that has been implicated in susceptibility toL. amazonensis infection and the development of infectionchronicity [47,48]. Experimental studies that have neutral-ized TGF-� have shown increases in the IL-12-induced secre-tion of IFN-� and an increase in the percentage of CD4+

T cells [49,50]. However, it is not clear why not all ani-mals vaccinated/treated with ONO-4007 plus antigens werepdgsdaw

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in BALB/c mice [57]. Our results support the hypothesis thatsusceptibility to L. amazonensis infection is due to a failureto mount a strong type-1 cytokine response, rather than thedevelopment of a Th2 [24].

ONO-4007 injected alone in BALB/c mice in both theprophylaxis and therapy studies suppressed footpad-swellingbut did not cause sterile immunity as shown by a high pro-portion of mice footpads that remained Leishmania parasite-positive by culture. It is possible that protective immunitymay require the vaccine combination to generate a more‘parasite-focused’ immune response. Similar observationshave been made for another lipid A-derived adjuvant, MPLwhen injected alone [45].

Protective immunity appeared to be long-lasting asBALB/c mice that were immunized with the combinationwere protected even when re-challenged 8 weeks post-vaccination. Long-lasting immunity is observed in humansfollowing natural infection and injected with live para-sites “leishmanization”, and is observed also followinginfectious challenge in experimental murine leishmaniasis[31,53,58,59].

In conclusion, by using a vaccine that combined thenovel lipid A-analogue ONO-4007 and L. amazonensis-amastigote-antigens, we were able to demonstrate strongimmune response against L. amazonensis infection which isassociated with 60% of protection at 5 weeks of follow-up intahnatmwct

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rotected/cured. Individual mice receiving the combinationemonstrated L. amazonensis-specific IFN-� production sug-esting that the failure to protect was not secondary to incon-istent priming of recipients. Other external factors for thisisparity are unlikely because mice in each experiment werege and sex-matched, were essentially the same weight, andere inoculated in comparable sites by the same investigator.IL-12 plays a central role in protective immunity against

urine leishmaniasis [51–53]. Increased levels of IL-12 haveot been reported previously in the L. amazonensis/murinenfection model [54], but we detected significant levels of IL-2 soon after several intradermal inoculations of ONO-4007Fig. 3) corroborating previous studies that have demon-trated the capacity of ONO-4007 to induce IL-12 produc-ion [18–20]. ONO-4007 may stimulate the maturation and

igration of dendritic cells (DCs) – the most important cellource of IL-12 – into the lymph nodes, since LPS has beenhown to be a major stimulus for DC migration from skin55] and to be a potent inductor of IL-12p70 in DCs ofALB/c mice [56]. These data in conjunction with enhanced

evels of IFN-� observed, suggest that the protective immu-ity observed in both models may be attributable to theapacity of ONO-4007 to enhance the early production ofro-inflammatory cytokines (e.g., TNF-�), and IL-12 andhus initiating an adaptive Leishmania antigen-specific type-cytokine response.Furthermore, ONO-4007, in this study appeared to sup-

ress IL-4 production (compared to the levels seen in saline-njected mice). A previous study has shown that ONO-4007nhibits the differentiation of IL-4 producing CD4+ T cells

he immunoprophylaxis model and, 40% of clinical an par-sitological cure in the immunotherapeutic model describedere. Given the expense and toxicity of available antileishma-ial drugs, coupled with relative ease of culturing amastigotesnd the preparation of antigens, our results would encouragehe testing of this candidate combination vaccine in experi-

ental models for other Leishmania species. ONO-4007 isell tolerated in humans at doses of up to 100 mg [60] and

ould be considered for use as an adjuvant in human vaccinerials against New World CL.

cknowledgments

This investigation was supported by the Ministry of Edu-ation, Science, Culture and Sports of Japan (MOMBUSHO)y Grant nos. 10041177 and 10037385. We thank ONO Phar-aceutical Co., Osaka, Japan, for providing ONO-4007.

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