Combinatorial Approach of Antigen Delivery Using M Cell-Homing

Article pubs.acs.org/molecularpharmaceutics

Combinatorial Approach of Antigen Delivery Using M Cell-Homing Peptide and Mucoadhesive Vehicle to Enhance the Efficacy of Oral Vaccine Bijay Singh,†,‡,∥ Sushila Maharjan,†,‡,∥ Tao Jiang,†,‡ Sang-Kee Kang,†,§ Yun-Jaie Choi,*,†,‡ and Chong-Su Cho*,†,‡ †

Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea § Research Institute of Eco-friendly Animal Science, Institute of Green-Bio Science and Technology, Seoul National University, Kangwon-Do 232-916, Republic of Korea ‡

ABSTRACT: Orally ingested pathogens or antigens are taken up by microfold cells (M cells) in Peyer’s patches of intestine to initiate protective immunity against infections. However, the uptake of orally delivered protein antigens through M cells is very low due to lack of specificity of proteins toward M cells and degradation of proteins in the harsh environment of gastrointestinal (GI) tract. To overcome these limitations, here we developed a pH-sensitive and mucoadhesive vehicle of thiolated eudragit (TE) microparticles to transport an M cell-targeting peptide-fused model protein antigen. Particularly, TE prolonged the particles transit time through the GI tract and predominantly released the proteins in ileum where M cells are abundant. Thus, oral delivery of TE microparticulate antigens exhibited high transcytosis of antigens through M cells resulting in strong protective sIgA as well as systemic IgG antibody responses. Importantly, the delivery system not only induced CD4+ T cell immune responses but also generated strong CD8+ T cell responses with enhanced production of IFN-γ in spleen. Given that M cells are considered a promising target for oral vaccination, this study could provide a new combinatorial method for the development of M-cell-targeted mucosal vaccines. KEYWORDS: M cells, microparticles, mucosal vaccine, mucoadhesive vehicle, oral delivery

1. INTRODUCTION Mucosal surfaces are the most common route of entry of many bacteria and viruses for infection. In mucosal tissues, epithelial microfold cells (M cells), specifically located on the follicleassociated epithelium (FAE) in Peyer’s patch of the gut, capture and transport these invaders across the epithelial barrier to underlying gut-associated lymphoid tissues to induce protective immunity.1 A simple way to combat these infections is to induce immunity using vaccination. However, parenteral vaccination fails to generate an adequate local mucosal immune response against the intestinal infections. Therefore, oral vaccination, among the mucosal route vaccinations, has received much attention due to its easy handling, high patient compliance, and induction of mucosal immunity. However, the development of oral subunit vaccines holds major challenges due to physical, chemical, and biological barriers in delivering the antigens to the gastrointestinal (GI) tract. While the impermeable GI epithelium acts as a physical barrier, the harsh gastric pH and enzymes in the GI tract degrade the antigens, resulting in a low bioavailability.2 The next barrier is the cellular uptake of antigen by M cells due to lack of specificity of antigens toward M cells. If these barriers were overcome, the antigens could be delivered through M cells to underlying organized lymphoid follicles where they © XXXX American Chemical Society

encounter with antigen presenting cells. Therefore, the initiation of antigen-specific mucosal immune responses depends on the successful uptake of the antigens and their presentation by these antigen presenting cells. Previous studies of mucosal immunity have mainly focused on the roles of mucosal immune cells paying less attention to M cells. Although it was well-known that M cells actively transcytose macromolecules and microbes, it was less known whether the transcytosis of antigens by M cells occurs through specific receptors that bind to antigens with certain molecular patterns.3 To identify the molecular recognition of peptides with M cells, we recently discovered an M cell-homing peptide (CKSTHPLSC) by phage display technique.4 The peptide showed high affinity toward M cells and facilitated the transport of chitosan nanoparticles across the M cells to enter the FAE of Peyer’s patch demonstrating the potency of M cell-homing peptide as M cell-targeting ligand. Therefore, targeted delivery of vaccines to M cells would be an approach of oral vaccination to induce high mucosal immune responses. Received: August 9, 2014 Revised: August 11, 2015 Accepted: September 22, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00265 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

was dialyzed overnight against water (pH 7.9) at 4 °C, freezedried, and stored at −20 °C until use. Synthesis of Thiolated Eudragit. Thiolated Eudragit (TE) was synthesized according to a method described previously.18 Briefly, Eudragit L-100 (4 g) was dissolved in DMSO (100 mL). DCC (9 g) and NHS (5 g) were added into the Eudragit solution, and the reaction was stirred at room temperature for 24 h under nitrogenous condition. Byproducts were filtered, and the filtrate was further reacted with L-cysteine hydrochloride for 48 h under similar condition. Byproducts were filtered, and the filtrate was initially dialyzed against DMSO and then against distilled water. The final product was freeze-dried and stored at −20 °C until use. The amount of cysteine in TE was estimated by using Ellman’s reagent (Thermo Scientific) for the determination of free thiol. Synthesis of DAPI-Labeled Polymers. DAPI-labeled Eudragit polymers were synthesized similar to the method described above. Briefly, Eudragit or TE (0.1 g) was dissolved in DMSO (5 mL). DCC (0.2 g) and NHS (0.1 g) were added into the Eudragit or TE solution, and the reaction was stirred at room temperature for 24 h under nitrogenous condition. Byproducts were filtered, and the filtrate was further reacted with DAPI for 4 h under similar condition. Byproducts were filtered, and the filtrate was initially dialyzed against DMSO and then against distilled water. The final product was freeze-dried and stored at −20 °C until use. Labeling of Antigens with FITC. FITC-labeled antigens were prepared as follows. Twenty milligrams of protein was dissolved in 700 μL of carbonate-bicarbonate buffer (pH 9.5). One milligram of FITC was dissolved in 300 μL of DMSO. Protein solution was stirred with FITC solution for 4 h in dark at room temperature. The reaction product was dialyzed in water for 24 h in dark at 4 °C. The purified product was lyophilized. The amount of FITC conjugated on antigens was calculated by observing the absorbance at 492 nm. Preparation of Antigen-Loaded MPs. Eudragit microparticles (EMPs) and thiolated Eudragit microparticles (TEMPs) were prepared as mentioned in the previous study.15 Briefly, organic solution of Eudragit and TE were prepared by dissolving 50 mg of each in 5 mL of dichloromethane/ethanol (1:1) and dichloromethane, respectively. An aqueous solution of 10% Pluronic F-127 solution (100 μL) was mixed with BmpB or M-BmpB antigen (100 μL) to prepare an internal aqueous phase. The aqueous phase was emulsified with the organic solution of polymer (5 mL) using an ultrasonic processor (Sonics, Vibra cells). The primary emulsion was further added to external aqueous phase of PVA (1% w/v), and the mixture was homogenized with Ultra Turrax (T25, IKA, Germany) at 11,000 rpm for 4 min to prepare water-in-oil-in-water (W/O/W) emulsion. The emulsion was then stirred for 6−8 h at room temperature to allow the organic solvent to evaporate. Antigen-loaded MPs thus formed were collected, washed with distilled water, freeze-dried, and stored at −20 °C until use. Following the same procedures, FITCBmpB-loaded DAPI-labeled EMPs or TEMPs were prepared. Preparation of Coumarin 6 (C6)-Loaded MPs. In the case of C6-loaded MPs, C6 (5 mg) was dissolved into organic solution of polymer (100 mg) dissolved in dichloromethane (5 mL). An oil-in-water (O/W) was prepared by mixing the above polymer solution into PVA (1% w/v) following homogenizing method described before. Further processes were followed similar to the preparation of the antigen-loaded MPs, as described above.

