Colloids and Surfaces B: Biointerfaces ...

Colloids and Surfaces B: Biointerfaces 74 (2009) 37–44

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Enhancement of immune response and protection in BALB/c mice immunized with liposomal recombinant major surface glycoprotein of Leishmania (rgp63): The role of bilayer composition Ali Badiee a , Mahmoud R. Jaafari a,∗ , Ali Khamesipour b , Afshin Samiei a , Dina Soroush a , Masoumeh Tavassoti Kheiri c , Farzaneh Barkhordari c , W. Robert McMaster d , Fereidoun Mahboudi c,∗∗ a School of Pharmacy, Biotechnology Research Center and Pharmaceutical Research Center, Mashhad University of Medical Sciences, P.O. Box 91775-1365, Mashhad, Iran b Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran c Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran d Medical Genetic Department, University of British Columbia, Vancouver, Canada

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Article history: Received 21 February 2009 Received in revised form 14 June 2009 Accepted 16 June 2009 Available online 23 June 2009 Keywords: Vaccination Liposome rgp63 Leishmaniasis L. major

a b s t r a c t Development of new generation vaccines requires adjuvants to elicit the type and intensity of immune response needed for protection. Liposomes have been shown to be an effective adjuvant formulation. In this study, the role of liposome bilayer composition with different phase transition temperature (Tc ) to induce a T helper 1 (Th1) type of immune response and protection against leishmaniasis in BALB/c mice was assessed. Liposome formulations with different bilayer compositions consisting of egg phosphatidylcholine (EPC, Tc < 0 ◦ C), dipalmitoylphosphatidylcholine (DPPC, Tc 41 ◦ C), or distearoylphosphatidylcholine (DSPC, Tc 54 ◦ C) were prepared. All liposomes were contained rgp63 as a recombinant antigen and used to immunize mice subcutaneously 3 times in 3-week intervals. Evaluation of lesion development and splenic parasite burden after challenge with L. major, evaluation of Th1 cytokine (IFN-␥) and Th2 cytokine (IL-4), and titration of IgG isotypes were carried out to assess the type of generated immune response and extent of protection. The results indicated the generated immune response in mice was influenced by the bilayer composition of liposomes, so that mice immunized with liposomes consisting of EPC induced a Th2 type of immune response while liposome consisting of DPPC or DSPC induced Th1 type of immune response. It seems that liposomes prepared with higher Tm phospholipids are suitable formulation to induce Th1 type of immune response and protection, and so might be used for further investigations to develop an effective vaccine against leishmaniasis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The idea of using liposome as a vehicle for the presentation of antigens was introduced more than 30 years ago when it was shown that diphtheria toxoid incorporated in liposome is more immunogenic than free diphtheria toxoid antigens [1]. Since then, the influence of different liposomes’ characteristics on the immune response; such as lipid composition, liposome charge and size, rigidity of bilayer, the association of antigen with liposome (in the bilayer or in the aqueous phase) and targeting to cellular receptors via the inclusion of appropriate molecules (e.g. mannan, Fc-␥ or

∗ Corresponding author. Tel.: +98 511 8823252; fax: +98 511 8823251. ∗∗ Co-corresponding author. Tel.: +98 21 66480780; fax: +98 21 66465132. E-mail addresses: [email protected] (M.R. Jaafari), [email protected] (F. Mahboudi). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.06.025

antibodies) on the exterior surface of the liposome has been studied [2,3]. Liposome stability might be improved by the substitution of underivatized PC (Tc −10 ◦ C) with phospholipids having high transition temperature (Tc ) such as DMPC (Tc 23 ◦ C), DPPC (Tc 41 ◦ C), and DSPC (Tc 54 ◦ C) [4]. It has been shown that a correlation exists between the Tc of phospholipids and generated immune response (humoral/cellular) for membrane associated high molecular mass antigens [2]. However, it is known that a phospholipid composition that induces a strong immune response to a specific antigen may not necessarily induce the same type or intensity of immune response to other antigens. Hence, there is a need for designing a liposomal vaccine tailored for a specific antigen [5,6]. Leishmaniasis caused by different species of Leishmania is a health problem in some foci of 88 endemic countries [7]. Recovery and protection against further infection in leishmaniasis at least in animal model are mainly depend upon induction of a Th1 type of

