Parasite Immunology, 2011, 33, 335–348
DOI: 10.1111/j.1365-3024.2011.01289.x
Cysteine proteinase type I, encapsulated in solid lipid nanoparticles induces substantial protection against Leishmania major infection in C57BL⁄6 mice D. DOROUD,1,2* F. ZAHEDIFARD,1* A. VATANARA,2 A. R. NAJAFABADI2 & S. RAFATI1 Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran, 2Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 1
SUMMARY Appropriate adjuvant, proper antigen(s) and a suitable formulation are required to develop stable, safe and immunogenic vaccines. Leishmanial cysteine proteinase type I (CPB) is a promising vaccine candidate; nevertheless, it requires a delivery system to induce a potent immune response. Herein, solid lipid nanoparticles (SLN) have been applied for CPB [with and without C-terminal extension (CTE)] formulation to utilize as a vaccine against Leishmania major infection in C57BL ⁄ 6 mice. Therefore, SLNCPB and SLN-CPB)CTE formulations were prepared from cetyl palmitate and cholesterol, using melt emulsification method. After intraperitoneal vaccination and subsequent L. major challenge, a strong antigen-specific T-helper type 1 (Th1) immune response was induced compared to control groups. Lymph node cells from immunized mice displayed lower parasite burden, higher IFN-c, IgG2a and lower IL-4 production, indicating that robust Th1 immune response had been induced. Our results revealed that CTE is not necessary for inducing protective responses against L. major infection as the IFN-c ⁄ IL-4 ratio was significantly higher, whereas IgG1 responses were lower in the SLN-CPB)CTE vaccinated group, post-challenge. Thus, SLN-CPB)CTE was shown to induce specific Th1 immune responses to control
Correspondence: Sima Rafati, Molecular Immunology and Vaccine Research Lab, Pasteur Institute of Iran, Tehran, Iran (e-mail:
[email protected] or
[email protected]) and Abdolhossein R. Najafabadi, Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (e-mail:
[email protected] or Roholami@ sina.tums.ac.ir). Disclosures: None. Received: 16 January 2011 Accepted for publication: 9 March 2011 *Equally participated as first author. Equally participated as corresponding author. 2011 Blackwell Publishing Ltd
L. major infection, through effective antigen delivery to the peritoneal antigen presenting cells. Keywords cysteine proteinase type I, delivery systems, Leishmania major, solid lipid nanoparticle, vaccination
INTRODUCTION Leishmaniasis is an important vector-borne parasitic infection which can cause a spectrum of diseases, ranging from a clinically silent to a fatal progressive disease in human and is a major public health problem in many Mediterranean, Asian, African, Central and South American, Caribbean, Near and Middle East countries including Iran (World Health Organization website, http://www.who.int/ vaccine_research/diseases/soa_parasitic/en/index3.html). In the recent years, much interest has been directed towards finding a vaccination as effective prevention is not available, and current curative therapies are costly, often poorly tolerated and not always effective. Furthermore, drug resistance exists in endemic areas. The existence of 14 million clinical cases of leishmaniasis worldwide and 350 million people living at risk of infection directs essential research and trials towards vaccination to stop the incidence of more than 2 million newly infected cases annually (World Health Organization website, http://www.who.int/vaccine_ research/diseases/soa_parasitic/en/index3.html). However, over the years, even though there has been research on different vaccine generations ranging from killed, live-attenuated to recombinant, synthetic and even naked DNA vaccines, there is no routine vaccine available against leishmaniasis worldwide (1). Immunity against Leishmania is dependent on the development of a strong T-cell response (mainly Th1) involving the production of cytokines such as IFN-c and IL-12 that stimulate the microbicidal activity of macrophages.
