The mechanism of action of acid-soluble chitosan as an adjuvant in the formulation of nasally administered vaccine against HBV

Abstract

Recently, numerous attempts have been made to evaluate the potential of chitosan as an adjuvant; however, few have explored the mechanism underlying the adjuvant activity of chitosan. Here, we used hepatitis B surface antigen (HBsAg) as a model antigen for the formulation of an intranasal chitosan-based vaccine against hepatitis B. In vitro and in vivo assays were conducted to evaluate its efficacy and explore the possible underlying mechanism of action. Our results showed that HBsAg was efficiently encapsulated within chitosan particles, and that the strong adhesive effect of the positive charges on the particle surface prolonged the residence in the nasal cavity. The insolubility of chitosan particles in physiological pH enabled the formation of a sustained-release depot in extracellular fluids, whereas their solubility in aqueous acids facilitated the escape of the encapsulated antigen from lysosomes, thereby enabling cross-presentation. Chitosan was able to open epithelial-cell tight junctions, thus allowing the entry of free antigens. In addition, chitosan enhanced the uptake of antigens by dendritic cells and promoted their maturation. In vivo results showed that intranasal delivery of chitosan particles induced significantly higher levels of cellular and mucosal immunity than did injected alum-based vaccine. These findings are highly useful for the rational development of new and improved vaccines.

---

1. Introduction

Vaccines play an important role in controlling the spread of the hepatitis B virus (HBV). The safety of second-generation recombinant HBV vaccines is significantly higher than that of first-generation plasma-derived vaccines. At present, the recombinant HBV vaccine based on aluminum adjuvants is the most widely used. However, it has several limitations including a low response rate of 90–95%,^1,2 necessitating enhancement of the protective effects of the vaccine. This injection-type vaccine induces relatively strong humoral immunity; however, the cellular immunity it generates is far from satisfactory. In addition, the injection causes pain and discomfort, giving rise to poor compliance. Numerous attempts have been made to improve the efficacy and compliance rate of the recombinant HBV vaccine by using various adjuvants such as 3-O-desacyl-4′-monophosphoryl lipid A (MPL), CpG oligonucleotides, saponin QS21, immune stimulating complexes (ISCOMs),^3–5 alternative vaccination routes such as mucosal administration via particle carriers,^6–8 improvement of the immunogenicity of HBsAg by the addition of preS1 and preS2,^9,10 and identification of novel antigens to replace the recombinant HBsAg, e.g., HBV core antigen (HBcAg).¹¹

Chitosan, a unique biopolymer with remarkable properties, is highly useful as a vaccine adjuvant; it possesses good biological compatibility and enhances antigen uptake by antigen-presenting cells (APCs). When used as a particle-delivery system, chitosan additionally protects the antigen from proteolytic degradation at mucosal surfaces and enables the formation of an antigen depot at the inoculation site, prolonging antigen residence in the body.^12,13 Therefore, chitosan possesses numerous advantages as an antigen delivery system.^14–17 Numerous studies have attempted to evaluate its effects as an adjuvant;^3,18–22 however, few have focused on the underlying mechanisms.^23,24

The chemical structure of chitosan is shown in Fig. 1A. Modification of the hydroxyl or amino groups enables the synthesis of derivatives with varying biological or physical properties. As different antigens may have different properties, the adjuvant effects of chitosan and its derivatives may be antigen-specific. In the present study, HBsAg, used as a model antigen, was mixed with acid-soluble chitosan to formulate a HBV vaccine for intranasal delivery. In vitro and in vivo experiments were performed to systematically evaluate the adjuvant effect of the acid-soluble chitosan, and its underlying mechanism of action was investigated.

Fig. 1 The chemical structure of chitosan (A). Arrows indicate the most heavily modified sites. Particle size (B) and zeta potential (C) of HBsAg, vaccine (HBsAg mixed with chitosan), and vaccine mixed with mucin; the results are expressed as means ± SDs (n = 3). Various forms of combined HBsAg and chitosan: encapsulated, absorbed, and free HBsAg (D); the percentages of each form were analyzed (E); results are expressed as means ± SDs (n = 3).

2. Materials and methods

2.1 Mice

Specific pathogen-free (SPF) 4–6-week-old female BALB/c mice were purchased from Beijing Vital River Laboratory Animal, Inc. (Beijing, China). The mice were maintained in an SPF environment under standardized conditions. Studies involving animals were approved by and conducted in accordance with the guidelines of the Animal Research Ethics Committee of the China National Vaccine and Serum Institute.

2.2 Reagents

Endotoxin-free pharmaceutical-grade chitosan (M_w 150–300 kDa) with a deacetylation degree of 72.1% and a purity of 99.5% was obtained from Heppe Medical Chitosan GmbH (Halle, Germany). Recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) and HBV vaccine with aluminum phosphate as the adjuvant (HBsAg–aluminum–vaccine) were produced under Good Manufacturing Practice (GMP) conditions by Beijing Tiantan Biological Products Co., Ltd. (Beijing, China). Mucin from bovine submaxillary glands was obtained from Sigma-Aldrich (St. Louis, MO, USA). The TRITC antibody labeling kit, which was used to label HBsAg in the antigen penetration assay, was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The CF488A succinimidyl ester protein labeling kits used in the antigen uptake assays as well as the near-infrared (NIR) dye CF680R succinimidyl ester used for in vivo fluorescence imaging, were obtained from Biotium Inc. (Hayward, CA, USA). Granulocyte-macrophage colony-stimulating factor (GM-CSF) used to generate bone marrow-derived dendritic cells (BMDCs) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). Antibodies used for flow cytometry including FITC anti-mouse CD40 (clone: 11M40-3), FITC anti-mouse CD80 (clone: 16-10-A1), and APC anti-mouse CD86 (clone: GL-1) were purchased from Biolegend (San Diego, CA, USA). The IL-2 and IFN-γ ELISpot kits were obtained from Mabtech (Nacka Strand, Sweden). The synthetic 12-mer S28–39 peptide “N”-IPQSLDSWWTSL-“C” of HBsAg was synthesized by SBS Genetech Co., Ltd. (Beijing, China).

The HBsAg quantitative CLIA kit, used for quantitative analysis of HBsAg, was purchased from Autobio Diagnostic Co., Ltd. (Zhengzhou, China) and the HBsAb quantitative ELISA kit was purchased from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. (Beijing, China).

