Iranian J Parasitol: Vol. 5, No.1, 2010, pp. 47-54 Iranian J Parasitol Open access Journal at http:// ijpa.tums.ac.ir Tehran University of Medical Sciences Iranian Society of Publication Parasitology http:// tums.ac.ir http:// isp.tums.ac.ir Original Article The Role of Liposomal CpG ODN on the Course of L. major Infec- tion in BALB/C Mice H Hejazi1, M Tasbihi1, MR Jaafari2, A Badiee2, N Pestechian1, A Javadi3, *A Khamesi- pour4* 1- Department of Parasitology, Isfahan University of Medical Sciences, Isfahan, Iran 2- Biotechnology Research Center and Nanotechnology Research Center, School of Pharmacy, Mash- had University of Medical Sciences, Mashhad, Iran 3- Department of Social Medicine, Qazvin University of Medical Sciences, Qazvin, Iran 4- Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sci- ences, Tehran, Iran (Received 21 Sep 2009; accepted 22 Jan 2010) Abstract Background: Historically, leishmanization is the most effective protective measure against Cutaneous Leishmaniasis (CL), CL lesion induced by leishmanization sometimes takes a long time to heal. Ma- nipulation of leishmanization inoculums needed to induce a mild and acceptable CL lesion. The aim of this study was to explore if liposomal form of CpG ODN (Cytosin phosphate Guanin Oligodeoxynu- cleotides) mixed with Leishmania major would induce a milder lesion size in Balb/c mice. Methods: This study was performed in Biotechnology Research Center, Mashhad, and Center for Re- search and Training in Skin Diseases and Leprosy, Tehran, Iran during 2008-2009. Different groups of BALB/c mice were subcutaneously (SC) inoculated with L. major mixed with liposomal form of CpG ODN, or L. major plus free CpG ODN, or L. major mixed with empty liposomes or L. major in PBS. The lesion onset and the size of lesion were recorded; the death rate was also monitored. Result: Footpad thickness was significantly (P<0.01) smaller, death rate was also significantly (P<0.05) lower in the mice received L. major mixed with liposomal CpG ODN or free CpG ODN than control groups received L. major in PBS or L. major plus liposomes, also mice which received L. ma- jor mixed with CpG ODN in soluble form showed a significantly (P < 0.001) smaller lesion size than control groups. Conclusion: CpG ODN seems to be an appropriate immunopotentiator mixed with Leishmania stabi- late in leishmanization. Key words: CpG ODN, Liposome, Leishmania major, BALB/c * Corresponding Author: Fax: +9 8 21 8897 0658. E-mail [email protected] 47 Hejazi et al.: The Role of Liposomal Cpg ODN… Introduction C utaneous Leishmaniasis (CL) is a ma- done in Biotechnology Research Center, jor health problem in some endemic Mashhad, Mashhad, Iran and animal experi- areas, including Iran (1). Search for an ments were done in Center for Research and effective vaccine against leishmaniasis seems Training in Skin Diseases and Leprosy, Te- to be the sole control measure. Various forms hran University of Medical Sciences, Tehran, of Leishmania antigens, such as; recombinant Iran. Leishmania antigens and live attenuated para- sites, have been used to immunize against Animals murine model of leishmaniasis (2-4). Induc- Forty female BALB/c mice aged 6-8 weeks tion of protection in animal model of old were purchased from Pasteur Institute leishmaniasis is possible, but today there is no (Tehran, Iran). The mice were maintained in vaccine available against any form of animal house of Center for Research and leishmaniasis, regardless of global attempts Training in Skin Diseases and Leprosy and (5-7). Leishmanization showed to be the most fed with tap water and standard laboratory effective tool against CL (5, 8, 9). It was diet. Animals were housed in a colony room stopped due to lack of standardization, and with a 12-h-12-h light-dark cycle at 21ºC with unexpected prolonged lesion at the site of free access to water and food. leishmanization (10, 5). Bacterial DNA en- hances innate and adaptive immune responses Parasites (11, 12). Stimulation of immune responses by Leishmania major (MRHO/IR/75/ER) was bacterial DNA is due to the presence of un- grown in Novy-MacNeal-Nicole (NNN) me- methylated Cytosine-Guanine nucleotides dium, and for mass production, the promas- motif in DNA sequences. Synthetic oligode- tigotes were subcultured in RPMI (Sigma, oxynucleotides containing unmethylated gua- Germany) supplemented with 0.2 mM L- nine cytosine motif (CpG ODN) mimic the glutamine, 100 U/ml penicillin, 100 µg stimulating effect of bacterial DNA (11, 12). streptomycin and 15% fetal bovine serum. Using CpG ODN as an adjuvant mixed with The promastigotes were harvested at station- L. major ribosomal proteins or promastigote ary phase (stationary phase was estimated by antigens or recombinant Leishmania protein