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Although live vaccines can induce these protective longlasting immunity responses, vaccination using this strategy is being suspended because of undesirable side effects and clinical complications. On the other hand, Leishmania vaccines based on recombinant proteins and pDNAs encoding leishmanial antigens are often not sufficiently immunogenic, and it is critical to co-administer an effective safe T-cell adjuvant and ⁄ or delivery system (2). One of the vaccine candidates studied by our research group is cysteine proteinase (CP) type I or CPB. This enzyme is produced by a variety of organisms, including Leishmania, and belongs to the papain superfamily and has been reported to be an attractive target for drug development (3). Another important point is the presence of an unusual C-terminal extension (CTE) that differentiates CPB from the other CPs in the papain superfamily (3). Comparison of Leishmanial CPBs showed that the CTE is highly variable (4). In some cases, the CTE is glycosylated and may be partially removed by proteolytic cleavage during processing of the enzyme to its mature form (5). Hence, the CTE fragment is not essential for enzyme activity and intracellular trafficking, but it has been postulated that it is highly immunogenic and responsible for immune evasion and plays a role in the diversion of the host immune responses (6). Previously, we investigated the potential role of L. major rCPB to elicit a protective immune response against infectious challenge in BALB ⁄ c mice. We demonstrated that immunization with rCPB in combination with Poloxamer 407 as an adjuvant could partially protect the mice against infectious challenge (7). Subsequent to these observations, we have had two major concerns. The first was utilizing Poloxamer 407 as an adjuvant, which has no relevant advantage for our forthcoming practical vaccination studies for the reason that it has been reported that a single intraperitoneal (i.p.) injection of this adjuvant produces marked physio-pathological changes in rodents such as mice (8,9). The second concern was about the production of a predominant IgG1 response that antigenic rCTE of L. infantum elicited in our previous studies which confirmed that rCTE is not protective as a vaccine candidate (10). Therefore, there was still a need to further explore and examine the protective potential of rCPB without the CTE fragment. On the other hand, rCPB, with or without CTE, in common with other subunit antigen requires a suitable adjuvant and ⁄ or delivery system to enhance and direct the induced immunity. The delivery system must not only protect the antigen from extracellular enzymatic degradation but also target it to the relevant immune cells. Polymeric particulate drug delivery systems as well as lipidic delivery
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systems, such as fat emulsions, liposomes and solid lipid nanoparticles (SLN), are particulate carriers that can deliver cargos such as proteins efficiently. It has been shown that SLN are highly biocompatible and have the ability to encapsulate antigens and release them in a controlled manner (11). In the present study, we investigated the preparation of L. major rCPB with and without CTE (rCPB)CTE) and their entrapment in SLN. The ability of formulations to induce the required immune responses for protective immunity against L. major infection in the resistant C57BL ⁄ 6 mice was evaluated via administration of vaccine through an i.p. route. This route of vaccine administration might be helpful because of high accessibility to antigen-presenting cells (APCs) in the peritoneal cavity and lymph nodes (LNs) (12) and has been reported in several studies for evaluation of protein delivery when using lipidic colloidal carriers (13,14).
MATERIALS AND METHODS Chemicals All solutions were prepared using MilliQ ultrapure (Milli-Q-System, Millipore, Molsheim, France) and apyrogenic water. Cetyl palmitate, Tween-80 and cholesterol were purchased from Merck (Darmstadt, Germany). Sodium dodecyl sulphate (SDS) was purchased from Sigma-Aldrich (Deisenhofen, Germany). The materials employed for SDS–PAGE gel electrophoresis were acquired from Sigma (Darmstadt, Germany) and those applied for PCR and enzymatic digestion were provided by Roche Applied Sciences (Mannheim, Germany). Cell culture reagents including fetal calf serum (FCS) and RPMI were sourced from Gibco (Gibco, Life Technologies GmbH, Karlsruhe, Germany) and Sigma, respectively.