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immune response [8,9]. gp63 is a zinc-metalloprotease membrane glycoprotein of Leishmania species [10]. The recombinant form of gp63, rgp63, lacks the sugar molecules but induces a protective immune response against leishmaniasis when used with an appropriate immunoadjuvant [11–13]. Our previous study showed that BALB/c mice immunized with liposomal rgp63 prepared with detergent solubilization method showed an increased protection rate and immune response against L. major infection compared with the mice immunized with rgp63 in soluble form [14]. Hence, in the current study to optimize the adjuvanticity of liposome formulation for rgp63 antigen, the role of phospholipids’ type in liposomal bilayer was studied on the type and intensity of generated immune response against leishmaniasis. Liposomal rgp63 consisted of different phospholipids (DSPC, DPPC or EPC) were prepared and the adjuvanticity of the prepared liposomes against challenge with L. major in BALB/c mice was assessed and compared.

tively the presence of antigen in liposomes after purification as described before [14]. Briefly, the gel was consisted of running gel (12%, w/v, acrylamide) and stacking gel (3%, w/v, acrylamide). Electrophoresis was carried out at 150 V constant voltages for 45 min. After electrophoresis, the gel was stained with silver stain for protein detection.

2. Materials and methods

The immunized mice (7 per group) were challenged at 3 weeks after the last booster SC into the left footpad with L. major promastigotes harvested at stationary phase (1.5 × 106 promastigotes in 50 ␮l volume), and as a control right footpad was injected with the same volume of PBS. The lesion development was recorded in each mouse by measurement of footpad thickness with a metric caliper (Mitutoyo Measuring Instruments, Japan). Grading of lesion size was done by subtracting the thickness of uninfected contralateral footpad from that of the infected one.

2.1. Animals, parasites, SLA, and rgp63

2.3. Immunization of BALB/c mice Different groups of mice, 10 mice per group, were subcutaneously (SC) immunized 3 times in 3-week intervals with one of the followings: DSPC/Chol./rgp63, DPPC/Chol./rgp63, EPC/Chol./rgp63, rgp63 in PBS, or PBS. The concentration of injected rgp63 was adjusted to 2 ␮g/50 ␮l liposome (or PBS)/mouse. 2.4. Challenge with L. major promastigotes

Female BALB/c mice 6–8 weeks old were purchased from Pasteur Institute (Tehran, Iran). The mice were maintained in animal house of Biotechnology Research Center and fed with tap water and laboratory pellet chow (Khorassan Javane Co., Mashhad, Iran). Animals were housed in a colony room 12/12 h light/dark cycle at 21 ◦ C with free access to water and food. Experiments were carried out according to Mashhad University of Medical Sciences, Ethical Committee Acts. L. major strain (MRHO/IR/75/ER) [15] used in this experiment is the one which has been used for experimental Leishmania vaccine and Leishmanin preparation in old world [16]. Soluble Leishmania antigen (SLA) was prepared from promastigotes of L. major as described previously [17] and stored in small aliquots at −70 ◦ C until use. The protein concentration of SLA was determined using Lowry protein assay method [18]. rgp63 was expressed in E. coli BL21 (DE3) [19] and purified as described previously [14]. Briefly, cells were harvested, disrupted and then was purified using DEAE Sepharose fast flow column. Protein concentration of solubilized rgp63 was determined by Lowry protein assay [18]. The purity of rgp63 was confirmed by SDS-PAGE.

