Intranasal immunisation of mice against Streptococcus equi using positively charged nanoparticulate carrier systems

Vaccine 30 (2012) 6551–6558

Contents lists available at SciVerse ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Intranasal immunisation of mice against Streptococcus equi using positively charged nanoparticulate carrier systems L. Figueiredo, A. Cadete, L.M.D. Gonc¸alves, M.L. Corvo, A.J. Almeida ∗ Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia da Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisboa, Portugal

a r t i c l e

i n f o

Article history: Received 6 January 2012 Received in revised form 23 July 2012 Accepted 20 August 2012 Available online 2 September 2012 Keywords: Streptococcus equi Liposomes Nanoparticles Chitosan Nasal vaccines Adjuvants

a b s t r a c t In order to potentiate a strong immune response after mucosal vaccination with a low immunogenic S. equi enzymatic extract, two positively charged particulate delivery systems (liposomes and nanoparticles) were created. Positively surface charged particles were expected to efficiently bind to negatively charged cell membranes and facilitate antigen uptake. Phosphatidylcholine–cholesterol–stearylamine liposomes encapsulating S. equi antigens were prepared and dimensionated to 0.22 ± 0.01 ␮m with a polydispersity index <0.242, zeta potential of +12 ± 4 mV and an encapsulation efficiency of 13 ± 3% (w/w). Chitosan nanoparticles were prepared by ionotropic gelation with sodium tripolyphosphate, presenting a particle size of 0.17 ± 0.01 ␮m with polydispersity index <0.362, zeta potential of +23 ± 8 mV and an encapsulation efficiency of 53 ± 6% (w/w). Both encapsulation methods were recognised as innocuous once antigens structure remained intact after incorporation as assessed by SDS-PAGE. Intranasal immunisation of mice with both formulations successfully elicited mucosal, humoral and cellular immune responses. Mucosal stimulation was confirmed by increased sIgA levels in the lungs, being the chitosan nanoparticles more successful in this achievement probably due to their different mucoadhesive properties. Both formulations share the ability to induce Th1-mediated immune responses characterised by IFN-␥ production and high IgG2a antibody titers as well as a Th2 immune response characterised mainly by IL-4 production and IgG1 antibodies. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Strangles is a highly contagious disease of the Equidae caused by Streptococcus equi subsp. equi [1,2]. Bacteria penetrate the oral and nasal mucosa, lodging at the pharyngeal and upper respiratory tract lymph nodes, forming painful abscesses that prevent horses from feeding and causing breathing difficulties, resulting in a general morbid condition [2,3]. The swelling of the lymph nodes may restrict the airway and it is this clinical feature that gave strangles its name [3]. Rare complications of the disease include guttural pouch empyema resulting from the rupture of abscessed lymph nodes into the guttural pouch or the extension of infection from the pharynx. Drying and hardening the purulent material in the guttural pouch, leads to formation of discrete masses known as chondroids [2]. In these circumstances, animals are asymptomatic but represent a high risk of contamination of other animals. Recovered animals exhibit a protective immunity mostly against the cell wall M-like protein (SeM), presenting both specific IgG and IgA in serum and nasal secretions. However, vaccines targeting SeM provide a limited protection, even when in association with different

∗ Corresponding author. Tel.: +351 217946400; fax: +351 217937703. E-mail address: [email protected] (A.J. Almeida). 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.08.050

adjuvants [4] A non-encapsulated strain of S. equi (Pinnacle IN® ) has been used as a nasal vaccine, but has not been licensed for sale in Europe due to safety concerns. A different vaccine with a live attenuated vaccine strain TW 928 has been on the market as Equilis StrepE® (Intervet) but provides a limited protection of 3 month duration [3]. Intranasal (i.n.) vaccination emerges as the logical choice in order to reach protective immunity. The locally produced nasopharyngeal antibodies will constitute a primary line of defence against S. equi invasion via the mucosal surface. In fact, the ineffectiveness in already marketed vaccines seems to correlate with the insufficient stimulation of local nasopharyngeal antibodies, which are present in convalescent horses [5]. Specific i.n. immunisation strategies, using particulate antigen delivery systems proved to trigger the local immune response. Such carrier systems are more efficiently absorbed at the mucosal epithelia than soluble antigens. Particulate carriers also protect the antigens against proteolytic enzymes and facilitate the access to the mucosal-associated lymphoid tissue (MALT). The type of immune response thus obtained may also be modulated by using different particulate formulations that will lead to a different antigen presentation [6,7]. The i.n. vaccination of mice with S. equi antigens associated to particulate carriers enhances not only the mucosal immunity but also systemic responses, as well as protection from

