Leishmanial antigens in liposomes promote protective immunityand provide immunotherapy against visceral leishmaniasis
via polarized Th1 response
Sudipta Bhowmick, Rajesh Ravindran 1, Nahid Ali ∗
Infectious Diseases Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India
Received 21 February 2007; received in revised form 14 May 2007; accepted 21 May 2007Available online 8 June 2007
bstract
Leishmaniasis affects 12 million people, and it is generally agreed that vaccination provides the best long-term strategy for its control. Andeal vaccine should be effective in both preventing and treating leishmaniasis. However, immunological correlates to predict vaccine efficacynd success of treatment in visceral leishmaniasis (VL) remain ill defined. Here, we correlated the vaccine efficacy of soluble leishmanialntigens (SLA) from Leishmania donovani promastigote membrane, entrapped in negative, neutral and positively charged liposomes withhe elicited immune responses to predict vaccine success in experimental VL. Production of both IFN-� and IL-4 with a dominance ofh1 response following immunization was required for optimum success against L. donovani infection in BALB/c mice. The best vaccine
ormulation, SLA in positively charged liposomes, was then used for immunotherapy. This vaccine induced more than 90% elimination of
arasites from both liver and spleen. The success of immunotherapy exhibited an immune modulation with surge in Th1 cytokines, IFN-�nd IL-12 with extreme down regulation of disease promoting IL-4 and IL-10. These findings suggest that an immune modulation towardsh1 is effective for both successful vaccination and immunotherapy.2007 Elsevier Ltd. All rights reserved.
Leishmaniases are distributed worldwide, being endemicn 88 countries with a prevalence of 12 million casesnd 350 million people at risk. The clinical manifestationsf the disease are traditionally divided into three major
yndromes cutaneous (CL), mucosal and visceral leishma-iasis (VL). World Health Organization (WHO) attributesigher priority to VL, as it is the main source of death
n leishmaniases in the absence of treatment [1,2]. Fur-hermore, risk factor for VL is increasing due to theeishmania-HIV coinfection [1]. Available chemotherapyor VL is far from satisfactory because antileishmanialrugs are costly and frequently have unpleasant side effects.n addition, drug resistance exists in various regions ofndemicity [3]. Vaccination would therefore to be a betterption for the development of an effective control strategyor VL. An increasing number of Leishmania moleculesith potential for vaccine development are being identi-ed [4]. Moreover, killed parasites and components theref have been assayed for vaccines and immunotherapy inumans and canines with varying success [5–11]. Since
ife long protection after exposure to and cure of naturalnfection involves multiple host immune factors associatedith the production of strong cellular T cell response [12]
nd antileishmanial agents also work in conjunction with
he host immune response mediated primarily through Tells [13,14], for development of a leishmanial vacciner an immunotherapeutic agent, understanding of the keymmunogical responses during protection and treatment areequired.
The Th1/Th2 paradigm of resistance/susceptibility wasargely determined using Leishmania major [15]. Severaltudies support that immunotherapy that shifts the balancerom interleukin (IL)-4 to interferon (IFN)-� would pro-ide the key to vaccine success in CL [16]. Protectivemmunity in Leishmania donovani is also dependent onn IL-12 driven Th1 and IFN-� production [17,18]. How-ver, an exclusive generation of a vaccine-induced Th1 isnsufficient to ensure protection and cannot be a predic-or of vaccine success in experimental VL [19,20]. Recenttudies demonstrate that IL-10 is the major immunosup-ressive cytokine in VL [14,16]. Although induction ofL-4 in infected BALB/c and noncuring models [21,18]as been reported, beneficial roles of IL-4 have also beenescribed for L. donovani resistance [16]. Thus, for preciseorrelates of protective immunity and requirements for anffective antileishmanial, immune responses in VL need to beefined.
Antigens alone are generally weak immunogens andequire an adjuvant to induce protective immunity. Again arotein must not only be protected from extracellular degra-ation but needs to be targeted to the relevant immune cells.iposomes serve the above criteria and proved its usefulnesss immunological adjuvants to several leishmanial antigens22–24]. The quality of the generated immune response, how-ver, depends on the combined action of the antigen andhysical nature of the liposomes including size, phospholipidomposition and surface charge [25]. Our prior observationsemonstrated that membrane antigens of L. donovani pro-astigotes (LAg) in liposomes of different charges could
nduce significant but varied levels of protection againstnfection in BALB/c mice [26–28]. The variation could be
result of an adjuvant-induced difference in the immuneesponses to LAg or the differential entrapment of the vari-us components of LAg. Soluble leishmanial antigens (SLA),artially purified from LAg, is a mixture of antigens with atriking resemblance to the immunodominant antigen pro-les of LAg in neutral and positively charged liposomes.oreover, components of SLA demonstrated equal reac-
ivity with the sera from mice immunized with LAg inifferent liposomes [29]. These observations prompted uso undertake the present study, using SLA, to explore thenfluence of differently charged liposomal vaccine formula-ions on immune responses and challenge outcome with L.onovani to identify key immunological correlates of pro-ection in VL. Since SLA in positive liposomes induced
aximum protection, and potentiated activation of immune
esponses, we further investigated the effect and requirementsf therapeutic success in BALB/c mice with estab-ished visceral infection following treatment with liposomalLA.
