Send Orders for Reprints to reprints@benthamscience.net Current Drug Therapy, 2014, 9, 9-20 9

Mucosal Delivery of Vaccine by M Cell Targeting Strategies

Tae-Eun Park1, Bijay Singh1, Sushila Maharjan1, Tao Jiang1, So-Yeon Yoon1, Sang-Kee Kang2, Jin-Duck Bok2, Yun-Jaie Choi1,2,* and Chong-Su Cho1,*

1Department of Agricultural Biotechnology & Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea; 2Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchanggun, Gangwondo 232-916, South Korea

Abstract: Mucosal vaccination is one of the most effective methods to prevent infectious diseases because most pathogens enter the body at mucosal surfaces. Mucosal vaccination induces not only systemic immune responses but also mucosal responses compared to parenteral vaccination that induces poor mucosal immunity. Another advantages of using mucosal vaccines are high patient compliance, low cost and easy other administration. Despite these advantages, very few mucosal vaccines are commercially available today. This is because mucosal vaccines are prone to degradation in the harsh conditions of the gastrointestinal (GI) tract lowering the bioavailability of antigens to induce immune responses. Therefore, protective and effective formulations are required for successful mucosal vaccination. Accordingly, the use of nano- and micro-polymeric particles has received much attention as delivery vehicles of antigens because they can protect the antigens from degradation in the GI tract and they also enhance the antigen uptake in mucosal-associated lymphoid tissue. Particularly, mucoadhesive polymeric carriers are the most promising vehicles for mucosal vaccine delivery because these carriers retain the vaccines on the mucosal tissues for longer period thus improving the bioavailability of the antigens. Most importantly, M cells on the follicle-associated epithelium of the Peyer’s patch play a key role in mucosal infection and immunity because they uptake and deliver antigens across mucosal epithelia to the lymphoid tissues via transcytosis. In this review, we dig the role and characteristics of M cells on mucosal immunization and explore the molecules of M cells for targeted delivery of antigens by polymeric particle system.

Keywords: M cell targeting, mucoadhesive polymeric carriers, mucosal immune system, polymeric particle system, vaccine.

1. INTRODUCTION

Mucosal surfaces are the most common portal of entry for pathogens, as they cover an enormous surface area (400 m2) in the human body [1]. Therefore, the mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory immunoglobulin A (sIgA) daily to protect the mucosal surface from pathogens [2]. Protective mucosal immune responses are most substantially induced by mucosal immunization. Despite this evidence, the majority of vaccines in use today are administered by injection although it does not induce an efficient production of antigen-specific sIgA. Vaccination through the mucosal route has several advantages over parenteral vaccination. First, mucosal immunization induces antigen-specific sIgA responses at the site of infection, which is main effector of mucosal immune system as the first line of defense against pathogens. Second, the immunization at one mucosal site yields specific responses at distant sites known as the common mucosal immune system due to the expression of

*Address correspondence to these authors at the (Y-J. C) Department of Agricultural Biotechnology & Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea; Tel: +82-2-880-4807; Fax: +82-2-875-7340; E-mail: cyjcow@snu.ac.kr (C-S. C) Department of Agricultural Biotechnology & Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea; Tel: +82-2-880-4868; Fax: +82-2-875-2494; E-mail: chocs@snu.ac.kr

mucosa-specific homing receptors by primed lymphocytes. Third, mucosal vaccination also induces systemic IgG responses for representing a further defense against invasion by microorganisms. Fourth, the mucosal immunization elicits cross-protection immunity mediated by sIgA and CD8+ cytotoxic T lymphocytes. Fifth, the mucosal administration of the vaccines is non-invasive and needle-free, which can avoid the problems of transmissible infections in blood. Finally, mucosal vaccination is relatively easy, patient compliance and cost-effective than injectable vaccines because it does not require trained medical personnel.

There are several routes of vaccine administration in mucosal immunization, such as oral, intranasal (IN), pulmonary, rectal, vaginal, and sublingual delivery. The advantages and disadvantages of several routes of mucosal immunization are summarized in Table 1.

