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ARTICLE IN PRESS Model

VAC 13016 1–8

Vaccine xxx (2012) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Vaccine

jou rn al h om epa ge: www.elsev ier .com/ locate /vacc ine

iposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce theame protection against the murine model of leishmaniasis

ahid Heravi Shargha, Mahmoud Reza Jaafari a,b,∗,1, Ali Khamesipourc, Iman Jaafari a, Seyed Amir Jalali a,zam Abbasia, Ali Badieea,∗,1

Nanotechnology Research Center, School of Pharmacy, Mashhad, University of Medical Sciences (MUMS), Mashhad, IranBiotechnology Research Center, School of Pharmacy, MUMS, Mashhad, IranCenter for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran

r t i c l e i n f o

rticle history:eceived 2 September 2011eceived in revised form 16 February 2012ccepted 16 March 2012vailable online xxx

eywords:accineiposomeLApG ODNseishmaniasis

a b s t r a c t

First generation Leishmania vaccines consisting of whole killed parasites with or without adjuvants havereached phase 3 trial and failed to show enough efficacy mainly due to the lack of an appropriate adjuvant.In this study, the nuclease-resistant phosphorothioate CpG oligodeoxynucleotides (PS CpG) or nuclease-sensitive phosphodiester CpG ODNs (PO CpG) were used as adjuvants to enhance immunogenicity andrate of protection against leishmaniasis. Due to the susceptibility of PO CpG to nuclease degradation, anefficient liposomal delivery system was developed to protect them from degradation. 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid was used because of its unique adjuvanticityand electrostatic interaction with negatively charged CpG ODNs. To evaluate the role of liposomal formu-lation in protection rate and enhanced immune response, BALB/c mice were immunized subcutaneouslywith liposomal soluble Leishmania antigens (SLA) co-incorporated with PO CpG (Lip-SLA–PO CpG), Lip-SLA–PS CpG, SLA + PO CpG, SLA + PS CpG, SLA or buffer. As criteria for protection, footpad swelling at the

site of challenge, parasite loads, the levels of IFN-� and IL-4, and the IgG subtypes were evaluated. Thegroups of mice receiving Lip-SLA–PO CpG or Lip-SLA–PS CpG showed a high protection rate comparedwith the control groups. In addition, there was no significant difference in immune response generationbetween mice immunized with PS CpG and the group receiving PO CpG when incorporated into the lipo-somes. The results suggested that liposomal form of PO CpG might be used instead of PS CpG in future

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vaccine formulations as a

. Introduction

Leishmania species are dimorphic homoflagellate intracellu-ar protozoan parasites of macrophage-dendritic cell lineage thatause a spectrum of diseases ranging from a self-healing cutaneousesion to potentially fatal visceral form of disease, known as leish-

aniasis [1,2]. The current control strategies are either ineffectiver hard to maintain in many foci, available drugs are expensive,eed multiple injections, accompany with side effects and are notlways effective [3–5].

Please cite this article in press as: Shargh VH, et al. Liposomal SLA co-incorpagainst the murine model of leishmaniasis. Vaccine (2012), doi:10.1016/j.v

It is well known for centuries that long lasting protectionnduces upon recovery from cutaneous leishmaniasis (CL) or leish-

anization [4,5]. Leishmania subunit vaccines has not reached

∗ Corresponding authors at: Nanotechnology Research Center, School of Phar-acy, Mashhad, University of Medical Sciences (MUMS), Mashhad, Iran. Tel.: +98

11 8823255; fax: +98 511 8823251.E-mail addresses: Jafarimr@mums.ac.ir (M.R. Jaafari), badieea@mums.ac.ir

A. Badiee).1 Both authors equally designed research.

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264-410X/$ – see front matter © 2012 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2012.03.040

cient adjuvant.© 2012 Published by Elsevier Ltd.

phase 3 while whole killed Leishmania with or without adjuvant

reached to phase 3 clinical trials. However, prophylactic studies

showed a limited efficacy mainly due to the limited Th1 inducer

adjuvant for use in human [4–8].

Synthetic oligodeoxynucleotides, containing unmethylated CpG

motifs, are extremely efficient inducer of Th1 immune response and

generation of cytotoxic T lymphocyte (CTL) which have shown to

induce protection against an extensive range of viral, bacterial and

some parasitic pathogens in animal models [9]. While human tri-

als have yielded promising results [10–12], clinical use of free CpG

ODNs still faces several challenges which limit their effectiveness.

