Mucosal Immunization with PsaA Protein, Using ... - Wiley Online Library

EXPERIMENTAL IMMUNOLOGY doi: 10.1111/sji.12267 ..................................................................................................................................................................

Mucosal Immunization with PsaA Protein, Using Chitosan as a Delivery System, Increases Protection Against Acute Otitis Media and Invasive Infection by Streptococcus pneumoniae J.-H. Xu*, W.-J. Dai*, B. Chen* & X.-Y. Fan†

Abstract *Department of Otology and Skull Base Surgery, Eye Ear Nose & Throat Hospital of Fudan University, Key Laboratory of Health Ministry for Hearing Medicine, Shanghai, China; and †Shanghai Public Health Clinical Center Affiliated to Fudan University, Shanghai, China

Received 22 August 2014; Accepted in revised form 11 December 2014 Correspondence to: W.-j. Dai, Department of Otology and Skull Base Surgery, Eye Ear Nose & Throat Hospital of Fudan University, No. 83 Fenyang Road, Shanghai 200031, China. E-mail: [email protected]

As infection with Streptococcus pneumoniae (mainly via the mucosal route) is a leading cause of acute otitis media, sinus and bacterial pneumonia, the mucosal immunity plays an important role in the prevention of pneumococcal diseases. Therefore, intranasal vaccination may be an effective immunization strategy, but requires appropriate mucosal vaccine delivery systems. In this work, chitosan was used as a mucosal delivery system to form chitosan–PsaA nanoparticles based on ionotropic gelation methods and used to immunize BALB/c mice intranasally. Compared to mice immunized with naked PsaA, levels of IFN-c, IL-17A and IL4 in spleen lymphocytes, the systemic (IgG in serum) and mucosal (IgA in mucosal lavage) specific antibodies were enhanced significantly in mice inoculated with chitosan–PsaA. Furthermore, increased protection against acute otitis media following middle ear challenge with pneumococcus serotype 14, and improved survival following intraperitoneal challenge with pneumococcus serotype 3 or serotype 14, was found in the mice immunized with chitosan– PsaA nanoparticles. Thus, intranasal immunization with chitosan–PsaA can successfully induce mucosal and systemic immune responses and increase protection against pneumococcal acute otitis media and invasive infections. Hence, intranasal immunization with PsaA protein, based on chitosan as a delivery system, is an efficient immunization strategy for preventing pneumococcal infections.

Introduction Acute otitis media (AOM) is one of the most prevalent illnesses in children. Approximately three quarters of children experience at least one episode of AOM by the 3 years of age [1]. Recurrent AOM affects 10–20% of children by the age of 1 year [2]. Streptococcus pneumoniae (pneumococcus) is the most common pathogen to induce AOM in children, and the condition is primarily treated with antibiotics. However, the high prevalence of antibiotic-resistant pathogens, such as penicillin-resistant pneumococcus complicates AOM management [3]. Therefore, prevention of pneumococcal AOM has become increasingly important. To this end, vaccines have been under investigation. The currently available polysaccharide vaccine is unable to induce protective effects in infants, while the currently available 7-valent pneumococcal conjugate vaccine induces

Ó 2015 John Wiley & Sons Ltd

serotype-specific protection against colonization and invasive disease, but lower protection against AOM and pneumonia. The study from the Northern California Kaiser Permanente has shown 97% efficacy for PCV7 serotypes against invasive disease, but only 7% against all episodes of AOM [4]. In another double-blind study [5], the 7-valent pneumococcal conjugate vaccine reduced 6% episodes of AOM from any cause, 34% episodes of AOM caused by pneumococcus and 57% episodes of AOM caused by the serotypes contained in the vaccine. However, AOM caused by other serotypes of pneumococcus increased by 33%. In addition, the high cost of these vaccines limits their wide use in developing countries [6]. Therefore, an effective vaccine against all infections caused by clinically relevant pneumococcal serotypes, including AOM, is needed. A vaccine approach that uses conserved pneumococcal protein antigens can be a potentially effective means of preventing pneumococcal diseases [7] and holds the