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5 (2007) 6544–6556 6545
. Materials and methods
.1. Mice
BALB/c mice were bred in the animal facility of Indiannstitute of Chemical Biology (Kolkata, India). All mice were–6 weeks old at the onset of the experiments. Mice wereandled in accordance with institutional guidelines, and theelevant committee approved the use of mice for this study.
.2. Parasite culture
L. donovani strain AG83 (MHOM/IN/1983/AG83) wasaintained by serial passage in hamsters and BALB/c mice.he amastigotes were isolated from the spleens of infectednimals and allowed to transform into promastigotes at 22 ◦Cn Medium 199 supplemented with l00 U/ml penicillin Godium, 100 �g/ml streptomycin sulfate and 10% heat inac-ivated fetal bovine serum (FBS) (Sigma–Aldrich, St. Louis,
O). Freshly transformed promastigotes were subculturedn the same medium at an average density of 2 × 106 cells/ml26].
.3. Preparation of antigens
LAg were prepared from L. donovani promastigotes asescribed earlier [26]. SLA extracted from L. donovaniromastigotes membranes, were prepared as follows [29].riefly, stationary-phase promastigotes, harvested after the
hird or fourth passage, were washed four times in cold0 mM phosphate buffered saline (PBS), pH 7.2 and resus-ended at a concentration of 1.0 g of cell pellet in 50 mlf cold 5 mM Tris–HCl buffer (pH 7.6), containing 5 �gf leupeptin/ml, 1 mM EDTA, 1 mM phenylmethylsulfonyluoride and 1 mM iodoacetamide (Sigma–Aldrich) (lysisuffer). The suspension was vortexed and centrifuged at310 × g for 10 min. The membrane pellet was resuspendedn 10 ml of lysis buffer and sonicated for 3 min by ultrasoundrobe sonicator (Misonix, Farmingdale, NY). The suspensionhus obtained was solubilised with 1% (w/v) octyl-�-d-lucopyranoside (Sigma–Aldrich) in the lysis buffer withvernight incubation at 4 ◦C, and was finally ultracentifugedor 1 h at 100,000 × g. The supernatant containing SLA washen dialyzed against 2 mM PBS and stored at −20 ◦C untilse. The amount of protein obtained from 1.0 g cell pellet, asssayed by the method Lowry et al. [30], was approximatelymg.
.4. Entrapment of SLA in liposomes
Neutral, positive and negatively charged liposomes wererepared with egg lecithin (27 �mol) and cholesterol (7:2
olar ratio) (Sigma–Aldrich) or with egg lecithin, cholesterol
nd either stearylamine (Fluka, Buchs SG) or phospha-idic acid (Sigma–Aldrich) (7:2:2 molar ratio), respectively,ccording to the method of Gregoriadis et al. with slight mod-
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fications, as reported earlier [26–28,31]. The lipid mixtureas dissolved in chloroform and the solvent was removednder reduced pressure by a rotary evaporator. The thin, drylm was dispersed in either 1 ml PBS or 1 ml PBS containingmg/ml SLA for the preparation of empty and SLA entrapped
iposomes, respectively. The mixture was vortexed and theuspension sonicated for 30 s by an ultrasound probe son-cator (Misonix). Liposomes with entrapped antigen wereeparated from excess free antigen by three successive wash-ng in PBS with ultracentrifugation (105,000 × g, 60 min,◦C). The amount of SLA associated per mg egg lecithinas 30, 35 and 25 �g for neutral, positive and negatively
harged liposomes, respectively, determined by the methodf Lowry et al., in the presence of 10% SDS [30].
.5. SDS-PAGE analysis of SLA and liposomal SLA
SLA were subjected to SDS-PAGE by the method ofaemmli [32]. Gels were loaded with proteins (amounts are
ndicated in the figure legends) of the SLA and SLA entrappedn liposomes of positive, neutral and negative charge. Pro-eins, separated on 10% polyacrylamide, were silver stained33].
.6. Trypsin treatment of liposomal SLA
To measure the encapsulated antigens in the differ-nt liposomes, SLA associated vesicles were divided intowo portions. The divided portions received either trypsin0.5 mg/ml) (Sigma–Aldrich) or PBS and were then incu-ated for 30 min at 10 ◦C. After addition of soyabean trypsinnhibitor (0.5 mg/ml) (Sigma–Aldrich), the mixtures wereltracentrifuged at 105,000 × g for 60 min at 4 ◦C. Trypsin-reated and untreated liposomes were then subjected torotein estimation by the method of Lowry et al., in theresence of 10% SDS [30].
.7. Characterization of liposomes by transmissionlectron microscopy (TEM)
Morphological analysis of liposomes was carried out byEM using FEI tecnai spirit (FEI Company, Hillsboro, Ore-on). A small drop of sample (10 �l) was placed on a polymerlmed copper grid and allowed to stand for 2 min. The excessample was removed by filter paper, followed by addition of0 �l of uranyl acetate. The grid was then allowed to stand fornother 2 min, excess solution removed, air dried and viewedt 60 kV under the microscope.