Mice and parasite Female C57BL ⁄ 6 mice (6–8 weeks old, weighting 20 € 5 g) were obtained from the Pasteur Institute of Iran and were housed in plastic cages with free access to tap water and standard rodent pellets in an air-conditioned room under a constant 12:12-h light–dark cycle and kept at room temperature of 25C with relative humidity (50– 60%). The appropriate review board ethics committee of the Pasteur Institute of Iran has reviewed the use of experiments involving animals. The L. major strain (MRHO ⁄ IR ⁄ 75 ⁄ ER) was kept in a virulent state by continuous passage in BALB ⁄ c mice. A homogenized lymph node from an infected BALB ⁄ c mouse was cultured in RPMI-1640 media supplemented with 10% FCS and 100 lg of gentamycin ⁄ mL. Five-day 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 335–348
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stationary-phase promastigotes were harvested by centrifugation at 3000 · g for 10 min at 4C. The pellet was washed with PBS (8 mM Na2HPO4, 1Æ75 mM KH2PO4, 0Æ25 mM KCl, 137 mM NaCl) and re-suspended at a concentration of 3 · 106 cells ⁄ 50 lL. Fifty microlitres of this preparation was injected subcutaneously in the left hind footpad of each mice, and the course of infection was monitored weekly by measurement of the footpad with a metric calliper. Sizes were compared to the size of the contra-lateral noninfected footpad.
Preparation of the gene constructs Construction of a pEGMII plasmid-encoding cpb gene and amplification conditions was the same as our previous report (15). To prepare cpb)CTE, the pGEM-cpb plasmids (15) encoding the open reading frame of cpb (950 bp) were utilized as templates for PCR amplification. Specific primer pairs were designed as sense H6KB: 5¢-CGGGATC CACCATGGATGCGGTGGACTGGCGCG-3¢ and antisense CpbE: 5¢-CTGAAGCTTCACATGCGCGGACAC GGG-3¢. The PCR product (650 bp) was digested with BamHI and HindIII, gel purified and cloned in pEGMII plasmid (named as pEGM-cpb)CTE). Plasmid DNA was purified (Qiagen Plasmid Midi kit, Hilden, Germany), and the sequence was confirmed using the dideoxy chain termination method on an automated sequencer.
Expression and purification of rCPB and rCPB)CTE Construct corresponding to pQE-cpb was produced in fusion form with an N-terminal histidine (His6-tag) for expression and purification of rCPB, as previously described (13). The cpb)CTE gene was subcloned into the cloning site of the bacterial expression vector pET-23a, downstream of the T7 promoter. The E. coli strain BL21 (DE3) was transformed with pET-cpb)CTE and grown at 37C in 100 mL LB medium supplemented with 100 lg ⁄ mL ampicillin and 25 lg ⁄ mL chloramphenicol. The culture was induced with 1 mM IPTG at an OD600 of 0Æ8 and grown for a further 4Æ5 h at 37C. Cells were centrifuged at 8000 · g for 20 min. Bacterial pellets were dissolved in a lysis buffer (50 mM Tris–HCl (pH 8), 100 mM NaCl and 1 mM EDTA) and frozen overnight at )20C. After centrifugation (10 000 · g, 15 min, 4C), the pellets were washed extensively with washing buffer [20 mM Tris– HCl (pH 8), 20 mM NaCl and 1 mM EDTA]. The inclusion bodies were purified by the imidazole-SDS-Zn reverse staining method. The purified recombinant protein was concentrated by ultrafiltration using Amicon Filter (MWCO: 10 kDa) and dialysed against PBS. The protein concentration was determined with BCA assay kit (Pierce, 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 335–348
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Rockford, IL, USA). The purified recombinant proteins were analysed by SDS–PAGE and Coomassie blue staining to assess the integrity and purity of proteins. These proteins were recognized by previously prepared rabbit anti-CPB antisera using Western blot technique (15,16).