2.3 Particle preparation and characterization

HBsAg-loaded chitosan particles were prepared using the self-assembly method according to the method published by Tafaghodi et al.²⁵ without the use of cross-linkers. Briefly, chitosan was dissolved in 0.1% (v/v) acetic acid and then, HBsAg was added to the chitosan solution and mixed thoroughly. The final concentrations of chitosan and HBsAg were 0.1% (w/v) and 100 μg mL⁻¹, respectively. This formulation was used throughout the study.

Following 10-fold dilution of the particles with water, the particle size distribution and zeta potential of the chitosan particles were determined using a Zetasizer Nano ZS90 (Malvern, UK). The size and zeta potential of HBsAg were also measured.

The relative proportions of various forms of HBsAg were determined as follows: after filtration through a 0.22 μm-pore-size low protein-binding PVDF membrane syringe filter (Millipore Corp., Bedford, MA, USA), the HBsAg present in the filtrate corresponded to free HBsAg, which had not been absorbed or packaged within particles. The total amount of antigen (free, absorbed, and packaged) was detected following the digestion of chitosan by incubation with chitosanase (final concentration of 0.2 U mL⁻¹) at 37 °C for 1 h. In the absence of chitosanase digestion, the directly measured HBsAg corresponded to the non-packaged HBsAg (free and absorbed). The amount of packaged HBsAg was calculated as the total amount of the antigen minus the amount of non-packaged HBsAg. Finally, packaging efficiency was calculated by dividing the amount of packaged HBsAg by the total amount of HBsAg.

2.4 Quantitative analysis of HBsAg

Standard curves were produced using the HBsAg reference standard purchased from the National Institutes for Food and Drug Control (NIFDC), which was serially diluted with 1% (w/v) BSA in PBS (pH 7.4). The test sample was diluted with the same buffer to obtain three different concentrations. The amount of HBsAg was measured using the HBsAg quantitative CLIA kit according to the manufacturer's instructions, with slight modification. Briefly, the precoated plate was brought to room temperature (25 °C) about 30 min before use. Then, the samples and standards were transferred onto the plate in duplicate, and the plate was incubated at 37 °C for 1 h after covering with plate scales. Then, the plate was washed three times. Finally, the chromogenic substrate was added and incubated at room temperature for 10 min; the colored product formed was detected with a microplate luminometer (Autobio, Zhengzhou, China).

2.5 In vitro release assay

An in vitro release assay was conducted to mimic antigen release in vivo. To create incubation conditions similar to those in extracellular fluid, the HBsAg-loaded chitosan particles were diluted with PBS buffer (0.1 M, pH 7.4)—both had been equilibrated to 37 °C—mixed thoroughly, and then incubated at 37 °C. Samples were withdrawn at various time points (10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 7 h, 24 h, and 48 h). The release of HBsAg was terminated by passing the samples through a 0.22 μm-pore-size low protein-binding PVDF membrane syringe filter. The amount of HBsAg in the filtrate minus the amount of free HBsAg before the assay represented the released amount. The morphology of the HBsAg-loaded chitosan particles, before and after incubation in PBS buffer for 30 min, was visualized by transmission electron microscopy (TEM) (JEM-1400Plus; JEOL Co., Japan), and the change in particle size was detected by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern, UK).

2.6 Mucoadhesion evaluation

Mucoadhesion of the particles was evaluated as described previously.²⁶ Briefly, the HBsAg-loaded chitosan particles were mixed with mucin (1 mg mL⁻¹) at a ratio of 10 : 1 and incubated for 1 h at room temperature. The particle size and zeta potential were recorded before and after incubation with mucin.

2.7 In vivo imaging

The residence time of CF680R succinimidyl ester-labeled antigen in the nasal cavity was evaluated by in vivo imaging according to the method of Hagenaars et al.²⁷ Mice were randomly divided into an experimental and a control group, with 4 mice in each group. After anesthesia with isoflurane, experimental and control mice received CF680R-labeled HBsAg with chitosan and CF680R-labeled HBsAg alone, respectively, via intranasal drops. The mice were immediately scanned using the IVIS SpectrumCT Preclinical in vivo Imaging System (PerkinElmer Inc., Waltham, MA, USA). The fluorescence intensity in the nasal cavity of the mice was regularly monitored over a period of 160 min. In between measurements, the mice were returned to the cages to recover from the anesthesia. CF680R-specific fluorescence was separated from the background autofluorescence using the Living Imaging software (version 4.5.2, Caliper Life Sciences, Hopkinton, MA, USA) and the fluorescence in the regions of interest within the nasal cavity was detected and quantified. The fluorescence intensity at 0 min was set as 100%, and the relative fluorescence intensities at different time points were calculated.

2.8 Effects of chitosan on epithelial cell tight junctions

The effects of chitosan on tight junctions were evaluated by measuring the transepithelium electrical resistance (TEER) and by immunofluorescence analysis of zonula occludens-1 (ZO-1) tight junction protein.

Caco-2 cells (ATCC no.: HTB-37) were purchased from ATCC and grown in Eagle's minimal essential medium (EMEM) with 20% fetal bovine serum in a humidified incubator with 5% CO₂ at 37 °C. For TEER measurement, cells were seeded in Transwell filters (Corning, NY, USA) with a pore size of 0.4 μm and used when resistance across the insert membrane was higher than 300 Ω cm². The cells were washed with Hank's balanced salt solution (HBSS, pH 6) and equilibrated with the solution for 40 min at 37 °C prior to testing. The samples were applied to the apical chamber and TEER was measured at various time points (0 min, 15 min, 30 min, 45 min, 1 h, and 1.25 h). The value at the beginning of the experiment was set as 100%. To monitor recovery, the test solution was replaced with culture medium after treating the cells with samples for 1.5 h, and TEER was measured again at 5 h and 48 h. All experiments were repeated at least 3 times in triplicate wells.