The number of viable L. major parasites in the spleen of each mouse was enumerated by a limiting dilution assay [20] as described previously [17]. Briefly, the mice were sacrificed at 14 weeks post-challenge; the spleens were aseptically removed and homogenized in RPMI 1640 supplemented with 10%, v/v, heat inactivated FCS (Eurobio, Scandinavie), 2 mM glutamine, 100 U/ml of penicillin and 100 ␮g/ml of streptomycin sulfate (RPMI-FCS). The homogenate was diluted with the media in 8 serial 10-fold dilutions and then was placed in each well of flat-bottom 96-well microtiter plates (Nunc) containing solid layer of rabbit blood agar in triplicate and kept at 25 ◦ C for 10 days. The number of viable parasite per spleen was determined using ELIDA software [21].

2.2. Liposomes preparation and characterization

2.6. In vitro spleen cells responses

Liposomes containing rgp63 were prepared by dehydrationrehydration vesicle (DRV) method as described before [17]. Briefly, the lipid phase consisting of DSPC, DPPC, or EPC (16 ␮mol/ml; Avanti Polar lipids, USA) and cholesterol (Avanti Polar lipids, USA) (2:1 molar ratio) was dissolved in chloroform:methanol (2:1, v/v) in a round-bottom flask. The lipid film was then hydrated and dispersed in distilled water using vortex above Tc of each phospholipids. The resulting empty multilamellar vesicles (MLV) were converted to 100 nm small unilamellar vesicles (SUV) using the Mini Extruder (Avestin, Canada). rgp63 was then added to empty SUV liposomes, dried with freeze-drier overnight and rehydrated as described before [17]. Unentrapped rgp63 was separated from entrapped ones using centrifugation and subsequently washed three times with PBS. Optical microscope (Olympus, Germany) and particle size analyzer (Klotz, Germany) were used to study the morphological features and mean diameter of the liposomes, respectively. The efficiency of rgp63 incorporation (% entrapment) in the liposome was determined with analysis of unentrapped rgp63 in the supernatants using Lowry protein assay as described previously [14]. The polyacrylamide gel electrophoretic analysis (SDS-PAGE) was carried out to characterize the antigen and to determine qualita-

Three mice from each group were sacrificed at 3 weeks after the last booster (at the same time as challenge experiment), the spleens were aseptically removed and cell suspension was obtained by homogenization of the tissue. The erythrocytes were lysed using ammonium chloride lysis buffer. The splenocytes were washed and resuspended in RPMI 1640-FCS and seeded at 2 × 106 /ml in 96well flat-bottom plates (Nunc). The spleen cells were stimulated in vitro with either rgp63 (5 ␮g/ml) or SLA (10 ␮g/ml) or Con A (2.5 ␮g/ml), and incubated at 37 ◦ C in 5% CO2 for 72 h. The supernatants were collected and the level of IFN-␥ and IL-4 was titrated using ELISA method according to the manufacturer’s instructions (Bender MedSystems GmbH, Vienna, Austria).

2.5. Quantitative parasite burden after challenge

2.7. Antibody isotype assay Blood samples were collected from the mice before and 14 weeks after challenge and the sera were isolated and kept frozen until being used to titrate anti-rgp63 or anti-SLA IgG total, IgG1 and IgG2a antibodies by ELISA method as described previously [22]. Briefly, 96-well micro titer plates (Nunc) were coated with 50 ␮l of 0.5 ␮g/ml of either rgp63 or SLA overnight at 4 ◦ C. Plates were washed and blocked for 1 h at 37 ◦ C with 200 ␮l of 1% bovine serum