6552

L. Figueiredo et al. / Vaccine 30 (2012) 6551–6558

experimental infection [8–10]. For example, the use of ISCOMs, a particulate system with proven adjuvant properties, associated with recombinant S. equi antigens resulted in protective vaccination of mice and horses [11]. Encapsulation of S. equi antigens, in polymeric and biodegradable poly(lactic acid) (PLA) and poly-␧-caprolactone (PCL) nanoparticles has recently been studied in our group for immunisation purposes. The study included the modification of nanoparticle surface with different adjuvants and permeation enhancers, such as chitosan (CS), spermine and oleic acid. The resulting formulations were able to induce mucosal (SIgA) and balanced Th1/Th2 systemic responses in mice [9,10,12,13]. Particularly, serum specific IgG, IgG1 and IgG2a antibody responses were induced and increased amounts of Th1 cytokines were observed [9,10]. Although immunisation experiments performed with mice may have limited application to equids–the natural host of S. equi [4,14] the mouse is still the only preclinical model for strangles vaccines. On the other hand, our previous results suggest that particulate carriers should be further studied as antigen delivery systems and immunological adjuvants for S. equi antigens. For example, liposomes present vaccine adjuvant properties inducing both humoral and cell-mediated immune responses upon being efficiently endocytosed by antigen-presenting cells (APC). Variation of parameters such as composition, particle size and surface charge, will influence the in vivo fate of liposomes as well as antigen presentation [7,15]. Liposomal formulations composed of cationic lipids have shown to be immunostimulatory being greatly captured by dendritic cells [16]. Intranasally administered cationic liposomes encapsulating protein antigens have demonstrated high proficiency in inducing humoral and cellular immune response as well as a local immunity in mice, leading to protection [17,18]. Also chitosan has been extensively studied as a mucosal delivery system of antigens and as an immune response enhancer due to its bioadhesive and penetration enhancement properties [19]. It has the power to increase permeability of the nasal epithelium when used as a nasal vaccine adjuvant through the opening of the tight junctions between cells, which facilitates paracellular transport [20]. Due to its characteristics and chemical structure chitosan also decreases the mucociliary clearance allowing a prolonged antigen presentation [21]. Therefore, chitosan nanoparticles, with a positive superficial charge, and positive liposomes are potential good S. equi antigen particulate carriers. The aim of the present study was to investigate the efficiency of positively charged liposomes and chitosan nanoparticles as nasal delivery systems and mucosal adjuvants for S. equi antigens, able to trigger suitable local and systemic immune responses in an experimental mouse model. 2. Materials and methods 2.1. Materials Egg-phosphatidylcholine (PC) was obtained from Lipoid (Germany). Cholesterol (Chol), stearylamine (SA), chitosan low molecular weight (LMW) with degree of deacetylation 75-85%, sodium tripolyphosphate (TPP), ciabacron brilliant red 3B-A, glycine, sodium chloride and hydrochloric acid were all obtained from Sigma–Aldrich (Spain). 2.2. Animals Female BALB/c mice (n = 5/group), 6–8weeks old provided from Charles River, (Spain). Animals were fed with standard laboratory food and water ad libitum. All in vivo studies using animals were

carried out with the authorization of the local ethics committee and in strict accordance with the Declaration of Helsinki, the Directive 2010/63/EU that replaces the previous Directive 86/609 EEC), the relevant Portuguese laws D.R. no. 31/92, D.R. 153 I-A 67/92, and all following legislation. 2.3. Antigen preparation Antigens were prepared as described by Florindo et al. [12] with slight modifications. Briefly, inactivated Streptococcus equi subsp equi cells (ATCC 53186) were washed with phosphate buffered saline (PBS) 50 mM at pH 6.0 and homogenised by sonication. Lysozyme (0.75 g), mutanolysin (5KU) and sucrose (12.8 g) by each 30 g of cells, all from Sigma–Aldrich (Spain), were added to the homogenised cells and incubated at 37 ◦ C overnight, under constant agitation. Finally, protoplasts were separated by centrifugation at 30,000 × g and the extract was ultra-filtered by a 30 kDa membrane in order to remove sucrose residues. The resulting protein solution was then frozen at −20 ◦ C and freeze-dried to obtain a dry powder containing ca. 2.7% (w/w) of SeM among many other proteins [12]. 2.4. Liposome preparation Positively charged liposomes were prepared according to a modification of the method described by Corvo et al. [22]. Briefly, thin films were obtained by rotary evaporation of a mixture of the appropriate amounts of phosphatidylcholine, cholesterol and stearylamine in a molar ratio of 7:3:1 in chloroform, with a lipid concentration of 40 mM. The lipid films were kept under a nitrogen stream until being completely dried. One film was then dispersed with 30 mL of 5 mg/ml enzymatic extract solution and the other with 0,145 NaCl/10 mM citrate buffer, pH 5.6. Then, the same buffer was added to the lyophilised powder in a volume amounting to 1/10 of that of the original dispersion. This hydration step lasted 30 min and, subsequently, citrate buffer was added to adjust the volume to the starting amount. Obtained liposomes were extruded sequentially through polycarbonate filters with pore sizes ranging from 0.8, 0.4 to 0.2 ␮m. Non-encapsulated protein was separated from the liposome by dilution and ultracentrifugation at 300,000 × g for 180 min at 10 ◦ C in a Beckman L8–60 M ultracentrifuge. Finally, liposomes were resuspended in a 0.145 M NaCl/10 mM citrate buffer pH 5.6. 2.5. Preparation of CS–TPP nanoparticles Nanoparticles were prepared by ionotropic gelation of chitosan with TPP, according to a slight modification of a method described elsewhere [23]. Chitosan and TPP were dissolved in ultrapurified water in order to obtain chitosan/TPP weight ratio of 5/1. Nanoparticles were formed by the addition, under magnetic agitation, of TPP to the chitosan solution. For the preparation of the antigen-containing nanoparticles, the S. equi enzymatic extract was incubated with a TPP solution of a concentration suitable to maintain the volume ratios of the blank nanoparticle formulations. Non-encapsulated protein was separated by centrifugation of the samples at 29,000 × g for 30 min. 2.6. Particle characterization Mean particle size, polydispersity index (P.I.) and zeta-potential of liposomes and chitosan nanoparticles were assessed by photon correlation spectroscopy and laser Doppler anemometry, using a Zetasizer Nano-S and Zetasizer 2000 (Malvern Instruments, UK), respectively. For particle size measurements, the samples were diluted in 0.22 ␮m filtered purified water or appropriate buffer