One of the limiting factors in the success of oligonucleotide-based

immunotherapeutics is rapid degradation of unmodified ODNs (PO

CpG) within the body. This problem is diminished by modifica-

tions such as the replacement of non-bridging oxygen with sulfur

in phosphate linkages to prepare nuclease-resistant phosphoroth-

orated with PO CpG ODNs or PS CpG ODNs induce the same protectionaccine.2012.03.040

ioate analogs (PS CpG) [13]. Despite backbone stabilization of CpG 50

ODNs, PS-modified ODNs are still susceptible to nuclease degrada- 51

tion, albeit at a lower rate [14]. More critically, phosphorothioate 52

modification is associated with inherent disadvantages including 53

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ARTICLEVAC 13016 1–8

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on-sequence specific toxicity, unfavorable pharmacokinetic (PK)nd bio-distribution (BD), poor cellular uptake and lack of speci-city for target cells [15–18]. Administration of high doses of PSpG has been demonstrated to result in a significant acute toxic-

ty in primates due to transient complement activation and otheremodynamic changes which, in extreme cases, may result in car-iovascular collapse and death [14]. Moreover, PS CpG causes longerm severe side effects in mice such as induction of arthritis [19],ransient splenomegaly [20], lymphoid follicle destruction [21], andS CpG-specific IgM production [22] depending on the CG sequencend backbone modification. There are evidences that PS-modifiedDNs do not closely mimic the interaction of natural PO CpG with

oll like receptor 9 (TLR9) [17,23–25]. Based on the mentioned rea-ons and the lower price of PO CpG in comparison with PS CpG, wehose PO CpG as the main adjuvant in combination with solubleeishmania antigens (SLA) into the cationic liposomes.

One strategy to protect and extend the activity of PO CpG isncapsulation of CpG ODNs into the liposomes [26,27]. Liposomesave been used as a delivery vehicle to provide a close associationf CpG ODNs with antigens, and enhance the immune responses28]. Lipid based delivery systems are also developed to protecthe CpG ODNs payload, alter their pharmacokinetic characteris-ics and enhance immune cell targeting and facilitate intracellularptake [9]. Induction of cell-mediated immune response to poorly

mmunogenic Ags is possible through encapsulation of Ags into theiposomes, particularly cationic ones [29,30].

We have previously assessed the role of CpG ODNs in enhance-ent of immune response against two recombinant antigens

rgp63 or rLmSTI1), when entrapped into the liposomes [31,32].onsidering the promising results obtained from Leishmania crudeaccine [8], in this study, SLA was used as a model of first gen-ration vaccine with PS or PO CpG either in free forms or wheno-incorporated in liposomes to immunize BALB/c mice againsteishmaniasis.

. Materials and methods

.1. Ethics statement

The protocol was approved by the Institutional Ethical Com-ittee and Research Advisory Committee of Mashhad University

f Medical Sciences (Education Office dated March 31, 2010; pro-osal code 88527), based on the Specific National Ethical Guidelinesor Biomedical Research issued by the Research and Technology,eputy of Ministry Of Health and Medical Education (MOHME) of

ran, issued in 2005. Animals were kept in cages and provided withood and water ad libitum.

.2. Animals, parasites, SLA and CpG ODNs

Female BALB/c mice 6–8 weeks old were purchased from Pas-eur Institute (Tehran, Iran). The mice were maintained in animalouse of Pharmaceutical Research Center and fed with tap waternd laboratory pellet chow (Khorassan Javane Co., Mashhad, Iran).nimals were housed in a colony room 12/12 h light/dark cycle at1 ◦C with free access to water and food. Experiments were carriedut according to Mashhad University of Medical Sciences, Ethicalommittee Acts.

Leishmania major strain (MRHO/IR/75/ER) used in this exper-ment is the one which was used for preparation of experimentaleishmania vaccine, leishmanin, and Leishmania for leishmanization

Please cite this article in press as: Shargh VH, et al. Liposomal SLA co-incorpagainst the murine model of leishmaniasis. Vaccine (2012), doi:10.1016/j.v

33–35].The preparation of SLA was carried out using protocol developed

y Scott et al. [36] with some modifications. Briefly, the para-ites were harvested at stationary phase and washed 4 times using

PRESS xxx (2012) xxx– xxx

HEPES buffer (10 mM, pH 7.5). Then the number of promastigotes

was adjusted to 1.2 × 109/ml in buffer solution containing enzyme

inhibitor cocktail, 50 �l/ml (Sigma, St. Louis, MO, USA) and the

preparation was incubated in ice-water bath for 10 min and then

the parasites were lysed using freeze–thaw method followed by

probe sonication in an ice bath. The supernatant of centrifuged

lysate parasites was collected, dialyzed against buffer solution, ster-

ilized using a 0.22 �m membrane and stored at −70 ◦C until use. The

protein concentration of the SLA was determined using BCA protein

assay kit (Thermo Scientific, USA).

The PS and PO CpG (Microsynth, Balgach, Switzerland) used in

this study were a 20-mer (5′-TCCATGACGTTCCTGACGTT-3′) with

fully nuclease-resistant phosphorothioate or nuclease-susceptible

phosphodiester backbones, respectively, which contains two CpG

motifs with known Th1 type immunostimulatory effects in murine

model [37,38].