.8. Zeta potential measurements
Liposomes were diluted with 20 mM PBS and surfaceharges on the vesicles were measured via analysis of zetaotential using ZetaMeter system 3.0+ (ZetaMeter Incorpo-ated, Staunton, VA).
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5 (2007) 6544–6556
.9. Vaccination and immunotherapy
The experimental groups consisted of 4–6 weeks oldALB/c mice. Mice (12 mice per group) were immunizedy three intraperitoneal injections at 2-week intervals with5 �g of SLA in PBS or incorporated in different lipo-omes (500, 429 and 600 �g of egg lecithin for neutral,ositive and negatively charged liposomes) in a total vol-me of 200 �l. Animals receiving PBS or empty liposomeserved as controls. Ten days after the last booster, serum sam-les were collected, and spleens were removed aseptically forhe analysis of humoral and cellular responses after immu-ization. Ten days after the final immunization mice werehallenged with 2.5 × 107 freshly transformed stationary-hase promastigotes in 200 �l PBS injected intravenously viahe tail vein [26]. After 2 and 4 months of challenge infec-ion, the mice were sacrificed to determine the parasite loadn liver and spleen. The course of infection was monitored byhe microscopic examination of Giemsa-stained impressionmears of liver and spleen. The parasite load was expressed aseishman-Donovan units and was calculated by the following
ormula: number of amastigotes per 1000 cell nuclei × organeight (mg) [34].For immunotherapy, mice were infected as described ear-
ier and 60-day infected mice (five mice per group) wereiven three intraperitoneal doses of 15 �g of SLA, either inBS or incorporated in positively charged liposomes at 2-eek intervals. Animals receiving PBS or empty liposome
erved as controls. Ten days after the last injection, the miceere sacrificed to determine the parasite load in liver and
pleen.
.10. Assessment of delayed type hypersensitivityesponse (DTH)
After the last vaccination and 10 days after the last vaccinereatment, delayed type hypersensitivity (DTH) was deter-
ined as an index of cell mediated immunity. The responseas evaluated by measuring the difference in the footpad
welling at 24 h following intradermal inoculation of the testootpad with 50 �l of LAg (800 �g/ml) and the swelling ofhe control (PBS injected) footpad with a constant pressurealiper (Starrett Company, Athol, MA) [26].
.11. Determination of antibody response
Serums from immunized, infected and treated animalsere analyzed by ELISA for the presence of SLA specific
ntibodies. In brief, 96-well Microtiter plates (Maxisorp,unc) were coated with SLA (15 �g/ml) diluted in 0.02 Mhosphate buffer (pH 7.5) overnight at 4 ◦C. The platesere blocked with 1% BSA in PBS at room temperature
or 3 h to prevent nonspecific binding. After washing withBS containing 0.05% Tween 20 (Sigma–Aldrich) (PBST)
he plates were incubated overnight with 1:1000 dilution ofice sera at 4 ◦C. Next day, the plates were again washed
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Fig. 2 shows the outcome of challenge infection inBALB/c mice following vaccination with SLA or entrapped
Table 1Vesicle size and zeta potential of liposomes of different charges with orwithout SLA
ith PBST and incubated further for 3 h at room tempera-ure with horseradish peroxidase conjugated goat antimousegG (Sigma–Aldrich) diluted 1:5000 in blocking buffer.or isotype analysis parrallely plates were incubated withorseradish peroxidase conjugated goat antimouse IgG1 andgG2a (BD Pharmingen) in 1:1000 dilution for 3 h at roomemperature. The plates were washed and substrate solutiono-phenylene diamine dihydrochloride, 0.8 mg/ml in 0.05 Mhosphate-citrate buffer, pH 5.0, containing 0.04% H2O2)100 �l) was added for 30 min and the absorbance was readn an ELISA plate reader (Thermo, Waltham, MA) at 450 nm26].
.12. Spleen cell proliferation and cytokine assays
The spleens were aseptically removed from the immu-ized, 2 and 4 months infected, and treated BALB/cice and single cell suspension was prepared in RPMI
640 supplemented with 10% FBS, penicillin G sodium100 U/ml), streptomycin sulfate (100 �g/ml) and 50 �M �-ercaptoethanol (Sigma–Aldrich). RBCs were removed by
ysis with 0.14 M Tris buffered NH4Cl. The remaining cellsere washed twice with culture medium and viable mononu-
lear cell number was determined by counting Trypan bluenstained cells in a hemocytometer. Then the cells wereultured in triplicate in a 96-well flat bottom plate (Nunc,aperville, IL) at a density of 2 × 105 cells/well in a finalolume of 200 �l and stimulated with SLA (6 �g/ml). Theells were incubated for 96 h at 37 ◦C in a humified chamberontaining 5% CO2. Cells were pulsed with 1 �Ci of [3H]-hymidine (Amersham Biosciences, Buckinghamshire, UK)er well 18 h before they were harvested on glass fiberaper. Thymidine uptake was measured in a �-scintillationounter (Beckman Instruments, Fullerton, CA) [29]. After2 h incubation, culture supernatants were collected and theoncentration of IFN-�, IL-4, IL-12 and IL-10 were quanti-ated using an Opt EIA kit (BD Pharmingen, San Diego, CA)n accordance with the manufacture’s instructions [35].