Current oral vaccines are based on attenuated or inactivated bacteria or viruses. Although both formulations provide a high level of antigen exposure, their immunogenicity and stability still remain a problem.5 In particular, attenuated vaccines are highly reactogenic and controversial. Therefore, the use of a subunit or peptide-based vaccine is appealing because it obviates potential safety concerns.6 Although recombinant vaccines produced by recombinant DNA techniques are comparatively safer than traditional vaccines, they are often poorly immunogenic and usually require multiple doses and effective mucosal adjuvants.7,8 To counteract these problems, biodegradable polymeric microparticles (MPs) have been widely used as vaccine carriers/adjuvants.7,9−11 Particularly, thiolated polymers are applied as protein and peptide carriers for oral delivery because of their mucoadhesive nature.12,13 The mucoadhesive property is derived from the stable interaction between the reactive thiol groups of the polymers and the mucus glycoproteins in mucosal surfaces.14,15 Besides, thiolated polymers have been demonstrated to show a strong permeation enhancing effect for the uptake of peptide drugs from mucosal membranes.16 In this study, we synthesized a mucoadhesive vehicle to deliver a membrane protein of pathogenic intestinal spirochete Brachyspira hyodysenteriae (BmpB) as an oral vaccine, and we investigated the efficacy of the vehicle to induce both mucosal and cellular immunity in order to develop an efficient oral vaccine delivery system.

2. EXPERIMENTAL SECTION Materials. Eudragit L-100 was obtained from Röhm Pharma (Weiterstadt, Germany). Dimethyl sulfoxide (DMSO), N,N′dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), L-cysteine hydrochloride monohydrate, poly(vinyl alcohol) (PVA), Pluronic F-127, bovine serum albumin (BSA), dichloromethane, 4′,6-diamidino-2-phenyindole dilactate (DAPI), fluorescein isothiocyanate isomer I (FITC), and coumarin 6 (C6) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-glycine-PAG precast SDS gel was from Komabiotech (Seoul, Korea). Amersham ECL prime Western blotting detection reagent was obtained from GE healthcare (Buckinghamshire, UK). Horseradish peroxidase (HRP)conjugated goat antimouse IgA, IgG, IgG1, and IgG2a; and HRP-conjugated goat antirabbit IgG antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Isolation and Purification of Protein Antigens. BmpB and M cell-homing peptide fused BmpB (M-BmpB) proteins were purified as follows. Seed culture of E. coli harboring gene encoding for BmpB or M-BmpB protein17 was inoculated in LB broth (800 mL) supplemented with ampicillin (100 μg/mL) and incubated at 37 °C while shaking at 200 rpm for 4 h. The culture was induced with IPTG (1 mM) and incubated for 12 h more. E. coli cells were collected by centrifuging at 6000g for 10 min and washed with phosphate buffered saline (PBS) (200 mL) twice. The cells were resuspended in His-binding buffer (25 mL) and sonicated. The soluble lysate fraction was separated from the cell debris by centrifuging at 12000g for 30 min. His-tagged recombinant protein in the cell lysate was finally purified using Talon metal-affinity resin according to the manufacturer’s instructions (Clontech). Endotoxins were removed by Detoxi-gel endotoxin removing columns (Thermo Scientific Pierce, USA) according to the manufacturer’s instructions. The purity of the protein was observed in sodium dodecyl sulfate polyacrylamide gel (12%). The purified protein B


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Molecular Pharmaceutics Morphology Observation. Prior to observation of the morphology of MPs, the MPs were coated with gold using coating chamber (CT 1500 HF, Oxford Instruments Oxfordshire, UK). The coated MPs were observed through field-emission scanning electron microscope (FE-SEM; Supra 55VP; Carl Zeiss, Oberkochen, Germany). Average size of the microspheres was calculated from SEM images. Observation of Mucoadhesive Property of MPs. To evaluate the mucoadhesive property of the MPs to the intestine, C6-loaded MPs (1 mg/mL) were injected into the closed ileal loop of mouse intestine. After 1 h of retention, the closed ileal loop was excised and washed with cold PBS. The tissue samples were then fixed with 4% (v/v) paraformaldehyde and freezesectioned. Sections of the samples were stained with DAPI and observed through confocal laser scanning microscopy (CLSM) (Carl Zeiss LSM710, Carl Zeiss, Inc.). Determination of Loading Efficiency and Loading Content. Loading efficiency of the antigen was determined using the ratio of the actual amount of loaded antigen over the total amount of antigen used for the preparation of the MPs. Loading content was determined as follows. The MPs (2 mg) were dispersed into 1 mL of His-binding buffer and dichloromethane (1:3) mixture. The suspension was incubated in a water bath at 37 °C for 1 h with shaking. Following centrifugation, 200 μL of the supernatant was withdrawn, and the absorbance of protein was measured at 280 nm using Eppendorf BioPhotometer. A standard calibration curve was plotted to calculate the concentrations of the loaded or released proteins. Release of Proteins from the Protein-Loaded MPs. The protein-loaded MPs (2 mg/mL) were suspended in two physiological buffer conditions; simulated gastric fluid (pH 2.0) and simulated intestinal fluid (pH 7.2). The suspended MPs were agitated with 100 rpm at 37 °C to observe the release of proteins from the protein-loaded MPs with time. After a given time interval, the MPs were centrifuged at 6000g for 1 min, and the supernatant was withdrawn to measure the absorbance of released protein at 280 nm using Eppendorf BioPhotometer. The experiments were performed in triplicates. Structural and Functional Integrity of the Antigens. The structural integrity of protein antigens before and after loading into MPs was assessed by SDS-PAGE, Western blot, and circular dichroism (CD). The proteins were separated by a 4−20% SDS gel under reducing condition using XCell SureLock Mini-Cell (Life Technologies, USA) at 130 V for 2 h. After electrophoresis, the protein gel was electro-transferred to nitrocellulose membrane (Protran nitrocellulose membrane, Whatman, UK) using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, USA) at 10 V for 60 min. The membrane was then blocked with 5% skim milk in Trisbuffered saline-Tween (TBST) for 60 min at room temperature and then washed with TBST. The membrane was incubated with an antibody against BmpB on a shaker at 4 °C overnight, followed by washing with TBST. The membrane was further incubated with HRP-conjugated goat antirabbit IgG antibody in TBST at room temperature for 1 h. After washing with TBST, the membrane was treated with enhanced chemiluminescence detection system (GE Healthcare, UK) and exposed to Gel Doc XR system (BioRad, USA) to capture chemiluminescent signal on the Western blot. CD measurements were performed using a Chirascan-plus CD Spectrometer (Applied Photophysics Ltd., Leatherhead, UK). Far-UV CD spectra were measured in the range of 260−