daily enumeration of parasite number). induced protection and even curative effect on CpG ODN: L. major infection (13-15). CpG ODN 1826 (Microsynth, Balgach, Swit- Liposomes are artificial closed vesicles com- zerland), was a 20-mer (5'-TCC ATG ACG posed of concentric lipid bilayers, separated TTC CTG ACG TT-3') with a nuclease-re- by aqueous domains and utilized as delivery sistant phosphorothioate backbone containing systems for drugs, peptides, proteins and two CpG motifs (marked as bold) known to DNA (16, 17). Encapsulation of CpG ODN show an immunostimulatory effect on Th1 into liposomes extends the duration of CpG response in murine model (19). ODN activity (18). In this study, L. major promastigotes were co- Encapsulated of CpG ODN in liposome inoculated with CpG ODN encapsulated in Liposomes containing CpG ODN were pre- liposomes or in free form subcutaneously into pared by the dehydration-rehydration vesicle susceptible BALB/c mice. The lesion size and (DRV) method (20). The lipid phase consist- the death rate were evaluated in immunized ing of 1,2-distearoyl-sn-glycero-3-phospho- mice and compared with the control groups. choline (DSPC), 1,2 dioleoyl propyl 3 trimethylammonium bromide (DOTAB) and Materials and Methods cholesterol dissolved in chloroform:methanol (2:1:1,v/v) in a round bottom flask. The sol- vent was removed by rotary evaporation re- In this experimental study, performed during sulting in deposition of a thin lipid film on the 2008-2009, preparation of liposoms and en- flask’s wall. The lipid film was freeze-dried capsulation of CpG ODN into liposomes were overnight to ensure total removal of the sol- 48 Iranian J Parasitol: Vol. 5, No.1, 2010, pp. 47-54 vent. The lipid film was then hydrated and < 0.05 were considered statistically signifi- dispersed in distilled water using vortex at cant. 55°C. The resulting empty multilamellar vesi- cles (MLVs) were converted to 100 nm small Ethical considerations unilamellar vesicles (SUVs) using the mini- Animal experiments were carried out accord- extruder (Avastin, Canada). The CpG ODN ing to Tehran University of Medical Sciences, were then added to empty SUVs liposomes, Ethical Committee Acts and were approved dried with freeze-drier overnight and rehy- by the TUMS Ethical Committee. drated by distilled water. CpG ODN, which remained not encapsulated, was removed Results from encapsulated ones by centrifugation at 14,000 × g for 15 min at 4 °C. Liposome characterization Optical microscope (Olympus, Germany) was The liposomes were morphologically multi- used to study the morphological features of lamellar vesicles, as observed under optical liposomes. The particle size distribution and microscope. The mean diameters calculated mean diameter of liposomes were determined by particle size analyzer were 1.01 ± 0.45 and by a particle size analyzer (Malvern, UK). 1.3 ± 0.3 µm (n=3) for liposomal CpG ODN and empty liposome, respectively. The en- Encapsulation efficiency of CpG ODN into trapment of CpG ODN in liposomes was es- liposomes timated to be more than 95%. The efficiency of incorporation (% entrap- ment) of CpG ODN in liposome was deter- Lesion size mined using UV absorption at 260 nm. The Footpad swelling in mice received L. major analysis was performed on supernatants fol- mixed with liposomal form of CpG ODN was lowing PBS washes. The percentage of en- significantly (P<0.01) smaller than control trapment was calculated as described before groups which received L. major in PBS or L. (17). The concentration of CpG ODN in the major mixed with empty liposomes. In the liposomes was adjusted to 10 mg/50 ml after group of mice, received L. major mixed with purification and calculation of percent of en- liposomal form of CpG ODN, only induration trapment. was induced and no ulcer was seen in the footpad of any of the mice. The mean average Induction of lesion in BALB/c mice of footpad swelling in this group showed to BALB/c mice (10 per group) were inoculated increase up to 6 weeks after inoculation with SC in the right footpad with 2 × 106 L. major, maximum thickness of 1.14 mm and then the promastigotes harvested at stationary phase lesion size started to decreased thereafter mixed with either CpG ODN (10µg), lipo- which ultimately reached to 0.23 mm at week somal CpG ODN (10µg CpG ODN), empty 14 (Fig. 1). liposome or PBS in final volume of 60µl. The The average footpad thickness in the group of development of lesion was monitored and re- mice that received L. major mixed with free corded in each mouse by weekly measure- CpG ODN was significantly (P < 0.001) ment of footpad thickness using a metric cali- smaller than groups received either L. major per. Grading of lesion size was done by sub- in PBS or mixed with empty