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178 Chitosan–PsaA Particles and Pneumococcal Infection J.-h. Xu et al. .................................................................................................................................................................. promise of overcoming issues related to serotype-dependent vaccines. Pneumococcal surface adhesin A (PsaA), a lipoprotein expressed by all serotypes of pneumococcus, is one of the most promising candidates [8]. Mucosal immunization with PsaA in several studies has been shown to protect mice against pneumococcal colonization, pneumonia and septicaemia [9]. However, the ability of PsaA immunization to protect against AOM has not been reported. Pneumococcus colonizes the human nasopharynx and induces AOM via the Eustachian tube route. In addition, pneumococcus can also induce bacteremia and sepsis through the bloodstream. Consequently, vaccination strategies against pneumococcus must involve both systemic and mucosal immunity. Compared with parenteral immunization, intranasal vaccination is the most effective approach to induce both mucosal and systemic immune responses [10]. However, the permeability of epithelial cell membranes is low. Therefore, an appropriate mucosal delivery system is also required to increase mucosal absorption of the vaccine. Chitosan is a biodegradable, biocompatible, cationic polysaccharide and has benign mucoadhesive features. Moreover, it has been shown to be non-toxic both in experimental animals and in humans [11]. These advantages make chitosan an ideal mucosal delivery candidate [12]. Vaccines using chitosan as a delivery system have proven effective in several animal experiments [12–16]. In our previous study, chitosan was used as a pneumococcal DNA vector and the efficiency against pneumococcal nasopharyngeal colonization has been proved [17]. However, chitosan was used as a pneumococcal protein vaccine delivery system has not yet been studied. Thus, in this work, chitosan was used as the PsaA protein vector to form chitosan–PsaA nanoparticles. Then, the mice were intranasally inoculated with the nanoparticles. The efficiency of this approach to induce mucosal and systemic immunity and protection against AOM and invasive pneumococcal infection was assessed.

Materials and methods Bacterial strains and growth conditions. Pneumococcus (serotype 14, number 31226) was obtained from the National Center for Medical Culture Collections, and pneumococcus (serotype 3, ATCC6303) was obtained from the American Type Culture Collection (Manassas, VA, USA). Both strains were grown in Todd-Hewitt broth (Sigma, St Louis, MO, USA) added with 0.5% yeast extract (THY). Escherichia coli (E. coli) strains DH5a and BL21 used for cloning and protein expression were grown in Luria broth medium containing 50 lg/ml kanamycin. Recombinant PsaA expression in E. coli BL21. The 864-bp psaA sequence was amplified from pneumococcus genomic DNA by PCR using the forward (F) and reverse (R)