200 nm in 1 nm steps and a 0.5 s sampling time at each wavelength, using Quartz cuvettes (0.1 cm path length). Cellular Uptake of Antigens through M Cells. To ascertain the efficiency of M cell homing peptide to induce the cellular uptake of antigens through M cells, an equivalent amount of FITC-labeled M-BmpB or BmpB, i.e., antigen with or without M cell homing peptide fusion, was injected into the closed ileal loop of mouse intestine. After 1 h of retention, the closed ileal loop was excised and washed with cold PBS. The tissue samples were then fixed with 4% (v/v) paraformaldehyde and freeze-sectioned. Sections of the samples were stained with DAPI and observed through CLSM. Uptake of Antigens Loaded MPs and/or Antigens through M Cells. To determine the cellular uptake of MPs and/or antigens through M cells in vivo, an equivalent amount of FITC-BmpB-loaded DAPI-labeled EMPs or TEMPs were delivered orally to individual mouse. After 8 h of delivery, the mice were sacrificed; intestines were cut and washed with PBS. The intestinal samples were then fixed with 4% (v/v) paraformaldehyde and freeze-sectioned. Sections of the samples were stained with rhodamine and observed through CLSM. The degree of uptake of protein antigens and MPs through M cells in the Peyer’s patch was done by using ImageJ v1.36b software (http://rsb.info.nih.gov/ij/). Measurement of Cytokine Induction in Response to Protein Antigens. The murine macrophage cell lines, RAW 264.7, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). The murine dendritic cell lines, JAWS II, were maintained in HyClone minimal essential medium alpha modification (Thermo Scientific) with 20% FBS and recombinant mouse granulocyte macrophage colony stimulating factor (5 ng/mL). The cell lines were treated with protein antigens (1, 5, and 10 μg/mL) and incubated at a given interval of time according to a method described previously15 to test the release of TNF-α and IL-6 cytokines from RAW 264.7 and JAWSII cells, respectively. The amounts of released cytokines were analyzed by using mouse TNF-α and IL-6 ELISA kits (Thermo Scientific) according to the manufacturer’s instructions. Oral Immunization. Female BALB/c mice, 6 weeks of age, were obtained from Samtako, Co. Ltd. (Osan, Korea). The mice, maintained under standard pathogen-free conditions, were provided with free access to food and water during the experiments. The experiments were performed in accordance with the guidelines for the care and use of laboratory animals under the approval of animal ethics committee at Seoul National University (SNU-130520-7). The mice were divided into five cohorts (five mice per cohort). Each mouse was given an oral gavage of an amount equivalent to 200 μg of protein in MPs suspended in PBS (200 μL). Each group received a total of six doses of the vaccines (two priming and four boosting) as shown in the Scheme 1. To monitor immune responses, serum and fecal samples were taken before immunization and 4 weeks after first immunization. The blood samples were centrifuged at 6000g for 10 min, and the serum was collected and stored at −70 °C until analysis for antibody levels. Similarly, fecal pellets from each mouse were soaked in PBS and incubated for 30 min at room temperature. The fecal samples were centrifuged at 6000g for 10 min, and supernatants were collected and analyzed for the presence of antigen-specific IgA. Samples were stored at −70 °C until analysis. Evaluation of Antibody Production. The induction of specific antibodies was measured by enzyme-linked immunoC


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were expressed as mean ± SD. The statistical significance of the differences was calculated by two-tailed unpaired Student’s t test. Statistical significance is denoted by *P < 0.05, **P < 0.01, and ***P < 0.001.

Scheme 1. Schematic Representation of Oral Immunization Schedulea

3. RESULTS Preparation of Protein Antigens. Recent advances in recombinant DNA technology have made it easy to express, isolate, and purify the desired protein fragment of pathogen in order to utilize it as a subunit vaccine for oral delivery. Therefore, the gene encoding for membrane protein (BmpB) of intestinal pathogen B. hyodysenteriae was amplified and cloned into an expression vector by recombinant DNA technique. Similarly, gene encoding for M cell-homing peptide was fused with the BmpB gene at its N-terminal end and cloned into the expression vector in order to express M-BmpB protein. The clones were transformed into E. coli. Individual clone of E. coli harboring gene encoding for BmpB or M-BmpB protein was cultured, induced, and incubated for protein expression as described in the Experimental Section. The proteins were then isolated and purified by metal affinity chromatography. LPS contamination in the protein isolates were removed by Detoxigel endotoxin removing columns. The purified proteins were dialyzed prior to lyophilization. Finally, the size and the purity of the proteins were analyzed by SDS-PAGE (Figure 1).

a

Each group of mice received a total of six doses of the vaccines (two priming and four boosting). To monitor immune responses, serum and fecal samples were taken before immunization and 4 weeks after first immunization.

sorbent assay (ELISA) using BD OptEIA ELISA kit (BD Biosciences, California, USA) according to the manufacturer’s instructions. Briefly, microwell plates were coated with BmpB antigen (25 μg/mL) diluted in coating buffer (100 μL) and incubated at 4 °C overnight. The plates were washed three times with wash buffer and blocked with assay diluent (200 μL) for 1 h. The plates were washed again. Serum (1:2000) or fecal samples (1:1000) diluted in assay diluent (100 μL) were loaded into the plate and incubated for 2 h. Following washes, each sample was incubated with HRP-conjugated goat antimouse antibody (100 μL) for 1 h. After several washings, the samples were reacted with substrate reagent (100 μL) for 30 min in the dark, and the reaction was stopped with stop solution (50 μL). Finally, the absorbance of the samples was measured at 450 nm using an Infinite 200 PRO multimode reader (Tecan, Switzerland). A standard calibration curve was plotted to calculate the concentrations of the antibodies (fold increase) using the absorbance value in ELISA. End point titers were measured as the highest serum dilution that resulted in an absorbance value (OD). Flow Cytometric Detection of Antigen-Activated CD8+ and CD4+ T Cells in Spleen. Spleens from mice were aseptically collected 2 weeks after the final immunization, and the spleens were homogenized on a 70 μm cell strainer with a grinder in RPMI supplemented with 10% FBS. The cells were collected by centrifugation and incubated with ACK lysis buffer for 5 min to lyse the RBCs. Finally, the cells were pelleted and then suspended in RPMI with 10% FBS. A 96-well round-bottomed plate was seeded with 2 × 106 cells/well and stimulated with antigen for 5 days. After centrifugation, cells were washed twice with PBS and stained with cell-specific (CD8+ and CD4+) antibodies (BD Pharmingen) and analyzed by FACS. Flow Cytometric Detection of Intracellular IFN-γ and IL-4 in Splenocytes. Cells from spleens of immunized mice were isolated as described above. Splenocytes were seeded (2 × 106 cells/well) in 96-well round-bottomed plates and stimulated with cell stimulation cocktail (plus protein transport inhibitor) (eBioscience) for 16 h according to the manufacturer’s protocol. The cells were collected by centrifugation and washed twice with PBS. The cells were fixed with formalin, permeablized with cytofix/cytoperm solution (BD Biosciences), and stained with intracellular cytokine-specific (IFN-γ and IL-4) antibodies (BD Pharmingen). Finally, the cells were sorted by FACS. Statistical Analysis. Unless otherwise stated, independent experiments were run at least in triplicate (n = 3). The results