2.3. Liposomes preparation and characterization

Liposomes containing SLA and CpG ODNs (Lip-SLA–PO CpG or

Lip-SLA–PS CpG) were prepared using lipid film method. Briefly,

the lipid phase consisting of 1,2-dioleoyl-3-trimethylammonium-

propane (chloride salt) (DOTAP) (4 �mol/ml; Avanti Polar lipids,

USA) and cholesterol (Avanti Polar lipids, USA) (1:1 molar ratio)

was dissolved in chloroform in a sterile tube. The solvent was then

removed by rotary evaporation (Hettich, Germany) resulting in

deposition of a thin lipid film on the tube’s wall. The lipid film was

freeze-dried (Taitec, Japan) overnight to ensure complete removal

of the solvent. The lipid film was then hydrated and dispersed in

sterile HEPES buffer containing SLA (0.5 mg/ml) at 45 ◦C. The result-

ing multilamellar vesicles (MLVs) were converted to 200–400 nm

vesicles using the bath sonicator (Branson 5510, USA). The PS or PO

CpG (200 �g/ml) was then added slowly to liposomes containing

SLA using mild shaking tube. The sonication of liposomal formula-

tions was done before injection into the mice to reduce the size of

liposomes.

Particle size analyzer (Nano-ZS, Malvern, UK) was used to deter-

mine the mean diameter and zeta potential of the liposomes. The

concentration of SLA encapsulated in liposomes was determined

using BCA protein assay kit (Thermo Scientific, USA).

2.4. SDS-PAGE analysis of SLA and liposomal SLA

Analytical SDS-PAGE was carried out to characterize and esti-

mate qualitatively the concentration of SLA encapsulated in the

prepared Lip-SLA after purification by Vivaspin6® centrifugal con-

centrators (300 kDa) (Sartorius Stedim Biotech S.A., France). The gel

was consisted of running gel (10.22%, w/v, acrylamide) and stack-

ing gel (4.78%, w/v, acrylamide). The gel thickness was 1 mm. The

electrophoresis buffer was 25 mM Tris, 192 mM glycine, 0.1% SDS,

pH 8.3. Electrophoresis was carried out at 140 V constant voltage

for 45 min. After electrophoresis the gels were stained with silver

for protein detection.

2.5. Immunization of BALB/c mice

Different groups of mice, 8 mice per group, were subcutaneously

(SC) immunized in their left hind footpad 3 times at 2 weeks

intervals with one of the following formulations: Lip-SLA–PO

CpG (25 �g SLA-10 �g PO CpG/50 �l liposome/mouse), Lip-SLA–PS

CpG (25 �g SLA–10 �g PS CpG/50 �l liposome/mouse), SLA (25 �g

orated with PO CpG ODNs or PS CpG ODNs induce the same protectionaccine.2012.03.040

SLA/50 �l buffer/mouse), SLA (25 �g SLA/50 �l buffer/mouse) plus 169

PO CpG (10 �g PO CpG/50 �l buffer/mouse), SLA (25 �g SLA/50 �l 170

buffer/mouse) plus PS CpG (10 �g PS CpG/50 �l buffer/mouse) or 171

HEPES buffer (HEPES 10 mM, sucrose 10%, pH 7.5) alone. 172

IN PRESSG Model

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accine xxx (2012) xxx– xxx 3

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Table 1Particle size, polydispersity index (PDI), and surface charge of various liposomalformulations. Results denote mean ± SD (n = 3).

Z-average (nm) PDI Zeta-potential (mV)

Empty Lipa 224.5 ± 82.1 0.4 ± 0.2 64.0 ± 13.7Lip-SLA 361.0 ± 84.1 0.4 ± 0.1 45.6 ± 11.4Lip-PS CpG 529.3 ± 52.3 0.6 ± 0.2 72.5 ± 4.8Lip-PO CpG 501.2 ± 48.9 0.5 ± 0.1 76.6 ± 2.3Lip-SLA–PS CpG 359.4 ± 85.7 0.3 ± 0.1 63.9 ± 2.7Lip-SLA–PO CpG 351.5 ± 86.8 0.4 ± 0.1 63.8 ± 6.0

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PAGE (Fig. 1). SDS-PAGE analysis of soluble SLA revealed several 252

protein bands with a range of molecular weight between 10 and 253

70 kDa (Fig. 1). The SDS-PAGE analysis of liposomal SLA after 254

ARTICLEVAC 13016 1–8

V.H. Shargh et al. / V

.6. Challenge with L. major promastigotes

The immunized mice and control group (five per group) werehallenged SC, at week 2 after the last booster, in the left footpadith 1 × 106 L. major promastigotes harvested at stationary phase

n 50 �l volume. Lesion development was recorded in each mousey measuring the footpad swelling using a metric caliper (Mitutoyoeasuring Instruments, Japan). Grading of lesion size was done by

ubtracting the thickness of uninfected contralateral footpad fromhat of the infected one.

.7. Quantitative parasite burden after challenge

The number of viable L. major parasites in the spleen of differentroup of mice were estimated using limiting dilution assay methods described previously [39]. Briefly, the mice were sacrificed ateek 9 post-challenge; the spleens were aseptically removed andomogenized in RPMI 1640 supplemented with 10% (v/v) heat inac-ivated FCS (Eurobio, Les Ulys, France), 2 mM glutamine, 100 U/mlf penicillin and 100 �g/ml of streptomycin sulfate (RPMI-FCS). Theomogenate was diluted with the media in 8 serial 10-fold dilutionsnd then was placed in each well of flat-bottom 96-well microtiterlates (Nunc, Denmark) containing solid layer of rabbit blood agar

n tetraplicate and kept at 25 ± 1 ◦C for 7–10 days. The number ofiable parasite per spleen was determined using ELIDA software, atatistical method for limiting dilution assay [40].