.13. Statistical analysis
One-way ANOVA statistical test was used to assess theignificance of the differences among various groups. In casef significant F-value multiple comparison Tukey-Kramerest was used to compare the means of different groups.esults with p < 0.05 were considered to be statistically sig-ificant.
. Results
.1. Characterizations of SLA in liposomes of different
harges by SDS-PAGE and trypsin treatment
SDS-PAGE analysis (Fig. 1A) revealed that SLA is a mix-ure of approximately eight (72, 63, 51, 45, 43, 41, 36 and
SS
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5 (2007) 6544–6556 6547
1 kDa) polypeptides. Interestingly, silver stained data illus-rate that all of the polypeptides of SLA were entrapped iniposomes of positive, neutral and negative charges with-ut any preferences. Amount of empty liposomes equal tohe amounts of liposomes with SLA were also subjected toDS-PAGE and no bands were observed (data not shown).or further characterization of liposomal SLA, the vesiclesere treated with trypsin to digest the protein associated with
he outer surface of the liposomes and entrapped proteinsere measured by the method of Lowry et al. and observed
hrough SDS-PAGE (data not shown). Although the totalmount of associated SLA per mg egg lecithin was highestn positively charged liposomes (35 �g) followed by neutral30) and negatively charged liposomes (25 �g), the antigensntrapped within the liposomes were highest in negativelyharged liposomes (81%) followed by positive (75%) andeutral liposomes (65%).
.2. Characterization of liposomal SLA of differentharges by TEM and zeta potential measurements
Liposomes were negatively stained and viewed under elec-ron microscope to characterize the morphology and sizeistribution of liposomal SLA. These liposomes were mul-ilamellar vesicles (MLV) with a uniform size distributionTable 1; Fig. 1B–D). Further, liposomes of different chargeshowed no significant differences in size. Zeta potentialnalysis of the differently charged liposomes showed a netositive charge in positively charged liposomes, whereas neu-ral and negatively charged liposomes showed neutral andegative surface charges (Table 1). Free and SLA entrappediposomes showed no significant difference in the measuredize and zeta potential, suggesting that entrapment of anti-ens made no significant impact on the physical attributes ofhe liposomes.
.3. SLA in positively charged liposomes confers almostomplete protection against L. donovani, whereas free orLA in neutral and negative liposomes are partially
LA in neutral liposomes 178 ± 23 4.3 ± 0.6LA in negative liposomes 170 ± 16 −33.5 ± 3.1
esicle size was measured by TEM. Liposomes were dispersed in PBS andeta potential was measured using a ZetaMeter system 3.0+. Results denoteean ± S.D. of 10 samples.
6548 S. Bhowmick et al. / Vaccine 25 (2007) 6544–6556
Fig. 1. SDS-PAGE analysis and transmission electron micrographs of liposomal SLA. Silver-stained SDS-PAGE gel of SLA and in different liposomes (A).L 3–5, SLc wn at tl electro
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anes: 1, SDS-PAGE molecular weight standards (Bio-rad); 2, SLA (6 �g);harged (lane 5) liposomes (6 �g). The molecular weight standards are shoiposomes contained SLA were stained with uranyl acetate and viewed in an
n liposomes of different charges. Mice immunized with SLAlone could induce partial protection in liver at 2 (51%) and 4onths (68%) in comparison to PBS-and empty liposomes-
mmunized (controls) animals (p < 0.001) (Fig. 2A). Miceeceiving SLA entrapped in neutral and negatively chargediposomes were also partially protective, comparable to thatfforded by free SLA. In comparison, mice vaccinated withLA in positive liposomes demonstrated highest resistance88% at 2 months) to hepatic infection with almost com-
lete protection (93%, p < 0.001, compared to controls) at 4onths, which was significantly higher than that afforded
y free and other liposomal SLA formulations (p < 0.05).ince L. donovani persists in the spleen, causing consider-
pwca
A entrapped in positively charged (lane 3), neutral (lane 4) and negativelyhe left in kilodaltons. Positive (B), neutral (C) and negatively charged (D)n microscope (20,500×).
ble organ-specific pathology in mice similar to that seenn human kala-azar, we evaluated the impact of vaccinationn this organ. Insignificant protection in spleen was obtainedith free SLA (35%) at 2 months (Fig. 2B). In contrast, vacci-ation with SLA in neutral and negatively charged liposomesemonstrated better protection at 2 months (61% and 58%,espectively, p < 0.001, in comparison to controls). How-ver, free and SLA in neutral and negative liposomes werequally protective at 4 months (83%, 81%, 72%, respectively,
< 0.001, compared to controls). Importantly, immunizationith SLA in positively charged liposomes conferred almost
omplete protection also in the spleen (93% and 98% at 2nd 4 months), compared to controls (p < 0.001).