Figure 1. Analysis of size and purity of the proteins by SDS-PAGE. The calculated size of BmpB and M-BmpB protein is 28 and 29 kDa, respectively.

Preparation of Mucoadhesive Vehicles. Mucosal vehicles offer the advantages of not only being carriers or adjuvants but they also show strong permeation effect for the delivery of therapeutic agents through the intestinal mucosa. Due to these advantages, a mucoadhesive polymer was synthesized by thiolation of Eudragit as described in the Experimental Section. Eudragit is a pH-sensitive polymer that dissolves at pH > 6, and therefore, it is used for enteric coating to protect protein drugs from the acidic environment in stomach. Incorporation of thiol group in Eudragit was confirmed by NMR (Figure 2). A standard Ellman’s test was used to verify the presence of thiol group in TE. Hence, the composition of the cysteine in TE was 3.42 wt %. Preparation of Protein-Loaded MPs. The proteins for oral vaccination must be protected from the harsh environment of GI tract. Therefore, the proteins are usually concealed with pH-sensitive polymers to protect them from enzymatic degradation and acidic pH in GI tract. In this study, the D


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Figure 2. Confirmation of the synthesis of thiolated Eudragit by 1H NMR (DMSO-d6, 600 MHz). Conjugation of cysteine group in Eudragit is depicted by the protons of −NH−C(H) of cysteine and −O−C(H3) of Eudragit in circles.

protein antigens were loaded into MPs using conventional double emulsion method. As shown in Figure 3, the MPs have spherical shapes with smooth surface. The sizes of the particles were around 1−10 μm. It is well-known that phagocytotic cells such as macrophages and M cells are able to ingest MPs with diameters between 1 and 5 μm,19 and therefore, targeting these cells in the GI tract allows the use of larger particles. The loading content and loading efficiency of protein antigens in EMPs and TEMPs are shown in Table 1. Stability Test of Protein Antigens in Protein-Loaded MPs. Unlike chemical drugs, proteins fold into secondary, tertiary, and quaternary structures based on the intramolecular bonding between functional groups. Loss or breakage of these structures may lead to loss of protein activity.20 To retain their intrinsic property, it is necessary to maintain the stability of protein antigens during the loading into MPs. Therefore, the structural integrity of the protein antigens loaded into the MPs was evaluated by releasing the proteins from the protein-loaded MPs and analyzed by SDS-PAGE (Figure 4A) and Western blot (Figure 4B). As shown in Figure 4A,B, the proteins released from the protein-loaded MPs had similar size to native proteins (Figure 1) in SDS gel. Similarly, the binding of antibodies to the protein antigens in Western blot analysis confirmed that the protein antigens retain their structures without alterations during the loading into the MPs. Furthermore, the structural integrity of protein after releasing from MPs was confirmed by CD. CD spectra of the released proteins from the MPs are shown in Figure 4C,D. The far UVCD spectra were identical with molar ellipticity minima at 225 and 215 nm indicating that β- sheet structure of proteins released from MPs remained intact. Mucoadhesive Property of MPs. During oral delivery, the first and foremost requirement of an antigen delivery vehicle is mucoadhesive property to retain and release its contents at the mucosal sites as mucosal immune responses are elicited by direct contact between the antigen and the mucosa.21 Therefore, the mucoadhesive property of the MPs was evaluated by ileal loop assay in mouse intestine ex vivo as described in the Experimental Section. Immunohistochemical analysis of frozen sections of the small intestine demonstrated that the number of TEMPs adhered to the intestine was higher than the EMPs (Figure 5). The results are consistent with our previous experiments of mucoadhesive study of these MPs on

Figure 3. Analysis of shape and size of MPs. The MPs were prepared by double emulsion method to load the antigens or coumarin 6. Panels A−D are analyzed by SEM (scale bar: 2 μm), while panels E−H are analyzed by CLSM (scale bar: 5 μm). (A) M-BmpB-TEMPs; (B) BmpB-TEMPs; (C) M-BmpB-EMPs; (D) BmpB-EMPs; (E) C6TEMPs; (F) C6-EMPs; (G) FITC-BmpB-DAPI-EMPs; (H) FITCBmpB-DAPI-TEMPs.

Table 1. Loading Content and Loading Efficiency of Antigen-Loaded MPs microparticles (MPs) M-BmpB-TEMPs M-BmpB-EMPs BmpB-TEMPs BmpB-EMPs

loading content (wt %) 16 14 18 13

± ± ± ±

2.1 1.3 0.5 2.3

loading efficiency (%) 75.2 69.1 73.1 66.1

± ± ± ±

1.2 1.7 0.6 3.1

pork intestine in vitro, where 82% of the TEMPs of the initial feeding adhered in the porcine intestine, while the adherence capacity of EMPs was only the half of the TEMPs.22 E


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Figure 5. Immunohistochemical analysis of the mucoadhesive property of the MPs in small intestine by CLSM. To validate the mucoadhesive property of the MPs to the intestine, C6-loaded MPs (1 mg/mL) were injected into the closed ileal loop of mouse intestine. After 1 h of retention, the closed ileal loop was excised and washed with cold PBS. The tissue samples were then fixed with 4% (v/v) paraformaldehyde and freeze-sectioned. Sections of the samples were stained with DAPI and observed through CLSM. (A) C6-loaded TEMPs and (B) C6-loaded EMPs.

Figure 4. Analysis of the proteins released from the protein-loaded MPs. (A) SDS-PAGE and (B) Western blot. The protein-loaded MPs (2 mg/mL) were suspended in His-binding buffer and sonicated to release the proteins. The proteins were isolated and observed in SDS gel. The proteins in the gel was transferred to nitrocellulose membrane and incubated with an antibody against the protein antigens. The membrane was further incubated with HRP-conjugated goat antirabbit IgG antibody and treated with chemiluminescence detection system to capture chemiluminescent signal on the Western blot. (1) BmpBTEMPs; (2) BmpB-EMPs; (3) Marker; (4) M-BmpB-TEMPs; (5) MBmpB-EMPs. (C,D) Confirmation of structural integrity of the proteins released from the protein-loaded MPs by circular dichroism.