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albumin in PBS–Tween per well. Serum samples were diluted to 1:200 with PBS–Tween and applied to the plates. The plates were then treated with anti-mouse IgG isotype according to the manufacturer’s instructions (Zymed Laboratories Inc., USA). 2.8. Statistical analysis One-way ANOVA statistical test was used to assess the significance of the differences among various groups. In the case of significant F value, Tukey–Kramer multiple comparisons test was carried out as a post-test to compare the means in different groups of mice. Results with P < 0.05 were considered to be statistically significant. 3. Results 3.1. Liposome characterization Liposomes were morphologically multilamellar vesicles and heterogeneous in size, as observed by light microscope. The mean liposome size calculated by particle size analyzer was 1.1 ± 0.34, 1.23 ± 0.25 and 1.13 ± 0.31 ␮m (n = 3) for DPPC/Chol./rgp63, DSPC/Chol./rgp63 and EPC/Chol./rgp63, respectively. Entrapment efficiency of rgp63 in DPPC/Chol./rgp63, DSPC/Chol./rgp63 and EPC/Chol./rgp63 liposome was 70 ± 8%, 65 ± 9% and 48 ± 7%, respectively. 3.2. Characterization of the purified rgp63, and liposomal rgp63 by SDS-PAGE SDS-PAGE analysis of the purified rgp63 revealed a single protein band with MWt of 55 kDa (Fig. 1, lane 2). Since the MWt of rgp63, which lacks the sugar molecules of native gp63, is 54–58 kDa [23] the purified protein is confirmed to be rgp63. Also, the SDS-PAGE analysis of the DPPC/Chol./rgp63, EPC/Chol./rgp63, and DSPC/Chol./rgp63 after removing free rgp63 in the supernatant revealed rgp63 incorporation into the liposomes (Fig. 1, lanes 3, 5, and 7, respectively). As shown in Fig. 1 (lanes 4, 6, and 8), there was no free antigen in the supernatant of liposome samples after three times washing with PBS. The SDS-PAGE indicated that rgp63 remains stable during liposome preparation and purification.

Fig. 2. Footpad swelling in BALB/c mice immunized SC, 3 times in 3-week intervals, with DSPC/Chol./rgp63, DPPC/Chol./rgp63, EPC/Chol./rgp63, rgp63 in PBS or PBS alone after challenge with virulent L. major promastigotes. The mice were challenged in the left footpad with 1.5 × 106 L. major promastigotes, 3 weeks after the last booster. The footpad thickness of mice was then measured on both footpads for 14 weeks. Each point represents the average increase in footpad thickness ± SEM (n = 7).

opment in the footpad was recorded weekly (Fig. 2). There was a significant (P < 0.001) difference between mice received PBS and other immunized groups at week 5 post-challenge. The results indicated that the size of footpad swelling in group of mice immunized with EPC/Chol./rgp63 liposome showed no significant difference with the size of footpad swelling in the group of mice immunized with rgp63 in PBS. At week 13 post-challenge, there was no significant difference in footpad swelling of mice immunized with DSPC/Chol./rgp63 and mice immunized with DPPC/Chol./rgp63 liposomes but they showed a significant difference compared with the mice immunized with EPC/Chol./rgp63 liposome (P < 0.05) or mice received rgp63 in PBS (P < 0.001). The results showed that immunization with DSPC/Chol./rgp63 or DPPC/Chol./rgp63 liposomes induces protection in mice more efficiently than immunization with EPC/Chol./rgp63 liposome.

3.3. Challenge results To investigate the extent of protection, the immunized mice were challenged with L. major promastigotes and the lesion devel-

Fig. 1. SDS-PAGE analysis of purified rgp63, Lip-rgp63 with different phospholipids formulation, Lane 1, low-range protein standard (Sigma, USA); Lane 2, purified rgp63; Lane 3, DPPC/Chol./rgp63; Lane 4, supernatant of DPPC/Chol./rgp63 after three times washing with PBS; Lane 5, EPC/Chol./rgp63; Lane 6, supernatant of EPC/Chol./rgp63 after three times washing with PBS; Lane 7, DSPC/Chol./rgp63; Lane 8, supernatant of DSPC/Chol./rgp63 after three times washing with PBS.

Fig. 3. Parasite burden in the spleen of BALB/c mice immunized SC, 3 times in 3-week intervals, with DSPC/Chol./rgp63, DPPC/Chol./rgp63, EPC/Chol./rgp63, rgp63 in PBS or PBS alone after the challenge with virulent L. major promastigotes. A limiting dilution analysis was performed at 14 weeks after the challenge on the cells isolated from the spleen of individual mice (n = 4) and cultured in triplicate in the RPMI-FCS for 10 days at 25 ◦ C in serial 8-fold dilutions. The wells were assessed microscopically for L. major growth, and the number of viable parasite per spleen was determined by ELIDA software based on limiting dilution assay method. The bar represents the average score ± SEM.