S. Bhowmick et al. / Vaccine 25 (2007) 6544–6556 6549
Fig. 2. Clinical outcome following L. donovani challenge in immunized BALB/c mice. Kinetics of liver (A) and spleen (B) parasite burden of mice immunizedintraperitoneally three times at 2-week intervals with PBS, empty liposomes of different charges, SLA and SLA entrapped in liposomes of different charges.A ously wm the wet hose fro
3h
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t 10 days after the last immunization, the mice were challenged intravenice per group were sacrificed, and their parasite loads were determined by
issues. The mean value ± S.D. is indicated for each group. The results are t
DTH, an index of cell-mediated immunity, evaluated inice 10 days after the last immunization, demonstrated direct
orrelation with the observed protection in the different vac-ination groups. Vaccination of mice with SLA in positiveiposomes induced the highest level of DTH (p < 0.001)Fig. 3A), whereas free, and SLA in neutral and negative
tioi
ig. 3. DTH and specific antibody responses in mice after immunization and L. donays after the last immunization LAg specific DTH responses were measured (A)hickness of the test (LAg-injected) and control (PBS-injected) footpads at 24 h. Riffer significantly from control groups are indicated by ***(p < 0.001). Ten days afteollected immediately after last booster and 2 and 4 months after infection and assf 1:1000. Each sample was examined in duplicate. Data are presented as the absoresignated time points, representative of two experiments.
ith 2.5 × 107 promastigotes of L. donovani. At the designated times fouright and microscopic examination of impression smears of liver and spleenm one experiment representative of two performed.
iposomes vaccinated groups exhibited lower responses. Tonvestigate the induction of humoral responses, mice seraere assayed for SLA specific IgG levels through ELISA.nexpectedly, sera from mice immunized with free SLA
ould stimulate a substantial IgG response, compared to con-
rols (p < 0.001) (Fig. 3B). Immunization with SLA entrappedn negative liposomes enhanced the response significantlyver free SLA (p < 0.05). Immunization with SLA in pos-tive liposomes however, stimulated the maximum level of
ovani infection. Mice were immunized three times at 2-week intervals. Ten. The response is expressed as the difference (in millimeters) between theesults are shown as the mean ± S.D. of four mice per group. Means whichr immunization mice were challenged with L. donovani. Sera samples wereayed for SLA specific IgG antibodies by ELISA (B) with a serum dilutionbance at 450 nm and are mean ± S.D. of four individual mice per group at
6550 S. Bhowmick et al. / Vaccine 2
Table 2Specific IgG2a:IgG1 levels after challenge infection
Mice were immunized and infected as described in Section 2. Serum sam-ples were collected after immunization, 2 and 4 months after infection.IgG2a:IgG1 levels represent the ratio of the absorbances at 450 nm of spe-cs*
I(dlltdbgadmlsadc
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oiiLower but similar ratios were observed in mice immunizedwith free SLA and SLA in neutral and negative liposomes,reflecting the IgG2a:IgG1 described above (1.51 ± 0.26,
Fig. 4. Cytokine levels in immunized mice before and after L. donovaniinfection. Splenocytes were cultured, stimulated with SLA and 72 h laterconcentrations of released IFN-� (A) and IL-4 (B) in the culture supernatantswere determined after immunization and challenge infection. IL-12 level (C)
ific antibodies from each group with 1:1000 diluted serums by ELISA. Eachample was examined in duplicate and the experiment was done twice.** p < 0.001, vs. the control groups.
gG, significantly higher than the other vaccinated groupsp < 0.001). With progressive infection, IgG levels did notiffer significantly between controls and other groups. IgG2aevels are dependent on IFN-�, whereas IgG1 levels corre-ate with IL-4. We, therefore, analyzed the isotype responseso SLA following immunization and challenge infection andetermined the ratio of IgG2a:IgG1 as a measure of Th1:Th2alance. As shown in Table 2, SLA in positive liposomesroup had the highest ratio (1.11) after immunization andfter infection (1.51 and 1.91 at 2 and 4 months, respectively)emonstrating a skewing towards Th1 response. In contrast,ice immunized with free and SLA in neutral and negative
iposomes had similar ratios lower than SLA in positive lipo-omes revealing a lack of Th1 dominance. Thus, at the level ofntibody responses the Th1:Th2 bias appeared not only pre-ictive of clinical outcome following vaccination, but alsoomparable with the extent of protection.