For analysis of immunofluorescence, Caco-2 cells were cultured as described above to form a monolayer and then treated with chitosan or 0.9% NaCl for 1 h. Then, the cells were fixed with 1% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 30 min. Blocking solution (5% bovine serum albumin) was used to block non-specific interactions for 30 min. For ZO-1 staining, the cells were incubated with anti-ZO-1 antibody (BD BioSciences, San Jose, CA, USA) at a concentration of 5 μg mL⁻¹ for 2 h at 37 °C. Subsequently, the cells were incubated with secondary antibody (TRITC-labeled anti-mouse IgG), purchased from Sigma, for 1 h at 37 °C. Nuclei were stained with DAPI and visualized by confocal laser scanning microscopy (CLSM) (FluoView FV1000MPE; Olympus, Tokyo, Japan).

2.9 Nasal uptake studies

Fluorescence microscopy was used to visualize the penetration of HBsAg into the nasal mucosa following nasal administration. TRITC-labeled HBsAg was mixed with chitosan to form particles. Dye-loaded particles were administered intranasally to anesthetized mice. After 30 min, the mice were sacrificed. The nasal cavity was washed with PBS solution, and the nasal bone was removed and cut into pieces that were sectioned by microtomy. The sections were viewed under the CLSM (FluoView FV1000MPE). In the control group, an equivalent amount of free TRITC-labeled HBsAg in PBS was administrated intranasally following the same protocol.

2.10 Effects of chitosan on DC antigen uptake and maturation

Flow cytometry was used to evaluate the effects of chitosan on antigen uptake by DCs and on DC maturation. CF488A succinimidyl ester was used to label the antigen, which was then mixed with chitosan to form particles. DC2.4 cells were established from bone marrow cells of C57BL/6 mice purchased from ATCC, and cultured in 24-well culture plates at 10⁶ cells per well in RPMI 1640 medium with 10% fetal bovine serum. The cells were incubated with the labeled HBsAg, in the presence or absence of chitosan, at 4 °C or 37 °C. Then, the cells were harvested at various time intervals and washed 3 times with 0.1% FCS/PBS. Data were acquired using a BD FACSCalibur flow cytometer (BD Biosciences) and analyzed using the TreeStar FlowJo software (TreeStar, San Carlos, CA, USA).

To evaluate the effects of the HBsAg-loaded chitosan particles on DC maturation, BMDCs were isolated and cultured according to the protocol described by Madaan et al.²⁸ Briefly, BALB/c mice were euthanized and their femur bones were isolated, and a bone marrow suspension was prepared. The cells were seeded in RPMI-1640 culture medium with 10% fetal bovine serum and 20 mM penicillin/streptomycin and stimulated with GM-CSF. After culturing for 6 days, non-adherent and loosely adherent immature BMDCs were harvested. The immature BMDCs were seeded in 24-well culture plates, at 10⁵ cells per well, in RPMI 1640 medium with 10% fetal bovine serum, and incubated with HBsAg in the presence or absence of chitosan. Twelve hours later, the cells were harvested and washed 3 times with 0.1% FCS/PBS. The cells were double stained with PE-labeled CD11c antibody and either FITC-labeled CD40 antibody, FITC-labeled CD80 antibody, or APC-labeled CD86 antibody. The expression of CD40, CD80, and CD86 in CD11c-gated cells was determined using a BD FACSCalibur flow cytometer.

2.11 Subcellular localization of HBsAg

CLSM was used to determine the subcellular localization of HBsAg after uptake by DCs. DC2.4 cells were seeded in Fluorodish Petri dishes (World Precision Instruments, Sarasota, FL, USA) at a density of 10⁵ cells per dish, and allowed to adhere overnight in RPMI 1640 with 10% fetal bovine serum. Cells were washed and incubated with CF488A-labeled HBsAg, in the presence or absence of chitosan. Three hours later, cells were carefully washed and stained with LysoTracker Red, in RPMI 1640 medium, at 37 °C for 20 min. After washing with PBS, cells were fixed with 4% paraformaldehyde and the nuclei were stained with DAPI. Finally, cells were visualized by CLSM (FluoView FV1000MPE).

2.12 In vivo immunization study

Female BABL/c mice were randomly divided into 4 groups of 10 mice each: mice in group 1 were intranasally administered 0.9% NaCl solution as a negative control, mice in group 2 received plain HBsAg solution intranasally (2 μg), mice in group 3 received HBsAg-loaded chitosan particles (HBsAg amount: 2 μg), and mice in group 4 were injected with 100 μL of alum-based vaccine (HBsAg amount: 2 μg) as a positive control. The detailed immunization procedures are shown in Fig. 7A.

One week after the final immunization, the mice were euthanized and serum was obtained by centrifugation of the whole-blood samples. Anti-HBs antibody in the serum was quantified using HBsAb quantitative ELISA kits, according the manufacturer's instructions.

Spleens were harvested from 5 mice in each group for isolation of splenocytes. The IL-2 and IFN-γ ELISpot kit was used to determine the levels of antigen-specific IL-2- and IFN-γ-producing cells, following the manufacturer's guidelines, with slight modification. Briefly, cells were seeded in 96-well plates precoated with primary antibody (anti-IL-2 monoclonal antibody or anti-IFN-γ monoclonal antibody) at 5 × 10⁵ cells per well. The cells were cultured in RPMI-1640 culture medium with 10% fetal bovine serum for 18 h in the presence or absence of 0.5 μg of the HBsAg S28-39 CTL epitope. The plates were washed and incubated with the corresponding biotinylated secondary antibody at 37 °C for 2 h. Then, the plates were washed again and streptavidin–alkaline phosphatase conjugate was added. The plates were then incubated for 1 h at room temperature. Finally, the plates were washed and a chromogenic substrate (BCIP/NBT) was added. The plates were then incubated in the dark for 10 min, and the reaction was terminated by washing with water. The spots were counted using a CTL ImmunoSpot Analyzer (Cellular Technology Limited, Shaker Heights, OH, USA).