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3.4. Splenic parasite burden The number of viable L. major parasites was quantified in the spleen of different groups of mice at 14 weeks after challenge (Fig. 3). Mice immunized with DSPC/Chol./rgp63 or DPPC/Chol./rgp63 liposome showed the lowest number of live parasite which was significantly (P < 0.05) lower than the other groups. However, there was no significant difference between mice received DSPC/Chol./rgp63 and mice immunized with DPPC/Chol./rgp63 liposome. Interestingly, there was no significant difference in the parasite number between mice immunized with EPC/Chol./rgp63 liposome and mice immunized with rgp63 in PBS. 3.5. In vitro cytokine production by splenocytes The splenocytes of mice were separated the day before challenge and restimulated in vitro by either mitogen Con A as a positive control or SLA or rgp63 as a recalled antigen (Fig. 4). The culture supernatants were analyzed for the level of IFN-␥ and IL-4 using ELISA method. The highest level of IFN-␥ (∼6000 pg/ml) and IL-4 (∼850 pg/ml) were observed in cells stimulated with Con A which is an indicator of the fact that the cultured cells are live and response to stimulation correctly. The mice immunized with DPPC/Chol./rgp63 liposome showed the highest level

of IFN-␥ production in cell culture supernatant where rgp63 or SLA were used as recall antigens but there was no significant difference between the mice immunized with DPPC/Chol./rgp63 and the mice received DSPC/Chol./rgp63 liposome. The lowest level of IL-4 production was seen in the mice immunized with DPPC/Chol./rgp63 or DSPC/Chol./rgp63 liposome compared to the other groups (P < 0.001) when SLA was used as a recalled antigen. However, the lowest level of IL-4 production was observed in group of mice immunized with DSPC/Chol./rgp63 liposome when rgp63 was used as a recalled antigen (P < 0.05). 3.6. Antibody response To assess the type of immune response generated in immunized mice, the anti-gp63 and anti-SLA-specific IgG, IgG1 and IgG2a antibodies were titrated before (Figs. 5A and 6A) and after (Figs. 5B and 6B) challenge. As shown in Fig. 5A, there was a significant (P < 0.001) difference in IgG total antibody titer in the sera of mice immunized with liposome formulations compared with mice immunized with rgp63 in PBS. Moreover, the level of specific IgG2a antibody isotype against gp63 antigen in sera of mice immunized with DSPC/Chol./rgp63 showed a significant (P < 0.001) difference compared with the other groups. The level of IgG2a antibody in the sera of mice immunized

Fig. 4. Splenic cell responses of BALB/c mice immunized SC, 3 times in 3-week intervals, with DSPC/Chol./rgp63, DPPC/Chol./rgp63, EPC/Chol./rgp63, rgp63 in PBS or PBS alone. At 20 days after the last booster, the spleens were removed and the splenocytes were stimulated in vitro with either rgp63 (5 ␮g/ml) or SLA (10 ␮g/ml), Concanavalin A (2.5 ␮g/ml), or medium alone. Production of IFN-␥ (A) and IL-4 (B) were assessed by ELISA method in supernatants collected at 72 h of culture. Cells from 3 mice per group were pooled. Values are the mean ± SEM of triplicate wells.

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Fig. 5. Levels of anti-gp63 antibodies (IgG, IgG2a, and IgG1) in sera of BALB/c mice immunized SC, 3 times in 3-week intervals, with DSPC/Chol./rgp63, DPPC/Chol./rgp63, EPC/Chol./rgp63, rgp63 in PBS or PBS alone. Blood samples were collected 3 weeks after the last booster (A) and 14 weeks after the challenge (B). The rgp63-specific IgG, IgG2a and IgG1 levels were assessed using ELISA method. Panel (C) indicates the ratio of IgG2a/IgG1 based on absorbance. The assays were performed in triplicate with 200-fold diluted serum samples. Values are the mean ± SD.