.8. Antibody isotype assay

Blood samples were collected from mice before and at week 9fter the challenge and the sera were isolated and kept frozen untileing used to assess anti-SLA IgG total, IgG1 and IgG2a antibodies byLISA method as described before [39]. Briefly, 96-well micro titerlates (Nunc, Denmark) were coated with 50 �l of 10 �g/ml of SLAvernight at 4 ◦C. The plates were washed and blocked by adding00 �l per well of 1% of bovine serum albumin in PBS-Tween and

ncubated for 1 h at 37 ◦C. Serum samples were diluted to 1:200,:2000 or 1:20,000 with PBS-Tween and 50 �l applied to the plates.he plates were then washed and treated with HRP-rabbit anti-ouse IgG isotype according to the manufacturer’s instructions

Zymed Laboratories Inc., USA). Optical density (OD) was deter-ined at 450 nm using 630 nm as the reference wavelength.

.9. In vitro spleen cells responses

Three mice from each group were sacrificed at week 2 afterhe last booster injection and their spleens were asepticallyemoved. Mononuclear cells were isolated by Ficoll–Hypaque den-ity centrifugation method from spleen cell suspension. The cellsere washed and resuspended in RPMI 1640-FCS and seeded at

× 106/ml in 96-well flat-bottom plates (Nunc, Denmark). Theells were stimulated in vitro with either SLA (5 �g/ml), Con A2.5 �g/ml), or medium alone, and incubated at 37 ◦C in 5% CO2or 72 h. The culture supernatants were collected and the levels ofL-4 and IFN-� were checked using ELISA method according to the

anufacturer’s instructions (MabTech, Sweden).

.10. Statistical analysis

One-way ANOVA statistical test was used to assess the sig-ificance of the differences among various groups. In the case of

Please cite this article in press as: Shargh VH, et al. Liposomal SLA co-incorpagainst the murine model of leishmaniasis. Vaccine (2012), doi:10.1016/j.v

ignificant F value, Tukey–Kramer multiple comparisons test wasarried out as a post-test to compare the means in different groupsf mice. Results with P < 0.05 were considered to be statisticallyignificant.

a Liposomes (Lip) were composed of DOTAP and cholesterol in a molar ratio of1:1.

3. Results

3.1. Liposome characterization

Liposomes used for in vivo study were 265–445 nm vesi-

cles with mean diameters 359.4 ± 85.7 nm (PDI: 0.3 ± 0.1)

and 351.5 ± 86.8 nm (PDI: 0.4 ± 0.1) and the zeta potentials

of + 63.9 ± 2.7 and + 63.8 ± 6.0 mV as calculated by particle size ana-

lyzer for Lip-SLA–PS CpG and Lip-SLA–PO CpG, respectively. In

terms of particle size and surface charge, there was no significant

difference between PS and PO CpG in liposomal form. The parti-

cle size results for all the formulations showed a slightly high PDI

values due to the used preparation method.

As shown in Table 1, the presence of SLA in formulations

decreased the surface charge of empty liposomes, Lip-PO CpG, and

Lip-PS CpG. However, the presence of CpG ODNs in the formula-

tions did not considerably affect the surface charge compared with

empty liposomes.

The entrapment of SLA in Lip-SLA–PS CpG or Lip-SLA–PO CpG

formulations was 45% and 50%, respectively. The entrapment of CpG

ODNs in liposomes was more than 90% based on the absorption at

260 nm because of their electrostatic interaction with DOTAP. The

concentration of SLA and CpG ODNs in liposomes was adjusted to

25 �g and 10 �g per 50 �l just before injection, respectively.

Identification of SLA and liposomal SLA was performed by SDS-

orated with PO CpG ODNs or PS CpG ODNs induce the same protectionaccine.2012.03.040

Fig. 1. SDS-PAGE analysis of SLA alone and liposomal SLA. Lane 1, low-range proteinstandard (Sigma, USA); lane 2, SLA (2.5 �g); lane 3, SLA (5 �g); lane 4, liposomal SLA(2.5 �g); lane 5, liposomal SLA (5 �g).

ARTICLE IN PRESSG Model

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4 V.H. Shargh et al. / Vaccine xxx (2012) xxx– xxx

Fig. 2. Footpad swelling in BALB/c mice immunized SC, three times in 2-week inter-vals, with Lip-SLA–PO CpG, Lip-SLA–PS CpG, SLA + PO CpG, SLA + PS CpG, SLA orHEPES buffer after challenge with 106 L. major promastigotes in the left hind foot-pads. The footpad thickness was measured weekly for 8 weeks. Each point representstiw

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Fig. 3. Spleen parasite burden in BALB/c mice immunized SC, 3 times in 2 weekintervals, with Lip-SLA–PO CpG, Lip-SLA–PS CpG, SLA + PO CpG, SLA + PS CpG, SLAor HEPES buffer after challenge with L. major promastigotes. A limiting dilutionanalysis was performed 9 weeks after challenge on the cells isolated from the spleenof individual mice and cultured in tetraplicate in serial 8-fold dilutions. The numberof viable parasite per spleen was determined by ELIDA software based on limitingdilution assay method. The bar represents the average score ± SEM (n = 2). **P < 0.01when the mice immunized with Lip-SLA–PO CpG or Lip-SLA–PS CpG are comparedwith the mice received SLA or HEPES buffer.