For the analysis of secretory IgA (sIgA), female BABL/c mice were randomly divided into 3 groups of 10 each, one group of mice received plain HBsAg solution intranasally (2 μg) as a negative control, the second group received HBsAg-loaded chitosan particles (HBsAg amount: 2 μg), and the third group was injected with 100 μL of alum-based vaccine (HBsAg amount: 2 μg). Immunization was done as described above. One week after the final immunization, bronchoalveolar lavage fluids (BAL) were collected²⁹ and the specific anti-HBs sIgA in the BAL was determined by ELISA as reported previously.^30,31

2.13 Statistical analysis

The results were presented as means ± standard deviations (SDs); multiple comparisons were made using analysis of variance (ANOVA) and comparisons between two groups were made using the one-tailed Student's t-test. Error bars indicate SD and asterisks indicate the degree of significance; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3. Results

3.1 Chitosan generates positive charges on HBsAg and enables packaging of the antigen into particles

Preconditioning was performed to ensure the loading of HBsAg onto chitosan particles. The acid dissociation constant (pK_a) of chitosan is 6.5 and the isoelectric point of HBsAg is 5.2–5.5;^32,33 chitosan carries positive charges and HBsAg carries negative charges at pH values of 5.5–6.5. The pH of the final formulation was 5.6; at this pH, the self-assembly of chitosan and HBsAg into particles may be established via electrostatic attraction. We measured the average particle size and zeta potential of HBsAg, with or without the addition of chitosan. As shown in Fig. 1B and C, both parameters were found to increase following the addition of chitosan; the particle size increased from 98.9 ± 1.0 nm to 397.1 ± 8.3 nm, and the zeta potential increased from −7.43 ± 0.76 mV to 17.0 ± 1.0 mV, demonstrating the occurrence of self-assembly. The particles had a narrow size distribution with a polydispersity index (PDI) value 0.26 ± 0.01. The results showed that the chitosan particles could be obtained on the basis of electrostatic interactions alone, without the need for a crosslinker or organic solvent.

As chitosan assembles spontaneously, 3 forms of HBsAg, namely, chitosan-encapsulated HBsAg, HBsAg absorbed on the surface of the chitosan particle, and free HBsAg, are present in the vaccine (Fig. 1D). To measure the relative proportions of these different forms and to evaluate the encapsulation efficiency of HBsAg by chitosan, the vaccine was fractionated as described in the Methods section before detection. The results showed that the percentage of free antigens in the vaccine formulation was lower than 0.02%, the fraction of non-packaged antigens lower than 5%, and that of packaged antigens larger than 95% (Fig. 1E). These data indicate that chitosan is able to package HBsAg with high efficiency, confirming its utility in the formulation of a high-efficacy vaccine against HBV.

3.2 Chitosan increases the residence time of HBsAg in the nasal cavity

Mucoadhesives are added to prolong vaccine residence in the nasal cavity. In the absence of mucoadhesives, subunit antigens are generally rapidly cleared following administration due to the movement of the mucosal cilia, thus reducing antigen absorption.³⁴ As chitosan imparts positive charges to HBsAg, this might promote its adhesion to the negatively charged mucosa of the nasal cavity.

The adhesivity of the vaccine was evaluated in in vitro and in vivo experiments. Mucoadhesion was evaluated in an in vitro assay.²⁶ As shown in Fig. 1B, the average particle size in the experimental group (with addition of mucin, 579.5 ± 10.3 nm) was higher than that in the control group (without addition of mucin, 397.1 ± 8.3 nm) owing to the association of the particles with mucin. However, the negative charge of mucin was found to decrease the zeta potential of the particles from 17.0 ± 1.0 mV to 14.3 ± 1.1 mV (Fig. 1C). These findings demonstrate that the chitosan-based intranasal HBV vaccine adheres to negatively charged protein, enabling the adsorption of the vaccine onto negatively charged surfaces.

A NIR fluorescent probe was used to label the antigen and in vivo small animal-imaging techniques were used to investigate the antigen residence time in the nasal cavity. In both the groups with and without chitosan, the strength of fluorescence in the nasal cavity decreased by 50% within 160 min of intranasal administration (Fig. 2). However, the rate of decrease was lower in the HBsAg plus chitosan group, demonstrating that chitosan prolongs the nasal residence time of the vaccine.

Fig. 2 Residence time in the nasal cavity as analyzed by in vivo small animal imaging techniques, using CF680R to label HBsAg. After intranasal administration, the fluorescence intensity in the nasal cavity of the mice was regularly monitored over a period of 160 min (A). The initial fluorescence in the nasal cavity was set as 100%; the relative fluorescence intensity is shown in (B). Data are expressed as means ± SDs (n = 4).

3.3 Effects of chitosan on epithelial tight junctions

The mucosal surface of the nasal cavity consists of only a thin layer of pseudostratified epithelial cells connected by tight junctions. Human colonic carcinoma-derived Caco-2 cells form monolayers of differentiated cells with tight junctions, and are widely used as a model of paracellular transport of compounds across the epithelial cell monolayer. Chitosan was incubated with a monolayer of Caco-2 cells to evaluate its effect on the junctions. As shown in Fig. 3A, the TEER of the monolayer was decreased after incubation with HBsAg in the presence of chitosan, but not in the group without chitosan, indicating that chitosan is capable of opening tight junctions. After the removal of chitosan, TEER recovered gradually, demonstrating that the chitosan-mediated junction opening is a reversible process.

Fig. 3 Relative TEER, defined as the percentage of the initial TEER, of Caco-2 cells after incubation with various samples were analyzed; the arrow indicates the time point at which the samples were withdrawn (A); values are expressed as means ± SDs (n = 3). Immunofluorescence staining of Caco-2 cells with anti-ZO-1; (B) Caco-2 cells treated with 0.9% NaCl as control, (C) Caco-2 treated with chitosan. The penetration of HBsAg (labeled with TRITC) into nasal mucosa following nasal administration was analyzed by fluorescence microscopy (D and E).

Immunofluorescence staining of ZO-1 was used to visualize chitosan-mediated tight junction opening. ZO-1 staining became discontinuous following treatment with chitosan (Fig. 3C). These findings showed that chitosan induces the reorganization or translocation of proteins constituting the tight junctions,³⁵ corroborating that chitosan mediates the opening.

3.4 Chitosan promotes the penetration of HBsAg into nasal mucosa

The effect of chitosan on antigen transportation was assayed in vivo. TRITC-labeled HBsAg was administered intranasally and the localization of the vaccine was monitored by CLSM after 30 min. No fluorescent particles were observed in the mucosal tissues of the control group (Fig. 3D); however, numerous fluorescent particles were observed in the mucosal tissues of the nasal cavity of the group with added chitosan (Fig. 3E).