Despite the several advantages of each mucosal site of vaccine delivery, the routes of administration appear to be limited to oral and nasal for human use because of their simplicity. The oral route of mucosal immunization has several benefits [3]. First, immune responses are induced at the site of primary colonization of pathogens. Second, it elicits mucosal immune response to other sites such as upper respiratory tract, mammary gland. Third, it demonstrates safety, efficacy and high patient compliance rates because the vaccines are administered by the natural route of infection. Fourth, the vaccine formulation of higher stability such as solid tablets is available for oral administration.

2212-3903/14 $58.00+.00 ©2014 Bentham Science Publishers

10 Current Drug Therapy, 2014, Vol. 9, No. 1 Park et al.

IN administration also has unique advantages [4-7]. First, it is effective to protect from airborne pathogen infection which starts from nasal cavity. Second, it can avoid the degradation of the vaccine caused by digestive enzymes and bacterial proteases. Third, it requires a smaller antigen dose because of easier access to mucosal immune induction sites. Fourth, it can generate immune response on respiratory, gastric and genitourinary tracts. Finally, IN administration poses potential for direct-to-central nervous system delivery because of the proximity of nasal mucosa to the brain which is beneficial to brain targeted vaccine [8].

For induction of the immune responses by orally or nasally administered vaccines, dendritic cells (DCs) should capture antigens and present them to T cells. At the sites of organized mucosal lymph nodes from gut-associated lymphoid tissue (GALT) or nasal-associated lymphoid tissue (NALT), antigens easily translocate across the epithelial barrier through specialized M cells and are presented to adjacent mucosal-T cell area after being captured by underlying DCs. The antigens also can be sampled by DC under the epithelial layer or between the epithelial cells. However, most of orally or nasally administered antigens cannot reach to the inductive sites for mucosal immune response or rather elicit the immune tolerance because of their lower bioavailability under harsh environment. The pre-systemic enzymatic degradation in the GI tract, low pH in the stomach and the intestinal membrane as a physical barrier are challenges associated with oral administration [9]. Moreover, the short residence time within the nasal cavity due to mucociliary clearance and low permeability of the

vaccine formulation are major limitations of IN administration [10-12]. Despite the obvious advantages of mucosal vaccinations, only limited vaccines have been approved for clinical uses [13].

To address the challenges, mucosal vaccines have been designed to include components that promote the immuno- genicity. In particular, much efforts have been directed toward the development of mucosal delivery system for protection of antigen from mucosal environment and access to immune inductive sites [14]. Here, we review the polymeric nano- and micro- carriers available in oral and nasal vaccine delivery and M cell targeting strategies recognizing the presence of mucosal-associated lymphoid tissue (MALT) on gut and nasal cavity.

2. POLYMERIC CARRIERS FOR MUCOSAL DELIVERY OF ANTIGENS

It is now apparent that antigens delivered in carriers are better recognized by innate immune system than soluble antigen and less affected by harsh mucosal environment. An ideal delivery system requires efficient encapsulation and protection of antigens from enzymatic degradation and ability to cross over epithelium through M cells for access to MALT. In this section, polymeric particles as adjuvant will be covered for oral and nasal delivery of antigens.

Recently, the use of polymeric nano- and micro- particles as delivery vehicles for mucosal vaccination has received much attention because they facilitate the vaccine uptake by M cell and promote the recognition by mucosal DCs in

Table 1. Advantages and disadvantages of delivery routes of mucosal immunization.

Route Advantages Disadvantages

Oral Safe and easy administration High patient compliance without pain No need of trained medical personnel

Easy soak up Immune responses in systemic and mucosal sites

Induction of tolerance in some animals Requirement of large dose of antigens

Instability of antigens against the harsh condition of the GI

Nasal Easy administration No need of needles and syringes

No need of trained medical personnel Avoidance of degradation of antigens by digestive enzymes

Generation of cross-protective immunity in the gut Immune responses in systemic and mucosal sites

Side effects of Bell’s palsy Damage to olfactory nerves and nasal epithelium

Low bioavailability of antigens Occurrence of induction of immunological tolerance

Pulmonary Humoral and cellular immune responses in systemic and mucosal sites Administration of dry powder or liquid formulations

Requirement of device Risk of exacerbation of respiratory infections

Difficulty in administration to congested patients and infants Difficulty in controlling dose of antigens