Fig. 4. Levels of anti-SLA-specific IgG1 (A), IgG2a (B), and IgG (C) in pooled sera ofdifferent group of BALB/c mice immunized SC, three times in 2-week intervals, withLip-SLA–PO CpG, Lip-SLA–PS CpG, SLA + PO CpG, SLA + PS CpG, SLA or HEPES buffer.Blood samples were collected from the mice 2 weeks after the last booster. The SLA-

he average increase in footpad thickness ± SEM (n = 5). (*) P < 0.05 when the micemmunized with SLA + PS CpG, Lip-SLA–PS CpG or Lip-SLA–PO CpG are compared

ith the mice received SLA alone.

urification by Vivaspin6® centrifugal concentrators showed allhe same bands as in the soluble SLA but corresponding to theiriposomal entrapment efficiency.

.2. Challenge results

Lesion development was monitored by weekly measurement ofootpad thickness (Fig. 2). The lesion size progressed at a more rapidate in control groups which received either HEPES buffer or SLAlone in comparison with the group of mice immunized with Lip-LA–PS CpG or Lip-SLA–PO CpG (P < 0.05) at week 7 after challenge.t week 8 after challenge, the lesion size in mice immunized withLA + PS CpG, Lip-SLA–PS CpG or Lip-SLA–PO CpG was significantlyP < 0.05) smaller than the control groups. There was a significantifference between the group received SLA + PO CpG and the groupf mice received HEPES buffer (P < 0.01) from week 4 to week 8 afterhallenge. In control groups, the lesion thickness reached a plateaufter 6 weeks (Fig. 2) but the infection progressed by metastasis tother organs and the mice lost their foot.

.3. Splenic parasite burden after challenge

The number of viable L. major was estimated in the spleen ofifferent groups of mice at week 9 after challenge (Fig. 3). Inter-stingly, live parasites were not detected in mice immunized withip-SLA–PS CpG or Lip-SLA–PO CpG but the mice immunized withLA + PO CpG or SLA + PS CpG showed live parasites in their spleens.he number of parasites in the spleen of mice immunized withLA + PO CpG or SLA + PS CpG was significantly (P < 0.01) lower thanhe control groups. However, there was no significant differenceetween the mice immunized with SLA + PO CpG or SLA + PS CpGnd the groups of mice immunized with Lip-SLA–PS CpG or Lip-LA–PO CpG.

.4. Antibody response

In order to determine the type of immune response generatedn immunized mice, the anti-SLA IgG antibody and IgG1, IgG2aubclasses were assayed before (Fig. 4A–C) and after (Fig. 5A–C)hallenge with L. major promastigotes.

Please cite this article in press as: Shargh VH, et al. Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protectionagainst the murine model of leishmaniasis. Vaccine (2012), doi:10.1016/j.vaccine.2012.03.040

The sera of mice immunized with Lip-SLA–PS CpG or Lip-SLA–POpG before challenge showed significantly higher levels of IgG1nd IgG2a antibody compared with the other groups (P < 0.001).he ratio of IgG2a/IgG1 in sera of mice immunized with SLA + PS

specific IgG1, IgG2a and IgG levels were assessed using ELISA method. The assayswere performed in triplicate at 200-, 2000- or 20,000-fold dilution for each serumsample. Values are the mean ± S.D. (***) P < 0.001 when the mice immunized withLip-SLA–PS CpG or Lip-SLA–PO CpG are compared with the other groups.

ARTICLE IN PRESSG Model

JVAC 13016 1–8

V.H. Shargh et al. / Vaccine xxx (2012) xxx– xxx 5

Fig. 5. Levels of anti-SLA-specific IgG1 (A), IgG2a (B), and IgG (C) in pooled seraof different group of BALB/c mice immunized SC, three times in 2 weeks intervals,with Lip-SLA–PO CpG, Lip-SLA–PS CpG, SLA + PO CpG, SLA + PS CpG, SLA or HEPESbuffer. Blood samples were collected from the mice 9 weeks after challenge. The SLA-specific IgG1, IgG2a and IgG levels were assessed using ELISA method. The assayswere performed in triplicate at 200-, 2000- or 20,000-fold dilution for each serumsample. Values are the mean ± S.D. (*) P < 0.05 and (***) P < 0.001 when the miceig

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Table 3IgG2a:IgG1 ratio in different group of immunized mice at week 9 after challenge.