Encapsulation of the recombinant proteins into SLN A modified hot melt microencapsulation technique (also called Melt dispersion) (17,18) was used to prepare the SLN-CPB and SLN-CPB)CTE formulations. First, a lipid melt (containing cetyl palmitate, cholesterol; 100 mg) was prepared, and the protein solution (1Æ6 mg of rprotein in isotonic PBS, pH 7Æ4) was incorporated into the lipid melt by vigorous vortex-mixing for 3 min. Afterwards, liquid nitrogen was added to the primary emulsion and the melt dispersion was grinded in a mortar. The preparation was emulsified into a small volume of hot PBS (65C) containing Tween-80 (5 mL, 1% w ⁄ v). The mixture was then homogenized by ultra-turrax T25 at 15 000 · g for 60 s (IKA-Labortechnik, Staufen, Germany). The lipid particles were precipitated by adding 10 mL water to the initial emulsion. The resulted suspension was stirred at (4–5C) for 30 min until the particles were completely formed. The samples were filtered by 0Æ45-lm filters. The SLNs were washed by centrifugation (6000 · g, 10 min, three times) using the Amicon Ultra centrifugal filters 100 kDa (Millipore, Schwalbach ⁄ Ts, Germany) to purify the suspension from excess surfactants. Finally, to assist nanoparticle separation from dispersion by centrifugation, the pH value of nanoparticles dispersion was adjusted to 1Æ2 by addition of 0Æ1 M hydrochloric acid. Afterwards, the particles were isolated by centrifugation at 20 000 · g for 1 h. The formulations were appropriately diluted in 5 mM PBS, pH 7Æ4, and stored at 4C until further use. The size distribution, zeta potential, polydispersion index (PI) and the solution pH were monitored after preparation and 1 month later. In addition, the blank SLN (without rCPB or rCPB)CTE) was prepared according to the same method. Meanwhile, the free rprotein solutions were also used as control.
Particle size, zeta potential and differential scanning calorimetry (DSC) analysis Particle size was analysed by photon correlation spectroscopy (PCS). The samples were diluted with Milli-Q-water to suitable concentration, and the size and zeta potentials were measured with a Malvern Zetasizer 5000 (Malvern Instruments, Worcestershire, UK). All measurements were performed in triplicate. Differential scanning calorimetry thermograms of the SLN samples were obtained using a
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Mettler instrument (Mettler-Toledo AG, Greifensee, Switzerland) (19).
Process yield determination For the calculation of the SLN-CPB ⁄ or CPB)CTE production yield, the suspensions were centrifuged at 20 000 · g for 1 h, and the supernatant was kept for loading efficiency assessment. The eppendorf tubes containing the sediment were air-dried in a desiccator for 24 h, and the difference in the theoretical solid weights (solid lipid materials + rprotein) and the actual dried nanoparticles’ weight was determined.
rCPB and rCPB)CTE encapsulation efficiency (EE) The encapsulation efficiency was determined directly and indirectly. For indirect analysis, the amount of rprotein entrapped into SLN was calculated by the difference between the total amount of rprotein used to prepare the nanoparticles and the amount of rprotein that remained in the aqueous phase (supernatant) after SLN separation. The protein concentration in the supernatant was determined by BCA assay. In a parallel experiment, blank SLNs were prepared and protein loading was measured using BCA assay to see whether the protein loading calculations show any interference in BCA assay. EE ¼
Total amount of rCPB - FreerCPB in supernatant 100 Total amount of rCPB
To determine the protein loading directly, approximately 5 mg of washed precipitated particles was measured and dissolved in 1 mL hexane. Then, 1 mL of citrate buffer (pH = 3Æ0) containing 2% SDS was added. The two-phase system was agitated gently for 4 h on a horizontal shaker at 4C, the aqueous phase was removed and rCPB concentration was determined by BCA assay.
Protein release estimation from the nanoparticles To study the in vitro rprotein release from SLN, a dialysis method was applied using dialysis bag of 100 000 MWCO (Sigma). Approximately 25 mg of the formulation was dispersed in 0Æ5 mL of PBS containing 0Æ02% sodium azide as a bacteriostatic agent. This dispersion was then transferred to a presoaked dialysis bag, completely fixed and placed in a receiving compartment containing 10 mL PBS (pH = 7Æ4) and SDS (2%). The whole set was shaken at 200 · g inside a 37C shaker incubator. At predetermined time intervals (2, 4, 6, 8, 10, 24 and 48 h up to 7 weeks), the protein content from withdrawn samples was measured via BCA assay in duplicate. The release profile
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was described in the terms of cumulative release (% w ⁄ w) against the incubation time. A solution containing 150 lg rCPB and a blank SLN dispersion were also used as positive and negative controls, respectively.