Rectal Response of specific antibodies and T lymphocyte in small animals Modest levels of local IgG and IgA in human Difficulty in quantification of antibodies in rectal sites

Vaginal Ability to by-pass first pass metabolism Easy administration

Poor absorption across the vaginal epithelium Local irritation

Personal hygiene

Sublingual Quick absorption of antigens Induction of IgG at systemic sites No need of needles and syringes

Difficulty in controlling dose of antigens Difficulty in formulation of antigens

Lack of strong adjuvants

Mucosal Delivery of Vaccine by M Cell Targeting Strategies Current Drug Therapy, 2014, Vol. 9, No. 1 11

addition to the protection of antigen [15]. Generally, the polymeric carriers should be biocompatible, and biodegradable for pharmaceutical applications [16]. For optimal vaccine formulation, the physicochemical properties such as particle size and surface charge of the polymeric carriers should be considered. Generally, the particle uptake rate of M cells increases as the particle size decreases [17] although no definite boundaries for particle size have been established [18]. It has been suggested that positive zeta potential of the particles is better for M cell transport because the M cell membrane is negatively charged, however, it may provide non-specific interaction with mucus

and epithelial cells which carry negative charges [13]. Furthermore, biophysical, biochemical and physiological characteristics of antigens such as their biological half-life, molecular size, immunogenicity, conformation stability, dose, site and rate of administration, pharmacokinetics and pharmacodynamics should be considered for the development of effective polymeric carrier-based vaccine delivery [19].

A number of polymeric particles made from chitosan (CS), poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their copolymers are in use for vaccine delivery because they can protect the antigen against degradation on mucosal surfaces and enhance antigen uptake in MALT [20].

Table 2. Polymer-based particles for oral vaccination.

Polymer Method Antigen Size (µm) Characteristics Ref.

Chitosan and chitosan derivatives

Chitosan and N-trimethyl chitosan

(TMC)

Ionic gelation Ovalbumin (OVA)

0.290 - TMC nanoparticles has intrinsic adjuvant effect [24]

Chitosan Coacervation/precipitation Bovine serum album (BSA)

0.135-0.435 - Encapsulation of BSA-loaded CS nanoparticles into vesicles and higher serum IgG titers

[25]

Chitosan Ionic gelation No antigen 1.7 - Construction of M cell model by coculture of Caco-2 with Raji-cells

[26]

Chitosan Coacervation/precipitation OVA 4.3 - Only microparticles smaller than 10 µm can be taken by M cells

[27]

Chitosan Ionic gelation Tetanus toxoid (TT)

-Strong enhancement of systemic and local immune responses

[28]

Chitosan Coacervation/precipitation Diphtheria toxoid (DT)

4.7 - Enhancement of both systemic and local immune responses

[29]

Chitosan/alginate Ionic gelation BSA 1.0 - Prevention of immediate release of antigen by coating with alginate

[21]

Chitosan/alginate Coacervation/precipitation OVA 0.3-0.6 - Increased stability of CS particles [20]

TMC/alginate Ionic gelation BSA/urease 1-5 - Higher antibody titers of IgG and IgA [22]

Chitosan/Eudragit Ionic gelation OVA 47.9-161.1 - More efficient induction of IgA by coating with Eudragit [30]

Chitosan/thiolated Eudragit

Ionic gelation BSA 1.83 - Improvement of mucoadhesiveness and residual time at the target site

[31]

Chitosan/albumin Coacervation/precipitation Typhoid Vi® 9 - Induction of systemic and mucosal immune response [32]

PLA, PGA and their copolymers

PLGA W/O/W OVA 0.2-4.1 - Carboxymethylethylcellulose-stabilized microparticles induced higher IgG and IgA antibodies

[38]

PLGA W/O/W Hepatitis B surface(HBS)

10 - Induction of serum IgG and IgM [39]

PEG-PLA-PEG W/O/W HBS 0.1-0.2 - Effective humoral immune response along with the mucosal and cellular immune response

[40]

PLGA W/O/W OVA 0.32 - Systemic and mucosal immune response by codelivery of OVA and immunostimulant

[41]

PLGA W/O/W HBS 0.39 - Lectin-anchored nanoparticles increased four folds interaction with bovine submaxillary mucin

[42]

Polymer-based particles for mucosal vaccination are summarized in Table 3.