with DPPC/Chol./rgp63 showed no difference with mice immunized with rgp63 in PBS. However, the level of IgG1 in mice immunized with DPPC/Chol./rgp63 was significantly (P < 0.001) lower than mice immunized with rgp63 in PBS. Interestingly, the sera of mice immunized with EPC/Chol./rgp63 liposomes showed the

significantly (P < 0.001) lowest titer of IgG2a antibody compared with the other groups (P < 0.001). The sera of mice immunized with DPPC/Chol./rgp63 or DSPC/Chol./rgp63 liposome showed that the significantly highest ratio (P < 0.001) of IgG2a/IgG1 compared with the other groups (Fig. 5C). The mice immunized with rgp63 in

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Fig. 6. Levels of anti-SLA antibodies (IgG, IgG2a, and IgG1) in sera of BALB/c mice immunized SC, 3 times in 3-week intervals, with DSPC/Chol./rgp63, DPPC/Chol./rgp63, EPC/Chol./rgp63, rgp63 in PBS or PBS alone. Blood samples were collected 3 weeks after the last booster (A) and 14 weeks after the challenge (B). The SLA-specific IgG, IgG2a and IgG1 levels were assessed using ELISA method. Panel (C) indicates the ratio of IgG2a/IgG1 based on absorbance. The assays were performed in triplicate with 200-fold diluted serum samples. Values are the mean ± SD.

PBS showed the lowest ratio of IgG2a/IgG1 in immunized groups (Fig. 5C). Fig. 6A shows the level of SLA-specific antibodies in the sera of mice before challenge. A significant (P < 0.001) difference in IgG total antibody was shown in the sera of mice immunized with liposome formulations compared to the mice

immunized with rgp63 in PBS (Fig. 6A). There was no significant difference between the level of IgG2a antibody in the sera of immunized mice but in case of IgG1 antibody, the mice immunized with DSPC/Chol./rgp63 or DPPC/Chol./rgp63 liposomes showed the lowest level of IgG1 antibody (P < 0.001) compared to the

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other groups. Thus, as shown in Fig. 6C, the mice immunized with DSPC/Chol./rgp63 or DPPC/Chol./rgp63 liposomes induced the highest ratio of IgG2a/IgG1 (P < 0.001). As shown in Figs. 5B and 6B, challenge with L. major promastigotes induced elevation of IgG, IgG1 and IgG2a antibodies in all groups of mice compared with the antibody titers before challenge. Interestingly, the sera of mice in control group (PBS) showed a high level of IgG1 antibody after challenge. The sera of mice immunized with DSPC/Chol./rgp63 showed a significantly (P < 0.05) higher levels of anti gp63-specific IgG2a antibody isotype compared with the other groups (Fig. 5B). The sera of mice immunized with DPPC/Chol./rgp63 liposome showed significantly (P < 0.001) the lowest level of gp63-specific IgG1 antibody isotype (Fig. 5B). On the other hand, the mice immunized with DPPC/Chol./rgp63 liposome showed the highest ratio of IgG2a/IgG1 after challenge infection compared to the other groups (Fig. 5C). As shown in Fig. 6B, the mice immunized with DPPC/Chol./rgp63 showed significantly (P < 0.001) the highest level of SLA-specific IgG2a but there was no significant difference between this group and the mice received DSPC/Chol./rgp63 liposome. The mice immunized with EPC/Chol./rgp63 or DSPC/Chol./rgp63 liposome showed significantly (P < 0.001) the highest and lowest level of IgG1 antibody isotype, respectively. Moreover, the mice immunized with DSPC/Chol./rgp63 showed significantly (P < 0.05) the highest ratio of IgG2a/IgG1 after challenge compared to all other groups (Fig. 6C). 4. Discussion In this study, the effect of phospholipid composition on adjuvanticity and protection extent of liposomal rgp63 against murine model of leishmaniasis was assessed. The results showed that liposomes prepared with DSPC were as efficient as DPPC in the entrapment of rgp63 and in their ability to potentiate a Th1 immune response to some extent; by contrast, liposomes prepared with EPC showed to be a Th2 type of immune response inducer as it is shown in Figs. 2–6. Liposomes prepared in the current study with using DRV method [4,24] were morphologically multilamellar vesicles as observed with light microscope and heterogeneous in size as detected by particle size analyzer. Preparation of liposomes via DRV method consists of mixing an aqueous solution of the solute (rgp63) with a suspension of empty SUV liposomes and freeze-drying the resulting mixture. The intimate contact of flattened liposomal membrane structures and solute molecules in a dry environment and the fusion of membranes caused by dehydration facilitate the incorporation of solute during the controlled rehydration steps. Upon the controlled addition of water, up to 80% of the water-soluble solute might be entrapped into the formed liposomes. DRV method is simple, easy to scale up, usually gives high yields of solute entrapment [24] compared to some other methods such as detergent solubilization which was used in our previous study [14]. In this study, entrapment efficiency of rgp63 in different liposome formulations was variable between 40 and 80% depended on liposome formulation. Liposomes prepared with DSPC or DPPC showed higher entrapment efficiency probably because of more bilayer rigidity than liposomes prepared with EPC. Resistance to leishmaniasis is associated with a predominant Th1 response with IFN-␥ production from the antigen-specific CD4+ T lymphocyte population. In addition, activation of CD8+ T cell population has been shown to play a critical role in protection after recovery from L. major infection and in effective vaccination against experimental murine leishmaniasis [25]. By contrast, Th2 immune responses which are characterized by IL-4 production are associated with susceptibility and exacerbation of the disease [25]. The results showed that liposomes prepared with DSPC were as efficient as DPPC to potentiate a Th1 immune response to some extent; by