TEMPs increased with incubation time, and their release was higher at pH 7.2 compared to pH 2.0 due to pH-sensitive nature of TE (dissolution pH > 7) that allows antigen release in distal region of small intestine where M cells are abundant. Notably, the release of M-BmpB from the protein-loaded TEMPs was slightly higher than the release of BmpB at both pH conditions. Cellular Uptake of Antigens through M Cells. The released antigens in the mucosal sites enter into the common or specialized epithelial cells (M cells) to enter the Peyer’s patch for immune responses. To confirm and compare the ability of antigens with or without M cell homing peptide to enter the M cells, the antigens were labeled with FITC. The cellular uptake of these FITC-labeled antigens was evaluated by ileal loop assay

Release of Protein Antigens from Protein-Loaded MPs. After successful retention in the gut, the antigen delivery vehicle should be able to release its contents into the mucosal sites in intestine. Most importantly, the vehicle should be refrained from gastric pH as it passes from stomach to intestine. Therefore, the release of protein antigens from protein-loaded MPs was tested in two different pH conditions (Figure 6). The results demonstrated that the release of protein antigens from F


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Figure 7. Localization of M-BmpB and BmpB in Peyer’s patch of mouse small intestine. FITC-labeled antigens were injected into closed ileal loops and their localization was monitored under fluorescencemicroscopy. The green fluorescent signals of FITC-labeled M-BmpB on the FAE and PP regions are higher due to the interaction of M cell homing peptide with the M cells in FAE.

TEMPs in subepithelial dome under the FAE (Figure 8). The result also highlighted the higher efficiency of mucoadhesive MPs to enter from the mucus layer of intestine to inner layer of Peyer’s patches. When quantified by imageJ analysis, the transcytosis of TEMPs was 2.1-fold higher than that of EMPs. Immune Response to Protein Antigens by Antigen Presenting Cells. After transcytosis of antigens through M cells in Peyer’s patch, the antigens are phagocytosed by antigenpresenting cells (dendritic cells and macrophages), which produce specific cytokines to activate T cells or B cells that will result in a successful immune response depending upon the nature of antigens.23,24 To ascertain that the recombinant antigens could induce expression of cytokines associated with T cell or B cell proliferation, BmpB and M-BmpB antigens were treated with RAW 264.7 (macrophages) and JAWSII (dendritic cells). The immune responses from these cells after the treatment of recombinant antigens were evaluated by measuring the amount of cytokine secretions as described in the Experimental Section. It was found that both antigens were equally capable of stimulating IL-6 (B cell proliferation) from JAWSII cells, while the secretion of TNF-α (T cell proliferation) from RAW 264.7 was higher with M-BmpB than BmpB (Figure 9). Moreover, the secretion of both cytokines from these cells increased significantly with time. Oral Immunization and Immune Responses. Groups of mice were vaccinated three times orally with the protein antigens (BmpB or M-BmpB) using vehicles with or without mucoadhesive property. The vaccine schedule is shown in the Scheme 1. The successful delivery of oral vaccine kicks off with the stimulation of secretory IgA (sIgA) antibodies in mucosal sites. To determine the level of sIgA production after immunization, excreta samples from the intestine were collected and analyzed by ELISA. The results demonstrated that the oral delivery of antigens with TEMPs showed significantly higher level of BmpB-specific sIgA antibody responses in feces compared with those delivered with EMPs or PBS (Figure 10A). In particular, the mice treated with M-BmpB-TEMPs showed high level of BmpB-specific sIgA antibody responses compared to the mice treated with BmpB-TEMPs (P < 0.05). Overall, 4 weeks after their first immunization, the mice treated with M-BmpBTEMPs and BmpB-TEMPs displayed 1.52- and 1.68-fold high

Figure 6. Analysis of antigen release test from protein-loaded MPs at different pH conditions. The protein-loaded MPs (2 mg/mL) were suspended in two physiological buffer conditions: simulated gastric fluid (pH 2.0) and simulated intestinal fluid (pH 7.2). The suspended MPs were agitated with 100 rpm at 37 °C. After a given time interval, the amount of released proteins was calculated by taking absorbance at 280 nm. The experiments were performed in triplicates. (A) Antigens released from TEMPs and (B) antigens released from EMPs.

in mouse intestine ex vivo as described in the Experimental Section. Immunohistochemical analysis of frozen sections of Peyer’s patch region demonstrated that the adherence of FITClabeled M-BmpB in FAE region was higher compared to FITClabeled BmpB (Figure 7). As a consequence, the number of FITC-labeled M-BmpB entered into Peyer’s patch was higher. The result is a clear indication of higher transcytosis of M− BmpB antigens through M cells due to interaction of M cell homing peptide in M-BmpB with the M cells in the FAE region. When quantified by imageJ analysis, the transcytosis of M-BmpB through M cells was 1.6-fold higher than that of BmpB. Uptake of MPs through M Cells in Vivo. We also examined the transcytosis of MPs and/or protein antigens through M cells. Eight hours after oral delivery of FITC-labeled antigen loaded DAPI-labeled EMPs or TEMPs in mouse, the intestine samples were isolated and freeze-sectioned as described in the Experimental Section. Immunohistochemical analysis of frozen sections of Peyer’s patch region revealed that the MPs could enter through M cells. The amount of TEMPs was relatively higher in Peyer’s patch with greater abundance of G


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Figure 8. Simultaneous uptake of antigen and MPs in Peyer’s patch of mouse small intestine. FITC-labeled BmpB-loaded DAPI-labeled TEMPs or EMPs were orally administered to mice and their distribution in Peyer’s patch was monitored after 8 h by immunohistochemical analysis using confocal microscopy. The blue fluorescent dots represent the MPs and the green fluorescent signals represent the antigen. Follicle-associated epithelium (FAE), subepithelial dome (SED), and Peyer’s patch (PP).