12 Current Drug Therapy, 2014, Vol. 9, No. 1 Park et al.

Particularly, CS and its derivatives have been used as carriers for the mucosal delivery of vaccines because they have biocompatibility with cationic charges, mucoadhesiveness and the ability to open the tight junctions guarding paracellular pathway, thus facilitating cellular permeability of antigens [18, 20]. Besides, hybrid particles that contain alginate in addition to CS have been developed [20-22] to improve the stability and to enhance pharmacological performance than conventional CS particles. CS-based particles can be easily prepared by ionic gelation with negatively charged polyanions in aqueous solution which provides the mild conditions for the loading of antigens [23]. CS-based particles have been developed for oral vaccination of several antigens [20-22, 24-32].

PLA, PGA and their copolymers have been used as carriers for the oral delivery of vaccines because they have biocompatibility, biodegradability and controlled release capacity from several days to months [33]. They are already approved for a number of clinical applications in humans by FDA with a number of marketed products, although there is no licensed vaccine system using these polymers [15]. The surface modifications of the polymers were performed by conjugation of ligands with functional groups of the polymers to improve the active targeting properties [34-36]. The most widely used method for the preparation of PLA, PGA and their copolymers particles is water-in-oil-in-water (W/O/W) double emulsion method using dichloromethane as the solvent. The aqueous antigen solution is emulsified into the polymer solution by sonication followed by homogenization of the water-in-oil emulsion in poly(vinyl alcohol) as the emulsifier [37]. Nanoprecipitation and oil-in-water emulsification have been also used for the loading of antigens [18]. PLA, PGA and their copolymers-based particles have been developed for oral vaccination of various antigens [38-43]. Polymer-based particles for oral and nasal vaccination are summarized in Tables 2 and 3 [44-64], respectively.

3. MUCOADHESIVE POLYMERS

3.1. Role of Mucoadhesive Polymers on Mucosal Immune Responses

Mucoadhesive polymeric carriers are one of the promising candidates for mucosal vaccine delivery because they have several advantages, such as reduction of degradation of the vaccine by enzymes at the absorption membrane, provision of controlled release of antigens and reduction of frequency of antigen administration, and provision of a prolonged retention time of carriers on mucosal tissue, leading to antigen absorption for an extended period of time and thus improving bioavailability of the antigens [9, 65]. Mucoadhesion of the polymers to tissues may be achieved by physical bonds, and/or secondary chemical bonds come from hydrophobic interaction, Van der Waals interaction and hydrogen bonds. Generally, the hydrophilic functional groups responsible for forming hydrogen bonds are carboxyl groups, amine groups and hydroxyl groups [66].

New generation of mucoadhesive polymers, called as thiomers or thiolated polymers [67], have stronger mucoadhesiveness due to covalent bonding of the thiol groups of the mucins and thiolated polymers by forming stable disulfide brides. They increase local concentrations of

glutathione by decreasing oxidized-glutathione in the intestine epithelia and the glutathione inhibits protein tyrosine phosphate involved in the closure of tight junctions by dephosphorylation of occluding by forming disulfide bonds with an active enzyme [68]. The inhibition of protein tyrosine phosphate results in prolonging the opening of tight junctions and cell-cell adhesions, and accompanied by enhanced permeability of the antigens [69].

3.2. Application of Mucoadhesive Polymers for Vaccine Delivery

Mucoadhesive polymers are classified into synthetic and naturally derived polymers. The synthetic mucoadhesive polymers include carbopol, polycarbophil, poly(acrylic acid), polyacrylate, poly(methylvinylether-co-methacrylic acid), poly(2-hydroxyethyl methacrylate), polymethacrylate, polyalkylcyanoacrylate, poly(vinyl pyrorolidone), poly(vinyl alcohol) and thiol-containing synthetic polymers [9]. The naturally derived polymers include carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, methylhydro- xyethyl cellulose, alginate, dextran, starch, hyaluronic acid, chitosan, guar, xanthan, and thiol-containing natural polymers [9]. The mucoadhesive polymers should have ideal characteristics [66], such as high molecular weight, optimum surface tension, hydrophilicity, hydrogen bonding capacity, sticky to mucus glycoproteins, non-toxicity and non-allergenicity, chemical inertness and cost-effectiveness. Mucoadhesive particles used in mucosal vaccine delivery are summarized in Table 4.