primers: F: 50 -AACGGATCCGCTAGCATGGGAAAAA AAGATACAACTTC-30 and R: 50 -CGCAAGCTTTTAT TTTGCCAATCCTTCAG-30 , containing BamHI and HindIII restriction enzyme sites, respectively. Then, the psaA gene fragment was cloned into the pGEM-T vector (Promega, Madison, WI, USA) and further subcloned into pET28a via the BamHI and HindIII sites to generate pET28a-psaA. The pET28a-psaA recombinant plasmid was transformed into E. coli BL21. The recombinant PsaA (rPsaA) protein was induced in E. coli BL21 using isopropylthiogalactoside (IPTG). The rPsaA protein fused to a sixhistidine (His) tag was analysed by Western blotting as previously we reported [17]. The rPsaA protein was purified from the soluble fraction by affinity chromatography using Ni2+-charged resin (Qiagen, Dusseldorf, Germany). Preparation of chitosan–PsaA particles. Chitosan, of about 390 kD, was purchased from Sigma. Chitosan–PsaA complexes were prepared according to a previously reported procedure [18], based on the ionotropic gelation of chitosan with tripolyphosphate (TPP, 500 ll, 0.6%, w/v) anions. In brief, PsaA (250 ll, 1.5 mg/ml) was mixed with TPP. Then, the volume was diluted to 1 ml with water. Subsequently, the chitosan (3 ml, 0.2%, w/v) was slowly added to the mixture under magnetic stirring (1000 rpm/ min) and the mixture was subsequently continually stirred for 1 h. Particles were separated by centrifugation at 16,000 9 g for 30 min. Supernatants were thrown away, and particles were resuspended in water for their immunization. Particle size, zeta potential and encapsulation efficiency of chitosan–PsaA nanoparticles. The size and zeta potential of the particles were determined by a Malvern Zetasizer (3000HSA, Malvern, UK). To assess the encapsulation efficiency of PsaA, the particles were first centrifuged at 16,000 9 g for 30 min. The naked PsaA in the supernatants was quantified using a BCA protein quantitative kit (Beyotime Institute of Biotechnology, Nantong, China). The encapsulation efficiency (EE) was calculated as follows: EE (%) = (the total amount of PsaAthe amount of naked PsaA)/total amount of PsaA 9 100. Intranasal immunization with chitosan–PsaA and sample collections. Specific pathogen-free (SPF) female BALB/c mice (SIPPR/BK Lab Animal Ltd., Shanghai, China) at 6- to 8-week-old (six animals per group) were inoculated intranasally with chitosan–PsaA (15 lg) or naked PsaA protein (15 lg) in a volume of 20 ll, without general anaesthesia. A group of control mice was inoculated with chitosan only. Mice were immunized twice a week for three consecutive weeks, according to the methods described previously [19]. Two weeks after the last booster immunization, serum, nasal washes (NW), bronchoalveolar fluids (BALF) and middle ear lavages (MEL) were collected according to the previously reported methods [17]. All animal procedures were approved by Laboratory Animals Care and Use Committees, Fudan University.

Scandinavian Journal of Immunology, 2015, 81, 177–185

182 Chitosan–PsaA Particles and Pneumococcal Infection J.-h. Xu et al. .................................................................................................................................................................. A

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Figure 4 Analysis of inflammatory responses on 3 and 7 days after middle ear challenge with pneumococcus. (A) Analysis of diameter of tympanic membrane on 3 days after middle ear challenge; (B) analysis of inflammatory cells on 3 days after middle ear challenge; (C) analysis of diameter of tympanic membrane on 7 days after middle ear challenge; (D) analysis of inflammatory cells on 7 days after middle ear challenge. (H&E staining; original magnification: 9200; bar = 100 lm).

Increased survival in the presence of invasive pneumococcal infection

The potential of intranasal vaccination with chitosan– PsaA to enhance survival from invasive pneumococcal infection was investigated. BALB/c mice were challenged intraperitoneally with 7 9 102 CFU/mouse of pneumococcus serotype 3 or 1.7 9 107 CFU/mouse of pneumococcus serotype 14 at 2 weeks after the last booster immunization and were monitored for survival for a further 21 days. All of the mice immunized intranasal with chitosan–PsaA nanoparticles survived invasive infection with pneumococcus serotype 3, compared to only 60% of those in the naked PsaA group (P < 0.05; Fig. 5A) and 20% of those in the chitosan alone group (P < 0.05; Fig. 5A). In addition, 100% of mice immunized intranasal with chitosan–PsaA nanoparticles survived invasive infection with pneumococcus serotype 14 compared to only 40% of those in the naked PsaA group

(P < 0.05; Fig. 5B), while no mice survived in the chitosan alone group.