.5. Induction of IFN-γ , IL-4 and IL-12 inLA-liposomes vaccinated mice
As strong DTH, an indicator for cellular response, wasbserved in vaccinated mice, we analyzed the supernatants ofLA stimulated splenocyte cultures for IFN-� and IL-4 from
hese animals to understand the immune responses correlat-ng with the observed protections. Splenocytes from miceaccinated with SLA in positive liposomes secreted signif-cantly higher levels of IFN-� than controls (p < 0.001) andree antigen immunized mice (p < 0.001) (Fig. 4A). Interest-ngly, IL-4 was also highest when SLA was entrapped inositively charged liposomes (p < 0.001, in comparison toontrols) (Fig. 4B). Lower levels of IFN-� and IL-4 wereeleased by splenocytes from free and SLA in neutral andegative liposomes immunized mice. With challenge infec-
ion, the levels of IFN-� and IL-4 were maintained in SLA inositive liposomes immunized whereas both the levels wereimultaneously increased in free SLA and SLA in neutral andegative liposomes immunized animals. Again as a measure
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5 (2007) 6544–6556
f Th1:Th2 bias, the IFN-�:IL-4 ratio was highest in SLAn positive liposomes immunized mice (4.12 ± 0.35 aftermmunization, 3.65 ± 0.78 at 2 and 3.55 ± 0.29 at 4 months).
as measured from supernatants of SLA stimulated cells of immunized andontrol mice. Each sample was examined in duplicate. Results are showns the mean ± S.D. (four mice per group at each designated time points),epresentative of two experiments. Means which differ significantly fromontrol groups are indicated by *(p < 0.05).
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S. Bhowmick et al. / V
.07 ± 0.52, 1.48 ± 0.16, respectively, after immunization,
.89 ± 0.59, 1.66 ± 0.42, 1.39 ± 0.35, respectively, at 2 and
.4 ± 0.19, 1.89 ± 0.1, 1.34 ± 0.42, respectively, at 4 months)uggesting that levels of both the cytokines and their ratiosrovide a further predictive correlate of vaccine medi-ted protection against VL. The finding that free SLA isighly immunogenic and protective, even in the absencef any adjuvant, led us to investigate whether immuniza-ion of free and SLA in positive liposomes could induceroduction of IL-12. Interestingly, similar levels of IL-12ignificantly higher than controls (p < 0.05) were detectedn splenocyte cultures of both the immunization groupsFig. 4C).
.6. Immunotherapy with SLA in positively chargediposomes for the treatment of VL
SLA in positive liposomes vaccination provided highestevel of protection both in liver and spleen against L. dono-ani infection in BALB/c mice. The antibody response andytokine analysis data also suggested that positively chargediposomal SLA was the best formulation for the vaccinationgainst VL. In addition, the property of free SLA to stimu-ate the production of IL-12 indicates that SLA is an antigenhat merits further study as an immunotherapeutic againstL. To determine the therapeutic potential of SLA, 60-day
nfected mice were treated intraperitoneally with three dosesf free and positive liposomes entrapped SLA, empty lipo-omes and PBS. As judged by comparing parasite burdenst 60 and 100 days, PBS and empty liposomes treated miceailed to reduce parasite loads. In contrast, liposomal SLAreated animals showed a significant reduction in parasite loadn both liver (91%, p < 0.001) and spleen (95%, p < 0.001),ompared to controls (Fig. 5A and B). The reduction in par-
sitic load was also statistically higher than that by free SLAp < 0.01), which by itself could induce partial reduction inoth liver (51%) and spleen (49%) parasite burden, comparedo controls (p < 0.01).
cloA
ig. 5. Infection levels in BALB/c mice after immunotherapy with liposomal SLA.ice were given SLA alone or in association with positive liposomes intraperitonea
iposomes. Hundred days after infection mice were sacrificed and the course of infor five mice per group are shown. The results are representative of two experiment
We further analyzed the treated mice sera for SLA spe-ific IgG1 and IgG2a antibody isotypes, convenient surrogatearker of Th1 and Th2 CD4+ T cell differentiation. ControlBS and empty liposomes treated groups showed high lev-ls of IgG1 and low levels of IgG2a (Fig. 6A). SLA vaccinereated mice produced reduced levels of IgG1 (p < 0.01) andncreased levels of IgG2a (p < 0.001), compared to controlsnd the levels were equal for both isotypes. In contrast, lipo-omal SLA treated group elicited high levels of IgG2a andow levels of IgG1. In this group, IgG2a level significantlyncreased and IgG1 level decreased from controls (p < 0.001).hus, the effective stimulation of IgG2a after immunotherapyith liposomal SLA could modulate the immune response
owards Th1 type, which confers resistance to VL.
.8. Induction of cellular responses in liposomal SLAmmunotherapy
To analyze the cellular responses elicited by liposomalLA immunotherapy, DTH and splenocyte proliferation weretudied 60 and 100 days after infection. Low DTH and pro-iferative responses were observed in 60-day infected mice.nimals receiving liposomal SLA demonstrated enhanced
nd highest DTH response after immunotherapy, in compar-son to controls (p < 0.001) and free SLA (p < 0.05)(Fig. 6B).ree SLA induced lower DTH correlating with the partialesistance acquired through this agent. Again mice treatedith liposomal SLA exhibited significant in vitro prolifer-
tion when compared to controls (p < 0.001) and free SLAnjected mice (p < 0.001) (Fig. 6C). Lower but significantroliferation was also observed with free SLA (p < 0.01,
ompared to controls). Analysis of the cytokines revealedow levels of IFN-� and IL-12 with pronounced stimulationf IL-4 and IL-10 by splenocytes of 60-day infected mice.fter immunotherapy, splenocytes from mice that received
Mice were infected with L. donovani and 60 days after challenge infectionlly, three times at 2-week intervals. Control groups received PBS or emptyection determined in the liver (A) and spleen (B). The mean values ± S.D.s.