Vaccination studies Immunization of mice and sampling schedules Four groups, each including 12 female mice, were studied. The first and second groups (G 1, 2) were immunized with the vaccine formulation corresponded to 18 lg of the rCPB and rCPB)CTE antigens, respectively. Control groups received unloaded SLN formulation (G 3) or PBS alone (G 4). We didn’t assign a group for inoculation of rCPs alone, as we have previously observed that the immune response in vaccinated mice with the antigen without any adjuvant was negligible (7). Two intraperitoneal (i.p.) injections were given at 3-week intervals. Endotoxin concentration in the formulations was determined as 0Æ314 EU ⁄ 50 ug by limulus amoebocyte lysate assay (LAL kit, Charles River Endosafe, Wilmington, MA, USA). All mice were challenged 3 weeks after the booster injection, and progress of infection was followed until the 17th week after challenge, as described in the Mice and parasite section. Blood samples were taken from the eye vein of each individual mouse before and 12 weeks post-challenge. The sera were pooled in each group and kept at )20C. Cytokine assay Prior to challenge and 12 weeks after challenge, three mice from each group were sacrificed and LNs were excised, homogenized and washed. Cells were cultured at 106 per well in RPMI 5% (RPMI 1640 supplemented with 5% FCS, 1% L-glutamine, 1% HEPES, 0Æ1% 2ME, 0Æ1% gentamicin) and stimulated against soluble Leishmania antigen (SLA), rCPB, rCPB)CTE and ConA (concanavalin A). Antigens and ConA were prepared at 10 and 5 lg ⁄ mL concentrations, respectively. Cell culture plates were incubated for 5 days at 37C in 5% CO2 humidified atmosphere. Production of IFN-c and IL-4 was measured in the supernatant of the stimulated lymph node cultures by sandwich-based Elisa kits (R&D, Minneapolis, MN, USA) according to manufacturer’s instruction. The lower detection limits of IFN-c and IL-4 were 2 and 7 pg ⁄ mL, respectively. All tests were performed twice in triplicate. Determination of serum IgG1, IgG2a Pooled sera were prepared from each group of mice, before and 8 weeks after challenge. Production of IgG1 and IgG2a antigen-specific antibodies were measured against SLA, rCPB)CTE and rCPB. Briefly, 96-well plates 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 335–348
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were coated with rCPB, rCPB)CTE or SLA antigen (10 lg ⁄ mL in PBS) and incubated at 4C overnight. After three washes with washing buffer (PBS plus 0Æ05% Tween20), plates were blocked with blocking buffer (1% BSA in PBS) for 2 h at 37C, then 100 lL of pooled sera was added and incubated for another 2 h at 37C (1 : 400 dilution). Anti-mouse IgG1 (1 : 10 000; Zymed, Burlington, ON, Canada) and IgG2a (1 : 500; Southern Biotech, Birmingham, AL, USA) were added and incubated for 2 h at 37C. Streptavidin-horseradish peroxidase was added to biotinylated anti-antibodies and incubated for 1 h at 37C. Conjugate binding was visualized with O-phenylenediamine, the reaction was stopped by sulphuric acid (4N) and the absorbance was measured at 492 nm. All tests were performed twice in triplicate. Parasite burden Three mice from each group were sacrificed 12 weeks after challenge, and parasite burdens were determined in triplicate as follows: LNs were excised, weighed and then homogenized with a tissue grinder in 2 mL of Schneider’s Drosophila medium supplemented with 20% heat-inactivated FCS and gentamicin (0Æ1%). Under sterile conditions, serial dilutions ranging from 1 to 10)20 were prepared in wells of 96-well microtitration plates. After 7 and 15 days of incubation at 26C, the plates were examined with an inverted microscope at a magnification of 40·. The presence or absence of mobile promastigotes was recorded in each well. The final titre was the last dilution for which the well contained at least one parasite (20,21). The number of parasite per gram was calculated through the following formula: Parasite burden = )log10 (parasite dilution ⁄ tissue weight) Statistics Statistics were performed using GraphPad Prism 5.0 for Windows (GraphPad Software Inc 2007, San Diego, CA, USA). All the data were analysed with one-way ANOVA (multiple comparisons Tukey post-test) when required, with the exception of size and zeta potential measurements which were analysed with a Student’s t-test. A P-value of £0Æ05 was considered significant.