surfaces, serum-derived immunoglobulin G (IgG) also contributes significantly to immune defense.25 The BmpBspecific IgG antibody responses in serum after immunization are shown in Figure 10B. The mice treated with M-BmpBTEMPs showed the highest level of BmpB-specific IgG response. As in the case of sIgA responses, the level of IgG responses using TEMPs was significantly higher than EMPs or PBS. Besides, the IgG production was clearly higher in response to the M-BmpB antigens as compared to their BmpB counterparts. All together, the delivery of BmpB and MBmpB with TEMPs, when compared to their EMPs counterparts, showed 1.98- and 2.33-fold high level of IgG production, respectively. Additionally, the serum isotype levels of IgG1 and IgG2a were determined to reveal the type of immune response. While the production of IgG1 isotype (indicative of humoral immune response) is associated with a Th2-type response, IgG2a isotype (indicative of cellular immune responses) is associated with a Th1-type response. The ratio of IgG2a/IgG1 is used to indicate the Th1 or Th2 bias of the generated immune response.26 The BmpB-specific IgG1 and IgG2a antibody responses in serum after immunization are shown in Figure 10C. Consistent to the previous results, the mice treated with M-BmpB-TEMPs showed higher levels of both IgG1 and IgG2a antibody responses than other MPs treated groups. Strikingly, delivery of M-BmpB induced higher IgG2a response while BmpB induced higher IgG1 response. Altogether, the results of the assay indicated that the antigens in these MPs have potential to induce both Th1 and Th2 immune responses in vivo. Stimulation of CD8+ and CD4+ T Cells in the Spleen of Immunized Mice. The final reaction of effective antigen delivery will result in the accumulation of memory T cells as adaptive immunity for future protection.27 Generally, initiation of cellular or humoral response to antigens is largely dependent on the number of CD8+ or CD4+ T cell populations. To determine the efficacy of antigen delivered by MPs to induce specific T cell response, splenocytes were isolated from mice immunized with antigen followed by stimulation with the respective antigen in vitro and stained by CD8+ or CD4+ T cell specific antibody. Antigen-specific CD8+ or CD4+ T cell populations were subsequently analyzed by flow cytometry. The results demonstrated the dominance of CD8+ T cell subsets over CD4+ T cell populations in all the immunized mice (Figure 11). The mice immunized with TE(M-BmpB) had the highest amount of CD8+ T cells (72.4%), whereas the

Figure 9. Secretion of cytokines from immune cells in response to antigens. (A,B) Cell lines were treated with protein antigens (1 μg/ mL). Secretion of cytokines, such as TNF-α from RAW 264.7 (A) and IL-6 from JAWSII cells (B) were evaluated in a time-dependent manner.

levels of sIgA antibody production respectively than their EMPs counterparts. While sIgA antibodies are the predominant immunoglobulins to display the adaptive humoral immune defense at mucosal H


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Figure 10. Analysis of BmpB-specific antibody responses in serum and feces after oral immunization. Each mouse was given an oral gavage of an amount equivalent to 200 μg of protein in MPs suspended in PBS (200 μL). Each group received a total of six doses of the vaccines. To monitor immune responses, serum and fecal samples were taken before immunization and 4 weeks after first immunization. (A) IgA titer; (B) IgG titer; (C) IgG2a and IgG1 titer.

groups. Remarkably, we observed the higher induction of IFN-γ production by M-BmpB than BmpB in both EMPs and TEMPs consistent with the higher induction of CD8+ T cells by M-BmpB than BmpB.

mice immunized with TE(BmpB) demonstrated comparably lower amount of CD8+ T cells (67.6%). However, CD4+ T cells (27.3%) of TE(M-BmpB) group was lower than CD4+ T cells (31.2%) of TE(BmpB) group. While the amount of both CD8+ and CD4+ T cells in E (M-BmpB) group was slightly lower than TE(M-BmpB) group, the amount of both CD8+ and CD4+ T cells in E(BmpB) group was drastically low. Detection of Intracellular IFN-γ and IL-4 in Splenocytes. The intracellular detection of IFN-γ and IL-4 in splenocytes allows the frequency of cells producing the respective cytokine and thus can evaluate the persistence of cellular or humoral immune responses. To determine the intracellular production of IFN-γ and IL-4, splenocytes were isolated from mice immunized with antigen and stimulated in vitro with a cell stimulation cocktail plus protein transport inhibitor. The cells were subsequently stained with intracellular cytokine antibodies followed by flow cytometric analysis. We found that administration of TE(M-BmpB) resulted in a marked increase of IFN-γ+ splenocytes along with the highest percentage of IL-4+ splenocytes (Figure 12). While the magnitude of IFN-γ+ splenocytes was in the order of TE(BmpB) > E(M-BmpB) > E(BmpB), the magnitude of IL4+ splenocytes was same among these groups. However, the percentage of IFN-γ+IL-4+ splenocytes varied among the

4. DISCUSSION Indeed, recombinant DNA technology has made tremendous breakthrough in the discovery of various vaccines or therapeutic antigens. With the advent of this technology, significant progress has also been made in the antigen delivery with the use of a fragment of the pathogen to trigger specific immune responses, rather than presenting a whole organism to the immune system. Examples include the subunit vaccine against hepatitis B virus, which is composed of only the surface proteins of the virus. These vaccines, prepared from the viral/ microbial proteins or their fragments or the nucleic acid sequences, have been attractive because of their stability, noninfectious nature, homogeneity, and cost-effectiveness. Swine dysentery is a contagious mucohemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete B. hyodysenteriae. Recently, a surface lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies I


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Figure 12. Flow cytometric detection of intracellular IFN-γ+ and IL-4+ splenocytes. Spleens were collected aseptically from the antigen immunized mice 2 weeks after final immunization. Splenocytes were stimulated with cell stimulation cocktail (plus protein transport inhibitors) for 16 h. Following restimulation, cells were fixed, permeabilized, stained with IFN-γ and IL-4 specific antibodies, and analyzed by FACS. The percentage of positive cells is indicated.

study. For selective targeting of M cells, we selected an M celltargeting ligand by phage display method, and the ligand facilitated the entry of chitosan nanoparticles across the M cells. Similar M cell-targeting peptide ligand (Co1) not only promoted antigen delivery through M cells but also induces antigen-specific immune responses in mucosal vaccination.29 Particularly, oral delivery of Co1-fused antigen induced efficient systemic and mucosal immune responses against the pathogenic antigen.30 In accordance with these consequences, we fused M cell-homing peptide to BmpB to obtain M-BmpB protein by recombinant DNA technique. Although recombinant DNA technology have made it easy to express, isolate, and purify the required proteins or antigens in order to use as oral vaccines, oral delivery of these proteins in GI tract encounters two major barriers; mainly, enzymatic barrier that is responsible for rapid degradation of proteins, while absorption barrier is responsible for poor absorption of proteins from GI membranes. Usually, these barriers are overcome by using enzyme inhibitors31 and permeation enhancers.32 In this study, we modified Eudragit by thiolation to synthesize TE polymer that has dual nature, in part being pH-sensitive and in part mucoadhesive. While the pH-sensitive part protects the proteins from enzymatic degradation and acidic pH in GI tract, the mucoadhesive property enhances the retention of microparticulate antigens through GI membranes. Moreover, the M cell-homing peptide is introduced to our model antigen to facilitate the transcytosis of antigens through M cells. It is well-known that structural properties of proteins are closely correlated to their biological activity and stability. It is well documented that various physical, thermal, or chemical activities during manufacture, storage, and formulation can cause structural changes to protein antigens leading to aggregation or degradation.33,34 That is the reason why the

Figure 11. (A) Flow cytometric detection of CD8+ and CD4+ T cells in spleen. Spleens were collected aseptically from the antigen immunized mice 2 weeks after final immunization. Splenocytes were restimulated with antigen and corresponding T cell populations were analyzed by FACS. The percentage of each population is indicated. (B) Comparison between CD8+ and CD4+ T cells in spleen.