Groups Serum dilutions

1/200 1/2000 1/20,000

Buffer 0.792 0.578 0.369SLA 0.835 0.848 1SLA + PS CpG 1.259 1.453 1.849SLA + PO CpG 1.005 1.004 1.162

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mmunized with Lip-SLA–PS CpG or Lip-SLA–PO CpG are compared with the otherroups.

pG, Lip-SLA–PS CpG or Lip-SLA–PO CpG was higher than the otherroups (Table 2). Mice immunized with SLA + PS CpG produced aignificantly higher level of IgG2a (P < 0.001) than the group immu-

Please cite this article in press as: Shargh VH, et al. Liposomal SLA co-incorpagainst the murine model of leishmaniasis. Vaccine (2012), doi:10.1016/j.v

ized with SLA + PO CpG but there was no significant difference ingG1 level. There was no significant difference in IgG1 or IgG2a level

able 2gG2a:IgG1 ratio in different groups of mice at 2 weeks after last booster.

Groups Serum dilutions

1/200 1/2000 1/20,000

Buffer 0.661 0.333 0SLA 0.508 0.338 0.091SLA + PS CpG 1.387 1.396 1.286SLA + PO CpG 0.591 0.382 0.217Lip-SLA–PS CpG 1.041 1.054 1.107Lip-SLA–PO CpG 1.211 0.95 0.889

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Lip-SLA–PS CpG 1.559 1.759 1.848Lip-SLA–PO CpG 1.632 1.35 2.379

between the group of mice immunized with Lip-SLA–PS CpG and

the group received Lip-SLA–PO CpG.

Challenge with L. major promastigotes induced elevation ofIgG1, IgG2a and IgG antibody levels in control groups received

SLA or HEPES buffer compared with the results of before challenge

(Figs. 5A–C). The sera of mice immunized with Lip-SLA–PO CpG

or Lip-SLA–PS CpG (1/200 serum dilution) produced a significantly

(P < 0.001) elevated levels of specific IgG2a antibody compared with

the other groups but there was no significant difference in IgG1

level. The ratio of IgG2a/IgG1 in sera of mice immunized with Lip-

SLA–PO CpG, Lip-SLA–PS CpG or SLA + PS CpG was higher than

the other groups (Table 3). Although the level of IgG2a in sera

of mice immunized with SLA + PO CpG was significantly (P < 0.01)

higher than those received SLA or HEPES buffer (1/200 and 1/20,000

serum dilution), but it was (P < 0.05) lower than the group received

SLA + PS CpG. There was no significant difference in IgG1 or IgG2a

production between mice immunized with Lip-SLA–PS CpG or Lip-

SLA–PO CpG (1/200 or 1/2000 serum dilutions).

3.5. In vitro cytokine production by splenocytes

The supernatant of cultured splenocytes collected at week 2

after the last booster injection were re-stimulated in vitro withSLA and analyzed to determine the levels of IFN-� and IL-4,

cytokines indicative of Th1 and Th2 response, respectively. Theresults showed that the group of mice immunized with Lip-SLA–PO

CpG produced significantly (P < 0.05) higher levels of IFN-� than

all the other groups. Interestingly, the level of IL-4 detected in

mice immunized with Lip-SLA–PS CpG or Lip-SLA–PO CpG was high

and there was no significant difference in IL-4 production between

these groups and mice received SLA. Although the level of IFN-�

detected in mice immunized with Lip-SLA–PS CpG was higher than

those received SLA but it was not significant. The mice immunized

with HEPES buffer or SLA + PO CpG produced significantly (P < 0.05)

lower level of IFN-� than all the other groups. The level of IL-4

detected in mice immunized with SLA + PO CpG, SLA + PS CpG or

HEPES buffer was significantly (P < 0.001) lower than all the other

groups (Fig. 6A and B).

4. Discussion

First generation Leishmania vaccines have been reached to

phase 3 trials. However, the meta-analysis of results showed that

although vaccines are safe, efficacy in some cases was limited

mainly due to the lack of an appropriate adjuvant [5]. A review of

adjuvants for vaccine candidates against leishmaniasis showed the

vaccine efficacy is strongly improved by using an appropriate adju-

vant [41]. Therefore, in this experiment, SLA as a first generation

vaccine and CpG ODNs as an immunostimulatory adjuvant were

co-entrapped in liposomes and used to immunize BALB/c mice. The

orated with PO CpG ODNs or PS CpG ODNs induce the same protectionaccine.2012.03.040

main goal of this study was to compare the adjuvanticity of PS CpG 344

with PO CpG in mice model. Moreover, the rate of protection and 345

generated immune response were analyzed and compared with 346

control groups received HEPES buffer or SLA in soluble form. 347

ARTICLE ING Model

JVAC 13016 1–8

6 V.H. Shargh et al. / Vaccine

Fig. 6. Cytokine levels in different groups of immunized mice at week 2 after thelast booster injection. Mononuclear splenocytes were cultured in the presence ofSLA (5 �g/ml) and the released IFN-� (A) or IL-4 (B) in the culture supernatantswere detected using ELISA method. Results are shown as the mean ± SEM (n = 3).*wC

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P < 0.05, part (A) when the mice immunized with Lip-SLA–PO CpG are comparedith the other groups. **P < 0.01, part (B) when the mice immunized with SLA + PSpG, SLA + PO CpG or HEPES buffer are compared with the other groups.