3.5 HBsAg–chitosan particles exhibit sustained release of antigen

Several adjuvants serve as depots to enable the slow release of encapsulated antigens,^36,37 thereby providing sustained immune stimulation. To evaluate the effect of chitosan on sustained release, an in vitro release experiment was conducted. As shown in Fig. 4B, under conditions of pH, osmotic pressure, and temperature similar to those in the extracellular fluid, the release was rapidly initiated, and then stabilized. After 2 days, the amount of HBsAg released was only ∼20%, indicating that chitosan particles enable sustained antigen release. DLS and TEM revealed that at pH 7.4, the acid-soluble chitosan particles (Fig. 4C) aggregated and became insoluble (Fig. 4D), and the particle size increased significantly (Fig. 4A, C and D).

Fig. 4 Changes in particle morphology upon incubation at pH 7.4. (A) The significant increase in particle size, (B) the slow release of antigen at pH 7.4, (C) and (D) the various morphologies of chitosan particles at pH 5.6 and pH 7.4; the data in (A) and (B) are expressed as means ± SDs (n = 3).

3.6 Chitosan promotes antigen uptake by cells

Antigen uptake by DCs was evaluated in vitro. HBsAg, alone or mixed with chitosan, was incubated with DCs at 37 °C. After a period of incubation, HBsAg associated with cells was quantified by flow cytometry. As cell-surface adhesion of HBsAg may affect the results, the cells were incubated at 4 °C to ensure that they were in a resting state and to inhibit energy-dependent uptake, so that only physical absorption occurred. As shown in Fig. 5A and B, DCs incubated at 4 °C showed a lower percentage of fluorescence-positive cells than those incubated at 37 °C, indicating that cell-associated fluorescence at 37 °C was caused mainly by the uptake of HBsAg rather than by adhesion to the DC membrane.^38,39 Additionally, at 37 °C, the group administered with HBsAg mixed with chitosan showed a higher percentage of fluorescence-positive cells than the group receiving HBsAg without chitosan, indicating that chitosan promotes the uptake of HBsAg by DCs.

Fig. 5 Uptake of antigen by DCs. DC2.4 cells were incubated with CF488A-labeled HBsAg in the presence or absence of chitosan for 30 min, 1 h, 2 h, 3 h, and 4 h, at 4 °C (A) or at 37 °C (B). The percentage of CF488A-HBsAg-positive cells was analyzed; the results are expressed as means ± SDs (n = 3). The intracellular location of antigen in DCs (C), HBsAg alone, or HBsAg mixed with chitosan incubated with DC2.4 cells was detected by CLSM. Lysosomes were labeled with LysoTracker (red points), HBsAg with CF488A (green points), and nuclei with DAPI (blue). Merged images showing co-localization: the red, green, and yellow points indicate empty lysosome, escaped antigen, and co-localized antigen, respectively.

No significant difference was found between both groups upon incubation at 4 °C. Chitosan, which carries positive charges in acidic solution, interacts with negative cell surfaces; however, this interaction does not occur in alkaline medium. These results indicate that the uptake-enhancing effect of chitosan depends on the shape of the chitosan particle formed after encapsulation with HBsAg, not on the ability to adhere to cell surfaces.

3.7 Chitosan promotes endosomal escape of HBsAg

The distribution of HBsAg in DCs was examined using CLSM. CF488A (green) was used to label the antigen and LysoTracker (red) and DAPI (blue) were used to label lysosomes and nuclei, respectively. After incubation, HBsAg and lysosomes were clearly visualized by CLSM. As shown in Fig. 5C, a larger amount of HBsAg was taken up by cells treated with HBsAg plus chitosan than by those receiving HBsAg alone, confirming that chitosan promotes the uptake of antigens. HBsAg colocalized with lysosomes in cells receiving HBsAg alone; however, in cells treated with HBsAg plus chitosan, most of the antigen did not colocalize with lysosomes. These findings indicated that most of chitosan-encapsulated HBsAg escaped from the lysosome and was released into the cytoplasm.

3.8 Chitosan effectively promotes the maturation of DCs

DCs, which represent the most powerful antigen-presenting cells, play a key role in initiating and regulating immune responses.⁴⁰ Costimulatory molecules, such as CD40, CD80, and CD86, which are expressed on the surface of mature DCs, serve as major signals for activated specific T lymphocytes, while CD11c is a DC-specific marker.

To study the effect of chitosan on DC maturation, BMDCs were incubated with HBsAg alone or with HBsAg plus chitosan. The expression of CD40, CD80, and CD86 on the surface of CD11c-positive cells was studied. The percentage of double-positive cells, including CD11c⁺CD40⁺, CD11c⁺CD80⁺, and CD11c⁺CD86⁺ cells, in the BMDC population was determined to evaluate the influence of various components on DC maturation. Compared with the cells treated with HBsAg or 0.9% NaCl solution, the cells receiving chitosan exhibited significant upregulation of the expression of CD40 (P < 0.0001), CD80 (P < 0.0001), and CD86 (P < 0.01) on DCs, indicating that chitosan promotes the maturation of DCs (Fig. 6).

Fig. 6 Effects of the chitosan particles on DC maturation. Samples were incubated with immature BMDCs for 12 h and the expression of CD40 (A), CD80 (B), and CD86 (C) in CD11c-gated cells was analyzed. Results are expressed as means ± SDs (n = 3).

3.9 Chitosan-encapsulated HBsAg effectively induces immune responses

HBV vaccines with alum adjuvant induce essential humoral immune responses, but poor cellular and mucosal immune responses. To investigate the effects of administration of HBsAg plus chitosan as a new HBV vaccine candidate, in vivo immunization studies were conducted. Mice were divided into 4 groups, which were administered 0.9% NaCl solution (negative control group), alum-based vaccine (positive control group), plain HBsAg, and HBsAg mixed with chitosan (experimental groups). Except for animals receiving the alum-based vaccine, which was administered by intramuscular injection, each group was immunized intranasally. One week after the final vaccination, blood, BAL, and spleens were collected.

Anti-HBs antibody levels were measured in the serum. Quantitative analysis revealed significant differences between groups (F = 39.58, P < 0.0001) (Fig. 7B). There was no significant difference between the groups receiving plain HBsAg and 0.9% NaCl (P > 0.05) (Fig. 7B); however, when animals were administered HBsAg mixed with chitosan, the level of anti-HBs increased significantly (P < 0.01) (Fig. 7B), demonstrating that chitosan facilitates the entry of HBsAg. While the alum-based vaccine performed slightly better than the chitosan-based based vaccine with regard to the antibody titer, the difference was not significant (P > 0.05) (Fig. 7B).