recognizing the native BmpB of B. hyodysenteriae.28 Due to its successful application, we selected the recombinant BmpB protein as a protein antigen model for oral delivery in this J


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ing adherence and entry into epithelial cell transport pathways.35 To evaluate the effect of vehicle on the delivery of antigen, we delivered the same antigen by two different vehicles. Interestingly, the production of BmpB-specific mucosal IgA antibodies was relatively higher when antigens were delivered through TEMPs than EMPs. We observed similar tendency of high serum IgG antibodies production using TEMPs compared to EMPs. Consistent to our results, mucoadhesive carbohydrate biopolymers such as chitosan and gellan have been demonstrated to enhance the local and systemic antibody response to mucosally administered vaccine antigens.38 It seems that the delivery of antigens with mucoadhesive vehicle retains the antigens on mucosal surfaces for longer duration increasing the possibility of antigen uptake and presentation processes. Accordingly, the retention of vaccine antigens on mucosal surfaces by delivery with mucoadhesive chitosan has shown to increase antigen uptake and immune responses.39 More specifically, the relative contribution of cellular and humoral immune can be evaluated by dissecting the relative populations of CD8+ and CD4+ T cells and B cells in the spleen of immunized mice. During infection, antigen presenting cells specifically bind to CD8+ or CD4+ T cells, depending upon the antigens presentation, to initiate the particular immune responses.40 Basically, CD8+ T cells are very effective in direct lysis of infected cells by producing a range of cytokines, such as IFN-γ and TNF-α, while CD4+ T cells produce an array of cytokines that stimulate cells of innate immune system. Based on their cytokine production, CD4+ T cells are generally divided into two polarized subsets, Th1 and Th2 cells.41 Th1 cells primarily produce IFN-γ and are critical for the immune responses to intracellular bacterial and viral infections. Thus, these CD4+ T cells are critical constituents of the cell-mediated immune responses. Conversely, Th2 cells predominantly produce IL-4 cytokines triggering humoral (antibody) responses critical in the defense against extracellular pathogens.42 While Th2 cells are associated with IgG1 production in mice, Th1 cells induce IgG2a. Therefore, the nature of immune response, whether humoral or cell-mediated, can be also determined by analyzing the specific IgG isotypes. Our data suggested that both EMPs and TEMPs are capable of stimulating strong CD8+ T cells with enhanced production of IFN-γ indicating the higher induction of cell-mediated immunity with our delivery systems. More specifically, MPs loaded with M-BmpB induced higher antigen-specific antibody responses of IgG2a isotype as compared to IgG1, indicating a Th1 type antibody dominated response. Conversely, MPs loaded with BmpB induced higher antibody responses of IgG1 isotype than IgG2a, indicating a Th2 type antibody dominated response. The higher amount of TNF-α from macrophages and greater amount of IFN-γ from splenocytes induced by M-BmpB are consistent with Th1 dominant response. Another possible explanation for higher magnitude of Th1 responses by MBmpB might be due to optimal dose of vaccine antigen internalized through the M cells for the particular immune reaction. The consequence needs further study for verification. In summary, the elevated level of mucosal as well as cellular immunity is attributed to synergistic effect of M cell-homing peptide and mucoadhesive vehicle. As related to particlemediated mucosal vaccine delivery, mucoadhesive particles can enhance mucosal residence in the respiratory or intestinal mucosa.19 This observation is also supported by our experiment that TEMPs largely bind to intestinal surface, in comparison to

protein antigens in the MPs must retain their structural integrity and hence immunogenicity. Both protein antigens (BmpB and M-BmpB) had their molecular weight intact in the MPs, as confirmed by SDS-PAGE, while Western blot analysis demonstrated the binding ability of these protein antigens with the antibodies used for the detection in the same way that native antigens could bind. Further, CD analysis confirmed the retention of similar secondary structures of the proteins in the respective MPs. Thus, we confirmed the structural integrity of these protein antigens in the MPs. To ascertain whether the recombinant antigens after phagocytosis by antigen presenting cells could induce cytokines production to facilitate downstream immune responses (T cell or B cell activation),35 we treated BmpB and M-BmpB antigens independently to the antigen presenting model cell lines. The cytokine assay elucidated that both antigens are equally immunogenic to induce the cytokines, IL-6 from JAWSII cells and TNF-a from RAW 264.7 cells. It is well-documented that IL-6 is a B cell differentiation factor that induces B cell proliferation leading to humoral immune responses, whereas TNF-α induces T cell proliferation that increases the capacity of macrophages to phagocytose and kill the pathogens through cellular immune responses.36,37 It was therefore expected that these antigens would evoke the effective in vivo immune responses after oral immunization to the same extent as shown in their in vitro activity. During oral delivery of protein vaccine through GI tract, the MPs prevented protein release in acidic pH in stomach due to pH-sensitive nature but partially released the proteins in ileum (pH ≥ 7.2), which did not affect the protein integrity. Considering the abundance of M cells in ileum, the released antigens from MPs would have easy access to enter Peyer’s patch through M cells. Moreover, we demonstrated that the M cell targeting peptide conjugated to antigen has high affinity to enter M cells than only antigen (Figure 7). To further determine the simultaneous uptake of MPs and antigens through M cells in vivo, FITC-labeled antigen in DAPI-labeled MPs were orally delivered to mice. After 8 h of delivery, Peyer’s patches were isolated, freeze-sectioned, and observed through confocal microscopy. As shown in Figure 5, the MPs were spherical particles in the lumen while FITC-labeled antigens appeared diffused in the images after entering into Peyer’s patch (Figure 7). Since both MPs and antigens entered into Peyer’s patches after oral delivery, the confocal images showed both particle-like and dispersed fluorescent signals of microparticulate antigens (Figure 8). Importantly, this experiment confirmed the combinatorial delivery of antigen and MPs to Peyer’s patches that would exert synergistic effect to evoke the potential immune response against the antigen. The main goal of this study was to investigate the differential immune responses of the protein antigens with or without M cell-homing peptide using oral delivery vehicles with or without mucoadhesive property. Therefore, we delivered the antigenloaded vehicles to mice through oral administration and evaluated the antigen-specific mucosal IgA and serum IgG responses. Oral delivery of M-BmpB through TEMPs and EMPs both induced higher antigen-specific antibody production compared to the oral delivery of BmpB through respective vehicles. This result clearly elicited a connection between M cell-homing peptide fused antigen and higher uptake by M cells to induce the antibody response. The coupling of antigen with proteins that themselves are adherent to epithelial surfaces has enhanced mucosal immune responses, presumably by promotK