In the current experiment, immunization with SLA in solubleorm induced no protection in BALB/c mice based on foot-ad swelling and splenic parasite burden results. The ratio of

gG2a/IgG1 (a marker for the induction of Th1-like response)n mice immunized with SLA was less than 0.5 before thehallenge and the high level of IL-4 production was detectedhich was parallel with the footpad swelling and parasite bur-en results. In agreement with our results, SLA’s componentsere shown to possibly be a candidate vaccine for future studies

f L. donovani antigens if used in liposomal form [42]. Non-hosphatidylcholine liposomes (Escheriosomes) prepared of E.oli lipids containing L. donovani promastigote soluble antigensnduced parasite-specific CD8+ and CD4+ T cell immune responsend protected both hamsters and BALB/c mice against visceraleishmaniasis [43]. Moreover, the intraperitoneal administration ofligomannose-coated liposomes containing SLA strongly inducedn antigen-specific Th1 response against L. major infection inALB/c mice [44]. Hence, liposomal form of SLA has been used forur study to check the role of immunostimulatory adjuvants.

Based on our previous studies, PS CpG 1826 was used as a Th1mmunostimulatory adjuvant [31,32]. A significant shift in anti-ody isotypes response with an increase in the IgG2a/IgG1 ratio∼2-fold; Table 3) was shown when SLA was co-administered withS CpG which was in agreement with the other studies [45–49].ALB/c mice vaccinated with DCs pulsed ex vivo with the com-ination of a peptide portion of kinetoplastid membrane proteinnd CpG ODNs induced parasite-specific cellular responses andeduced efficiently the L. infantum load in visceral organs [50]. Also,ALB/c mice vaccinated with Leishmania ribosomal proteins pluspG ODNs were protected against primary L. major infection [48]

Please cite this article in press as: Shargh VH, et al. Liposomal SLA co-incorpagainst the murine model of leishmaniasis. Vaccine (2012), doi:10.1016/j.v

nd also developed a resistant phenotype to parasite associatedisease at a secondary site of infection (ear dermis) [49]. Hence,

t seems that the enhanced Th1 type of immune response becausef CpG ODNs is essential for counteracting intracellular pathogens

PRESS xxx (2012) xxx– xxx

[51]. CpG ODNs through TLR9 augment both activation and matu-

ration of DC as well as the induction of proinflammatory cytokines

[52]. The endogenous production of IL-12, IL-18, and other soluble

mediators from activated DC induced by CpG ODNs are likely to

result in a more physiologic cognate interaction between DC and

T cell, resulting in both a qualitatively and quantitatively different

type of CD4+ and CD8+ T cell response [46].

In the current study, PO CpG was used as the main adjuvant in

combination with SLA because of the drawbacks of PS CpG. Earlier,

Lee et al. [53] demonstrated that a potent PO CpG, namely MB-ODN

4531(O), has functional effects as a powerful adjuvant. PO CpG also

upregulate the expression of proinflammatory cytokines (TNF-�

and IL-12) [22,54]. However, rapid degradation of PO CpG within

the body is one of the limiting factors for their use in vivo. As shown

in Fig. 2, PO CpG in soluble form did not protect mice against chal-

lenge test compared with PS CpG and interestingly this was nottrue for the liposomal form of it. Therefore, this might be due totheir susceptibility to nuclease degradation.

The need for intracellular uptake into endosomal compartments

is another barrier since charged macromolecules such as free DNAhave difficulty crossing hydrophobic membranes [9]. Datta et al.

[55] investigated the effect of intracellular targeting of CpG ODNs

by incorporating it in mannose-coated liposomes and showed that

it was more effective in enhancing its therapeutic potential in redi-

recting curative Th1 responses in visceral leishmaniasis. We found

previously that the lesion size in mice which received rgp63 plus

PS CpG encapsulated in cationic liposomes (Lip-rgp63-PS CpG) was

significantly (P < 0.001) smaller than the control group up to 14

weeks after challenge [32]. In the current study, although there was

no considerable difference between mice immunized with SLA + PS

CpG and Lip-SLA–PS CpG (Fig. 2), the latter group produced signif-

icantly (P < 0.001) higher IgG2a antibody before or after challenge

test. Nevertheless, there was a significant difference in generated

immune response between the mice immunized with Lip-SLA–PO

CpG and those received SLA + PO CpG. Hence, our results showed

that the liposomal form of PO CpG may increase the efficacy of

vaccine formulation against leishmaniasis.