L. Figueiredo et al. / Vaccine 30 (2012) 6551–6558

and measured. For the zeta potential measurements, samples were diluted in 0.22 ␮m filtered purified water or buffer and measured in automatic mode. Results were expressed as mean ± standard deviation (S.D.). 2.7. Phospholipid determination Phospholipid concentration in liposomal formulations was determined based on the colorimetric quantification of inorganic phosphate as described by Rouser and Yamamoto [24]. 2.8. Protein determination Protein quantification incorporated in liposomes was determined by a modified Lowry method with prior disruption of liposomes with Triton X-100 and sodium dodecylsulphate [25]. Total protein content incorporated in nanoparticles was quantified indirectly using the BCA protein assay (Pierce, USA) to determine the free protein concentration in the supernatants. Calibration curves were made with corresponding solutions of blank nanoparticles. All samples were measured in triplicate. Encapsulation efficiency (E.E.) for liposome formulation was calculated as follows: Encapsulation efficiency(%) =

(Incorporated protein) × 100 (Total protein)

The encapsulation efficiency of the incorporated protein in nanoparticles was determined by difference as follows: Encapsulation efficiency(%) =

(Total protein) − (Free protein) × 100 (Free protein)

2.9. Structural integrity of S. equi antigens The integrity of protein antigens before and after encapsulation in liposomes and nanoparticles was assessed by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) by comparison with molecular weight reference marker (molecular weight 6.0–181 kDa, Invitrogen, UK) and by Western Blot as previously described [9,10] with some modification namely the primary antibody used was horse serum anti-Streptococcus equi and the secondary antibody used was mouse anti-horse IgG conjugated to phosphatase alkaline (Sigma Aldrich Co., UK). Samples were loaded onto NuPAGE 10% polyacrylamide gel (Invitrogen, UK). Proteins were visualised by Coomassie blue staining (SimplyBlueTM SafeStain solution, Invitrogen, USA). 2.10. Liposome stability studies The stability of liposomes was assessed by incubating the liposomal suspensions at 4 ◦ C for 72 h and at 22 ◦ C for 24 h. Briefly, 0.3 mL samples were collected in triplicate at different time intervals t = 0, 18, 24, 48 and 72 h for the 4 ◦ C study, and at t = 0; 2.5; 5 e 24 h for the 22 ◦ C study. Non-encapsulated protein was separated by size exclusion chromatography (Sephadex G-200). Liposomal stability was evaluated for protein/lipid ratio and for mean size. Antigen structural stability was assessed by SDS-PAGE stained with silver nitrate (Sigma–Aldrich, Spain). 2.11. Immunisation studies Four groups of female BALB/c mice (25 g; n 5/group) were immunised by intranasal (i.n.) route on day 1 and boosted on day 21, using a micropipette tip to administer 50 ␮l of sample (25 ␮l per nostril) containing S. equi antigen equivalent to 10 ␮g of SeM. The formulations were delivered slowly onto nostrils, so that the mice could

6553

inhale. Controls consisted of empty liposomes and empty nanoparticles. All formulations were freshly and aseptically prepared in a biological safety cabinet, immediately prior dosing. Blood samples were collected from the tail vein after 2, 4, 6, 8, 10, 12, 14 and 16 weeks of immunisation. Sera were separated by centrifugation (18,000 × g, 5 min at 4 ◦ C, Allegra 64R, Beckman, USA) and stored at −20 ◦ C until tested by antigen specific enzyme-linked immunosorbent assay (ELISA) for IgG, IgG subclass 1 (IgG1) and IgG subclass 2a (IgG2a). In order to examine the mucosal immune response, lungs were collected from ethically sacrificed animals and IgA presence assessed by ELISA. Previous publications have repeatedly reported the consistently low immune response to the free antigen by the same administration route and using the same experimental design [9,10,12,13]. Taking into account that the new directives of European legislation and regulations covering animal experimentation (Directive 2010/63/EU that replaces the previous Directive 86/609/EEC), state that the implementation of principles of replacement, reduction and refinement in those projects (3Rs) must be rigorously applied to the animal experimentation, as also the evaluation of the ethics committee, it was decided not to include the group of treated with free antigen. Results herein obtained will be compared to those previously published. 2.12. Quantification of antigen-specific IgA, IgG and subtypes by ELISA The antibody responses (IgG, IgG1 and IgG2a) to S. equi cell extracted proteins were determined based on a previously reported method [8]. Plates (Microlon® , High binding flat bottom plates, Greiner, Germany) were coated overnight with 5.0 ␮g/mL protein enzymatic extract in 100 mM sodium carbonate buffer (pH 9.6), washed and afterwards blocked with a 5% (w/v) skimmed milk powder (Merck KGaA, Germany) dissolved in 10 mM PBS at pH 7.4 containing 0.05% (v/v) of Tween® 20 (PBST; Sigma–Aldrich, Spain). Plates were again washed and sera were tested by serial two-fold dilutions. Sera obtained from naive mice were used as a control. Horseradish peroxidase conjugate goat anti-mouse IgG, (Sigma, Pool Dorset, UK), IgG1 and IgG2a (Serotec, UK), diluted 1:1000, were applied as secondary antibody. Finally, the substrate OPD (SigmaFASTTM OPD Kit, Sigma–Aldrich, Spain) was used to develop the plates, the colour reaction was stopped after 15 min, by adding 2.5 N H2 SO4 to the wells, and absorbance was read at 490 nm. The titres reported are the reciprocal of serum dilutions that gave an optical density 5% higher than the strongest negative control group, i.e. empty nanoparticles and liposomes respectively. For analysis of lung IgA, the ELISA method was essentially the same, with slight modifications. The lungs were homogenised in buffer 0.9% sodium chloride with protease inhibitor PMSF 1 mM and 0.5% Tween® 20. The homogenization was done with ultrasound probe for 3 times of 5 min intermittent pulsed for 30 s each in an ice bath. The homogenised tissues were centrifuged at 20,000 × g for 30 min at 4 ◦ C (Allegra 64R, Beckman, USA) and the supernatant was frozen at −80 ◦ C until freeze–drying. After freeze–drying, proteins were reconstituted with 500 ␮l of sterile water and directly added, in triplicates, to the plate wells. The horseradish peroxidase conjugate goat anti mouse IgA (1:2000; Serotec, UK) was used to detect specific IgA antibody, and OPD substrate was then added as mentioned above. The mean OD was determined for each treatment group and directly used to compare mucosal response. 2.13. Splenocyte culture studies Spleens were aseptically removed 16 weeks after mice immunisation and cell homogenisates were prepared as previously reported [10,13]. Supernatants obtained 72 h after splenocyte