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(3) Kraehenbuhl, J. P.; Neutra, M. R. Epithelial M cells: Differentiation and function. Annu. Rev. Cell Dev. Biol. 2000, 16, 301−332. (4) Yoo, M. K.; Kang, S. K.; Choi, J. H.; Park, I. K.; Na, H. S.; Lee, H. C.; Kim, E. B.; Lee, N. K.; Nah, J. W.; Choi, Y. J.; Cho, C. S. Targeted delivery of chitosan nanoparticles to Peyer’s patch using M cellhoming peptide selected by phage display technique. Biomaterials 2010, 31 (30), 7738−7747. (5) De Smet, R.; Demoor, T.; Verschuere, S.; Dullaers, M.; Ostroff, G. R.; Leclercq, G.; Allais, L.; Pilette, C.; Dierendonck, M.; De Geest, B. G.; Cuvelier, C. A. beta-Glucan microparticles are good candidates for mucosal antigen delivery in oral vaccination. J. Controlled Release 2013, 172 (3), 671−8. (6) Poland, G. A.; Neff, J. M. Smallpox vaccine: problems and prospects. Immunol. Allergy Clin. North Am. 2003, 23 (4), 731−43. (7) O’Hagan, D. T. Recent advances in vaccine adjuvants for systemic and mucosal administration. J. Pharm. Pharmacol. 1998, 50 (1), 1−10. (8) Lycke, N. Recent progress in mucosal vaccine development: potential and limitations. Nat. Rev. Immunol. 2012, 12 (8), 592−605. (9) Singh, M.; O’Hagan, D. The preparation and characterization of polymeric antigen delivery systems for oral administration. Adv. Drug Delivery Rev. 1998, 34 (2−3), 285−304. (10) Rajapaksa, T. E.; Lo, D. D. Microencapsulation of Vaccine Antigens and Adjuvants for Mucosal Targeting. Current Immunology Reviews 2010, 6 (1), 29−37. (11) Liu, Y.; Yin, Y.; Wang, L. Y.; Zhang, W. F.; Chen, X. M.; Yang, X. X.; Xu, J. J.; Ma, G. H. Surface hydrophobicity of microparticles modulates adjuvanticity. J. Mater. Chem. B 2013, 1 (32), 3888−3896. (12) Bernkop-Schnurch, A.; Steininger, S. Synthesis and characterisation of mucoadhesive thiolated polymers. Int. J. Pharm. 2000, 194 (2), 239−247. (13) Bernkop-Schnurch, A. Thiomers: A new generation of mucoadhesive polymers. Adv. Drug Delivery Rev. 2005, 57 (11), 1569−1582. (14) Leitner, V. M.; Walker, G. F.; Bernkop-Schnurch, A. Thiolated polymers: evidence for the formation of disulphide bonds with mucus glycoproteins. Eur. J. Pharm. Biopharm. 2003, 56 (2), 207−214. (15) Lee, W. J.; Cha, S.; Shin, M.; Islam, M. A.; Cho, C. S.; Yoo, H. S. Induction of Th1 polarized immune responses by thiolated Eudragitcoated F4 and F18 fimbriae of enterotoxigenic Escherichia coli. Eur. J. Pharm. Biopharm. 2011, 79 (2), 226−231. (16) Bernkop-Schnurch, A.; Clausen, A. E. Biomembrane permeability of peptides: strategies to improve their mucosal uptake. MiniRev. Med. Chem. 2002, 2 (4), 295−305. (17) Singh, B.; Maharjan, S.; Jiang, T.; Kang, S. K.; Choi, Y. J.; Cho, C. S. Attuning hydroxypropyl methylcellulose phthalate to oral delivery vehicle for effective and selective delivery of protein vaccine in ileum. Biomaterials 2015, 59, 144−159. (18) Quan, J. S.; Jiang, H. L.; Kim, E. M.; Jeong, H. J.; Choi, Y. J.; Guo, D. D.; Yoo, M. K.; Lee, H. G.; Cho, C. S. pH-sensitive and mucoadhesive thiolated Eudragit-coated chitosan microspheres. Int. J. Pharm. 2008, 359 (1−2), 205−10. (19) Chadwick, S.; Kriegel, C.; Amiji, M. Nanotechnology solutions for mucosal immunization. Adv. Drug Delivery Rev. 2010, 62 (4−5), 394−407. (20) Kammona, O.; Kiparissides, C. Recent advances in nanocarrierbased mucosal delivery of biomolecules. J. Controlled Release 2012, 161 (3), 781−94. (21) van Ginkel, F. W.; Nguyen, H. H.; McGhee, J. R. Vaccines for mucosal immunity to combat emerging infectious diseases. Emerging Infect. Dis. 2000, 6 (2), 123−32. (22) Singh, B.; Jiang, T.; Kim, Y. K.; Kang, S. K.; Choi, Y. J.; Cho, C. S. Release and Cytokine Production of BmpB from BmpB-Loaded pHSensitive and Mucoadhesive Thiolated Eudragit Microspheres. J. Nanosci. Nanotechnol. 2015, 15 (1), 606−610. (23) Russell-Jones, G. Oral vaccine delivery. J. Controlled Release 2000, 65 (1), 49−54.

EMPs, extending the period of antigen release and delivery. Therefore, a prolonged period of antigen delivery due to mucoadhesive property and a greater amount of total dose absorbed through M cells due to M cell homing peptide in antigen could be attributed for the elevated production of antibodies using mucoadhesive MPs. Although the mechanism explaining the different behaviors of these mucoadhesive particles is a subject of study, the increased uptake and immune response resulting from the more mucoadhesive particles may be related to the ability of these particles to be transported to the underlying organized lymphoid follicles via transcellular and paracellular routes in addition to M cells uptake. These variations have important implications for future use in the development of polymeric formulations as well as vaccine administration route.

5. CONCLUSIONS To date, oral vaccines are less successful due to low bioavailability, no targeting specificity, and poor immunogenicity of the delivered antigens. These circumstances necessitate exploring novel ways of vaccine delivery to enhance immune response and efficacy of the vaccines. In this study, a combinatorial approach of antigen delivery with TE vehicle through oral route in mice succeeded to generate both mucosal and systemic immune response against the antigen. Moreover, this approach holds a great promise as a delivery system to achieve a balanced Th1/Th2 immune response fundamental for animal’s protection against pathogen infection. Future efficacy studies are needed to evaluate whether and/or how BmpB vaccine with mucoadhesive vehicle confer protection against swine infection.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: (82)-2-880-4807. Fax: (82)-2875-7340. *E-mail: [email protected]. Tel: (82)-2-880-4868. Fax: (82)-2875-2494. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0029416) and Animal Disease Management Technology Development (No. 313014-3), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. S.M. is supported by Brain Korea 21 Program for Leading Universities & Students (BK21 PLUS), South Korea. We also acknowledge National Instrumental Center for Environmental Management (NICEM) and National Center for Inter-University Research Facilities (NCIRF) of Seoul National University, South Korea.

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REFERENCES

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Combinatorial Approach of Antigen Delivery Using M Cell-Homing

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