Fig. 7 Detailed immunization procedure (A) (i.n., intranasal; i.m., intramuscular) and the results of in vivo immunization study. Anti-HBs (B) antibody levels in the indicated groups (NS denotes no significant difference between groups) is shown. Results are expressed as means ± SDs (n = 10). Numbers of antigen-specific IL-2- (C) or IFN-γ- (D) producing cells as measured by ELISpot assay. The data in (C) and (D) are expressed as the means ± SDs (n = 5).

As sIgA plays an important role in preventing virus entry into host cells, the sIgA level in BAL was measured. Chitosan (log₁₀ value of sIgA: 1.90 ± 1.10) could induce the level of sIgA as compared to alum (log₁₀ value of sIgA: 0.82 ± 0.16). No significant difference was observed between the groups receiving plain HBsAg (log₁₀ value of sIgA: 0.87 ± 0.24) and alum-based HBV vaccine. These data indicated that chitosan was superior to alum in inducing a strong mucosal humoral immune response. To evaluate the ability of the chitosan-based HBV vaccine to elicit specific cellular immune responses, the levels of IFN-γ- and IL-2-producing cells were determined by ELISpot assays. The results showed that there were no statistically significant differences between the alum–HBsAg vaccine-administered group and the groups receiving plain HBsAg or 0.9% NaCl, in terms of the number of IFN-γ- or IL-2-producing cells. However, mice immunized with HBsAg plus chitosan showed a significantly higher number of IFN-γ- and IL-2 producing-cells than the mice in the other groups (Fig. 7C and D). These results demonstrated that the chitosan-based HBV vaccine induces stronger cellular immune responses than the alum-based HBV vaccine.

4. Discussion and conclusions

Although traditional HBV vaccines have shown high efficacy in preventing and controlling the spread of HBV, these vaccines have failed to elicit preventive effects in certain individuals.^41,42 In addition, as the traditional vaccine is delivered via injection, the rates of non-compliance are high, particularly in children. Intranasal vaccine delivery avoids reactions such as pain, heating, and swelling associated with injection, which is expected to significantly improve compliance rates.

The utility of chitosan and its derivatives, which possess numerous properties that are highly suited to use as a drug carrier, has been evaluated in numerous vaccine formulations;^43–47 however, such studies have mostly focused on the impacts of different types of particles, and their characterization, biological properties, and effects in vivo. Few attempts have been made to elucidate the underlying mechanisms of action of chitosan and its derivatives, and the fact that different physicochemical properties and administration routes may involve different mechanisms has been largely ignored.

In the present study, an acid-soluble chitosan was used to design a novel chitosan-based vaccine. We investigated the effects of chitosan as an adjuvant in the HBV vaccine and attempted to elucidate the underlying mechanism of action. Our results showed that HBsAg is effectively packaged by chitosan or absorbed onto the surface of chitosan particles via self-assembly, which enables the transport of the antigen across the mucosal surface. Furthermore, chitosan was found to be capable of opening the tight junctions between epithelial cells. While it remains difficult for relatively large particles to penetrate the epithelium via paracellular transport,⁴⁸ the opening of tight junctions likely provides a channel for the entry of free HBsAg. In addition, the positive charges on the particle surface prolong the antigen residence in the nasal cavity. The in vivo assay demonstrated that chitosan could significantly enhance the penetration of HBsAg into nasal epithelial cells and thus, in the nasal mucosa. Large particles are likely transported via endocytosis by M-cells.^49,50 Our findings showed that chitosan was soluble in acidic solutions but not when transported into extracellular fluid; this enabled a slow release and consequently, enhanced antigen uptake by DCs. However, chitosan dissolved in the acidic environment of endosomes, facilitating endosomal escape of antigens, which could result in cross presentation followed by typical processing and presentation on MHC I. In addition, in vitro assays showed that the adjuvant promoted DC maturation and upregulated the expression of co-stimulatory molecules CD40, CD80, and CD86. A recent study by Carroll et al.²⁴ showed that chitosan induces mitochondrial oxidative stress, leading to DNA release. The DNA thus released into the cytoplasm activates the cGAS-STING pathway, promoting DC maturation and enhancing antigen-specific T helper 1 (Th1) responses. Based on all above findings, we propose an underlying mechanism of action as shown in Fig. 8.

Fig. 8 Schematic presentation of the possible mechanism underlying the adjuvant effects of acid-soluble chitosan. Chitosan is soluble and positively charged under acidic conditions (a), the positive charge on the surface of the particles and the ability of chitosan to adhere to the cell surface prolong the antigen residence time in the nasal cavity (b). Chitosan is able to open tight junctions and to enhance transmembrane transport of free antigens via the paracellular route (c) as well as to facilitate transcytosis of packaged antigens by M-cells (d). Chitosan is insoluble at physiological pH, which enables the slow release of the encapsulated antigens (e), and promotes antigen uptake by DCs (f). After uptake by DCs, chitosan activates the STING-cGAS pathway,²⁴ leading to DC maturation (g). Insoluble particles are absorbed by the cell through endocytosis; under acidic pH, chitosan becomes soluble and positively charged, which promotes antigen escape from lysosomes (h and i) into the cytoplasm for cross-presentation by the MHC-I pathway (k). The free antigen taken up by DCs is unable to escape from these cells and is routed to the MHC-II pathway (j).