The stabilized antisense lipid particles (SALP) encapsulated PO

CpG, exerted a Th1 effect even more potent than that measured

for the same sequence with a PS backbone [56]. Interestingly, the

activity of PO CpG in a human cell line was greatly enhanced when

they were encapsulated in a liposomal form [57]. Therefore, in this

study, liposomes were used as a delivery system to enhance the bio-

logical activity of CpG ODNs, particularly PO CpG. Liposomes may

mediate this activity enhancement in a number of ways; liposomes

protect CpG ODNs from nuclease activity and hinder the distribu-

tion of CpG ODNs into the tissues, consequently increasing CpG

ODNs half-life and enhancing adjuvanticity [58]. Liposomes also

facilitate the intracellular delivery of CpG ODNs into the cytoplasm

based on their formulations [59]. Improved adjuvant effect of CpG

ODNs when encapsulated into the liposomes might be due to the

fact that TLR9, the receptor for CpG motifs [52], localizes into endo-

somal compartments but not in the cell surface [60], so CpG ODNs

are targeted efficiently into the active site. The co-encapsulation of

CpG ODNs and antigen in the same liposomes presumably allows

antigen presentation and adjuvant stimulation to occur in the

same cell, which is critical for the induction of immune responses

[61].

The SDS-PAGE analysis of liposomal SLA after separation of free

SLA from liposomal form showed although all of the bands are

present, some of the proteins are lost and this would be the reason

for different intensity of each band which is due to the considerable

orated with PO CpG ODNs or PS CpG ODNs induce the same protectionaccine.2012.03.040

decrease in proteins amount during separation procedure, particu- 444

larly the one which has ∼66 kDa MW. This was in agreement with 445

the BCA protein assay which showed that around 50% of SLA is lost 446

during separation process. 447

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DOTAP as a cationic lipid was used in the liposome formulationsor two reasons; firstly, to prepare positively charged liposomeshich interact more efficiently with CpG ODNs and the negatively

harged molecules on the APCs’ cell surface and so target antigensor endocytosis, and secondly, to utilize the intrinsic adjuvanticityf the synthetic quaternary ammonium compound as shown by thethers [62]. Yan et al. [62] showed that DOTAP is an active stimu-ator of DC resulting in extracellular-signal-regulated kinase (ERK)ctivation and CC chemokine (or �-chemokine) induction. Cationicipids with a quaternary ammonium group are more immunopo-ent than those with a tertiary ammonium group [63].

Due to the presence of DOTAP in the formulations, the neturface charge of empty liposomes was highly positive (+64 mV).owever, liposomes containing SLA had a zeta potential of+46 mV (Table 1). It means that SLA’s proteins have a negative

harge at pH 7.5 and interact electrostatically with the cationicipid in liposome bilayers. Hence, the zeta potential of liposomes

ill decrease. The electrostatic interaction could be the reasonf high entrapment efficiency of SLA in the positively chargediposomes (∼50%) compared with neutral ones (∼30%; unpub-ished data). Moreover, due to the fact that the zeta potential ofmpty liposomes was greater than those loaded with SLA, it cane concluded that some proteins in SLA were not entrapped within

iposomes and these fractions might be responsible for the surfaceharge decreasing. However, the zeta potential of empty liposomeshowed no significant change in the presence of CpG ODNs becausef very low molar ratio of CpG ODNs/DOTAP (7.5 × 10−3).

To assess the protection rate, footpad swelling induced by L.ajor was measured in immunized mice and compared with that

f the control groups. The results indicated the superiority of lipo-omal form of CpG ODNs in order to stimulate a Th1 immuneesponse. An improvement of adjuvanticity of CpG ODNs wasreviously reported by encapsulation of CpG ODNs in liposomes55,64–66]. However, PS CpG in their free forms showed signifi-antly higher protection rate than PO CpG as shown in antibodynd cytokine assay results.

Interestingly, IL-4 was produced in the spleen cells of miceeceived Lip-SLA–PS CpG or Lip-SLA–PO CpG after immunizationFig. 6B). However, the level of IL-4 decreased significantly at week

post-infection compared to the control groups (data not shown).he evidences that early IL-4 is needed to drive Th1 differentia-ion [67], to enhance IFN-� secretion [68,69] and to prime PBMCor cytokine production [70] imply that early IL-4 production doesot hinder the Th1 response. It might be in line with observationsevealing that IL-4 is crucial for the priming of long-term CD8+ T-ell memory responses [71–73]. As proposed by Sacks et al. [74], iteems that early IL-4 response might not be required to promoteusceptibility and there are more complexities in the mechanismsesponsible for acquired immunity.

The current data generated in murine model of L. major infectionhowed promising role of CpG ODNs and DOTAP cationic liposomess adjuvants to enhance stronger immune response against SLAhich is a crude Leishmania antigen. It is interesting to note that

here was no significant difference between PS CpG and PO CpG inerms of their ability to induce Th1 response when encapsulatednto the liposomes. Therefore, our results suggested that liposomalorm of PO CpG might be used instead of PS CpG in future vaccineormulations as an efficient adjuvant.

cknowledgment

Please cite this article in press as: Shargh VH, et al. Liposomal SLA co-incorpagainst the murine model of leishmaniasis. Vaccine (2012), doi:10.1016/j.v

The financial support of the Nanotechnology Research Centernd Biotechnology Research Center, Mashhad University of Medicalciences (MUMS), and Center for Research and Training in Skin Dis-ases and Leprosy, Tehran University of Medical Sciences (TUMS)

[

[

PRESS xxx (2012) xxx– xxx 7

are gratefully acknowledged. This study was part of Pharm.D. thesis

of VHS that was done in Nanotechnology Research Center, MUMS,

Iran.

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