RESULTS Isolation of CP genes encoding rCPB and rCPB)CTE The CPB)CTE gene encoding mature CP type 1 without the last 108 amino acids (from amino acid 209 to 317) was amplified from the construct pGEM-cpb. The PCR product corresponding to the CPB)CTE (650 bp) gene was inserted into pEGM II plasmid and sequenced (Fig 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 335–348
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ure 1a,b). The L. major CPB)CTE nucleotide sequence was compared to the coding sequences available for L. major cpb (16) and revealed to be 100% identical.
Generation of the rCPB)CTE of Leishmania major The confirmed cpb)CTE gene was subcloned into pET-23a for expression in BL21 (DE3) pLysS E. coli (Figure 1c). Expression of the protein was then induced with 1 mM IPTG as described in Materials and Methods, and the bacterial lysates were analysed by SDS–PAGE. After staining the gel with Coomassie Blue, a 23-kDa band was observed (Figure 1c, lane 2) which had not been detected in the bacterial lysate before induction (Figure 1c, lane 1). This protein was recognized using previously prepared rabbit anti-CPB antisera on the immunoblot (data not shown).
Characterization of the purified rCPB, rCPB)CTE and SLN-rCPBs by SDS–PAGE SDS–PAGE analysis of the purified rCPB and rCPB)CTE revealed a protein band in expected sizes i.e. 40 kDa (16)
cpb gene: 950 bp (a)
CTE: 300 bp cpb-CTE, 650 bp
(b)
MW
1
2
(c)
30002500-
39·2 -
2000-
26·6 -
1500-
14·6 -
MW
1
2
(d) MW 1
2
3
39·2 26·6 14·6 -
1000-
750-
Figure 1 Different analyses of CPB)CTE as DNA construct and recombinant protein expressed in E. coli culture. (a) Schematic presentation of cpb gene (950 bp) and cpb)CTE. (b) Digestion confirmation of cpb and cpb)CTE in pGEM II as shown in line 1 and 2. (c) Induction and expression of CPB)CTE; CPB)CTE was cloned in pET23a expression vector, lane 1, crude lysate of bacterial culture before induction; lane 2, crude lysate of bacterial culture after induction with 1 mm IPTG for 4 h containing rCPB)CTE (23 kDa). (d) Lane 1, bacterial lysate before induction; lane 2, bacterial lysate after IPTG induction; lane 3, reverse staining purified rCPB)CTE (23 kDa) from the inclusion bodies. Molecular weight markers are shown on the left side of the SDS–PAGE in kDa. The gels were stained with Coomassie blue. CPB, cysteine proteinase type I; CTE, C-terminal extension.
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cles appeared to be smaller than SLN-rCPB particles, probably because of the lower molecular weight of the rCPB)CTE and its less negative grand average of hydropathicity value (Table 1) which suggests that the mentioned protein is more hydrophobic than that of rCPB; these reasons might be the cause for the better incorporation of rCPB)CTE into the lipidic matrix of SLN. However, there was no significant difference in the size values of these nanoparticles. The mean values of zeta potential were )8Æ24 € 0Æ1, )8Æ43 € 0Æ5 and )12Æ4 € 0Æ3 mV for unloaded, rCPB and rCPB)CTE-loaded SLN, respectively. The observed zeta potential revealed all carriers have a negative charge at pH 7Æ4. The zeta potential values are slightly lower than electric neutrality. The differences between the zeta potential values are not statistically significant (P > 0Æ05). This may show that the protein deposition was not on the surface of the SLN. Although immediately after production, the pH value of rproteinloaded SLN dispersions was found to be near 8 which is higher than the iso-electric point of rCPB)CTE (PI = 5Æ3), this difference might be responsible for higher negative charge of the SLN-CPB)CTE particles. The characteristics of the prepared nanoparticles are summarized in Table 2. The EE of rprotein in cetyl palmitate SLN was 36Æ2 € 6% and 48 € 3% for CPB and CPB)CTE respectively. The incorporation of rprotein into the nanoparticles
and 23 kDa, respectively (Figure 1d, lane 3). These values were consistent with the results obtained from the analysis of protein sequences and structures using Expert Protein Analysis System (ExPASy) proteomics server (Table 1) (22–24). Identical concentration of rproteins (8 lg) incorporated in SLNs were run on 15% SDS–PAGE and stained with Coomassie blue. The band patterns of incorporated rproteins from the SLN formulations were assessed, and the results revealed that rproteins were successfully incorporated into the nanoparticles (data not shown). Subsequently, there was no free rprotein in the supernatants after three washes of the nanoparticles with PBS.