liposomes. In the tracting the thickness of the uninfected con- group of mice, which received L. major tralateral footpad from that of the infected one mixed with free CpG ODN, the average of (18). footpad thickness increased up to 4 weeks after inoculation with maximum thickness of Statistical analysis 0.85 mm and thereafter the lesion size started Repeated measurement ANOVA statistical to decrease and reached to 0.20 mm at week test was used to assess the significance of the 14 post-infection. In this group, only one out differences among various groups. Bonfroni of 10 mice developed an ulcer on week 5, test was used to compare the means of differ- which was self-healing and completely healed ent treatment groups. Results with p values of by week 12. 49 Hejazi et al.: The Role of Liposomal Cpg ODN… Ulcer was seen in group of mice received L. at week 20 after infection and no more death major in PBS at week 5 post-inoculation with was seen up to 6 months after the inoculation. an average lesion thickness of 2.75 mm and In group of mice received L. major mixed from week 14, the infected foot started to fall. with liposomal form of CpG ODN, one The average thickness of lesion was 2.78 mm mouse died at week 15, two mice died at in mice received L. major mixed with lipo- week 18, one mouse died at week 19 and one somes. There was no significant difference in mouse died at week 23 post-infection there- footpad swelling in mice received L. major fore a total of 5 mice (50%) died up to 6 with empty liposome and the mice received L. months. major in PBS. The mice that received L. major in PBS, 2 There was no significant difference between mice died at week 14, 3 mice died at week 15, the average of footpad swelling in the group, one mouse died at week 16 and 4 mice died at which received L. major in CpG ODN, and week 17 post-infection, a total of 10 (100%) group, which received L. major in liposomal died up to week 17. form of CpG ODN (Fig.1). Concerning the mice that received L. major with empty liposome, 2 mice died at week 15, Death rate 2 mice died at week 16, 3 mice died at week In the group of mice, received L. major mixed 17 and 3 mice at week 18 post-infection and a with CpG ODN, only one (10%) mouse died total of 10 (100%) died by week 18 (Fig. 2). Fig. 1: Footpad swelling in BALB/c mice inoculated in the right footpad with 2 × 106 L. major promastigotes SC together with PBS, soluble CpG ODN, liposomal CpG ODN or empty liposomes. The footpad thick- nesses of mice were measured for 14 weeks. Each point represents the average increase in footpad thickness ± standard error of the mean (n =10) 50 Iranian J Parasitol: Vol. 5, No.1, 2010, pp. 47-54 Fig. 2: Death rate in BALB/c mice inoculated in the right footpad with 2 × 106 L. major promastigotes SC together with PBS, soluble CpG ODN, liposomal CpG ODN or empty liposomes Discussion Leishmanization is considered the most ef- duced protection against challenged with fective tool to protect against CL. It is also L. major at week 12 after immunization an effective tool to evaluate efficacy of (13). CpG ODN used in this study pro- Leishmania candidate vaccines (5,8-10). motes Th1 response, the type of response However, due to the problems associated required to induce protection in leishmani- with leishmanization such as lack of con- asis (19). Mendez et al. (26) showed that trol over the lesion development and heal- when C57BL/6 mice infected intrader- ing process, it was stopped except in Uz- mally with L. major mixed with 50µg CpG bekistan (5). CpG ODN as an immu- ODN with or without ALM showed a nostimulatory adjuvant was used as much milder lesions compared to the con- monotherapy against cancer (21), and in trol groups, in addition the experimental conjunction with an allergen to improve group of mice were protected against L. the immunogenicity of an antigen and at major challenge for up to 6 months. the same time to reduce its allergenicity BALB/c mice are highly susceptible to L. (22). CpG ODN is also used to induce major infection, upon infection the mice protection against infectious disease such develop skin lesions, which expand and as hepatitis B (23), listeriosis (24) and metastasize, and eventually every mouse malaria (25) .CpG ODN was used as an succumbed to the disease (27). In the pre- adjuvant mixed with different Leishmania sent study, susceptible BALB/c mice were antigens to induce protection in murine infected with L. major mixed with 10 µg of model of leishmaniasis (13,14). The CpG ODN encapsulated in liposomes or in administration of L. major ribosomal pro- free form, the lesion development and teins (LRP) along with CpG ODN induced death rate was compared with the control protection against L. major infection in groups. BALB/c