6554

L. Figueiredo et al. / Vaccine 30 (2012) 6551–6558

without stimulation and with stimulation with 5.0 ␮g/mL of SeM protein enzymatic extract were used for IFN-␥, IL-2, IL-4 and IL-6 determination by ELISA, using a commercially available RayBio® kit (RayBiotech, Inc, USA). The concentrations, expressed as pg/mL, were determined by reference to different cytokines standard curves. 2.14. Statistical analysis Results are expressed as mean values ± standard deviation (S.D.). Statistical analysis was performed on the data obtained in the in vivo studies by the Mann-Whitney test, with significance set at P-values <0.05. 3. Results and discussion 3.1. Characterization of S. equi antigen-loaded liposomes and chitosan nanoparticles Particulate adjuvants are emerging as a real alternative in order to amplify the local and systemic immune response to poor immunogenic antigens. Numerous polymers, particularly biodegradable polymers such as PLA and PLGA albumin, chitosan and alginate have been used in the preparation of nanoparticles for nasal immunisation [26]. Chitosan in particular has also been used in surface modification of PLA nanoparticles for nasal immunisation studies with promising results [9,10]. On the other hand, in vivo studies using negatively charged liposomes modified with chitosan confirmed the potential of this polymer in the nasal immunity stimulation process [27,28]. Although liposomes have been described as potent stimulators of mucosal immune response, immunisation studies with S. equi antigens encapsulated in liposomes have never been reported. In the present work, positively charged liposomes were prepared and the elicited immune response was compared to that obtained with the chitosan nanoparticulate vaccine. The encapsulation efficiencies were determined being 13 ± 3% (w/w) for the liposomes and 53 ± 6% (w/w) for chitosan nanoparticles (Table 1). The low incorporation efficiencies may be related with the nature of the encapsulated proteins. In fact, being a mixture of proteins each one has different physicochemical properties reflecting in a distinct behaviour during particle formation and encapsulation process with different particle localization, as previously reported [9,10,12,13]. Each protein will interact in a different manner with the different matrixes, explaining the differences observed between both particulate systems. The antigen-loaded liposomes had mean size of 0.22 ± 0.01 ␮m presenting a narrow size distribution with a P.I. less than 0.242, while chitosan nanoparticles show a mean size of 0.17 ± 0.01 ␮m, with a P.I. below 0.362. Particle size is a major aspect where particle trafficking to lymph nodes, antigen uptake and processing by APCs are concerned, although its real role is steel dubious [15]. Several i.n. immunisation studies have been carried out with liposomes and nanoparticles but results do not present consistent size-wise influence on the elicited immune response. If it is generally accepted that size will influence the uptake process, that 20–200 nm particles

are usually taken up by receptor-mediated endocytosis eliciting a cellular biased response, particles of 0.2–5.0 ␮m are predominantly phagocytosed eliciting a humoral response, no boundaries have yet been established for the uptake by NALT M cells [7,29,30]. Good systemic and mucosal antibody titres were reported for particulate formulations exceeding the 0.5 ␮m threshold being difficult to define the optimal particle size, suggesting that particle composition may also play an important role [28,31]. In the present study both formulations present a mean size below 0.5 ␮m thus being expected a straightforward uptake by APCs as described in earlier studies [32,33]. Such dimensions are also favourable to the uptake by nasalassociated lymphoid tissue (NALT) cells where they are taken up by phagocytic cells [34]. On the other hand the positive surface charge (+12 ± 4 mV for liposomes and +23 ± 8 mV for the chitosan nanoparticles) will probably influence particle uptake by APCs, as a result of the ionic interactions established between positively charged particles and the negative charge of cell membrane, resulting in enhanced uptake by the cells through adsorptive endocytosis [18]. Previous studies demonstrated an improvement of the antigen presentation as well as stronger antigen-specific immune responses for cationic liposomes as opposed to neutral and negative ones [35,36]. 3.2. Integrity of S. equi antigens Encapsulation of protein molecules in liposomes or nanoparticles while maintaining their structural integrity and preserving their bioactivity has been a major challenge in protein formulation. In order to guarantee effective immunisation it is essential that S. equi antigens retain their integrity after incorporation. Accordingly, evaluation of structural integrity was performed by SDS-PAGE and Western Blot (Fig. 1). Protein patterns of migration observed for both native and encapsulated S. equi antigens are identical presenting no additional bands, which excludes the presence of aggregates or fragments. The presence of SeM protein (58 kDa) in its native form before and after encapsulation demonstrates that remains undamaged after the enzymatic extraction process. 3.3. Stability of liposomal formulations Stability studies at 4 ◦ C/72 h and 22 ◦ C/24 h were carried out with the antigen-containing liposomes so that commonly described phenomena, such as antigen leakage and liposome fusion, which could affect vaccine efficacy, could be ruled out. It was found that the protein/lipid concentration ratio remained stable (11.2 ± 2.4 ␮g/␮mol; 1.1 ± 0.3 ␮g/␮mol) respectively at 4 ◦ C and 22 ◦ C during the observation period. A non-significant difference (P = 0.114) in vesicle size was observed confirming that liposomes remained stable at 4 ◦ C (0.18 ± 0.01 ␮m; P.I. <0.125) and at 22 ◦ C (0.19 ± 0.01 ␮m; P.I. <0.139). Structural integrity of S. equi antigens was also assessed by SDS-PAGE. The electroforetic profiles show that protein integrity is maintained revealing that S. equi antigens remain stable when stored under these conditions (Fig. 2). Therefore at the time of administration, S. equi antigens will be largely associated with

Table 1 Characteristics of antigen-loaded liposomes and chitosan nanoparticles (mean ± S.D.; n = 3).