Physical characterization of nanoparticles Solid lipid nanoparticles-rCPB and SLN-rCPB)CTE were successfully produced by a modified melt emulsification method using high-shear homogenization. As previously described, this SLN formulation was proven to be a stable carrier because of the presence of Tween-80 in the formulation that acts as a protective coating against aggregation (25). Particle size analysis by PCS revealed comparatively polydisperse nanoparticles (PDI 0Æ4) with an average size of about 253 nm (for blank) or 360–380 nm (for rprotein-loaded SLNs) were produced. SLN-rCPB)CTE parti-
Table 1 Physicochemical and structural model of rCPB and rCPB)CTE, based on their homology with L. major rCPB. The model of the proteins was built on the rCPB nucleotide sequence of L. major (GenBank: U43706) template, using automated Mode of the Swiss-Model protein structure homology-modelling server (22–24)
rprotein
Formula
Theoretical pI
CPB)CTE
C1007H1538N274O310S14
5Æ03
CPB
C2109H3273N593O637S29
8Æ04
Instability index
Grand average of hydropathicity
22Æ892 kDa
35Æ81 (stable)
)0Æ045
40Æ058 kDa
34Æ34 (stable)
)0Æ164
MW
Structural model*
CPB, cysteine proteinase type I; CTE, C-terminal extension.
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achieved. The amount of protein release from SLNCPB)CTE nanoparticles was found to be higher than SLNCPB nanoparticles (86% vs. 76%) (P > 0Æ05); however, a statistical analysis indicated that the protein release rates (i.e. slope) between the two vaccine formulations was not significantly different (P = 0Æ74). The presence of the third release phase from the second week indicated an incomplete continuous protein release from the formulations.
Evaluation of protective efficacy of rCPB and rCPB)CTE formulated with SLN Mice were challenged 3 weeks after the last booster with L. major stationary-phase promastigotes, and the course of the infection was followed until the 17th week after challenge when all groups were completely healed. The infection progressed similarly in vaccinated and control groups for the first 3 weeks (Figure 4a). As the primary measure of vaccine efficacy, we compared the footpad swelling in different groups. Vaccinated mice showed partial though stronger protection than the nonvaccinated mice. On the other hand, the vaccine formulations showed the ability to reduce the footpad swelling diameters significantly after the 9th week post-challenge (Figure 4a). As
rCPB loaded SLN
Heat flow (mW)
varied with the MW and grand average of hydropathicity of the antigen, with rCPB)CTE giving the higher entrapment value. Therefore, approximately 64% and 52% of rCPB and rCPB)CTE were solubilized by the surfactant molecules and therefore did not get incorporated into the nanoparticles. After incubation in PBS at 37C, none of the formulations showed a significant increase (P > 0Æ05) in size and PDI values, within 24 h. This means that there was no tendency for aggregation in both formulations. To characterize the degree of crystallinity of the lipid nanoparticles, a thermal analysis DSC was performed (Figure 2). The obtained results were further correlated with the encapsulation efficacies and release profiles of these formulations. The melting point of nanoparticles was lower than that of the lipid bulk material (