4. M CELLS TARGETING STRATEGY FOR MUCOSAL IMMUNIZATION

4.1. Function of M Cells

M cells located on the follicle-associated epithelium (FAE) of the Peyer’s patch (PP) follicle as shown in Fig. 1 as well as in Bronchus-associated lymphoid tissue (BALT) play a key role in mucosal infection and immunity. Because they have a unique ability to uptake and deliver antigens across mucosal epithelia to the underlying lymphoid tissues via transcytosis where protective immune responses are generated [83]. The proportion of M cells in FAE is species-dependent. The percentage of M cells out of the total cells of the FAE for the rabbits and mini-pigs are over 20% whereas that of the cells for rodents and humans is around 10% and below 5% respectively [84]. The M cells can be exploited by microorganisms as the port of entrance whereas the epithelium of the gut intestine provides an effective barrier to the entrance of most pathogens [3]. Although the origin of M cells is the subject of much debate, it is known that they originate from stem cells in crypts located between a villus and a PP dome. [85]. During migration up to the dome, stem-cells from crypts are differentiated into M cells with the assistance of B cells towards the optimum function in antigen translocation [14].

4.2. M Cell Targeting

The ability of the M cells in PP to take up diverse microorganisms to antigen-presenting cells makes M cells an ideal target for vaccine delivery to the immune system [2]. However, the existence of low numbers of M cells in the intestinal tract makes it necessary to target the specific

Mucosal Delivery of Vaccine by M Cell Targeting Strategies Current Drug Therapy, 2014, Vol. 9, No. 1 13

Table 3. Polymer-based particles for nasal vaccination.

Polymer Method Antigen Size (µm) Characteristics Ref.

Chitosan and chitosan derivatives

Chitosan Coacervation/precipitation DT 4.7 - Enhancement of both systemic and local immune responses

[29]

TMC Coacervation/precipitation Group C meningococcal

conjugated vaccine (CRM-MenC)

5 - Enhanced systemic and mucosal antibodies by mucosal adjuvant LTK 63

[44]

Chitosan Ionic gelation Bordetella bronchiseptica

dermonecrotoxin (BBD)

4.39 - Secretion of tumor necrosis factor (TNF) and nitric oxide in RAW264.7 cells

[45]

Chitosan Ionic gelation OVA, CT 0.4-3 - Induction of systemic immune response in rats [46]

Chitosan Ionic TT 0.35 - Increased and long-lasting humoral immune response by low molecular weight chitosan

[47]

Chitosan Complex coacervation Hepatitis B DNA 0.34 - Humoral (both systemic and mucosal) and cellular immune responses

[48]

TMC Complex coacervation HBS 0.29 - Induction of stronger humoral and mucosal immunity than alum

[49]

Carboxymethyl chitosan(CMC)/TMC

Complex coacervation TT 0.28 - Induction of both mucosal and systemic immune response

[50]

TMC Ionic gelation Influenza subunit 0.8 - Induction of high IgA and IgG titers [51]

Chitosan Ionic gelation BBD 5.23 - Induction of systemic IgA and IgG titers in sera [52]

Pegylated chitosan Ionic gelation BBD 5.73 - Improvement of stability of microparticle [53]

Mannosylated chitosan

Ionic gelation BBD 5.23 - Increase of immune stimulatory activity in vitroin vivo through specific targeting and

activation of macrophages

[54]

Chitosan/F127 Ionic gelation BBD 5.21 - Higher BBD specific IgA antibody responses due to enhanced delivery of antigen by F127

[55]

Chitosan/alginate Coacervation/precipitation HBS 0.64 - Th1-biased antigen-specific systemic immune response was obtained by codelivery of CpG

oligodeoxynucleotide

[56]

TMC/alginate Ionic gelation BSA 1.0 - Decrease of initial burst release of BSA [57]

PLA, PGA and their copolymers

PLA W/O/W Yersinia pestis V antigen

1.5 - Translocation into nasal associated lymphoid tissue

[58]