Liposomes Nanoparticles

E.E. (% w/w)

L.C. mg/mga

Zeta potential (mV)b

Mean size (␮m)

P.I.

13 ± 3 53 ± 6

0.011 ± 0.002 3.94 ± 1

+12 ± 4 +23 ± 8

0.22 ± 0.01 0.17 ± 0.01

<0.242 <0.362

P.I., polydispersity index; E.E., encapsulation efficiency; L.C., loading capacity. a Liposomal L.C. expressed as mg of protein per mg of total phospholipid and nanoparticles L.C. expressed as mg of protein per mg of chitosan. b Liposomal formulation diluted in saline citrate buffer (pH 5.6) and nanoparticles diluted in ultra-purified water.

L. Figueiredo et al. / Vaccine 30 (2012) 6551–6558

6555

Fig. 1. SDS-PAGE and Western Blot analysisof S. equi enzymatic extract before and after incorporation in nanoparticles and liposomes. Lanes in gel: (1) Molecular weight markers; (2) Free S. equi enzymatic extract; (3) S. equi enzymatic extract incorporated in nanoparticles; (4) S. equi enzymatic extract incorporated in liposomes; Lanes in Western Blot: (5) Molecular weight markers; (6) Free S. equi enzymatic extract; (7) S. equi enzymatic extract incorporated in nanoparticles; (8) S. equi enzymatic extract incorporated in liposomes.

liposomes, which are expected to be critical for their success as a nasal vaccine [37]. 3.4. Systemic IgG antibody immune response S. equi is a one host adapted pathogen and protection in mice does not necessarily correlate with protection in the horse [14]. Nevertheless, the mouse model has been widely used to study the immune response to potential vaccines and to test new antigens [11,38–41]. Two i.n. administrations were conducted in days 1 and 21 to confirm the immunogenicity of the positively charged liposomes and chitosan nanoparticles loaded with S. equi antigens (enzymatic extracts). As in our previous reports, animals were immunised with free protein producing a low and reproducible response with titre values ranging from 100 to1000 (week 2), ≈5000 (week 4) and 1000 to 5000 (week 12) [9,10]. Due to this fact, these

Fig. 2. SDS-PAGE of S. equi enzymatic extract encapsulated in liposomes stored at 22 ◦ C/24 h (A) and at 4 ◦ C/72 h (B). Lanes in gel A: (1) Molecular weight markers; (2) t = 0; (3) t = 2.5 h; (4) t = 5 h; (5) t = 24 h. Lanes in gel B: (1) t = 0 h; (2) t = 1 h; (3) t = 18 h; (4) t = 24 h; (5) t = 48 h; (6) t = 72 h.

Fig. 3. Serum anti-S. equi specific IgG (A), IgG1 (B) and IgG2a (C) titres after intranasal immunisation of female BALB/c mice with liposomes and nanoparticles containing S. equi antigens (mean ± S.D.; n = 5).

values were used to discuss those herein obtained. On the other hand, no S. equi-specific IgG antibodies were detected in serum of animals vaccinated with plain particles, which confirms the potential of positively charged particulate carriers as immunological adjuvants, as they have the ability to improve S. equi antigen immunogenicity [9,10]. Two weeks after vaccination, animals treated with both formulations started to produce serum specific anti-S. equi IgG (Fig. 3). The antibody titres thus produced are clearly higher (ca. 2–3×) than those elicited by the free antigen when compared to our previously reported data, in which the same experimental conditions were used [9,10]. The maximum IgG levels were reached at 8 weeks for both formulations. After this time-point a general decrease in serum IgG and IgG2a titres was detected, although the decrease in IgG2a immune response was maintained in mice immunised with chitosan nanoparticles (P = 0.01), confirming the ability of chitosan to generate prolonged IgG responses to S. equi antigens [9,10]. Taking into account that both particulate carriers are positively charged, the differences observed are probably related to the aforementioned properties of chitosan nanoparticles, such as mucoadhesion that increases the nasal residence time of the antigen and improves the resulting immune response [19].