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contrast, liposomes prepared with EPC showed to induce a Th2 type of immune response as it is shown in Figs. 2–6. It should be noticed that the immune response is a complex reaction to infection, and both Th1 and Th2 phenotypic cells can almost always be found during the immune response. The biological phenotype of the immune response is, therefore, determined by the predominance of one cell type over the other, not simply the presence or absence of Th1- or Th2-type immune cells [25]. In the current study, rgp63 in PBS did not show to induce a strong Th1 type of immune response and the predominant response was a Th2 type. Antigenic peptides derived from soluble exogenous antigens generally cannot be presented via the major histocompatibility complex (MHC) class I molecules because of their inability to reach the cytosol. As a consequence, most solution formulations of antigen are poor at priming CD8+ T cell responses or cell-mediated immunity (CMI) unless they are introduced into the cytoplasm via delivery systems with this capacity [3]. Vaccines against leishmaniasis need to induce a CD4+ and CD8+ T cell responses or CMI [26]. Hence, a soluble anti-leishmanial antigen needs to deliver via a particulate delivery system such as liposome to induce CMI. Liposomes were long ago been shown to be effective immunological adjuvants for protein and peptide antigens. They are capable of inducing both humoral and cellular immune responses towards the liposomal antigens [27]. However, the ability of liposomes to induce CMI is probably one of their most important features as an immunoadjuvant. Evidence for this has been provided by positive delayed-type hypersensitivity (DTH) reactions, the lymph node lymphocyte proliferative response test and the induction of cytotoxic T lymphocyte [27]. Liposomes with encapsulated protein or peptide antigen are phagocytosed by antigen presenting cells (APC) and eventually accumulate in lysosomes. Once in the lysosomes, degraded peptides are presented with MHC class II molecule on the macrophage surface. This results in the stimulation of specific T helper cells, and stimulation of specific B cells, which results in the subsequent secretion of antibodies. A fraction of the liposomal antigen can escape from endosomes into the cytoplasm and in this case the liberated antigen is processed and presented in association with the MHC class I molecule, which induces a CD8+ T cell response; this provides liposomes with certain benefits over traditional adjuvants (such as Freund’s adjuvant) that do not induce any significant CD8+ T cell response [28]. Recently, it has been reported that particulate vaccine adjuvants including mineral salts, liposomes, microparticles and emulsions, besides promoting antigen multimerization and internalization, play an important role as activators of innate immunity in vivo. The particulate adjuvants induce chemokine production in accessory cells like macrophages, monocytes, and granulocytes, leading to cell recruitment at injection site followed by the differentiation of monocytes into activated dendritic cells (DC). Alum and probably other particulate adjuvants activate a cytoplasmic Nod-like receptor called NLRP3 to form a protein complex called inflammasome. While it is clear that the inflammasome is activated by a number of particulate adjuvants, it remains to be clarified whether NLRP3 is generally necessary for in vivo adjuvanticity or whether it is required only for specific functions in a subset of vaccination protocols [29]. The evaluation of immune response in mice showed that the bilayer fluidity of liposomes influences the type of generated immune response. There was a significant difference (P < 0.05) between mice immunized with liposomes composed of phospholipids with high Tc (DSPC or DPPC) and the mice immunized with low Tc (EPC) liposomes in footpad lesion development, parasite load in spleen, IFN-␥ production and the ratio of IgG2a/IgG1 antibodies (Figs. 2–6). Liposomes prepared with DSPC or DPPC were efficient in their ability to stimulate Th1 response (based on smaller footpad lesion development and parasite burden, higher titer of IFN-␥