Discussion Pneumococcus is a leading cause of AOM, sinus, pneumonia, meningitis and sepsis in children. In 2005, the World Health Organization estimated that the number of child deaths caused by pneumococcus ranged from 700 000 to 1 million every year, worldwide, most of whom were living in developing countries [22, 23]. Pneumococcal vaccine is an effective measure for preventing pneumococcal infections and the ideal vaccine immunization strategy should not only cause a systemic immune response, but should also induce a broad mucosal immune response, including that in the lung, middle ear and nasal mucosa. Few immunocompetent cells are found in the middle ear mucosa [24], but most of T cells, B cells and other immunocompetent cells can migrate into the middle ear mucosa during AOM



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Figure 2 ELISA analysis of total anti-PsaA IgG (A) antibody in serum and IgA antibody in NW (B), BALF (C) and MEL (D). The serum was detected at 1:100 dilutions, then two-fold proportion dilutions for IgG. The NW, MEL or BALF were detected for IgA without dilutions. The data from serum samples are presented geometric mean titre standard deviation of six mice per group. The data from NW, MEL or BALF are presented as mean OD405 standard deviation of six mice per group. The reciprocal titre was considered the last dilution of serum that registered an optical density of 0.10. Statistical analysis was performed using one-way ANOVA. Statistical difference between the group immunized with chitosan–PsaA and that immunized with the naked PsaA (P < 0.05) is marked with an asterisk.

Production of anti-PsaA antibodies by intranasal immunization

To detect the immunogenicity of the chitosan–PsaA nanoparticles, mice were immunized intranasally twice a week for three consecutive weeks with chitosan–PsaA, naked PsaA or chitosan alone. Anti-PsaA IgG levels in the serum and anti-PsaA IgA levels in the NW, BALF and MEL were measured by indirect ELISA. Significantly, higher levels of total IgG antibodies were observed in sera of immunized mice with chitosan–PsaA than those of mice that received naked PsaA (P < 0.05; Fig. 2A). As shown in Fig. 2B–D, increased levels of IgA antibody were detected in the NW, BALF and MEL of mice vaccinated with chitosan–PsaA than in those vaccinated with naked PsaA (P < 0.05), suggesting that chitosan may enhance the mucosal immune responses of the protein. Cytokines production by splenocytes

Splenocytes were stimulated with rPsaA and then the concentrations of IFN-c, IL-17A and IL-4 cytokines in the supernatants secreted by splenocytes were assessed by sandwich ELISA. The production of IL-17A in the

chitosan–PsaA group was higher than in the naked PsaA group (P < 0.05; Fig. 3A). Meanwhile, compared to the naked PsaA group, the concentrations of IL-4 and IFN-c in the chitosan–PsaA group were also significantly increased (P < 0.05, Fig. 3B, C). Protective effects of chitosan–PsaA nanoparticles on AOM

The effect of intranasal vaccination with chitosan–PsaA nanoparticles on pneumococcal AOM was examined. Results from pneumococcus colonies counting in middle ear cavity (Table 1) show a significant reduction of pneumococcus colonization in the chitosan–PsaA group, when compared to the animals that received naked PsaA (P < 0.05) or chitosan alone (P < 0.05) on day 3 following middle ear challenge. However, there was no significant difference in colonies counting between the naked PsaA and the chitosan group (P > 0.05). In addition, on day 3, no pneumococcus was found to have colonized in two from a total of 10 mice in the chitosan–PsaA group. However, pneumococcus was seen in all mice in the naked PsaA and chitosan alone groups on day 3. On day 7, no pneumococcus was seen in any of the mice in the chitosan–PsaA


J.-h. Xu et al. Chitosan–PsaA Particles and Pneumococcal Infection 181 .................................................................................................................................................................. A

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Figure 3 ELISA detection of IL-17A (A), IL-4 (B) and IFN-c (C) in the supernatants of splenocytes from mice immunized with chitosan–PsaA, naked PsaA and chitosan. Splenocytes were isolated from immunized mice 2 weeks after the last immunization and then were incubated for 72 h with rPsaA (5 lg/ml). IL-17A, IL-4 and IFN-c in the supernatants were detected through sandwich ELISA. Each column represents mean concentrations standard deviation of six mice per group, and statistical analysis was performed using one-way ANOVA. Statistical difference between the group immunized with chitosan–PsaA and that immunized with the naked PsaA (P < 0.05) is marked with an asterisk.