PLA W/O/W Streptococcus equi 0.41 - Induction of Th1 and Th2 response after modification by nucoadhesive glycol chitosan

[59]

PLA-PEG block copolymers

W/O/W HBS 0.10-0.15 -PEG-PLA-PEG block copolymer induced high and prolonged immune response

[60]

PLA-PEG W/O/W TT 0.2, 1.5, 5, 10 - Nanosizes enhanced more absorption of antigen than microsize

[61]

PLGA/poly(epsilon-caprolactone) (PCL)

W/O/W DT 0.25 - Hydrophobic nanoparticles induced more immune response

[62]

PLGA/poloxamer (or poloxamine)

Solvent diffusion Beta-galactosidase encoding DNA

0.16-0.19 - Fast and strong immune response [63]

PLGA/chitosan (or TMC)

W/O/W HBS 0.47-0.49 - Increased immune response due to enhanced absorbed antigen by the mucoadhesive polymers

[64]

14 Current Drug Therapy, 2014, Vol. 9, No. 1 Park et al.

Table 4. Application of mucoadhesive particles for mucosal vaccine delivery (modified from ref. [55]).

Polymer Route Antigen Characteristics Ref.

Natural

Alginate Oral Rotavirus Induction of antigen specific intestinal IgA [70]

Alginate Oral Pasteurella multocida Induction of antigen specific nasal lavage IgA and protective immunity

[71]

Alginate Nasal TT Induction of antigen specific serum IgG, antitoxin titers and nasal lavage IgA

[72]

Chitosan Oral TT Induction of systemic and mucosal immune response [28]

Chitosan Oral Lymphocystis disease virus DNA Induction of antigen specific serum antibodies [73]

Dextran Nasal TT Induction of antigen specific serum IgG and antitoxin [74]

Carboxymethyl starch Oral F4 fimbriae with CpG Increase of F4-specific IgM and IgG in intestinal secretions [75]

Starch Peritoneal Plasmodium berghei apical major antigen

Reduction of parasitemia with an extension of life span [76]

Polyacryl starch Oral Human serum albumin(HSA) Induction of antigen specific serum IgM/IgG and fecal IgA [77]

Polyacryl starch Oral/nasal DT Induction of systemic and mucosal immune response [78]

Starch grafted with TS-PDMS

Oral HSA Induction of antigen specific serum IgG1 [79]

Hyaluronic acid Nasal Influenza virus Induction of antigen specific serum IgG, hemagglutination inhibition titers and nasal wash IgA

[80]

Synthetic

Carboxy vinyl polymer Gastrointestinal OVA Induction of antigen specific serum IgG and fecal IgA [81]

Carbopol Oral pseudorabies virus Induction of antigen specificserum IgG [82]

receptors on the apical surface of M cells to increase the uptake and presentation of antigens thereby initiating the immune response and inducing protection against infection [13]. In this section, we will discuss several ligands for M cell targeted vaccine delivery.

4.2.1. Lectin-Mediated Targeting

Lectins are proteins and glycoproteins which specifically recognize and reversibly bind to carbohydrate residues present on cell surface proteins or lipids. Most lectins originated from plant or bacterial origin are relatively resistant to intestinal degradation [86]. The most studied lectin is ulex europaeus agglutinin 1 (UEA-1), which binds to α -L-fucose residues expressed on the apical surface of mouse M cells [87]. When hepatitis B surface (HBS) antigen-loaded PLGA nanoparticles conjugated with UEA-1 were administered orally, they predominantly associated with M cells and elicited a significantly higher sIgA response than untargeted nanoparticles or intramuscular immunization [88]. Similarly, birch pollen allergen-loaded PLGA microparticles conjugated with Aleuria aurantia lectin protected the entrapped allergens from gastric degradation and induced birch pollen-specific IgG2a after oral immunization into BALB/C mice [89]. On the other hand, the plant lectins as targeting ligand for oral vaccine delivery

have several drawbacks, such as toxicity, anti-nutritional properties and potential susceptibility to degradation once coated on the surface of the particles [90]. Moreover, the use of plant lectins in vaccine delivery might be limited because the glycosylation pattern in the intestine differs between enterocytes and M cells at different intestinal locations, with age and between species [43].