6552 S. Bhowmick et al. / Vaccine 25 (2007) 6544–6556
Fig. 6. Induction of antibody isotypes and cellular immune responses by liposomal SLA immunotherapy. Sixty days after infection with L. donovani mice wereinjected PBS, empty liposomes, SLA or SLA in liposomes. Serum samples were collected 100 days after infection and assayed for SLA specific IgG1 and IgG2aantibodies (A). Each sample was examined in duplicate. Data are presented as the mean absorbance values ± S.D. of five mice per group, representatives of twoexperiments. DTH responses to LAg (B), an indicator of cell-mediated immune responses were expressed as footpads swelling at 24 h. Spleens were collectedand splenocytes from infected and vaccine treated mice, were stimulated in vitro for 72 h with SLA. Splenocyte proliferation (C) was determined by thymidineincorporation and expressed as counts per minute. Each sample was examined in triplicate. After 72 h supernatants from SLA stimulated spleen cells werecollected and assayed for IFN-� (D), IL-12 (E), IL-4 (F) and IL-10 (G) levels. Each sample was examined in duplicate. Results are shown as the mean ± S.D.for five individual mice per group, representative of two experiments. Means which differ significantly from control groups are indicated by **(p < 0.01) or***(p < 0.001).
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ree SLA vaccine treatment secreted significant levels ofFN-� and IL-12 (p < 0.01), compared to controls, and theevels of both the cytokines were significantly higher in lipo-omal SLA treated mice than that induced with free SLAreatment (p < 0.05) (Fig. 6D and E). Interestingly, mice withiposomal SLA immunotherapy showed significantly reducedevels of IL-4 and IL-10, compared to the controls (p < 0.01nd 0.001, respectively) (Fig. 6F and G). Splenocytes fromice treated with free SLA also exhibited a lower but sig-
ificant decrease in the IL-4 and IL-10 levels, compared toontrols (p < 0.05 and 0.01, respectively). These observationsherefore indicate that liposomal SLA immunotherapy is ableo induce strong cellular responses associated with a strik-ng polarization toward Th1 with a significant inhibition ofmmunosuppressive IL-4 and IL-10.
. Discussion
The virulence factors for L. donovani, which affect theonsequent pathology associated with disease as well as themmune response that mediate susceptibility or resistant tonfection, differ significantly from other species of Leshma-ia, especially L. major. While protective immunity in bothhe species is dependent on IL-12 driven Th1 response andFN-� production, the role of IL-4, Th2 archetypal cytokineemains elusive in L. donovani. Moreover, a role for IL-10n facilitating susceptibility to VL has been reported [16,36].urther, vaccines used successfully against L. major infec-
ion have often been reported to be unsuccessful againsturine VL, despite the elicitation of Th1 response [19,20].hus, vaccine development and success of immunotherapygainst VL requires understanding of the specific immuno-ogical responses towards L. donovani infection. To identifyhe precise correlates of protective immunity, we have eval-ated the protective efficacy of SLA in positively, neutralnd negatively charged liposomes and compared the immuneesponses for protection against L. donovani in a BALB/codel. As an ideal vaccine should be effective in both pre-
enting and treating leishmaniasis [37], we also studied themmunotherapeutic efficacy of the best vaccine formulation,o modulate the suppressive immune responses during vis-eral disease towards a protective one.
Earlier we showed that entrapment of LAg in liposomesf different charges demonstrated varied levels of protection26–28], which could be due to adjuvant-induced immunetimulation or preferential entrapment of various compo-ents of LAg to different liposomes with possibly betternteraction of the acidic leishmanial proteins with positivelyharged vesicles [38,39]. SLA is a mixture of antigens, par-ially purified from LAg, and in contrast to LAg almostll its components showed equal and strong reactivity with
he sera from mice immunized with LAg in different lipo-omes, suggesting SLA might be more immunogenic [29]. Aspposed to LAg, there was no preferential entrapment in SLAith all polypeptides being present in the differently charged
4spl
5 (2007) 6544–6556 6553
iposomal formulations. So, SLA could be used for betteromparison of immune responses generated by the threeifferently charged liposomes. Our present study demon-trates that immunization with SLA in positively chargediposomes induced almost complete protection, which wasigher than LAg entrapped in these vesicles, confirming itsigher immunogenicity. Interestingly, this was again reflectedhen SLA alone induced partial protection, which was higher
han free LAg [26]. Although the immunogenecity of LAgas enhanced in association with neutral liposomes [27],
he immunopotentiating capacity of these liposomes appearsow since they made no substantial impact on the activity ofree SLA. Protection by SLA alone in the absence of adju-ant may possibly be due to the presence of contaminatingPG, which has been reported to have pathogen associatedolecular pattern (PAMP) like activity [40]. However, PAS
taining of SLA (data not shown) revealed no band in theegion 21 kDa suggesting a lack of LPG or below detectableevels [41]. Although rare, a similar protective activity by aeishmania protein, devoid of adjuvant, has been reportedarlier [42].