Table 1 Bacterial clearance in the infected middle ear of vaccinated mice challenged with pneumococcus. Differences between the pneumococcal Log10 (CFU) were analysed by the Mann–Whitney U-test. Treatment and day postchallenge Day 3 Chitosan Naked PsaA Chitosan–PsaA Day 7 Chitosan Naked PsaA Chitosan–PsaA

No. of bacteria from ME washes

No. of culture-positive ME washes/total no. of mice (%)

2.3 9 105 (1.8–3.8 9 105) 2.9 9 104 (0.1–3.2 9 105) 7.5 9 103 (1.9–9.8 9 103)

10/10 (100) 10/10 (100) 8/10 (80)

8.8 9 102 (0–1.3 9 103) 0 (0–5.9 9 102) 0 (0)

6/10 (60) 4/10 (40) 0/10 (0)

group. However, pneumococcus was still seen in four mice (40%) in the naked PsaA group and in six mice (60%) of the chitosan group. Differences were significant between mice inoculated with the chitosan–PsaA and mice inoculated with the naked PsaA (P < 0.05) or chitosan alone Ó 2015 John Wiley & Sons Ltd

(P < 0.05), but were not significant between the naked PsaA and chitosan groups (P > 0.05). The results from the histopathological examination showed that the thickness of the tympanic membrane was reduced in the chitosan–PsaA group compared to that of the naked PsaA (P < 0.05; Fig. 4A) or chitosan alone groups (P < 0.05; Fig. 4A), and the number of inflammatory cells in the middle ear cavity was less in the chitosan– PsaA group than that in the naked PsaA (P < 0.05; Fig. 4B) or chitosan alone groups (P < 0.05; Fig. 4B) on day 3. The inflammatory responses in the tympanic membrane had virtually disappeared in the chitosan–PsaA group by day 7, and the thickness of the tympanic membrane was reduced compared to that in the naked PsaA (P < 0.05; Fig. 4C) and chitosan groups (P < 0.05; Fig. 4C). The effusion in the middle ear cavity was also significantly reduced in the chitosan–PsaA group and naked PsaA group, compared to that in the chitosan alone group. However, the number of inflammatory cells was less in the chitosan–PsaA group than that in the naked PsaA group (P < 0.05; Fig. 4D).


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Figure 4 Analysis of inflammatory responses on 3 and 7 days after middle ear challenge with pneumococcus. (A) Analysis of diameter of tympanic membrane on 3 days after middle ear challenge; (B) analysis of inflammatory cells on 3 days after middle ear challenge; (C) analysis of diameter of tympanic membrane on 7 days after middle ear challenge; (D) analysis of inflammatory cells on 7 days after middle ear challenge. (H&E staining; original magnification: 9200; bar = 100 lm).

Increased survival in the presence of invasive pneumococcal infection

The potential of intranasal vaccination with chitosan– PsaA to enhance survival from invasive pneumococcal infection was investigated. BALB/c mice were challenged intraperitoneally with 7 9 102 CFU/mouse of pneumococcus serotype 3 or 1.7 9 107 CFU/mouse of pneumococcus serotype 14 at 2 weeks after the last booster immunization and were monitored for survival for a further 21 days. All of the mice immunized intranasal with chitosan–PsaA nanoparticles survived invasive infection with pneumococcus serotype 3, compared to only 60% of those in the naked PsaA group (P < 0.05; Fig. 5A) and 20% of those in the chitosan alone group (P < 0.05; Fig. 5A). In addition, 100% of mice immunized intranasal with chitosan–PsaA nanoparticles survived invasive infection with pneumococcus serotype 14 compared to only 40% of those in the naked PsaA group

(P < 0.05; Fig. 5B), while no mice survived in the chitosan alone group.