4.2.2. Pathogen-Exploited Molecules

Among pathogens, microbial adhesins have been used as ligands to M cells because they can interact with integrin expressed on the apical surface of M cells [12] and they are relatively resistant to intestinal degradation [90]. It was reported that F4 fimbriae binds to specific F4 receptors present on the surface of porcine enterocytes and M cells and these F4 receptors mediate transcytosis of F4 fimbriae across the intestinal epithelium [91]. Hence, the conjugation of F4 fimbriae with antigens induced enhanced mucosal antigen-specific antibody responses after oral administration [92]. By tracking the routes that pathogens utilize to invade the body, many receptors on M cells have been identified. Recently, glycoprotein 2 (GP2) was identified as transcytotic receptor on M cells for selective binding of certain pathogens by recognizing FimH, a component of type I pili on the bacterial outer membrane [2]. Yersinia-derived invasin

Mucosal Delivery of Vaccine by M Cell Targeting Strategies Current Drug Therapy, 2014, Vol. 9, No. 1 15

showed enhanced antigen-targeting and antigen-specific immune responses through interaction with β1 integrin expressed on the apical surface of M cells [93].

Reovirus invades intestinal M cells in rodents and rabbits by the interaction of the outer capsid protein σ1 with α2,3-sialic acid expressed on the apical membrane [94]. Incorporation of recombinant σ1 into liposomes enhanced binding to rat PP [95]. Brucella abortus invades the host through M cells by prion protein (C) on the apical surface of M cells [96]. Similarly, Kim et al. [97] used complement C5a receptor to Co1 ligand and they confirmed its non-redundant role as a target receptor for dengue virus antigen to M cells through oral immunization. Lo et al. identified the Claudin 4 as an M cell target receptor and developed a Claudin 4 targeting peptide (CPE) from c-terminal domain of the Clostridium perfringens enterotoxin which mediates enhanced uptake by M cell in vitro and in vivo [98, 99]. The incorporation of CPE into PLGA nanoparticles mediated targeted delivery into both NALT and GALT systems [100] while CPE-hemagglutinin (CPE-HA) fusion antigen induced a significantly higher IgA response in both serum and fecal pellets [101].

Cholera toxin (CT) from Vibrio cholera and heat-labile enterotoxin from enterotoxigenic E. coli act as potent mucosal adjuvants and enhance antigen-specific mucosal and systemic immune responses to coadministered antigens

through oral immunizations in rodents and pigs [102]. Similarly, oral administration of Salmonella-derived flagellin-coated OVA-loaded polyanhydride nanoparticles enhanced their uptake by PP and provided higher systemic and mucosal antibody responses [103].

4.2.3. Antibody-Mediated Targeting

More than two decades ago, Pappo et al. [104] reported that polystyrene microparticles conjugated with anti-M- cell monoclonal antibody 5B11 were more efficiently internalized by M cells than native particles after inoculated into the rabbit intestinal loops. Nochi et al. [105] also reported that NKM 16-2-4 conjugated-botulinum toxoid with the mucosal adjuvant CT induced strong antigen-specific mucosal IgA responses and protective immunity against lethal challenge with the toxin. Hase et al. [2] identified GP2 as a receptor for some bacteria expressing FimH because anti-GP2 antibodies have shown to bind to both human and murine M cells, suggesting that it may be useful for oral antigen delivery.

4.2.4. Peptide-Mediated Targeting

The peptide ligands have several advantages because peptides can be synthesized by chemical methods on a large scale and the peptides can be easily conjugated with carriers. There are two methods to screen peptide libraries either by chemical synthesis or phage display. Receptor-mediated

Fig. (1). Illustration of M cell targeting mucoadhesive oral vaccine system functioning in mucosa-associated lymphoid tissue to promote the immunogenicity.