6556

L. Figueiredo et al. / Vaccine 30 (2012) 6551–6558

3.5. IgG subtype profiling Apparently the generation of a dominant Th1 profile as a consequence of animal vaccination may facilitate the eradication of the microorganism and consequently contribute for a successful prevention against strangles in equids [4,14]. In the present study two intranasal vaccines resulted in strong IgG1 and IgG2a immune responses in the mouse model, confirming that i.n. administered positively charged particulate vaccines act as potent adjuvants (Fig. 3). Non-significant differences (P = 0.306) were found throughout the experiment for the IgG2a/IgG1 ratio elicited by both particulate vaccines. Nevertheless, it was noticeably more balanced for the nanoparticles (0.500) than for the liposomes (0.208) at the end of the trial period, similar to that previously obtained with chitosan–PCL nanoparticles [10]. On the other hand, the liposomal formulation induced high IgG1 levels essentially after boosting, whereas chitosan nanoparticles induced high titres of IgG2a in the same period. Differences in physicochemical characteristics and in vivo stability as well as the mucoadhesive and penetration enhancing activity of chitosan were expected to cause a markedly different effect after i.n. immunisation when compared to liposomes. Moreover, previous studies have demonstrated that neutral and negative liposomal formulations present better results concerning the immunological response suggesting other factors beyond surface charge may be involved in immune response stimulation [17]. In fact, non-significant differences (P = 0.506) were found in the IgG immune responses elicited by the two formulations (Fig. 3) suggesting the immunostimulating effect may be due to prolonged residence of the antigen at the nasal mucosa and improved uptake of positively charged particulates by M-cells and APC. However, the higher (P = 0.005) IgG2a levels that were observed from week 4 in the group treated with the chitosan nanoparticles may be related to a better capacity of chitosan to efficiently enhance antigen uptake by the NALT besides its natural mucoadhesive properties. In theory positively charged liposomes will be only expect to act through electrostatic interactions between their positively charged surface and the negatively charged mucosal cells. In this way, liposomes attach to the mucosa prolonging the presentation of the antigen in nasal cavity raising the antigen chances of being taken up in nasal tissues. The stronger mucoadhesiveness of the chitosan nanoparticles is probably related with a better uptake at the nasal epithelium and NALT and subsequent access of the vaccine to sub-mucosal lymphoid tissues. In spite of the relative efficacy, i.n. administration of liposomes and chitosan nanoparticles associated to S. equi extract resulted in a consistent immune response emerging as promising agents for the cellular immune response stimulation.

Fig. 4. Secretory IgA (sIgA) levels in lungs homogenates of mice intranasally immunised with S. equi antigens encapsulated in chitosan nanoparticles and liposomes. NP – chitosan nanoparticles; LP – liposomes; PBS – phosphate buffered saline (mean ± S.D.; n = 5).

which is somewhat atypical, once these values are significantly different (P = 0.001) from those obtained with PBS only. Better results obtained with chitosan may be related with its absorption enhancer properties, which might have significantly increased particles uptake by M cells in the NALT and subsequently delivered the antigen to the peripheral lymphoid tissues. Chitosan being a bioadhesive polymer may resist the mucociliary clearance in the nose more efficiently than the positively charged liposomes. In fact, positive charge is not mandatory in order to take place mucoadhesion. Neutral particles have already been described as mucoadhesive, so ionic interaction may not be the only responsible for the interaction chitosan–mucosal cells. Interpenetration phenomena between the mucosal layer and the mucoadhesive polymer may well be involved in the interaction between these two components, relying mostly on the hydrogen bonds and Van der Walls interactions [42,43]. Furthermore, chitosan presents imunostimulating activity, leading to the macrophage accumulation and activation promoting resistance to infection and stimulating the cellular immune response [27]. It is important to notice that the application method is not optimal once the gap between the nostril size and the applied volume will originate losses. On the other side, part of the dose may be swollen which translates in an oral administration or may even get to the lungs, in cases where the administrated volume is enough. In order rule out oral administration it ought to be performed an assessment of IgA levels on the intestines. In this way it is safe to say that chitosan nanoparticles efficiently stimulated IgA-secreting cells of the NALT and/or draining lymph nodes in addition to their effective systemic stimulation. 3.7. Splenocytes response

3.6. Local IgA immune responses Intranasal vaccination against strangles should result not only in systemic protection but also and most importantly in mucosal immunisation. In spite of the immune response to S. equi being associated with high levels of specific anti-SeM systemic antibodies, these are not protective because a well-established local immune response plays a very important role in animal immunisation [7]. The local production of secretory IgA is believed to be very important, establishing a first defence line at the mucosal surface blocking the S. equi adhesion [3]. In order to assess the success of the mucosal immunisation, local sIgA levels present in lungs were evaluated. Results indicate a more successful local immunisation by the nanoparticle formulation, the only one presenting sIgA levels statistically higher (P = 0.0004) than those elicited by the placebo formulation (Fig. 4). Liposomes with S. equi antigens induced sIgA levels similar to those elicited by empty liposomes,

Antigen presentation by class I MHC to CD8+ T cell molecules will stimulate cellular immune response. The activation, growing and differentiation of cytotoxic T cells is dependent of cytokines released from Th cells, so their expression levels will be helpful to decode which response type is being stimulated. Helper T cells play an essential role in lymphocyte activation, growing and differentiation being therefore very important to verify any alteration at their expression levels. Helper T cells divided into Th1 and Th2 will express different cytokines, IL-2, IFN-␥ and IL-4, IL-6 respectively. However, there are some deficiencies in knowledge of the protective cell-mediated immune responses in the horse, although it appears an important component of acquired resistance to strangles and must be extensively studied [4,44]. Based on previous results obtained with the mouse model, we expected these vaccine formulations, through particulate system antigen presentation, would facilitate the intra-cytoplasmatic

L. Figueiredo et al. / Vaccine 30 (2012) 6551–6558

6557

Fig. 5. Concentrations of cytokines (IL-2, IL-4, IL-6 and IFN-␥) in supernatants of splenocytesafter 72 h culture, without stimulation (empty columns)and stimulation with S. equi antigens (filled columns). (A) liposomes; (B) nanoparticles(mean ± S.D.; n = 5).