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production and of IgG2a/IgG1 ratio) compared with liposomes prepared with EPC. By substituting of EPC with ‘high melting’ phospholipids such as DSPC or DPPC, or supplementing phospholipids with excess cholesterol, vesicle bilayers become rigid at 37 ◦ C (the body’s temperature) or have their phospholipid molecules packed and thus resist phospholipid loss to extracellular microenvironment. Liposomal integrity is therefore preserved and entrapped solutes (e.g. antigens) remain with the carrier for longer periods of time [30]. On the other hand, liposomes with low membrane permeability may have a higher in vivo stability and thus enhanced adjuvanticity for the entrapped antigen. Previous studies showed that bilayer composition has a pronounced impact on the interaction and uptake of liposomes by APC [31,32]. Mazumdar et al. showed the effect of phospholipid on the adjuvanticity and rate of protection of liposome vaccine carriers against visceral leishmaniasis (VL) in hamster model [33] and showed that DSPC liposomes are superior to DPPC or DMPC liposomes formulation of L. donovani vaccine based on DTH response and the level of parasitemia observed in the spleen of hamsters immunized with Leishmania Ag (LAg) in DSPC liposomes [33]. It was proposed by the authors that the superiority of DSPC liposome observed against experimental VL is due to prolonged circulation of DSPC liposomes, which enabling more effective delivery of the antigens to APC. The stimulation of stronger immune response was also obtained with membrane antigens when phospholipids with low Tc were replaced with DSPC [34] and strong vaccine potential of DSPC liposomes over DPPC or EPC liposomes was also observed earlier for L. major antigens [35]. However, in the current study there was no significant difference in induction of Th1 type of response between the mice immunized with DSPC/Chol./rgp63 and those immunized with DPPC/Chol./rgp63 liposomes. Although the mechanism(s) of bilayer fluidity on immunoadjuvantcity of liposomes is still not clearly known, it is proposed that at least two mechanisms involved at the same time; (a) the rate of antigen release from the vesicles at the site of injection and (b) the mode of liposomes interaction with APC. The rate of release and the interaction with APC are depended mainly on the liposomal bilayer fluidity. In the first case, it is known that solid liposomes become unstable in vivo at a slower rate than fluid ones and so they present antigens more efficiently to APC. Secondly, liposomal fluidity influences the extent of liposomes’ fuse, endocytosis and/or processing by APC that in this case, solid liposomes probably would not be a very suitable choice. Therefore, it seems that liposomes need to have an optimal fluidity to be taken up by APC. In summary, the current results showed that DPPC or DSPC liposomes are suitable to be used to carry rgp63 antigen to induce a CMI against leishmaniasis.

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