Immunization with SLA in positive liposomes elicitedgG1 and IgG2a antibodies, with a skewing toward Th1 withigh levels of IgG2a [43]. Such a bias was not observed forree or SLA in neutral and negatively charged liposomes.urthermore, vaccination with SLA in positive liposomes
nduced the highest level of IFN-�, a signature cytokine ofh1 response. Very strikingly, immunization with SLA inositively charged liposomes also enhanced the productionf IL-4, a Th2 cytokine. Free and SLA in neutral and neg-tive liposomes also induced production of both IFN-� andL-4. The coexistence of Th1/Th2 responses with SLA immu-ization is consistent with previous vaccine studies with L.onovani antigens [35,42,44]. Although entrapment in neu-ral and negatively charged liposomes do not enhance the
agnitude of the response of free SLA, conventional lipo-omes are known to be processed for antigen presentation tooth Th1 and Th2 subsets of CD4+ T cells via MHC classI pathway [45,46]. Such a response may be influenced alsoy the size of the vesicles (150–250 nm) used herein whichavour a mixed Th1/Th2 response [47].
In contrast to observation in murine CL where a polarizedh1 response is sufficient for protection [48] and a con-omitant Th2 abrogates even a strong Th1 function [49,50],ur results herein substantiate earlier observations that aixed Th1/Th2 response is essential for protection againstL [35,42,44]. The evidences that early IL-4 is needed torive Th1 differentiation [51], to maintain IFN-� production52] and to prime IL-12 production in VL [53], strongly sug-est that early IL-4 production in VL does not hinder theh1 response later on. In concurrence, although vaccinationith SLA in positively charged liposomes also elicited an IL-
response, a polarization of the mixed response towards a
trong Th1 bias was generated, and maintained for optimumrotection. It may be noted that although positively chargediposomes are toxic towards erythrocytes, the concentration
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f stearylamine used in vivo was far below the toxic dose54].
The observation that SLA alone is partially protective andnduced significant and comparable levels of IL-12 as SLAn positive liposomes, is of particular interest because IL-2 can promote the development of curative Th1 response55,56], suggesting the potential of SLA as a component of anmmunotherapeutic approach against VL [57]. Immunother-peutic clinical trials against leishmaniasis suggest that it isevoid of side effects and leads to effective immunomod-lation without administration of chemotherapy [58,9]. Inxperimental murine model, PSA-2 DNA vaccine showedherapeutic potential against L. major infection with shiftingf disease-promoting Th2 cytokine profile towards a host-rotective Th1 [59]. In VL, severe immunosuppression makesore difficult the control of the disease after the onset of
nfection. In experimental VL, acquired resistance is driveno completion by Th1-type products, including IL-12 and IL-2 induced IFN-�. But endogenous IL-4, a Th2-cell productnd IL-10, a pleotropic cytokine, can deactivate the Th1 cellechanism and promote intracellular Leishmania infection
13,14]. Infection of BALB/c mice with AG83 strain of L.onovani leads to a progressive visceral infection [60,61] anderves as a good experimental model of VL. The present studyemonstrates that BALB/c mice with established visceralnfection could be cured by liposomal SLA immunotherapyith almost complete elimination of parasites from both liver
nd spleen. Vaccine treatment with fucose manose ligandFML) and its component were earlier shown to be effectivegainst murine and canine VL [62,63,10,11]. However, theseurine studies were restricted to evaluation of parasitic load
n liver alone. L. donovani parasites persist in the spleen, andre more resistant to various immunological interventions andven T cell-dependent chemotherapy [64,65]. The observa-ion that immunotherapy with liposomal SLA could eliminatelmost 95% parasites from the spleen is thus noteworthy, ando our knowledge this is the first report of such a therapeu-ic success with a protein-based vaccine against deep-seated. donovani infection. In addition, analysis of humoral andellular immune responses revealed that therapy-induced res-lution of parasitism corresponded with elevation of IgG2a asell as IL-12 and IFN-�, and downregulation of IL-4 and IL-0. Thus, resistance to L. donovani infection by liposomalLA immunotherapy can be attributed to the switching of
mmune responses from disease promoting to disease resolv-ng Th1 type.
In conclusion, we demonstrate that these different levelsf protection induced by immunization with SLA alone andn negative, neutral and positive liposomes, correlate withhe production of both IFN-� and IL-4 with a skewing towardFN-� producing Th1 type response for maximum protection.
oreover, SLA in positively charged liposome vaccine was
lso useful for immunotherapy against established visceralnfection in murine model causing stimulation of Th1 andignificant inhibition of disease promoting IL-4 and IL-10or successful resistance.
[
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5 (2007) 6544–6556
cknowledgements
We thank Mr. Sailen Dey for transmission electronicroscopy and Dr. R.N. Basu for zeta potential measure-ents. We thank S.K. Bhattacharya and S. Roy, past and
resent directors of IICB, Kolkata for supporting this work.e gratefully acknowledge support from the CSIR and theST, Government of India.
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