presence of antigens, activating the endogenous pathway thus stimulating the cellular immune response leading to an equilibrated immune response [9,10,12,13]. Liposomes have also been reported as cytokine stimulators, which signified a shift from a humoral to a mixed type immune response [45,46]. The same correlates with nanoparticles and previous studies performed by our group demonstrated that polymeric nanoparticles loaded with S. equi antigens possess immunostimulatory properties being able of stimulate the humoral immune response and also substantial cellular immune response when compared with soluble antigen [9]. After splenocyte incubation with S. equi antigens, cytokine quantification revealed and effective stimulation in the groups treated with both particulate formulations (Fig. 5). Significant increases of IL-2 (P = 0.0122), IL-4 (P = 0.0002), IL-6 (P = 0.02) and IFN-␥ (P = 0.0104) were obtained with the liposomal formulation compared to those detected in non-stimulated splenocytes. Similarly, the group treated with antigen encapsulated in chitosan nanoparticles showed a general increase in all cytokine levels, compared to those detected in non-stimulated splenocytes, i.e. IL-2 (P < 0.0001), IL-4 (P < 0.0001), IL-6 (P < 0.0001) and IFN-␥ (P < 0.0001). Furthermore, IFN-␥ and IL-2 levels are substantially elevated when comparing liposomes and nanoparticles results with previous findings for soluble antigen [10]. These results confirm that both formulations successfully stimulated cellular immune response, meeting the previous results that pointed out a mixed immune response based on the IgG2a/IgG1 ratio. Positively charged liposomes are prone to this type of occurrence in opposition to neutral and negatively charged liposomes [47]. However, chitosan characteristics may play its favour by prolonging nanoparticles containing S. equi antigens residence time and facilitating contact with APCs.

4. Conclusions The results presented substantiate particulate systems as a promising technological platform for i.n. vaccination. S. equi antigens were successfully associated with liposomes and chitosan nanoparticles with no compromise of their molecular structure, resulting in a successful stimulation of the humoral and cellular immune responses. Both formulations share the ability to induce Th1-mediated immune responses characterised by IFN-␥ production and high IgG2a antibody titres, as well as a Th2 immune response characterised mainly by IL-4 production and IgG1 antibodies. Moreover mucosal stimulation was confirmed by increased sIgA levels, being chitosan nanoparticles more successful in this achievement, probably due to their mucoadhesive properties. Altogether these data, obtained with the mouse model, support particulate systems as antigen carriers for i.n. vaccination against S. equi.

Acknowledgements This work was supported by Fundac¸ão para a Ciência e Tecnologia (Portugal) and FEDER (POCI/BIO/59147/2004; PPCDT/BIO/59147/2004 and the strategic project PEstOE/SAU/UI4013/2011). References [1] Harrington DJ, Sutcliffe IC, Chanter N. The molecular basis of Streptococcus equi infection and disease. Microbes Infect 2002;4:501–10. [2] Taylor S, Wilson WD. Streptococcus equi subsp. equi (Strangles) infection. Clin Tech Equine Pract 2006;5:211–7. [3] Waller AS, Jolley KA. Getting a grip on strangles: recent progress towards improved diagnostics and vaccines. Vet J 2007;173:492–501. [4] Timoney JF, Qin A, Muthupalani S, Artiushin S. Vaccine potential of novel surface exposed and secreted proteins of Streptococcus equi. Vaccine 2007;25:5583–90. [5] Timoney JF. Equine strangles: 1999. AAEP Proc 1999;45:31–7. [6] Vicente S, Prego C, Csaba N, Alonso MJ. From single-dose vaccine delivery systems to nanovaccines. J Drug Del Sci Tech 2010;20:267–76. [7] Heurtault B, Frich B, Pons F. Liposomes as delivery systems for nasal vaccination: strategies and outcomes. Expert Opin Drug Deliv 2010;7: 829–44. [8] Azevedo AF, Galhardas J, Cunha A, Cruz P, Gonc¸alves LMD, Almeida AJ. Microencapsulation of Streptococcus equi antigens in biodegradable microspheres and preliminary immunisation studies. Eur J Pharm Biopharm 2006;64: 131–7. [9] Florindo HF, Pandit S, Alpar HO, Almeida AJ. New approach on the development of a mucosal vaccine against strangles: systemic and mucosal immune responses in a mouse model. Vaccine 2009;27:1230–41. [10] Florindo HF, Pandit S, Lacerda L, Alpar HO, Almeida AJ. The enhancement of the immune response against S. equi antigens through the intranasal administration of poly-␧-caprolactone-based nanoparticles. Biomaterials 2009;30:879–91. [11] Guss B, Flock M, Frykberg L, Waller AS, Robinson C, Smith KC, et al. Getting to grips with strangles: an effective multi-component recombinant vaccine for the protection of horses from Streptococcus equi infection. PLoS Pathog 2009;5:e1000584. [12] Florindo HF, Pandit S, Gonc¸alves LMD, Alpar HO, Almeida AJ. Streptococcus equi antigens adsorbed onto surface modified poly-␧-caprolactone microspheres induce humoral and cellular specific immune responses. Vaccine 2008;26:4168–77. [13] Florindo HF, Pandit S, Gonc¸alves LMD, Videira M, Alpar O, Almeida AJ. Antibody and cytokine-associated immune responses to S. equi antigens entrapped in PLA nanospheres. Biomaterials 2009;30:5161–989. [14] Waller AS, Paillot R, Timoney JF. Streptococcus equi: a pathogen restricted to one host. J Med Microbiol 2011;60:1231–40. [15] Henriksen-Lacey M, Korsholm KS, Andersen P, Perrie Y, Christensen D. Liposomal vaccine delivery systems. Expert Opin Drug Deliv 2011;8:505–19. [16] Vangasseri DP, Cui Z, Chen W, Hokey DA, Falo Jr LD, Huang L. Immunostimulation of dendritic cells by cationic liposomes. Mol Membr Biol 2006;23:385–95. [17] Joseph A, Itskovitz-Cooper N, Samira S, Flasterstein O, Eliyahu H, Simberg D, et al. A new intranasal influenza vaccine based on a novel polycationic lipidceramide carbamoyl-spermine (CCS) I. Immunogenicity and efficacy studies in mice. Vaccine 2006;24:3990–4006. [18] Guy B, Pascal N, Franc¸on A, Bonnin A, Gimenez S, Lafay-Vialon E, et al. Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine. Vaccine 2001;19:1794–805. [19] Arca HC¸, Günbeyaz M, S¸enel S. Chitosan-based systems for the delivery of vaccine antigens. Expert Rev Vaccines 2009;8:937–53. [20] Esmaeili F, Heuking S, Junginger HE, Borchard G. Progress in chitosan-based vaccine delivery systems. J Drug Del Sci Tech 2010;20:53–61. [21] Amin M, Jaafari MR, Tafaghodi M. Impact of chitosan coating of anionic liposomes on clearance rate, mucosal and systemic immune responses following nasal administration in rabbits. Colloids Surf B: Biointerfaces 2009;74:225–9.