Discussion Pneumococcus is a leading cause of AOM, sinus, pneumonia, meningitis and sepsis in children. In 2005, the World Health Organization estimated that the number of child deaths caused by pneumococcus ranged from 700 000 to 1 million every year, worldwide, most of whom were living in developing countries [22, 23]. Pneumococcal vaccine is an effective measure for preventing pneumococcal infections and the ideal vaccine immunization strategy should not only cause a systemic immune response, but should also induce a broad mucosal immune response, including that in the lung, middle ear and nasal mucosa. Few immunocompetent cells are found in the middle ear mucosa [24], but most of T cells, B cells and other immunocompetent cells can migrate into the middle ear mucosa during AOM


J.-h. Xu et al. Chitosan–PsaA Particles and Pneumococcal Infection 183 .................................................................................................................................................................. A

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Figure 5 Survival of vaccinated mice challenged i.p. with pneumococcus serotype 3 (A) and serotype 14 (B). Differences between survival rates of 10 mice per group were analysed by Kaplan–Meier survival curve. 100% of mice immunized i.n. with chitosan–PsaA nanoparticles survived invasive infection with pneumococcus serotype 3 or serotype 14 compared to only 60% in the naked PsaA group (P < 0.05) infected with pneumococcus serotype 3 and only 40% in the naked PsaA group (P < 0.05) infected with pneumococcus serotype 14.

[25]. In a previous study [26], Suenaga et al. found that B cells are the main lymphocytes in the normal middle ear mucosa in mice. In another study [27], Kodama et al. found a similar composition of T and B cell populations in the Eustachian tube, nasal passage and middle ear mucosa, which are effector sites; this was different from the nasalassociated lymphoid tissues, which are inductive site. These studies indicated that, following appropriate antigen stimulation, the middle ear mucosa can induce an immune response. In our study, enhanced antigen-specific IgA antibodies were induced in the nasal cavity, middle ear and bronchoalveolar space in mice intranasally inoculated with PsaA protein using chitosan as a delivery system. Moreover, systemic immune responses were elicited in the serum and spleen as well. PsaA is one of the important pneumococcal vaccine candidate antigens. Previous studies have demonstrated that intranasal vaccination with PsaA protein can induce specific immune response in mice and cholera toxin B was


used as mucosal adjuvant [19, 28]. However, the potential neurotoxicity of cholera toxin and cholera toxin B limits their use in humans [29]. Over the past decade, chitosan has been widely used as a non-toxic vector and adjuvant [30]. In our study, chitosan was used as a mucosal delivery system. Chitosan nanoparticles are formed by ionotropic gelation, and the protein is encapsulated during particle formation. The mean size of particles in our study was 691 nm with a +21.1 mv zeta potential. Previous studies have shown that the particles ranging in size from 150 nm to 4.5 lm could be phagocytosed and processed by the antigen-presenting cells [31, 32]. Moreover, antigens encapsulated in particles can prevent antigen degradation and extend antigen duration time on the mucosal surface [33]. In addition, chitosan may also specifically stimulate the immune system and possess adjuvant activity [34]. Our results also indicated that chitosan enhanced the mucosal and systemic immune responses via the intranasal route. There are two kinds of methods for inducing AOM in animals, including intranasal challenge and direct middle ear injection of live bacteria. The latter includes the transbullar and transtympanic route [20]. The method by which the nasal mucosa is challenged with bacteria is similar to the natural mode of middle ear infection, but induction of AOM is often variable and it is difficult to control the dose of bacteria entering into the middle ear. It is simple to inject pathogens into the middle ear via the tympanic membrane. However, the hole in the tympanic membrane increases the risk of contamination. Injection of pathogens into the middle ear via the bulla route reduces the chance of contamination. The volume of the middle ear in the mouse is only 5–6 ll [35]. In our study, to ensure delivery of a 5 ll volume of pneumococcus into the middle ear, two holes were made in the bulla to maintain the pressure balance. Then, the pneumococcus was injected into the bulla via one of the holes with a microsyringe. Next, the holes were sealed with bone wax to prevent spillover of pneumococcus. No mice died during the operation. In animal models, the induction of pneumococcal otitis media not only relates to the animal species, but also to the bacterial serotype, quantity and infection routes. Pneumococcus involves 91 serotypes, and different serotypes differ in pathogenicity. Although studies have confirmed that the most common serotypes of AOM include serotype 3, 6A, 6 B, 9V, 14, 19A, 19F and 23F [22] in humans, different animals have different sensitivities to pneumococcus. Melhus et al. [36] injected serotype 3 pneumococcus into the middle ear of BALB/c mice; all mice succumbed to sepsis within days. However, in another study [21], serotype 3 pneumococcus at 10-fold the concentration used for the middle ear injection was dripped into the nasal cavity; yet, no mice died. In this study, we also found that even when 10 CFU of pneumococcus serotype 3 was