Routine immunization with the traditional alum-based recombinant hepatitis B subunit vaccine induces the production of long-acting protective antibodies, resulting in high performance in terms of reducing the rates of hepatitis B as well as the incidence of, and mortality associated with, hepatitis B-related diseases. However, this vaccine suffers from a limitation in that a low rate of response, or no response, attributed to the abnormal function of T lymphocytes or reduced secretion of antigen-specific Th1 and Th2 cytokines, is observed.⁵¹ In vivo assays were performed to compare the effects of the chitosan-based vaccine, delivered intranasally, with those of an alum–HBsAg vaccine, which was administered via the intramuscular route. Although the presence of the mucosal barrier results in the delivery of a smaller amount of antigen via the intranasal route than by injection, the humoral immunity stimulated by the chitosan-based vaccine was similar to that induced by the alum vaccine. Furthermore, the cellular (significantly high activation of Th1 cytokine production) and mucosal immune responses induced by the chitosan-based vaccine were much stronger. This was attributed to differences in physiological conditions and in the distribution of immune-related cells between the nasal cavity and muscles. In addition, as an adjuvant, chitosan possesses several advantages over alum, such as the sustained release of antigen after packaging and sustained stimulatory effect on immune cells, as alum adjuvant-containing vaccines are unable to form slow-release depots.⁵² Furthermore, unlike alum-based adjuvants, chitosan promotes DC maturation and enhances antigen presentation.^53,54

Ionotropic gelation and chemical crosslinking, which require the use of cross-linkers, are widely used for particle preparation.^55–58 However, cross-linkers, e.g., tripolyphosphate or glutaraldehyde, have potential health hazards. Moreover, the organic solvents, heat, or vigorous agitation used in these methods can damage the proteins. Our method, which exploits the self-assembly property of chitosan, does not require the use of any cross-linkers and thus, reduces the risk of introducing endotoxin and is suitable for large-scale preparation. The results showed that the antigens could be successfully encapsulated in the particles and preserved good activity.

In recent years, numerous derivatives of chitosan, with varying chemical and physical properties, have been synthesized. In the present work, the adjuvanticity of acid-soluble chitosan was evaluated. Our results show that the insolubility of the present chitosan in physiological pH, and solubility in aqueous acids, facilitates the escape of encapsulated antigen from lysosomes and enables efficient MHC class I presentation. We suspect that the differences in solubility between various chitosan derivatives result in differences in pathways of antigen presentation, and consequently, varying adjuvanticity. In addition, the specific form in which the antigen is mixed with chitosan, e.g., encapsulated or absorbed, may elicit varying immune effects.⁵⁹ Further work is required to clarify the effects of various chitosan derivatives on the immune response. Our findings are expected to be highly useful for the rational development of new and improved vaccines.

Conflict of interest

The authors declare that they have no conflict of interests.

Acknowledgements

This study was financially supported by China National Science and Technology Major Project (grant no: 2012ZX10002-002).