6558

L. Figueiredo et al. / Vaccine 30 (2012) 6551–6558

[22] Corvo ML, Cruz ME, Crommelin DJA, Storm G. Superoxide dismutase entrapped in long-circulating liposomes: formulation design and therapeutic activity in rat adjuvant arthritis. Biochim Biophys Acta 2002;1564:227–36. [23] Cadete A, Figueiredo L, Lopes R, Calado CC, Almeida AJ, Gonc¸alves LM. Development and characterization of a new plasmid delivery system based on chitosan–sodium deoxycholate nanoparticles. Eur J Pharm Sci 2011;45:451–8. [24] Rouser G, Yamamoto A. Two-dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots values obtained in quadruplicate determinations of different amounts of phosphorus. Lipids 1969;5:494–6. [25] Wang C, Smith RL. Lowry determination of protein in the presence of Triton X-100. Anal Biochem 1975;63:414–7. [26] Oyewumi MO, Kumar A, Cui Z. Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses. Expert Rev Vaccines 2010;9:1095–107. [27] Khatri K, Goyal AK, Gupta PN, Mishra N, Mehta A, Vyas SP. Surface modified liposomes for nasal delivery of DNA vaccine. Vaccine 2008;26:2225–33. [28] Chiou CJ, Tseng LP, Deng MC, Jiang PR, Tasi SL, Chung TW, et al. Mucoadhesive liposomes for intranasal immunization with an avian influenza virus vaccine in chickens. Biomaterials 2009;30:5862–8. [29] Sharma S, Mukkur TK, Benson HA, Chen Y. Pharmaceutical aspects of intranasal delivery of vaccines using particulate systems. J Pharm Sci 2009;98:812–43. [30] Bachmann M, Jennings G. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol 2010;10:787–96. [31] Patel GB, Zhou H, Ponce A, Chen W. Mucosal and systemic immune responses by intranasal immunization using archaeal lipid-adjuvanted vaccines. Vaccine 2007;25:8622–36. [32] Nixon DF, Hioe C, Chen PD, Bian Z, Kuebler P, Li ML, et al. Synthetic peptides entrapped in microparticles can elicit cytotoxic T cell activity. Vaccine 1996;14:1523–30. [33] Huang Y, Donovan M. Large molecule and particulate uptake in the nasal cavity: the effect of size on nasal absorption. Adv Drug Deliv Rev 1998;29:147–55. [34] Csaba N, Garcia-Fuentes M, Alonso MJ. Nanoparticles for nasal vaccination. Adv Drug Deliv Rev 2009;61:140–57. [35] Thiele L, Rothen-Rutishauser B, Jilek S, Wunderli-Allenspach H, Merkle HP, Walter E. Evaluation of particle uptake in human blood monocyte-derived cells in vitro. Does phagocytosis activity of dendritic cells measure up with macrophages? J Control Release 2001;7:545–55.

[36] Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S. Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine 2004;22:1903–13. [37] Shahiwala A, Vyas TK, Amiji MM. Nanocarriers for systemic and mucosal vaccine delivery. Recent Pat Drug Deliv Formul 2007;1:1–9. [38] Meehan M, Nowlan P, Owen P. Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi. Microbiology 1998;144: 993–1003. [39] Chanter N, Ward CL, Talbot NC, Flanagan JA, Binns M, Houghton SB, et al. Recombinant hyaluronate associated protein as a protective immunogen against Streptococcus equi and Streptococcus zooepidemicus challenge in mice. Microb Pathogenesis 1999;27:133–43. [40] Flock M, Jacobsson K, Frykberg L, Franklin A, Guss B, Flock JI. Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horses. Infect Immun 2004;72:3228–36. [41] Flock M, Karlstrom A, Lannergard J, Guss B, Flock JI. Protective effect of vaccination with recombinant proteins from Streptococcus equi subspecies equi in a strangles model in the mouse. Vaccine 2006;24:4144–51. [42] Andrews GP, Laverty TP, Jones DS. Mucoadhesive polymeric platforms for controlled drug delivery. Eur J Pharm Biopharm 2009;71:505–18. [43] Sriamornsak P, Thongborisute J, Takeuchi H. Liposome-based mucoadhesive formulations for oral delivery of macromolecules. In: Bernkop-Schnürch A, editor. Oral Delivery of Macromolecular Drugs. New York: Springer; 2009. p. 169–93. [44] Jacobs AA, Goonerts D, Nuijten PJM, Hartford OM, Foster TJ. Investigations towards an efficacious and safe strangles vaccine: submucosal vaccination with a live attenuated Streptococcus equi. Vet Rec 2000;147:563–7. [45] Brgles M, Habjanec L, Halassy B, Tomasic J. Liposome fusogenicity and entrapment efficiency of antigen determine the Th1/Th2 bias of antigen-specic immune response. Vaccine 2009;27:5435–42. [46] Baca-Estrada ME, Foldvari M, Snider M, Harding K, Kournikakis B, Babiuk LA, et al. Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses. Vaccine 2000;18:2203–11. [47] Nakanishi T, Kunisawa J, Hayashi A, Tsutsumi Y, Kubo K, Nakagawa S, et al. Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins. J Control Release 1999;61:233–40.