Mucoadhesive polymer

M-cell targeting

ligandAntigen

Peyer’s patch

HEVMesentericlymph node

M cell

FAE

Lamina propria

Crypt

Mucus layer

sIgA

Dendriticcell

Plasma cell

HEV

16 Current Drug Therapy, 2014, Vol. 9, No. 1 Park et al.

endocytosis is a process that transports peptide ligands into target cells through a receptor-ligand specific interaction. The peptide ligands and their receptors accumulate in coated pits and are internalized as receptor-ligand complexes [106]. Higgins et al. [107] identified targeting peptide ligands for M cells using in vivo phage display screening. Among 30 unique peptide sequences bound to PP, human Caco-2 and rat IEC-6 epithelial cells by successive screenings across four cycles of selection, two peptides such as P8 (LETTCASLCYPS) and P25 (VPPHPMTYSCQY) were shown to bind to receptors on the surface of human intestinal tissue. Besides, D-peptide analog of mutant peptide P8 (YGCSYTMPHPPV) enhanced polystyrene nanoparticles to M cells in a mouse model.

Similarly, Yoo et al. [108] selected a M cell-homing peptide ligand, CKSTHPLSC (CKS9), using the phage display technique. The target specificity was confirmed by an in vitro transcytosis assay and in vivo assay. As depicted in Fig. 2, CKS9-conjugated chitosan nanoparticles (CKS9-CNs) were accumulated more specifically into PP regions than chitosan nanoparticles, suggesting that CKS9-CNs

could be used as an efficient oral vaccine carrier. They also loaded membrane protein of Brachyspira hyodysenteriae (BmpB) as a vaccine against swine dysentery into porous PLGA microparticles coated with CKS9-coupled chitosan [109]. As shown in Fig. 3, oral immunization of BmpB vaccine with CKS9-chitosan-PLGA microparticles in mice showed elevated sIgA responses in the intestine and excreta as well as systemic IgG antibody responses due to the enhanced M cell targeting and transcytosis ability of CKS9-chitosan-PLGA microparticles. These two studies suggest that particulate vaccine using M cell homing peptide-CKS9 will be a promising tool for targeted oral vaccine delivery.

Fievez et al. [110] identified M cell targeting peptide by phage display using an in vitro model of the human FAE. Among different peptide sequences, CTGKSC and LRVC sequences that occur at high frequency enhanced phage transport across M-like cells. In consequence, CTGKSC- or LRVG-coupled PCL/PEG nanoparticles were highly transported into the FAE than PCL/PEG themselves. Kim et al. [111] selected M cell targeting peptide through biopanning of a phage display library using an in vitro M-

Fig. (2). Effect of M cell-homing peptide on in vitro adhesion of chitosan nanoparticles by M cell model. Green and red fluorescent signals in each panel indicate the chitosan nanoparticles and in vitro M cell layer, respectively. Control is the in vitro M cell layer without treatment of CKS9-CNs or CNs [108].

Mucosal Delivery of Vaccine by M Cell Targeting Strategies Current Drug Therapy, 2014, Vol. 9, No. 1 17

like cells and produced the recombinant model antigen fused to the selected ligand. SFHQLPARSPLP as one of the selected peptide ligand promoted the binding of ligand-fused antigen to mouse M cells. Further, the ligand enhanced the uptake of fused antigen into immunogenic tissue in an ex vivo loop assay and the ligand-fused antigen increased antigen-specific responses compared with those of the antigen without ligand after oral immunization in mice, indicating that the selected ligand can be used for targeted antigen delivery.

CONCLUSIONS

Mucosal vaccination has several advantages over the parenteral vaccines because it can induce both systemic and mucosal immune responses. However, instability of antigens against the harsh conditions of GI tract through oral administration and low bioavailability of antigens through nasal administration should be overcome. Therefore, to develop effective mucosal vaccination, the use of polymeric particle system for protection of antigens, mucoadhesive polymeric system for a prolonged retention time of carriers on mucosal tissue and M cell targeting ligands for delivery of antigens to the lymphoid tissues via transcytosis would be of great advantages. In this review, we explained the current

state of knowledge of polymeric particle system, mucoadhesive polymeric system and M cell targeting ligands for the development of effective mucosal vaccines. Although polymeric carriers have received great attention in the field of vaccine, there is no particulate delivery system approved to be used in human mucosal vaccine due to its poor immunogenicity. We expect that the combined system of mucoadhesive and M cell targeted polymeric particle systems will allow the development of the next generation of mucosal vaccination.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

This research was supported by Animal Disease Management Technology Development, Ministry of Agriculture, Food and Rural Affairs.

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Received: February 26, 2014 Revised: April 09, 2014 Accepted: May 20, 2014