184 Chitosan–PsaA Particles and Pneumococcal Infection J.-h. Xu et al. .................................................................................................................................................................. injected into bulla, all mice died of sepsis (data not shown). Therefore, in this experiment, we chose the common serotype 14 pneumococcus to challenge mice; no mice died of systemic infection (data not shown). Previous studies have confirmed that the maximum inflammatory response in the middle ear induced by pneumococcus occurs at post-inoculation day 3 and declines thereafter. A common evaluation of the degree of inflammation is the number of infiltrating inflammatory cells present in the middle ear and the thickness of the tympanic membrane [37]. Therefore, in this experiment, we evaluated the protective effects of the vaccine at day 3 and day 7 after pneumococcal challenge. The results confirmed that the inflammatory response in the middle ear in the chitosan– PsaA group was not obvious, and on day 7, the tympanic membrane thickness had returned to virtually normal, and mucosal inflammatory cells had decreased significantly, while in the naked PsaA and chitosan groups, the tympanic membrane remained thick and more infiltrative inflammatory cells were present in the middle ear. Bacterial clearance from the middle ear is also one indication of vaccine efficacy [38]. In our experiment, the bacterial clearance rate was significantly higher in the chitosan–PsaA group than in the naked PsaA and chitosan groups. These results indicated that chitosan can increase the immune protection afforded by the PsaA protein against AOM. The protective effects afforded by PsaA immunization against pneumococcal nasopharyngeal colonization have been confirmed in various studies [9]. However, few studies confirmed that it can induce protection against systemic infection. Moreover, conclusions varied in different studies. Talkington et al. [39] found that when mice were immunized with PsaA protein, and were then challenged with pneumococcus strain WU2 via the intravenous route, the survival of the mice increased. However, in another study, when mice were challenged with pneumococcus strain A66.1 via the intraperitoneal route, no obvious protection was observed [40]. Wang et al. [41] found that when mice were immunized intranasally with PsaA protein, delivered using a live attenuated Salmonella vaccine, and were then challenged with pneumococcus strain WU2, the vaccine failed to induce protective immunity. Pneumococcus has opaque, intermediate and transparent phenotypes. AntiPsaA antibody cannot bind to opaque cells [42]. One reason for these discrepant results may be the different pneumococcus phenotypes. In addition, the antibody titre may also affect its protective effect [43]. In our study, mice were inoculated intranasally with chitosan–PsaA nanoparticles and were then intraperitoneally challenged with serotype 3 and serotype 14 pneumococcus; the mice had a 100% survival rate. The reason may be that the PsaA localized on the surface of the serotype 3 and serotype 14 pneumococcus where anti-PsaA antibody can bind to the PsaA. In addition, the chitosan–PsaA group produced a higher antibody titre, strengthening the binding capacity.

In conclusion, mucosal and systemic immune responses were enhanced by intranasal immunization with PsaA vaccines using chitosan as a delivery system; these results indicated that chitosan is an appropriate mucosal delivery system for pneumococcal protein. Increased protection against AOM and invasive infection by pneumococcus was achieved by mucosal immunization with chitosan–PsaA nanoparticles, indicating that intranasal immunization with chitosan–PsaA is an efficient immunization strategy.

Acknowledgment This work was supported by grants from National Natural Science Foundation (No. 81000406, No. 81200736).

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