References

1. J. N. Zuckerman, J. Med. Virol., 2006, 78, 169–177.
2. A. P. Vermeiren, C. J. Hoebe and N. H. Dukers-Muijrers, J. Clin. Virol., 2013, 58, 262–264.
3. O. Borges, A. Cordeiro-da-Silva, J. Tavares, N. Santarém, A. de Sousa, G. Borchard and H. E. Junginger, Eur. J. Pharm. Biopharm., 2008, 69, 405–416.
4. P. Vandepapelière, Y. Horsmans, P. Moris, M. Van Mechelen, M. Janssens, M. Koutsoukos, P. Van Belle, F. Clement, E. Hanon and M. Wettendorff, Vaccine, 2008, 26, 1375–1386.
5. G. Leroux-Roels, P. Van Belle, P. Vandepapeliere, Y. Horsmans, M. Janssens, I. Carletti, N. Garçon, M. Wettendorff and M. Van Mechelen, Vaccine, 2015, 33, 1084–1091.
6. A. Farhadian, N. M. Dounighi and M. Avadi, Hum. Vaccines Immunother., 2015, 11, 2811–2818.
7. D. J. Bharali, V. Pradhan, G. Elkin, W. Qi, A. Hutson, S. A. Mousa and Y. Thanavala, Nanomedicine, 2008, 4, 311–317.
8. D. Pawar and K. Jaganathan, Drug Delivery, 2016, 23, 185–194.
9. P. Rendi-Wagner, D. Shouval, B. Genton, Y. Lurie, H. Rümke, G. Boland, A. Cerny, M. Heim, D. Bach and M. Schroeder, Vaccine, 2006, 24, 2781–2789.
10. B. Yerushalmi, R. Raz, O. Blondheim, E. Shumov, R. Koren and R. Dagan, Pediatr. Infect. Dis. J., 1997, 16, 587–592.
11. C. S. Chong, M. Cao, W. W. Wong, K. P. Fischer, W. R. Addison, G. S. Kwon, D. L. Tyrrell and J. Samuel, J. Controlled Release, 2005, 102, 85–99.
12. V. W. Bramwell and Y. Perrie, J. Pharm. Pharmacol., 2006, 58, 717–728.
13. S. De Koker, B. N. Lambrecht, M. A. Willart, Y. Van Kooyk, J. Grooten, C. Vervaet, J. P. Remon and B. G. De Geest, Chem. Soc. Rev., 2011, 40, 320–339.
14. I. Van der Lubben, J. Verhoef, G. Borchard and H. Junginger, Adv. Drug Delivery Rev., 2001, 52, 139–144.
15. L. Illum, I. Jabbal-Gill, M. Hinchcliffe, A. Fisher and S. Davis, Adv. Drug Delivery Rev., 2001, 51, 81–96.
16. N. K Garg, S. Mangal, H. Khambete, P. K Sharma and R. K Tyagi, Recent Pat. Drug Delivery Formulation, 2010, 4, 114–128.
17. I. Jabbal-Gill, P. Watts and A. Smith, Expert Opin. Drug Delivery, 2012, 9, 1051–1067.
18. Z.-S. Wen, Y.-L. Xu, X.-T. Zou and Z.-R. Xu, Mar. Drugs, 2011, 9, 1038–1055.
19. R. Scherließ, S. Buske, K. Young, B. Weber, T. Rades and S. Hook, Vaccine, 2013, 31, 4812–4819.
20. C. Prego, P. Paolicelli, B. Díaz, S. Vicente, A. Sánchez, Á. González-Fernández and M. J. Alonso, Vaccine, 2010, 28, 2607–2614.
21. K. Khatri, A. K. Goyal, P. N. Gupta, N. Mishra, A. Mehta and S. P. Vyas, Vaccine, 2008, 26, 2225–2233.
22. L. Chen, J. Zhu, Y. Li, J. Lu, L. Gao, H. Xu, M. Fan and X. Yang, PLoS One, 2013, 8, e71953.
23. Y. Q. Wang, J. Wu, Q. Z. Fan, M. Zhou, Z. G. Yue, G. H. Ma and Z. G. Su, Adv. Healthcare Mater., 2014, 3, 670–681.
24. E. C. Carroll, L. Jin, A. Mori, N. Muñoz-Wolf, E. Oleszycka, H. B. Moran, S. Mansouri, C. P. McEntee, E. Lambe and E. M. Agger, Immunity, 2016, 44, 597–608.
25. M. Tafaghodi, V. Saluja, G. F. Kersten, H. Kraan, B. Slutter, J. P. Amorij and W. Jiskoot, Vaccine, 2012, 30, 5341–5348.
26. P. T. Wong, P. R. Leroueil, D. M. Smith, S. Ciotti, A. U. Bielinska, K. W. Janczak, C. H. Mullen, J. V. Groom II, E. M. Taylor and C. Passmore, PLoS One, 2015, 10, e0126120.
27. N. Hagenaars, M. Mania, P. de Jong, I. Que, R. Nieuwland, B. Slütter, H. Glansbeek, J. Heldens, H. van den Bosch and C. Löwik, J. Controlled Release, 2010, 144, 17–24.
28. A. Madaan, R. Verma, A. T. Singh, S. K. Jain and M. Jaggi, J. Biol. Methods, 2014, 1, e1.
29. C. Thomas, A. Rawat, S. Bai and F. Ahsan, J. Pharm. Sci., 2008, 97, 1213–1223.
30. A. Debin, R. Kravtzoff, J. V. Santiago, L. Cazales, S. Sperandio, K. Melber, Z. Janowicz, D. Betbeder and M. Moynier, Vaccine, 2002, 20, 2752–2763.
31. K. Khatri, A. K. Goyal, P. N. Gupta, N. Mishra and S. P. Vyas, Int. J. Pharm., 2008, 354, 235–241.
32. K. Jaganathan, P. Singh, D. Prabakaran, V. Mishra and S. P. Vyas, J. Pharm. Pharmacol., 2004, 56, 1243–1250.
33. K. Jaganathan and S. P. Vyas, Vaccine, 2006, 24, 4201–4211.
34. B. Slütter, N. Hagenaars and W. Jiskoot, J. Drug Targeting, 2008, 16, 1–17.
35. J. Smith, E. Wood and M. Dornish, Pharm. Res., 2004, 21, 43–49.
36. B. Y. Chua, T. Sekiya, M. Al Kobaisi, K. R. Short, D. E. Mainwaring and D. C. Jackson, Biomaterials, 2015, 53, 50–57.
37. D. Christensen, M. Henriksen-Lacey, A. T. Kamath, T. Lindenstrøm, K. S. Korsholm, J. P. Christensen, A.-F. Rochat, P.-H. Lambert, P. Andersen and C.-A. Siegrist, J. Controlled Release, 2012, 160, 468–476.
38. M. J. Copland, M. A. Baird, T. Rades, J. L. McKenzie, B. Becker, F. Reck, P. C. Tyler and N. M. Davies, Vaccine, 2003, 21, 883–890.
39. C. Barnier-Quer, A. Elsharkawy, S. Romeijn, A. Kros and W. Jiskoot, Pharmaceutics, 2013, 5, 392–410.
40. L. K. Petersen, L. Xue, M. J. Wannemuehler, K. Rajan and B. Narasimhan, Biomaterials, 2009, 30, 5131–5142.
41. S. Walayat, Z. Ahmed, D. Martin, S. Puli, M. Cashman and S. Dhillon, World J. Hepatol., 2015, 7, 2503–2509.
42. M. H. Sjogren, Am. J. Med., 2005, 118(suppl. 10A), 34s–39s.
43. J. K. Sahni, S. Chopra, F. J. Ahmad and R. K. Khar, J. Pharm. Pharmacol., 2008, 60, 1111–1119.
44. A. J. Mann, N. Noulin, A. Catchpole, K. J. Stittelaar, L. de Waal, E. J. B. Veldhuis Kroeze, M. Hinchcliffe, A. Smith, E. Montomoli, S. Piccirella, A. D. M. E. Osterhaus, A. Knight, J. S. Oxford, G. Lapini, R. Cox and R. Lambkin-Williams, PLoS One, 2014, 9, e93761.
45. Y. Q. Wang, Y. Liu, Y. X. Wang, Y. J. Wu, P. Y. Jia, J. J. Shan, J. Wu, G. H. Ma and Z. G. Su, Int. Immunopharmacol., 2016, 39, 84–91.
46. D. Pawar and K. S. Jaganathan, Drug Delivery, 2016, 23, 185–194.
47. K. Zhao, Y. Sun, G. Chen, G. Rong, H. Kang, Z. Jin and X. Wang, Carbohydr. Polym., 2016, 149, 28–39.
48. L. Illum, J. Pharm. Sci., 2007, 96, 473–483.
49. M. A. Clark, M. A. Jepson and B. H. Hirst, Adv. Drug Delivery Rev., 2001, 50, 81–106.
50. R. Kuolee and W. Chen, Expert Opin. Drug Delivery, 2008, 5, 693–702.
51. G. Kardar, M. Jeddi-Tehrani and F. Shokri, Scand. J. Immunol., 2002, 55, 311–314.
52. S. Hutchison, R. A. Benson, V. B. Gibson, A. H. Pollock, P. Garside and J. M. Brewer, FASEB J., 2012, 26, 1272–1279.
53. M. Kool, T. Soullié, M. van Nimwegen, M. A. Willart, F. Muskens, S. Jung, H. C. Hoogsteden, H. Hammad and B. N. Lambrecht, J. Exp. Med., 2008, 205, 869–882.
54. H. Sun, K. G. Pollock and J. M. Brewer, Vaccine, 2003, 21, 849–855.
55. J. A. Ko, H. J. Park, S. J. Hwang, J. B. Park and J. S. Lee, Int. J. Pharm., 2002, 249, 165–174.
56. S. R. Jameela, T. V. Kumary, A. V. Lal and A. Jayakrishnan, J. Controlled Release, 1998, 52, 17–24.
57. B. C. Thanoo, M. C. Sunny and A. Jayakrishnan, J. Pharm. Pharmacol., 1992, 44, 283–286.
58. F. L. Mi, S. S. Shyu, C. T. Chen and J. Y. Schoung, Biomaterials, 1999, 20, 1603–1612.
59. W. Zhang, L. Wang, Y. Liu, X. Chen, Q. Liu, J. Jia, T. Yang, S. Qiu and G. Ma, Biomaterials, 2014, 35, 6086–6097.

---