mice. Footpad swelling of LRP + The results showed that co-administration CpG-ODN immunized mice was signifi- of CpG ODN with L. major in susceptible cantly lower than the control group re- BALB/c mice even with a lower dose i.e. ceived LRP alone (14). In addition, immu- 10 µg compared with 50 µg used previ- nization of BALB/c mice with autoclaved ously by others (26), induced a signifi- L. major (ALM) along with CpG ODN in- cantly smaller lesion size with significantly 51 Hejazi et al.: The Role of Liposomal Cpg ODN… lower death rate. Liposomes as system for To increase immunostimulatory, CpG carrying drugs, proteins, and DNA protect ODN was encapsulated into liposomes, encapsulated content from damages caused phosphorothioate CpG ODN which is used by environmental enzymes like endonucle- in this study is resistant to in vivo degrada- ase (16). In a study, BALB/c mice immu- tion by endonuclease enzymes. Based on nized with rgp63 plus CpG ODN encap- this theory, it was anticipated that encap- sulated in liposomes or rgp63 mixed with sulation of CpG ODN in liposomes will be CpG ODN in soluble form, upon chal- resulted in a lower lesion size and a lower lenged with L. major, there was a signifi- death rate in mice. However, as shown in cant (p<0.05) difference between the lesion Fig. 1, there is no significant difference size in mice immunized with rgp63-CpG between mice received CpG ODN in lipo- ODN and the control group up to week 11 somal form and mice received CpG ODN after challenges but thereafter the lesion in soluble form, which might be due to the size was not significantly different, but the lipid used in liposome formulation (i.e. lesion size in group which received rgp63- DSPC). DSPC has a very high transition lip-CpG ODN up to 14 weeks post chal- temperature (Tm 55°C) and produces a lenge was significantly (P<0.001) smaller very rigid and stable bilayer structure in than the control group (18). liposome formulation (28) and as a result, In the present study, the group of mice re- it does not destroyed easily in phagosome ceived L. major mixed with CpG ODN en- of target cells. Hence, there would not be capsulated in liposome, only indurations enough free CpG ODN available in were seen in the experimental group of phagosome to interact with its specific re- mice and no ulcer was seen up to 6 months ceptor, i.e. TLR9, localized in phagosome after infection. No significant different was (29). In terms of mice, which received free seen between averages of footpad swelling form of CpG ODN, there is one theory that of the group which received L. major free CpG ODN may interact with TLR9 re- mixed with CpG ODN encapsulated in li- ceptor more effectively than those encap- posomes, and the group received L. major sulated in very rigid and stable liposomes, mixed with free form of CpG ODN. also phosphorothioate backbone in this The groups of mice that received L. major type of CpG ODN is not damaged by en- mixed with CpG ODN encapsulated in donuclease in vivo. The results of current liposomes, or L. major mixed with free study suggest that further studies are form of CpG ODN, a significantly needed to identify a suitable lipid to for- (P<0.05) lower death rate was seen com- mulate the liposomes, which release their pared with the control groups up to 6 content, i.e. CpG ODN, on an appropriate month period after inoculation; one death time to interact with TLR9. out of 10 mice in group which received L. In conclusion, the results showed that co- major mixed with CpG ODN in free form inoculation of CpG ODN with L. major and 5 death out of 10 mice in group re- induce a milder leishmanization lesion ceived L. major mixed with CpG ODN en- compared to the control group and might capsulated in liposome were seen. Al- be appropriate to use in combination with though the death rate was lower in group it. of mice received L. major mixed with CpG ODN in free form than the group which Acknowledgments received L. major mixed with CpG ODN encapsulated in liposome but there was no The authors would like to appreciate Dr M. significant difference in lesion size be- Nateghi Rostami and Mrs A. Miramin tween the two immunized groups. Mohammadi, for their valuable lab work, Center for Research and Training in Skin 52 Iranian J Parasitol: Vol. 5, No.1, 2010, pp. 47-54 Diseases and Leprosy and Isfahan Univer- of leishmanization in control of cuta- sity of Medical Sciences and Mashhad neous leishmaniasis. Bull Soc Path University of Medical Sciences for finan- Exot. 1983; 76:397-383. cial supports. The authors declare that they 9. Nadim A, Javadian E, Mohebali M. have no conflicts of interest. The experience of leishmanization in the Islamic Republic of Iran. 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